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Structure-Function Relationship of Channel Catfish (Ictalurus punctatus) Muscle Proteins Subjected to pH-Shift Processing

Permanent Link: http://ufdc.ufl.edu/UFE0021621/00001

Material Information

Title: Structure-Function Relationship of Channel Catfish (Ictalurus punctatus) Muscle Proteins Subjected to pH-Shift Processing
Physical Description: 1 online resource (238 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: byproducts, catfish, muscle, ph, rheology, texture
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Novel processing utilizing pH-shift technology applied to muscle-based systems modifies or increases raw product utilization. The use of pH-shift processing on muscle products and by-products allows for the separation of muscle proteins from lipids, collagen, skin, bones and other undesirable components. Undesirable materials are not easily separated from whole muscle or other comminuted muscle products such as surimi. During pH-shift processing, animal and fish muscle including channel catfish muscle is modified by the reduction of lipid and heme content, altering protein composition and thermal gelation properties. Reduction of lipid and heme content increases whiteness and oxidative stability of protein isolates. Changes in protein composition are associated with the removal of sarcoplasmic proteins including heme proteins, some endogenous enzymes, collagen and other protein components. Modification of thermal gel properties induced variable gel strengths. It was hypothesized that pH-shift processing will structurally modify channel catfish muscle proteins resulting in functional properties leading to the expansion, utilization and production of muscle based products. The model system in this study was developed to provide the basis for implementation of economical and environmental utilization of current seafood by-products by pH-shift processing. The results show that pH-shift processing modifies muscle proteins structurally and functionally. pH-shift processing reduced the relative content of alpha helix to beta structure of muscle proteins after isoelectric precipitation indicating a molten globular state. Muscle proteins in a molten globular state showed reduced myosin ATPase activity, altered protein surfaces, modified thermal sensitivity, increased susceptibility to enzymatic crosslinking and modified solubility. These changes lead to changes in the physical properties of pH-shift processed muscle proteins. Alkali processed catfish showed increased gel rigidity, gel strength and gel flexibility compared to acid processed catfish which exhibited inconsistent functional performance, increasing and decreasing in gel rigidity, gel strength and gel flexibility. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a broad range of applications by inducing structural modification of muscle proteins. This innovative process yields muscle proteins with good color, high oxidative stability and modified functionality, which can find many applications in formulated comminuted muscle food products. Furthermore, this technology can be an important contribution to better utilization of seafood and land animal by-products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Kristinsson, Hordur G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021621:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021621/00001

Material Information

Title: Structure-Function Relationship of Channel Catfish (Ictalurus punctatus) Muscle Proteins Subjected to pH-Shift Processing
Physical Description: 1 online resource (238 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: byproducts, catfish, muscle, ph, rheology, texture
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Novel processing utilizing pH-shift technology applied to muscle-based systems modifies or increases raw product utilization. The use of pH-shift processing on muscle products and by-products allows for the separation of muscle proteins from lipids, collagen, skin, bones and other undesirable components. Undesirable materials are not easily separated from whole muscle or other comminuted muscle products such as surimi. During pH-shift processing, animal and fish muscle including channel catfish muscle is modified by the reduction of lipid and heme content, altering protein composition and thermal gelation properties. Reduction of lipid and heme content increases whiteness and oxidative stability of protein isolates. Changes in protein composition are associated with the removal of sarcoplasmic proteins including heme proteins, some endogenous enzymes, collagen and other protein components. Modification of thermal gel properties induced variable gel strengths. It was hypothesized that pH-shift processing will structurally modify channel catfish muscle proteins resulting in functional properties leading to the expansion, utilization and production of muscle based products. The model system in this study was developed to provide the basis for implementation of economical and environmental utilization of current seafood by-products by pH-shift processing. The results show that pH-shift processing modifies muscle proteins structurally and functionally. pH-shift processing reduced the relative content of alpha helix to beta structure of muscle proteins after isoelectric precipitation indicating a molten globular state. Muscle proteins in a molten globular state showed reduced myosin ATPase activity, altered protein surfaces, modified thermal sensitivity, increased susceptibility to enzymatic crosslinking and modified solubility. These changes lead to changes in the physical properties of pH-shift processed muscle proteins. Alkali processed catfish showed increased gel rigidity, gel strength and gel flexibility compared to acid processed catfish which exhibited inconsistent functional performance, increasing and decreasing in gel rigidity, gel strength and gel flexibility. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a broad range of applications by inducing structural modification of muscle proteins. This innovative process yields muscle proteins with good color, high oxidative stability and modified functionality, which can find many applications in formulated comminuted muscle food products. Furthermore, this technology can be an important contribution to better utilization of seafood and land animal by-products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Kristinsson, Hordur G.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021621:00001


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STRUCTURE/FUNCTION RELATIONSHIP OF CHANNEL CATFISH (Ictalurus puncta~tus)
MUSCLE PROTEINS SUBJECTED TO pH-SHIFT PROCESSING




















By

MATTHEW PAUL DAVENPORT


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008


































O 2008 Matthew Paul Davenport




































To my Lord and Savior Jesus Christ, my wife Alyson and the past three generations of my
family, thank you for paying the way making this milestone possible and welcoming me to your
ranks









ACKNOWLEDGMENTS

I thank my supervising committee chair for his mentorship, his friendship, and for giving

me the opportunity to pursue a life long dream. I thank my supervisory committee (Jess F.

Gregory, W. Steve Otwell, and James F. Preston) for their valuable expertise, instruction, and

contributions to the scientific community and general public through exceptional scholarship and

genuine concern. I thank Southern Pride Catfish LLC for providing raw materials and extending

my experiences during this process.

I thank my Lord Jesus Christ for His stead fast love. I thank my parents Dr. Paul and

Cherith Davenport for a lifetime of support, encouragement and tolerance; though your acts of

love this accomplishment was not only possible but given true meaning. To my wife thank you

for your years of love, patients and the embodiment of love. Your devotion over the years has

allowed me to reach this point. To Jeremy Jacobson, Jeremy Hershey, Ross Dubose and my

many friends for the encouragement and j oyful times allowing me to reach this goal with some

sanity intact. I thank all of my lab peers for giving me their time, knowledge, adventures and

laughs.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................. ...............9..____ .....


LIST OF FIGURES .............. ...............10....


AB S TRAC T ............._. .......... ..............._ 14...


CHAPTER


1 INTRODUCTION ................. ...............16.......... ......


Background ................. ...............16.................
pH- Shift Processing ................. ...............18.......... ......
Protein Gelation ................. ...............21.................
Rheology ................. ........ _._ .... .... ......... ... .... .........2
Key Muscle Proteins of Interest for the pH-shift Process ................. ..........................31
Obj ectives ................. ...............33.......... .....
Experimental Design .............. ...............35....

2 METHODS .............. ...............40....


Raw Material ................... ... ...............40

Preparation of Protein Isolates ................. ...............40................
Protein Concentration ................. ...............41.................
Protein Surface Hydrophobicity .............. ...............41....
Circular Dichroism (CD) .............. .... ... .... .. ..........................4
Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HC1) ..........................42
Differential Scanning Calorimetry (DSC) ................ ...............43................
Reactive Sulfhydryl Groups .............. ...............43....
Total Sulfhydryl Groups ................. ...............44._._. ......
Myosin ATPase Activity Assay................ .......... ..............4
Susceptibility of Proteins to Transglutaminase-Induced Cross Linking .............. .... ............45
Oscillatory Rheology ............_. ...._... ...............46....
Torsion Gel Testing ............_. ...._... ...............46....
Punch Test .............. ...............47....
F01d Test ..........._... ... ....._ ...............47.....
Gel Water Holding Capacity .............. ...............48....
Cook Loss .............. .......... ..............4
Heating Rate Gelation Shtdies............... ...............48
Protein Solubility as a Function of pH ............................. ................. ..............49
Protein Solubility as a Function of Salt Concentration .............. ...............50....
Statistical Analysis............... ...............50












3 CHANNEL CATFISH (Ictalurus punctatus) MUSCLE PROTEIN ISOLATE
PERFORMANCE PROCESSED UNDER DIFFERING ACID-AIDED AND ALKALI-
AIDED pH VALUE S. ............. ...............53.....

Introducti on ................. ...............53.................
M ethods .............. ...............55....
Raw Material ....................... ...............55

Preparation of Protein Isolates ................. ...............55................
Protein Concentration ................. ...............56.................
Oscillatory Rheology ............._. ...._... ...............57....
Torsion Gel Testing ............._. ...._... ...............57....
Punch Test ............_. ...._... ...............58....
Fold Test ........._..... ..... ...._ ...............58.....
Gel Water Holding Capacity .............. ...............58....
Cook Loss ............._. ...._... ...............59....
Statistical Analysis .............. ...............59....
R e sults............._.. ..... .._ ...... ...._... ... ... .. ........ ........6
Rheological Changes in Protein Isolates During Thermal Gelation ............... ...............60
Gel Quality of Isolates as Assessed by Torsion Testing ................. ......._._ ...........63
Gel Quality of Isolates as Assessed by Punch Testing ................. ................ ....._.63
Gel Quality of Isolates as Assessed by Expressible Moisture ................. ................ ...64
Gel Quality of Isolates as Assessed Fold Test ................. ...............64..............
Gel Quality of Isolates as Assessed by Cook Loss................... ................ ........ .64
Discussion ................. ...............65.................
Conclusions............... ..............7


4 CHEMICAL PROPERTIES OF ACID AIDED AND ALKALI AIDED PROTEIN
ISOLATES FROM CATFISH (Ictahtrus punctatus) ......___ ..... ...._. ..........._.......13 3

Introducti on ........._..._.._ ...._._. ...............133....
M ethods .............. ...............136....
Raw Material ....................... ...............13

Preparation of Protein Isolates. ........._..._.._ ...............136...._._ .....
Protein Concentration ........._..._.._ ...._._. ...............137....
Protein Surface Hydrophobicity ................. ......... ...............137 .....
Reactive Sulfhydryl Groups .............. ...............138....
Total Sulfhydryl Groups............... ...............139
Myosin ATPase Activity Assay .............. ...............139....
Protein Solubility as a Function of pH ................... .................... ...........4
Protein Solubility as a Function of Salt Concentration ................. ........... ...........141
Statistical Analysis .............. ...............141....
Re sults ......._.................. ........_ ..........14

M yosin ATPase ................. ...............142......... ......
Surface Hydrophobicity ................. ...............142................
Total Sulfhydryl Groups............... ...............142
Reactive Sulfhydryl Groups ............... .... .. ...............143
Solubility of Proteins at Different Salt Levels ................. ...............143........... ..












Solubility of Proteins at Different pH Values ................. ...............143........... ..
Discussion ................. ...............143................
Conclusions............... ..............14


5 THE EFFECT OF pH-SHIFT PROCESSING ON THE STRUCTURAL AND
THERMAL PROPERTIES OF CATFISH (Ictalunts prnctatus) PROTEIN ISOLATES ..155


Introducti on ........._.__....... .__ ...............155...
M ethods .............. ...............156....
Raw Material ....................... ...............15

Preparation of Protein Isolates. ........._.___..... .__. ...............156...
Protein Concentration ........._.___..... .___ ...............157....
Circular Dichroism (CD) ................. ..... ..._._ ......... ...... ..... .......... .......15
Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HC1) .................158
Differential Scanning Calorimetry (DSC) ............... ... .... ......... ... ......... .........15
Susceptibility of Proteins to Transglutaminase-Induced Cross Linking ................... ....159
Statistical Analysis .............. ...............159....
Re sults ................. .. ....._.._.. ...............160......
Circular Dichroism (CD) .................. ............. ...... ....................16
Guanidine Hydrochloride Denaturation (Gu-HC1) ................. .......... ................1 60
Micro-Differential Scanning Calorimetry (DSC)............... ...............161
Susceptibility of Proteins to Cross-linking by Transglutaminase ................. ...............162
Discussion ................. ...............163................
Conclusions............... ..............16


6 THE EFFECT OF HEATING RATE ON pH-SHIFT PROCESSED CATFISH
(Ictaluruspunctatus) MUSCLE PROTEINS .............. ...............181....

Introducti on ................. ...............18. 1...............
M ethods ...................... ........... .......... 8
Production of Protein Isolates .............. ...............182....
Protein Composition ................. ...............182................
Torsion............... ...............182
Rheology ............ ...... ._ __ ...............183....
Statistical Analysis .............. ...............183....
Re sults............._. ...._... ...............184....
Torsion............... ...............184
Rheology ............._. ...._... ...............185....
Discussion ............._. ...._... ...............186....
Conclusions............... ..............18


7 GENE RAL DI SCU SSION ............_. ...._... ............... 197...


APPENDIX


STAT STICAL TABLES .............. ...............201....











REFERENCES .............. ...............232....

BIOGRAPHICAL SKETCH .............. ...............238....




































































8










LIST OF TABLES


Table


page


5-1: Thermal events (A cal) of isolates............... ..............18










LIST OF FIGURES


Figure page

1-1 Experimental design of the structure/function relationship of channel catfish. ...............36

1-2 Experimental design of the physical testing of channel catfish..........._...._ ...........__ ..37

1-3 Experimental design of the chemical testing of channel catfish............. ..............__ .3 8

1-4 Experimental design of the structural testing of channel catf ish. ................ ................ .39

2-1 The process used in acid and alkali pH shift processing. ............. .....................5

3-1 The storage modulus (G') of catfish protein isolates treated at pH 2.0 without NaC1. .....76

3-2 The loss modulus (G") of catfish protein isolates treated at pH 2.0 without NaCl ...........77

3-3 The tan delta (G"/G') of catfish protein isolates treated at pH 2.0 without NaCl .............78

3-4 The storage modulus (G') of catfish protein isolates treated at pH 2.5 without NaC1. .....79

3-5 The loss modulus (G") of catfish protein isolates treated at pH 2.5 without NaCl ...........80

3-6 The tan delta (G"/G') of catfish protein isolates treated at pH 2.5 without NaC1. ............81

3-7 The storage modulus (G') of catfish protein isolates treated at pH 3.0 without NaC1. .....82

3-8 The loss modulus (G") of catfish protein isolates treated at pH 3.0 without NaC1. ..........83

3-9 The tan delta (G"/G') of catfish protein isolates treated at pH 3.0 without NaC1. ............84

3-10 The storage modulus (G') of catfish protein isolates treated at pH 10.5 without NaC1. ...85

3-11 The loss modulus (G") of catfish protein isolates treated at pH 10.5 without NaC1. ........86

3-12 The tan delta (G"/G') of catfish protein isolates treated at pH 10.5 without NaC1. ..........87

3-13 The storage modulus (G') of catfish protein isolates treated at pH 11.0 without NaC1. ...88

3-14 The loss modulus (G") of catfish protein isolates treated at pH 11.0 without NaC1. ........89

3-15 The tan delta (G"/G') of catfish protein isolates treated at pH 1 1.0 without NaC1. ..........90

3-16 The storage modulus (G') of catfish protein isolates treated at pH 11.5 without NaC1. ...91

3-17 The loss modulus (G") of catfish protein isolates treated at pH 11.5 without NaC1. ........92

3-18 The tan delta (G"/G') of catfish protein isolates treated at pH 1 1.5 without NaC1. ..........93










3-19 The storage modulus (G') of untreated catfish muscle without NaC1. ............. ..... ........._.94

3-20 The loss modulus (G") of untreated catfish muscle without NaC1. ............. .............95

3-21 The tan delta (G"/G') of untreated catfish muscle without NaC1. ................ ................. 96

3 -22 The storage modulus (G') of catfish protein isolates treated at pH 2.0 with NaCl............97

3-23 The loss modulus (G") of catfish protein isolates treated at pH 2.0 with NaC1. ...............98

3 -24 The tan delta (G"/G') of catfish protein isolates treated at pH 2.0 with NaC1. .................99

3-25 The storage modulus (G') of catfish protein isolates treated at pH 2.5 with NaCl..........100

3-26 The loss modulus (G") of catfish protein isolates treated at pH 2.5 with NaC1. .............101

3-27 The tan delta (G"/G') of catfish protein isolates treated at pH 2.5 with NaC1. ...............102

3-28 The storage modulus (G') of catfish protein isolates treated at pH 3.0 with NaCl..........103

3-29 The loss modulus (G") of catfish protein isolates treated at pH 3.0 with NaC1. .............104

3-30 The tan delta (G"/G') of catfish protein isolates treated at pH 3.0 with NaC1. .............105

3-31 The storage modulus (G') of catfish protein isolates treated at pH 10.5 with NaCl........106

3-32 The loss modulus (G") of catfish protein isolates treated at pH 10.5 with NaC1. ...........107

3-33 The tan delta (G"/G') of catfish protein isolates treated at pH 10.5 with NaC1. .............108

3-.34 The storage modulus (G') of catfish protein isolates treated at pH 1 1.0 with NaCl........109

3-35 The loss modulus (G") of catfish protein isolates treated at pH 11.0 with NaC1. ...........110

3-36 The tan delta (G"/G') of catfish protein isolates treated at pH 1 1.0 with NaC1. .............11 1

3-37 The storage modulus (G') of catfish protein isolates treated at pH 1 1.5 with NaCl........1 12

3-38 The storage modulus (G") of catfish protein isolates treated at pH 1 1.5 with NaCl. ......1 13

3-39 The tan delta (G"/G') of catfish protein isolates treated at pH 1 1.5 with NaC1. .............1 14

3-40 The storage modulus (G') of untreated catfish muscle with NaC1. ................ ...............115

3-41 The loss modulus (G") of untreated catfish muscle with NaC1. ................ .................1 16

3 -42 The tan delta (G"/G') of untreated catfish muscle with NaC1. ................ ..................1 17

3-43 The storage modulus (G') before heating catfish protein without NaC1. ................... .....118










3 -44 The storage modulus (G') after heating catfish protein to 800C without NaCl. ..............119

3-45 The storage modulus (G') after cooling catfish protein to 50C without NaCl. ................120

3-46 The storage modulus (G') before heating catfish protein with NaCl ............... ... ............121

3-47 The storage modulus (G') after heating catfish protein to 800C with NaCl. ...................122

3-48 The storage modulus (G') after cooling catfish protein to 50C with NaCl. ................... ..123

3-49 The shear stress (torsion) catfish muscle with 2% NaC1. ............. ......................2

3-50 The shear strain (torsion) catfish muscle with 2% NaC1. ............. ......................2

3-51 The shear stress (punch test) catfish muscle with 2% NaCl............... .....................2

3-52 The shear stress (punch test) catfish muscle with 2% NaCl............... .....................2

3-53 The j elly strength (stress" strain, punch test) with 2% NaCl ................. .....................128

3-54 The expressible moisture of catfish muscle with 2% NaC1. ............. ......................129

3-55 The quality score (fold test) of catfish muscle with 2% NaCl.................. ................ ..130

3-56 The cook loss of catfish muscle with 2% NaCl............................ ........131

3-57 Torsion texture profile of pH-shift processed catfish muscle ................. ................ ...132

4-1 Myosin ATPase activity of catfish muscle. ............. .....................149

4-2 The surface hydrophobicity of catfish muscle. ........................... ........150

4-3 Total sulfhydryl content of catfish muscle............... .................151

4-4 Reactive sulfhydryl content of catfish muscle. .............. ...............151....

4-5 Solubility (NaC1) of catfish muscle. ............. .....................152

4-6 Solubility (pH) of acid-treated catfish muscle. ........................... ........153

4-7 Solubility (pH) of alkali-treated catfish muscle ................. ...............154........... ..

5-1 Circular dichroism of catfish muscle, acid-treated and control. ............. .....................170

5-2 Circular dichroism of catfish muscle, alkali-treated and control ................ .................1 70

5-3 a-helix, P-structureand random coil in catfish muscle ................. .........................171

5-4 Guanidine Hydrochloride (Gu-HC1, 0-2M) on catfish muscle, acid-aided and control. .172










5-5 Guanidine Hydrochloride (Gu-HC1, 0-2M) on catfish muscle, alkali aided and
control. ............. ...............173....

5-6 Thermograms (5-800C) of acid treated catfish muscle................. ............... 174

5-7 Thermograms (5-800C) of alkali treated catfish muscle. ................... ............... 174

5-8 Enzyme susceptibility (TGase) of catfish muscle ................. .............................175

5-9 Rheogram of pH treatment 2.0 with and without TGase treatment. ............ ................176

5-10 Rheogram of pH treatment 2.5 with and without TGase treatment. ............ ................176

5-11 Rheogram of pH treatment 3.0 with and without TGase treatment ........._.... ..............177

5-12 Rheogram of pH treatment 10.5 with and without TGase treatment. ........._..... .............177

5-13 Rheogram of pH treatment 1 1.0 with and without TGase treatment. ........._..... .............178

5-14 Rheogram of pH treatment 1 1.5 with and without TGase treatment. ........._..... .............178

5-15 Rheogram of the control with and without TGase treatment............_.._._ ........_.._. ....179

6-1 Stress response of acid- and alkali-aided catfish muscle. ............... ...................189

6-2 Strain response of acid- and alkali-aided catfish muscle ..........._...._ ........... ..........190

6-3 Rheological response of acid- and alkali-aided catfish protein isolates. ......................... 191

6-4 Rheogram of alkali treated catfish muscle heated at 20oC/minute, no hold. ................... 192

6-5 Rheogram of alkali treated catfish muscle heated at 20oC/minute, 20 minute hold........193

6-6 Rheogram of alkali treated catfish muscle heated at 0.5oC/minute, no hold. .................. 193

6-7 Rheogram of acid treated catfish muscle heated at 20oC/minute, no hold. ..................... 194

6-8 Rheogram of acid treated catfish muscle heated at 20oC/minute, 20 minute hold. .........194

6-9 Rheogram of acid treated catfish muscle heated at 0.5oC/minute, no hold. ........._.........195

6-10 Rheograms of acid and alkali treated catfish muscle heated at 20oC/minute. ................196









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE STRUCTURE/FUNCTION RELATIONSHIP OF CHANNEL CATFISH (Ictalirus
princtatus) MUSCLE PROTEINS SUBJECTED TO pH-SHIFT PROCESSING

By

Matthew Paul Davenport

May, 2008

Chair: Hordur G. Kristinsson
Major: Food Science and Human Nutrition

Novel processing utilizing pH-shift technology applied to muscle-based systems modifies

or increases raw product utilization. The use of pH-shift processing on muscle products and by-

products allows for the separation of muscle proteins from lipids, collagen, skin, bones and other

undesirable components. Undesirable materials are not easily separated from whole muscle or

other comminuted muscle products such as surimi. During pH-shift processing, animal and fish

muscle including channel catfish muscle is modified by the reduction of lipid and heme content,

altering protein composition and thermal gelation properties. Reduction of lipid and heme

content increases whiteness and oxidative stability of protein isolates. Changes in protein

composition are associated with the removal of sarcoplasmic proteins including heme proteins,

some endogenous enzymes, collagen and other protein components. Modification of thermal gel

properties induced variable gel strengths. It was hypothesized that pH-shift processing will

structurally modify channel catfish muscle proteins resulting in functional properties leading to

the expansion, utilization and production of muscle based products. The model system in this

study was developed to provide the basis for implementation of economical and environmental

utilization of current seafood by-products by pH-shift processing.









The results show that pH-shift processing modifies muscle proteins structurally and

functionally. pH-shift processing reduced the relative content of alpha helix to beta structure of

muscle proteins after isoelectric precipitation indicating a molten globular state. Muscle proteins

in a molten globular state showed reduced myosin ATPase activity, altered protein surfaces,

modified thermal sensitivity, increased susceptibility to enzymatic crosslinking and modified

solubility. These changes lead to changes in the physical properties of pH-shift processed

muscle proteins. Alkali processed catfish showed increased gel rigidity, gel strength and gel

flexibility compared to acid processed catfish which exhibited inconsistent functional

performance, increasing and decreasing in gel rigidity, gel strength and gel flexibility.

These results show that pH-shift processing of channel catfish muscle provides highly

functional isolates with a broad range of applications by inducing structural modification of

muscle proteins. This innovative process yields muscle proteins with good color, high oxidative

stability and modified functionality, which can find many applications in formulated

comminuted muscle food products. Furthermore, this technology can be an important

contribution to better utilization of seafood and land animal by-products.









CHAPTER 1
INTTRODUCTION

Background

Presently the world demand for seafood products exceeds the sustainable limits of the

available fisheries (Hultin and Kelleher 2000). The supply traditionally has been based on wild

caught products; however, the demand for high quality seafood products has not been fulfilled.

The investigation into economically viable alternative production methods and utilization of

currently harvested products is of interest to fill the pressing needs of the consuming public. The

advent of commercially viable aquaculture in the past 50 years has helped to alleviate some of

the stress on wild stocks (Harvey 2002). The supply of aquacultured seafood products has come

under fire for reducing the price of high value wild species such as salmon, shrimp and grouper

(Eaglea and others 2004). Aquaculture also has been criticized for unmanaged waste production,

increase in fish disease and damage to the aquifer (Barton 1997). Thus the need for value-added

products is of great interest to commercial producers as the profit margins are getting smaller and

the cost of production is rising (Harvey 2002). The utilization of value addition with modern

processing and preservation has allowed for increased commercial production of high value

products from lower value fish species and by-products, but many useable protein sources are

still diverted into animal feed or waste (Kristinsson and Rasco 2002).

One of the products commercially produced as a value addition product is surimi (Park

2005). Surimi is not a new product. Surimi originated centuries ago as a high value product in

Japan (Park 2005). A variety of structured products can be formed from surimi, including the

traditional kamaboko (Park 2005). Kamaboko was traditionally made by first producing surimi

by mincing freshly ground fish meat, washing it with pure water or water with low

concentrations of salt, dewatering the washed tissue, forming a sausage, and cooking the sausage









to form kamaboko. The value of quality surimi and kamaboko was high due to the requirement

to use fresh Hish and the very short shelf-life of either the raw or cooked material (Park 2005).

Commercialization of surimi production and subsequent products made it necessary to extend

their shelf-life. Freezing surimi was one of the prerequisites to its success as a global

commodity, but surimi is particularly sensitive to freeze thaw denaturation (Park 2005).

Cryoprotection increased protein stability to freeze thaw treatments by utilizing small

carbohydrates which stabilized the muscle proteins during freeze thaw processes. This has been

attributed to paying the way for the commercialization of surimi (Park 2005). The current surimi

production uses lower value white fish, primarily Alaskan Pollack and Pacific Whiting as the

base material. The use of Alaskan Pollack and Pacific Whiting for the production of surimi

allows for commercial processors to fi11 the commodities market with two products, surimi and

frozen fillet blocks (Park 2005).

Surimi processing does have drawbacks such as still mainly requiring fresh fish (Park

2005). This has led to the utilization of large trawlers which catch, process and freeze surimi

either on the water or on shore (Park 2005). Concurrently on these trawlers the alternative

product, frozen fish fillet blocks and mince also are produced (Park 2005). Even though surimi

can allow for increased utilization and profit margins from these species, there are many other

commercially harvested species which are not used for surimi and go to animal feed or other

lower value products (Hultin and Kelleher 2000). Traditional commercial surimi processing

cannot easily handle complex materials, such as whole fish, species rich in dark muscle, high in

fat or processing byproducts (Hultin and Kelleher 2000). Not only is it difficult to recover

proteins effectively from complex materials, but these materials lead to maj or color and

oxidation problems resulting in surimi with poor functionality (Hultin and Kelleher 2000). The









by-products from fish processing which contain the most muscle tissue are the frame and the

trimmings. The frame is the backbone and ribs left after the fillet is removed. The trimmings are

the parts of the fillet which are removed to increase the quality and acceptability of a fillet; a

useable trimming from catfish is the nugget or belly flap. Large amounts of unprocessed by-

products such as frames and trimmings are not used for human consumption but rather end up in

animal feed, bone meal or fish meal if used at all (Hultin and others 2000). Unutilized by-

products end up as waste material and add disposal costs and increase the biological effluent

from the processing plant (Hultin and others 2000). Usable meat left on the frame and other

processing by-products can be used for human consumption leaving enough remaining material

to satisfy the animal feed market (Hultin and Kelleher 1999). Therefore a maj or need exists to

develop and use alternative techniques to provide economical advantages and recover these

proteins for high value products to minimize the impact on wild stocks and also satisfy the large

demand for seafood based protein.

pH-Shift Processing

A recently developed technology in Hish and meat processing allows for the potential use

of discarded raw materials for human consumption. The primary drawback to using these

materials is the high level of pro-oxidants in particular heme proteins, collagen, phospholipids

and triacylglycerols all of which contribute to reduced consumer acceptability (Kristinsson and

Hultin 2003b). Removal of these components resulting in a relatively pure protein product made

possible by acid or alkali solubilization of muscle proteins with recovery at or near the average

isoelectric point of a muscle homogenate (Hultin and Kelleher 1999; Hultin and others 2000;

Kristinsson and others 2005b). Isoelectric recovery is done after unwanted components are

removed by either filtration or centrifugation at low or high pH (Hultin and Kelleher 1999;

Hultin and others 2000; Kristinsson and Hultin 2004b; Kristinsson and others 2005b). This "pH-









shift process" allows for the recovery of proteins with high functionality at a relatively low cost

to the producer (Hultin and Kelleher 1999; Hultin and others 2000; Kristinsson 2002; Kristinsson

and Ingadottir 2006; Kristinsson and others 2005b). pH-shift processing relies on electrostatic

repulsion between muscle proteins to impart solubility (Kristinsson and Hultin 2003b). This is

possible after the muscle has been thoroughly homogenized and diluted with water. Dilution

allows for the dispersion of proteins and reduction of viscosity to a sufficient degree facilitating

solubilization by allowing for the necessary molecular spacing to occur. HCI and NaOH are

used to adjust the pH to extreme low or high pH, however the effect of other acids and bases are

under investigation (Raghavan and Kristinsson 2007b). After the appropriate pH has been

reached, i.e. the pH necessary to achieve solubility, the insoluble material is removed (Hultin and

Kelleher 1999; Hultin and others 2000). If centrifugation is used, the insoluble material will

produce a three phase separation; a top layer and bottom sediment, with the soluble proteins

comprising the middle phase (Kristinsson and others 2005b). The top fat layer is comprised

primarily of triacylglycerides and some emulsified proteins (Kristinsson and others 2005b). The

sediment layer consists of phospholipids, collagen, bones, skin, scales and some poorly soluble

proteins, including a small percentage of the actomyosin complex, actin and myosin among other

proteins also found in the soluble fraction (Kristinsson and others 2005b). After separation, the

middle soluble layer is collected and adjusted to the average pl (~5.3-5.5) of the muscle protein

slurry (Hultin and Kelleher 1999; Hultin and others 2000). The proteins are recovered by

dewatering using centrifugation, filtration or screening (Hultin and Kelleher 1999; Hultin and

others 2000). This gives a partly dewatered protein isolate of 70-80% moisture. The proteins

retained, for the most part, are the myofibrillar proteins, although some of the sarcoplasmic

proteins are retained as well, increasing the recovery over conventional washing processes to









recover muscle proteins (e.g. surimi processing) (Hultin and Kelleher 1999; Hultin and others

2000). This is particularly the case for the acid-aided process where significant amounts of heme

proteins (denatured and oxidized) are retained whereas in the alkali-aided process very low

levels of heme proteins are retained and they remain in their native and reduced state

(Kristinsson and Hultin 2004a). The sarcoplasmic proteins include not only the heme proteins

but also endogenous enzymes and other small proteins which can have negative effects on the

quality of the final protein product (Kristinsson and others 2005a). The removal of the

sarcoplasmic proteins, especially the heme proteins increases the whiteness of the proteins

isolate, leads to less oxidation and improves gel strength (Kristinsson and others 2005a).

Research to date has shown that during pH-shift processing, protein structure is disrupted

and may be partially responsible for the effects on the functional properties of the pH-shift

process isolate as compared to conventional methods (e.g. surimi processing) (Choi and Park

2002; Kristinsson and Hultin 2003b; Shann-Tzong and others 1998; Yongsawatdigul and Park

2004). Conformational changes occurring during the pH-shift isolation process are linked to

differences observed between acid and alkali solubilization on the physical nature of the isolate

(Kristinsson and Hultin 2003a). When utilizing the acid solubilization process higher puncture

force and lower deformation values are observed than with conventional surimi. However

isolate gels from the alkali process show both higher puncture and deformation values than

isolate gels from the acid process and gels from conventional surimi processing (Choi and Park

2002; Perez-Mateos and others 2004; Undeland and others 2002). Kristinsson and Demir

demonstrated that among four species, catfish, mackerel, mullet and croaker, alkali solubilization

produced higher gel storage modulus (i.e. gel rigidity/firmness) after cooling than either surimi

or acid processing (Kristinsson and Demir 2003). In all three species except croaker, acid










processing produced a much lower storage modulus than the other processes (Kristinsson and

Demir 2003).

The acid and alkali-aided technology is expected to provide the seafood industry a new

powerful tool in the production of high quality muscle protein isolates from inexpensive sources

(Kristinsson and others 2005b). The effect isolation conditions have on muscle proteins is still

under investigation and is relatively unknown. There are changes noted in the function, structure

and conformation of single proteins (e.g. myosin) subj ected to the process as well as whole

protein isolates from various species compared to proteins subj ected to conventional processes

(e.g. surimi processing) or no processing (Hultin and Kelleher 2000; Kristinsson and Demir

2003; Kristinsson and Hultin 2003a; Undeland and others 2002). The pH-shift process has been

successful on many aquatic species as well as turkey and beef (Kristinsson and Hultin 2003b;

Kristinsson and Ingadottir 2006; Kristinsson and others 2005b; Liang and Hultin 2003; Mireles

Dewitt and others 2002; Undeland and others 2002; Yongsawatdigul and Park 2004). The

proteins extracted and concentrated during pH-shift processing, when compared to similar

processes such as surimi, show altered physical properties (Kristinsson and others 2005b).

Further understanding on changes that occur to muscle proteins during the pH-shift processing

and the mechanism responsible may lead to tailor-made protein isolates with the functionality

desired for specific products, additives or utilizations.

Protein Gelation

The primary functionality of interest for surimi and protein isolates is gelation. Gelation is

the interaction of molecules, primarily polymers giving rise to a three dimensional network

entrapping solvent within the network (Stone and Stanley 1992). Water is typically the solvent

in food polymers (Stone and Stanley 1992). Protein gelation can and does occur via different

mechanisms, leading to differences in texture and quality (Stone and Stanley 1992). During









protein gelation, especially fish protein gelation, there are three phases of the protein paste/gel

formation. Phase one involves the uncooked raw product or the sol which has little to no

detectable tensile strength. The next phase is variable depending on the treatment. "Suwari" is

the process when the sol is set at medium to high temperatures and this is also the point at which

protein cross-linking enzymes (e.g. transglutaminase) may be added to increase the setting effect

at the elevated temperature. "Suwari" setting temperatures are highly species dependent, but

usually do not exceed 500C. Then the final phase of fish protein gelation is kamaboko the final

cooked and cooled gel product (Stone and Stanley 1992).

The three main types of protein gels are reversible gels, chemically/enzymatically set gels

and thermally set gels (Lanier 2000). The latter two are irreversible (Lanier 2000). Reversible

gels are unique since they are initiated by heating during which the solution becomes highly fluid

and upon cooling the gel is formed (Lanier 2000). Collagen forms these type of gels due to its

unique structure imparted by the high percentage of proline and hydroxyproline (Kolodziej ska

and others 2004). Collagen structure is comprised of a super structure forming a triple helix with

three collagen strands interacting to make large long strands (Ockerman and Hansen 2000). The

collagen complex loses most of its structure during the heating process as its non-covalent

interactions are broken leading to an unfolded scrambled chain, then upon cooling the regions

rich in proline and hydroxyproline reform hydrophobic patches and associate, next hydrogen

bonds form and, thus a, cold set gel is formed (Oakenfull and others 1997). This gel is

reversible, and melts on heating, due to hydrophobic interactions and hydrogen bonding

providing the basis for the three dimensional structure (Oakenfull and others 1997).

Enzymatically and chemically set gels are formed with either enzymes or chelating agents

respectively. The enzymes and chelating agents will not, for the most part, independently form









a gel but are used in setting or curing prior to heating the protein paste or solution to complete

gel formation. Transglutaminase (TGase) is one of the most common enzymes used to increase

the strength of protein gels (Walsh and others 2003). TGase is a calcium dependent enzyme

which mediates an acyl transfer between glutamine and lysine bound in a peptide/protein

forming an e-(y-glutamyl) lysine cross-link. The covalent cross-linking of these amino acids

gives increased strength the network (Lee and others 1997; Walsh and others 2003).

The final and most common method of protein gel formation is thermally setting the gel.

Thermally setting a gel is done primarily by heating a protein solution. The heating process

unfolds the proteins which allows for interaction of hydrophobic groups and disulfide

interchanges. Upon cooling the protein solution, the hydrophobic interactions are further set and

reformation of hydrogen bonds in a different order from the native system leading to the three

dimensional network of the newly created the gel structure (Oakenfull and others 1997).

Many different factors affect protein-protein interaction during the gel forming process.

Muscle proteins specifically include ionic strength, ionic type, pH, protein hydrophobicity,

reactive groups on proteins (e.g. SH groups), protein source and type, post-mortem age, chemical

additives and thermal treatment regiment (Oakenfull and others 1997).

The need to solubilize muscle proteins with salt is a believed prerequisite of well dispersed

gel network formation (Feng and Hultin 2001). Solubility was attributed to the increased

binding and interaction of protein systems with water leading to increased protein-protein

interaction, water binding and reduced cook loss (Feng and Hultin 2001). High ionic strength is

not necessary and low ionic strength, depending on pH leads to favorable conditions improving

gel formation (Chang and others 2001). If the pH of a low ionic strength muscle protein system

is slightly alkaline (e.g. pH 7.2-7.6), enough electrostatic repulsion will form between the









proteins to produce a well dispersed, ordered protein network (Chang and others 2001) This

network will form a strong gel on heating and cooling, capable of binding high levels of water

due to the gel pressure formed because of strong repulsive forces between proteins making up

the gel network (Kristinsson and Hultin 2003c).

The pH of a sol paste is very important to form a good gel (Feng and Hultin 2001).

Characteristics of a good gel include incorporating water into the gelled network and retaining

the water after setting (Kristinsson and Hultin 2003c). The effect of pH on proteins is very

specific due to the ionic nature of proteins (Feng and Hultin 2001). The interaction of proteins

with both acid and base can result in different structural modifications and intermolecular

interactions greatly affecting the taste, texture, tensile strength and water-holding capacity of raw

and cooked gels (Kristinsson and Hultin 2003a, 2003b, 2003c). A change in pH leads to a

change in surface amino acid ionization of the protein (Feng and Hultin 2001). A good muscle

protein paste and gel has a proper balance between electrostatic repulsion and attraction (Feng

and Hultin 2001). If the pH of the system is reduced close to the isoelectric point (pl) of the

proteins, they will aggregate in the raw system and associate so closely that they have no water

holding capacity and will form a brittle, dry gel (Lanier and Lee 1992). As mentioned above, at

a higher pH there is enough electrostatic repulsion that the proteins will still associate via other

means and produce open areas which are filled by water, giving rise to the texture and mouth feel

desired in a good muscle protein gel (Feng and Hultin 2001). If the pH is changed too far above

or below the pl, the total surface charge increases repulsion between proteins and results in the

behavior of muscle proteins to the point that the interactions necessary to form a gel are not

possible even on cooking (Kristinsson 2003; Vega-Warner and Smith 2001).









Proteins have hydrophobic amino acid residues, normally associated in groups to produce

hydrophobic patches, which gives rise to a certain hydrophobicity of a protein molecule which

may vary depending on solution conditions (Lanier 2000). Hydrophobic residues play a very

important role in the development of all levels of protein structure. Interactions of hydrophobic

patches (and their exposure) during heating (and cooling) are one of the main driving forces of

protein gelation as they provide much of the initial interaction and association of proteins (Lanier

2000). Hydrophobic interactions are attributed to the increase in gel strength during heating

(Lanier 2000). Hydrophobicity and hydrophobic interaction of proteins are mediated by a) the

structure of the protein and b) by the external factors of the matrix and physical treatment (Lanier

2000). These factors include temperature, pH, salts, lipid content, lipid oxidation products, free

fatty acids and many others (Lanier 2000).

Protein source has a great influence on the gelation characteristics of the system by

dictating the types of proteins which are present to participate in gelation (Lanier 2000). Protein

source is not limited to species differences, be it between fish species or between land animal

and fish species (Lan and others 1995a; Lanier 2000; Luo and others 2001b). Protein source also

pertains to the location within the animal, the difference between dark and light muscle or

between muscle types (Vega-Warner and Smith 2001). The presence of specific proteins and

concentrations of specific proteins can vary greatly between muscle types (Nowsad and others

2000). Differences observed between proteins, isolated under the same conditions, have been

seen from various physical and chemical analyses done on the proteins, including gel strength,

solubility, temperature stability, hydrophobicity, and other chemical indicators (Oakenfull and

others 1997).









The postmortem age of the raw material from which proteins are recovered from is highly

important (Lanier 2000). Some animal species have a shelf life of 2-3 weeks, e.g. beef and pork,

and thus will still form an acceptable gel based product after that time, whereas some fish species

have a very short shelf life and their proteins can be negatively affected very quickly (Lan and

others 1995b). This is because of endogenous enzymes and the rigor state of the muscle (Lanier

2000). Processing fish muscle pre- vs. post-rigor can have an effect on the recovery and state of

the isolated proteins (Lanier 2000).

Additives such as sugars, polyols and polysaccharides are commonly added to minimize

muscle protein denaturation and retain functionality during freezing and thawing. These

additives allow for long term frozen storage and make large scale processing of muscle protein

ingredients, such as surimi possible. The addition of these compounds affects the gelation

characteristics of the muscle proteins (Lanier and Lee 1992).

Thermal input has a profound affect on the gelation and gel strength of muscle proteins

(Riemann and others 2004a). The effect of thermal input in a gelation system can be equated to

the D value during canning, i.e. there is a relationship between time and temperature and the gel

quality (Riemann and others 2004a). During heat induced gelation, it has been demonstrated that

the longer the time taken to reach the end point temperature the greater the gel strength (Riemann

and others 2004a). This is assumed to be due to the increased energy added to the system during

slow heating in contrast to rapid heating. Studies where gels have been heated rapidly but held at

the end point temperatures to give equivalent energy input as the slow heating rate have also

shown to give similar increases in gel strength as slow heating, supporting the energy input

theory (Riemann and others 2004a).









Rheology

Gelation and gel formation can be studied and described using theology (Steefe 1992).

Rheology is the study of the flow properties of a material (Owusu-Apenten 2005). Rheological

tests apply a rotational or tangential force to a material and the response of the material to this

force and its mode of application provide information which relates to its characteristics of flow

(Owusu-Apenten 2005). The three main parameters which are controlled in the determination of

flow are force of application, distance of head movement or degree of oscillation and the

frequency of application (Steefe 1992). Macro-rheology is the study of tensile properties prior,

during and after breaking and/or reforming of the material's three dimensional structure (Steefe

1992). Micro-rheology studies the same tensile properties as macro-rheology, however the

breakage/resolution of the three dimensional structure may not be involved (Steefe 1992). There

is a linear viscoelastic range of most if not all gels (Owusu-Apenten 2005). This region is the

area where there is deformation of the three dimensional network, however the bond interactions

are not broken (Owusu-Apenten 2005).

Macro-rheology is utilized as either an end-point determination or stop-point determination

of gel properties (Owusu-Apenten 2005). As macro-rheology is destructive to the material,

except in reformation or continuous flow studies, it renders the material unusable for future tests

(Owusu-Apenten 2005). The other method of macro-rheology, which is continuous testing, is

flow properties which may or may not render the sample unusable for future studies (Owusu-

Apenten 2005). Common theology parameters followed during testing are break point or shear

strength, initial viscosity, which is the force required to begin flow, and continuous viscosity

which his the force required to maintain flow (Steefe 1992). Two characteristics associated with

continuous flow tests are shear thinning, where viscosity is reduced, and shear thickening, where

viscosity is increased (Owusu-Apenten 2005). These tests provide very valuable information









and are the primary methods of quality control (QC) for many food products, especially in

starches (Ta 2001). Among products evaluated using this method are pork, surimi, food starches

and many others (Steefe 1992).

Micro-rheology determines different aspects of structure and change in structure than

macro-rheology (Owusu-Apenten 2005). The changes monitored utilizing micro-rheology occur

mostly during the formation of a gel (Steefe 1992). Micro-rheology is less characterized on a

broad scale for QC utilization. Micro-rheology is very sensitive in its measurements and values;

thus a greater degree of variation may be seen between samples and instruments (Ta 2001).

Micro-rheology, when dealing with food polymers, remains the predominant method of choice in

basic research of materials (Steefe 1992). Micro-rheology is, however, also used in QC for other

polymer research pertaining to fluids, adhesives and rubbers (Ta 2001). The crossover for food

polymer analysis on a basic level to QC requires not a greater understanding of micro-rheology

but a standardization and data bank development of micro theological data for specific materials

and the correlation of those materials to currently held standard values for the other tests used in

QC labs (Ta 2001).

The basis for all theological testing is on the deformation of a material when under stress,

ie., the stress strain relationship. The differences between micro-rheology and macro-rheology

are the use of shear stress and shear strain or the force and distance a material can withstand at

failure or when the structure breaks. Macro-rheology utilizes the shear stress and shear strain

where as micro-rheology utilizes stress and strain parameters which may not shear the material.

To do this in micro-rheology, oscillatory stress is used in place of continuous stress. The

parameters which are then calculated in oscillatory stress testing are different that those observed

in continuous stress tests. The total response of the material to oscillatory stress is made up of









the two components of the stress applied the in-phase stress, that is the stress applied moving

away from the zero point and the out-of-phase stress which is the removal of the stress from the

material or the probe moving back to the zero position. The in-phase stress is referred to as the

storage modulus (G') and can be viewed as the resistance of the material to movement or the

solid fraction of a material. The out-of-phase stress is referred to as the loss modulus (G") and is

considered the liquid phase of the material. The G' represents the material as it is resistant to

motion, this resistance to an applied force, when a small enough stress and strain are used, which

does not break, shear, the three dimensional structure of the material is testing the intramolecular

strength of the material to tangential compression. As the probe returns to the zero position the

G" is testing for the compliance of the material to a non-fracture stress. This is the ability of the

material, though neither the stress nor strain is large enough to induce flow or breakage of the

structure, to move into space created by the positive stress applied by the probe and thus the

resistance of the material to the negative stress. The effect of the sinusoidal movement of the

head, G' and G", the resistance of the material to the movement of the head both forward and

back alter sin wave during the motion of the probe. The alteration of the sin wave is represented

at the tan delta. Tan delta ranged from 0-1 (G"/G'), or is the tangent of the degrees of change of

the sin wave during the motion of the probe, O being a pure solid and 1 being a pure liquid or 0

difference being there is no modification of the sin wave G' and G" are equal and 1 being there

is no G" pressure of the material or no space filling occurring. This relationship and the analysis

of tan delta in theological studies allows for the determination of the phase change of a material

as it transitions from a more liquid like material to a more solid like material or vice versa. This

transition period of a material from a more liquid like to a more solid like material is also

determined by the change of G' and G" or the transition from greater out-of-phase resistance









(G") to greater in-phase resistance (G'). As the G' increases and the G" decreases or vice versa,

if these two values cross during a treatment, this point is known as the gelation point, or where

the material is equally solid and liquid. The transition of the material at this point is either going

from a more solid to a more liquid material (melting) or a more liquid to a more solid material

(gelling) (Oakenfull and others 1997; Owusu-Apenten 2005; Steefe 1992; Stone and Stanley

1992; Ta 2001).

The micro-rheology as discussed above provides valuable information about what is

occurring during a treatment study. After the tests have been done to determine what is going on

in a system it is then necessary to test how strong the gel is. This is done using shear stress and

shear strain, causing the failure of the material and thus determining how strong it is after or at

some point during a treatment. This is done in one of two ways 1) torsional stress or 2)

tangential stress. That is either twisting or puncturing a material. The shear values of each of

these types of testing, both shear stress and shear strain, are dependent on the type of force

applied to a material. The torsional resistance of a material may not be the same as the tangential

resistance of a material as the structure of some materials are very directional sensitive. In food

systems the tangential and torsion resistance of a material has been shown to provide a good

indication of the mouth feel and texture. Both of these tests are also used as quality determinants

in muscle products, especially comminuted muscle products. The use of shear stress and shear

strain have lead to the development of texture profile analysis of foods and is a primary

component used to determine the grade and thus value of comminuted muscle products,

especially surimi (Oakenfull and others 1997; Owusu-Apenten 2005; Park 2005; Steefe 1992;

Stone and Stanley 1992; Ta 2001).









Key Muscle Proteins of Interest for the pH-shift Process

Myosin is the primary muscle protein comprising ~50% of the myofibrillar fraction of

muscle (Kristinsson and Hultin 2003a). Myosin is composed of a long helical rod, where most

of its secondary structure comes from, and a large bulky head group where most of its tertiary

structure is found. This structure of myosin allows it to be a good gel former (Kristinsson and

Hultin 2003a). The head is globular in nature with hydrophobic sites on the exterior of the

protein designed to interact with actin during muscle contraction (Ruppel and Spudich 1996).

These hydrophobic sites are blocked by meromyosins and troponin, primarily (Berne and others

2004). In a living system the release of calcium moves troponin and in the presence of ATP the

light chains are moved by a conformational shift in myosin, which looks like the myosin head

sitting up and making the exposed hydrophobic site on the S-1 fraction of the myosin head

available to actin (Berne and others 2004). The structural and functional design of the proteins

as they are in the living system still holds the predominant role in the mechanism of gelation. In

a non-living system when muscle is processed and cooked to form a gel, the sequence of events

is quite different.

The role of myosin in muscle gel formation is a cascade of events, the sequence of which is

vaguely proposed to be myosin-myosin head interaction at hydrophobic sites causing close

association between myosin molecules at lower temperatures as heat denaturation is taking place

(Hettiarachchy 1994). The heat denaturation moves and or removes both tropomyosin and

meromyosin from the hydrophobic patches on the myosin head allowing for this interaction

(Hettiarachchy 1994). After this association, disulfide interchanges, mediated by increasing

temperatures, are proposed to take place to some extent in the lower S-2 fragment of the myosin

head (Stone and Stanley 1992). These disulfide interchanges hold the myosin-myosin linkage,

created by the hydrophobic attraction, increasing the overall tensile strength of the system (Stone









and Stanley 1992). During the heating process, the myosin tail is partially uncoiled exposing the

buried hydrophobic patches in the tail portion as the myosin tail has on its surface predominantly

hydrophilic residues (Stone and Stanley 1992). The heat denaturation of the myosin tail causes a

scrambling effect in the myosin system allowing for the now exposed hydrophobic patches of the

myosin tail to interact and form new associations with other uncoiled myosin tails (Stone and

Stanley 1992). This rearrangement of the myosin tail along with the polymerization of the

myosin head is the strengthening effect of myosin gelation (Stone and Stanley 1992). This is

because of the increased disorder in the gel system which is a consequence of the disruption of

the natural organized tight packing of myosin, and other proteins, in the myofibrillar unit

(Hettiarachchy 1994).

Protein solubility based on pH and ionic strength is attributed to the effect of ionization of

the surface of the protein. Myosin has more than half of its amino acids as hydrophilic residues

and 80% of these are exposed to the solvent (Lanier and Lee 1992). The increasing

concentration of either positive or negative ions, depending on the pH and/or ionic strength of

the solvent, water in this case, proceed to interact on the surface of the protein, forming a

charged shell around the protein (Lanier 2000). This shell shields the ionic bridges between the

proteins, allowing for separation of the proteins and eventually solubilizing proteins. The ion

shell around the protein also increases the association of water around the protein surface,

resulting in soluble proteins (Lanier 2000). Once the salt bridges are weakened/broken by ion

shielding, increased water association is possible well beyond the hydration level of the ion

alone. This increased amount of water associated with the protein increases its total level of

hydration and, thus, that level of hydration is perceived as solubility of the protein (Stefansson

and Hultin 1994). This mechanism of solubility is more evident in the myofibrillar fraction of









muscle than in the sarcoplasmic fraction, as sarcoplasmic proteins are fairly soluble under most

conditions (Kristinsson and others 2005b). It is this aspect of sarcoplasmic proteins, mainly

hemoglobin and myoglobin, that allows for their separation (remaining soluble in the aqueous

phase) from the protein isolate during isoelectric precipitation where primarily myofibrillar

proteins aggregate (Hultin and Kelleher 1999; Hultin and others 2000). This removes off color

associated with pelagic surimi and increases the stability of the protein isolate as both myoglobin

and hemoglobin are active in lipid and protein oxidation (Hultin and Kelleher 2000).



Objectives

The overall obj ective of this proj ect was to investigate how different acid (pH 2.0, 2.5 and

3.0) and alkali (pH 10.5, 11.0, 11.5) treatments followed by isoelectric precipitation influenced

the structure, conformation and functionality of catfish muscle proteins. The ultimate goal of

this work was to attempt to build an understanding how specific pH unfolding and refolding

regimes affect the proteins and connect structural/conformational changes to functional changes.

We hypothesized that different pH treatments lead to differences in structure and conformation

which are responsible for changes in functionality (gelation). Basic work in this area is expected

to further our knowledge of muscle protein gelation and provide insight for production of muscle

proteins with specialized functional characteristics.

The specific objectives of this proj ect were:

1) Investigate the effect of different acid- and alkali-aided unfolding followed by isoelectric

precipitation on the gel forming properties protein isolates during thermal treatment.

2) Investigate the effect of different acid- and alkali-aided unfolding followed by isoelectric

precipitation on the strength, elasticity and quality of thermally set protein isolate gels.










3) Investigate the effect of different acid- and alkali-aided unfolding followed by isoelectric

precipitation on the chemical properties of protein isolates (surface hydrophobicity,

ATPase activity, sulfhydryl groups and protein solubility).

4) Investigate the effect of different acid- and alkali-aided unfolding followed by isoelectric

precipitation on the conformation, structure and structural stability of protein isolates.

5) Investigate the effect of heating rate on the gel formation, strength, elasticity and quality

of protein isolates made from acid- and alkali-aided unfolding followed by isoelectric

precipitation.









Experimental Design

The study of pH-shift processing in catfish muscle was conducted on catfish fillets. The

use of catfish fillets as the raw material was chosen to provide a stable, high quality raw material.

Catfish was chosen as the raw material due to its importance in the southeastern United States

and increasing consumption within this region and through out the entire United States. The

experiments used to study pH-shift processing, outlined in figures 1-1 through 1-4 and described

in the next chapter, were conducted due to their common use in meat and meat products. Each

method used to investigate the properties of pH-shift processed catfish muscle was replicated on

independent batches of catfish fillets. This was determined acceptable by power analysis

(a=0.05, P=0.80). The chemical analyzes used were specifically chosen for their previously

established correlation to the physical/textural properties of muscle products. The determination

of the physical properties of pH-shift processed catfish muscle was the initial focus, as previous

research had indicated that pH-shift processing will modify physical properties. The reason for

the modification of the physical properties (figure 1-2) would be further understood through the

investigation of the chemical properties (figure 1-3) and structural properties (1-4) of muscle

proteins. The combination of these three properties will provide a deeper look into how different

the physical/textural properties in meat are formed.










Catfish Fillet


Figure 1-1: Figure summarizing the overall experimental design of the structure/function
relationship of channel catfish (Ictalurus puncta~tus) muscle proteins subj ected to pH-
shift processing.



























Fold Test


Figure 1-2: Experimental design of the investigations into the physical properties of channel
catfish (Ictalurus punctatus) muscle proteins subj ected to pH-shift processing.




















Salt Solubility


Figure 1-3: Experimental design of the investigation into the chemical properties of channel
catfish (Ictalurus puncta~tus) muscle proteins subj ected to pH-shift processing.















Circu lar Dich roi sm












Structural Properties
Guanidine HCI Differential Scanning
Den atu ratio n I Calorimer









Susceptibility to
EzmtcCross-linin



Figure 1-4: Experimental design of the investigation into the structural properties of channel
catfish (Ictalurus punctatus) muscle proteins subj ected to pH-shift processing.











CHAPTER 2
IVETHOD S

Raw Material

The raw material used in these studies was fresh catfish fillets obtained 1-3 days post

harvest from a local supplier. Catfish fillets were only purchased which were determined to be

within 3 days of packaging. The catfish fillets were purchased and immediately transported on

ice to the laboratory and processed the same day.

Preparation of Protein Isolates

Protein isolates were prepared according to figure 2-1. Fresh fillets were initially ground

in an Oster heavy duty food grinder (Niles, Ill., U.S.A.) for the preliminary disruption and

collection of the muscle tissue. Following grinding, the comminuted meat was diluted 1:2 (w/v)

with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds.

Following homogenization, the resulting muscle tissue slurry was further diluted to give a final

dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manually stirred with a plastic

spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the

methods described below, using either 2N NaOH or 2N HCI as needed for the pH desired, with

continuous manual mixing. Upon reaching the desired pH, insoluble material was removed by

centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products,

Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at SoC. Following centrifugation, the

soluble middle layer was collected through a kitchen strainer with a mesh size of approximately

0.25 mm to minimize contamination with other separated materials. The soluble material was

readjusted to pH 5.5 as described above. After readjustment the solution was centrifuged to

remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The










precipitated protein was collected by decanting the supernatant containing the unprecipitated

proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH

was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate

was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing

until the moisture content was below 80%. Moisture content was determined using a Cenco

infrared moisture analyzer (CSC Scientific, Fairfax, Va., U. S.A.). Upon completion of manual

dewatering the protein isolation was complete. Preliminary unpublished investigation of protein

isolates in this laboratory found the shelf life of catfish protein isolates to be 5-7 days on ice. All

protein isolates were stored on ice at the precipitation pH and used within 5 days.

Protein Concentration

Protein concentration in the isolates and the subsequent solutions was determined using the

Biuret method, as described by Torten and Whitaker (1964), with of 10% w/v deoxycholic acid

in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any

remaining lipids in the samples. Protein concentration was measured based on a standard curve

based on BSA.

Protein Surface Hydrophobicity

Protein surface hydrophobicity was conducted according to Liang and Kristinsson (2005).

To measure surface hydrophobicity the isolate was diluted to give a stock solution of 10 mg/ml

in a 20 mM tris-HCI buffer, 0.6 M NaC1, pH 7.2. The stock solution was serially diluted to

obtain a concentration curve. Increasing volume (100 CIl, 200 CIl, 300 CIl, 400 Cll and 500 Cll) of

the stock solution were added to Tris-HCI buffer to a final volume of 4.0 ml. Then, 10 Cll of 6-

propionyl-2-(dimethylamino) naphthalene (PRODAN) (11.35 Clg/ml in methanol) was added and

the samples mixed for ~15 seconds. After mixing the samples were then incubated for 15 min in

the dark. All sample preparations and incubations were performed on ice in disposable test tubes.









After incubation, the sample was transferred to a fluorescence cuvette and the fluorescence

emission intensity scanned between 380-560 nm with excitation at 365 nm in a Perkin Elmer LS

45 Luminescence Spectrophotometer (Norwalk, CT). As the isolate is a collection of proteins,

the fluorescence peak of the samples is a relatively flat and broad peak between 430-460 nm.

The maximal fluorescence of the protein isolate was taken and used as the wavelength for

analysis. The surface hydrophobicity was calculated as the slope of the net fluorescence versus

protein concentration (mg/ml) of the samples.

Circular Dichroism (CD)

CD was done according to Kristinsson and Hultin (2003a). The isolate was diluted and

homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec

Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with 600 mM NaCl to a

concentration of 10 mg/ml. This protein stock solution of 10 mg/ml was prepared and diluted to

2 mg/ml 1 hr before analysis and held on ice. For analysis of secondary structure the sample was

scanned from 260-200 nm in a 0.1 cm quartz cuvette, 0.2 nm resolution scanned at 50 nm/min on

a Jasco J-500C circular dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room

temperature. The time duration of the scan was no more than 10 minutes. Differences in total

alpha helix beta structure and random coil structures present were determined from

DICHROWEB using the K2D analysis program (Lobley et. al., 2002).

Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HC1)

Catfish protein isolate and the control, untreated ground catfish muscle, were treated with

Gu-HCI over the range of 0-6M in 0.5 M increments. For assessment of denaturation, 222 nm

was used as an indicator wavelength of alpha helical content, scanning from 220-225 nm in a

0.10 cm quartz cuvette, 0.2 nm resolution scanned at 50nm/min on a Jasco J-500C circular

dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The protein









concentration was 1 mg/ml. Samples were allowed to incubate for 5 minutes on ice prior to

reading.

Differential Scanning Calorimetry (DSC)

DSC was conducted according to Fukushima and others (2003) on a MicroCal DSC

(MicroCal, LLC, North Hampton, MA). Isolate were diluted in sample buffer (20 mM tris-HC1,

pH 7.2 with 600 mM NaC1) to a protein concentration of 10 mg/ml and homogenized for 1

minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec Products Inc.,

Bartlesville, OK). The sample was degassed under vacuum for 5 min at SoC. After degassing,

0.6 ml was loaded into the sample cell, the reference cell contained sample buffer. The sample

was then linearly heated at loC/min from SoC-80oC. Analysis of the data was conducted on the

software provided by the manufacturer, Origin Pro 7.5, for determination of both exothermic and

endothermic events.

Reactive Sulfhydryl Groups

Reactive sulfhydryl groups were determined according to Kim and others (2003). The

isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer

(M133/1281-0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with

600 mM NaCl to 250 Clg/ml. After dilution, 80 Cll of 10 mM 5,5' -dithiobis(2-nitrobenzoic acid)

(DTNB) was added to the sample. This mixture was then incubated for 1 hr on ice. After

incubation the sample was read at 420 nm using an Agilent 8453 diode array UV-visible

spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany).

Micromolar determination of SH groups per gram in the sample were done using the following

equation:

SH/ g = [absorban2ce x dilution factor x 100]/[13600(mol/cm)x samp7C~leconentrationz~mg/ml)









Total Sulfhydryl Groups

Total sulfhydryl groups were determined according to Kim and others (2003) The isolate

was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-

0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with 600 mM

NaCl to 250 Clg/ml. After dilution, 0.5 ml of diluted isolate was mixed with 2.5 ml urea buffer.

The urea buffer contained 8 M urea, 0.2 M Tris-HC1, 2% SDS, 10 mM EDTA and was adjusted

to pH 8.5. After mixing with urea, 50 Cll of 10 mM DTNB was added to the sample. The

mixture was incubated in a water bath for 15 minutes at 400C. After incubation the sample was

read at 420 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent

Technologies Deutschland GmbH, Waldbronn, Germany). Total sulfhydryl groups were

calculated using the following equation.

SH/ g = [arbsorbanrce x d~ilurtion factor x 100]/[1 3600(mlol/cm)x samplconcentration( mg/mill)


Myosin ATPase Activity Assay

Myosin ATPase activity was determined by the method of Perry (1955). The buffers to

initiate the enzymatic hydrolysis of ATP by myosin were prepared prior to sample preparation.

The reaction buffers used in this assay were 0.2 M glycine-NaOH buffer (pH 9.0), 0.1 M calcium

chloride, 0.05 M ATP, sodium salt (pH 6.8) and 15% trichloroacetic acid (TCA). Liberation of

inorganic phosphate was monitored in this reaction as the determinant of enzymatic function.

For the estimation of inorganic phosphate the following buffers were used: 12% TCA, 10%

(w/v) ammonium molybdate stock solution in 10N sulfuric acid. This ammonium molybdate

stock was used in the preparation of the ferrous sulfate ammonium molybdate reagent which

was made fresh the day of analysis. For analysis of myosin ATPase activity the isolate was

diluted to 1 mg/ml in Tris-HCI buffer containing 600 mM NaCl pH 7.2 and homogenized









thoroughly. This was kept on ice until needed. In separate tubes for each reaction, prior to

isolate addition, the reaction buffer was prepared by adding 1.3 ml glycine-NaOH buffer, 0.2 ml

calcium chloride, 0.3 ml ATP. This mixture was then incubated at 25oC for 5 min to allow the

temperature to equilibrate. After temperature equilibration, 0.2 ml of the individual isolate

solution was added to each reaction tube with proper mixing and allowed to incubate at 25oC for

5 min. After 5 min, 1 ml of 15% TCA was added to each reaction tube to stop the reaction. This

was centrifuged for 10 minutes at 25,000 x g to precipitate the proteins from solution and leaving

liberated phosphate from ATP in solution. Next 0.5 ml of supernatant was added to 3.2 ml of 12

% TCA with good mixing and left to stand at 25oC for 10 minutes. This solution (3.0 ml) of this

solution was removed and mixed with 2 ml of the ferrous sulfate ammonium molybdate

reagent and incubated at room temperature for 1 min. After 1 min of incubation the solution was

transferred to a cuvette and read at 363 nm using an Agilent 8453 diode array UV-visible

spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The

color of this reaction intensifies with time so only one reaction was done at a time to standardize

the incubation time of all samples to 1 min. The results of myosin ATPase activity were assayed

as relative results to the control. The control absorbance was set to 100% and the percent

activity of the isolates was calculated from the absorbance of the samples relative to the control.

activity = pH treatedsample/lcontrolsample

Susceptibility of Proteins to Transglutaminase-Induced Cross Linking

The susceptibility of pH-shift processed catfish muscle to protein cross linking was

assayed using the commercial transglutaminase (TGase) Activia TI from Ajinomoto LLC

(Ajinomoto Food Ingredients LLC Chicago II). 0.2% TGase (w/w) was added to isolate diluted

to 10% solids in 20mM Tris-HCI buffer, 600mM NaC1. TGase activity was assayed by using an









AR2000 advanced research rheometer (TA Instrument, New Castle, DE) with a head with a flat

cross-hatched polyacrylic surface and thermally controlled plate. The gap was 1000 microns and

the head lowered onto the sample using the controlled speed function provided by the software.

The samples were tested in oscillatory mode under controlled frequency at 0.1 Hz, and strain

controlled at 0.01. TGase activity was monitored by the increase in G' over 1 hour at 30oC.

Activity was compared against samples with no TGase added. G' was plotted against time and

the slope of the line from linear regression was used to calculate the TGase activity. The activity

coefficient was calculated as the ratio of treated to untreated samples.

Oscillatory Rheology

For the gelation tests, the isolates were rehydrated to 10% protein and pH adjusted to 7.2

with 20 mM Tris-HCI and HCl/NaOH. For the added NaCl samples, NaCl was added at 2%

w/w. The sample was homogenized at speed 1 using an Ultra-Turrax T18 homogenizer. The

protein paste was then transferred to a peltier plate at 50C attached to an AR2000 advanced

research rheometer (TA Instrument, New Castle, DE) and a head with a flat cross-hatched

polyacrylic surface. The gap was 1000 microns and the head lowered onto the sample using the

controlled speed function provided by the software. The isolates were subjected to heating (5-

800C) and cooling (80-50C), with testing conducted in oscillation mode. The storage modulus

(G'), loss modulus (G") and tan delta (6) was followed as a function of heat and time. All

experiments were done under a controlled strain of 0.01 and controlled frequency of 0. 1 Hz.

Torsion Gel Testing

Protein paste was prepared as above, except at 20% protein concentration. Proteins were

stuffed in cylindrical tubes 10 cm long by 1.8 cm in diameter and cooked at 800C for 20 min in a

water-bath. The interior wall of the tube was coated with a lecithin based spray. The cooked

samples were then removed from the casing after cooling for 30 min on ice, held overnight on










ice, and subsequently cut into 2.54 cm long pieces. These pieces were glued to plastic disks (Gel

Consultants Inc., Raleigh, N.C.) using an instant adhesive, Krazy glue (Toagosel Co., LTD.

Nishi-shinnbashi Minato-Ku, Tokyo). Dumbbell shaped samples were then milled from each

sample to 1 cm diameter in a machine mill (Electro Sales Co., Somerville, Mass., U.S.A.),

wrapped in plastic wrap to prevent moisture loss, and brought to room temperature prior to

torsion testing. For testing, 4 gel specimens were vertically mounted and sheared to the point of

fracture at 2.5 rpm in a modified Brookfield viscometer (Gel Consultants Inc., Raleigh, N.C).

Stress (kPa) and strain dimensionlesss) at fracture were calculated with the manufacturer' s

software for each sample, corresponding to the strength and deformation of the gels, respectively

(Hamann and others 1990).

Punch Test

Punch test samples were prepared by adjusting protein isolates to 20% solids according to

the moisture content and stuffed into plastic tubing, 1.25 inches in diameter. These samples were

cooked for 20 min at 800C, and then cooled rapidly in ice water. Samples were air freighted the

same day they were cooked to the laboratory of Dr. Herbert O. Hultin in Gloucester MA

(University of Massachusetts Marine Station) where punch testing was conducted The samples

were cut to 1.0 inch length and 1.25 inch diameter. The sample was placed on the machine table

and the head adjusted to just above the sample surface. A 5 mm head was used at a speed of 1.0

cm/min with force and distance at failure reported. Punch testing was conducted on a Rheo Tex

Model AP-83 (Sun Science Co., Seattle, Wa).

Fold Test

A gel fold test was conducted according to Nowsad and others (2000). The test was

conducted by slicing 2-mm thick sample disc from the cylindrical gels, mentioned above, and

folding them into halves and quarters. The scales are: 5= no crack when folded into a quarter,









4=no crack when folded into half but crack when folded into quarter, 3=crack when folded into

half, 2=broke and split into halves when folded into half, 1=broke and split prior to being folded

into half.

Gel Water Holding Capacity

Gel water holding capacity was conducted according to Nowsad and others (2000).

Expressible moisture of the gels was determined by compressing a 1.0 g spherical gel slice

between 4 double layers of filter paper at a pressure of 20 Kg/cm2 for 1 min and calculated from

weight before and after the compression. Expressible moisture was determined as the percentage

of original weight after compression.

Cook Loss

Cook loss of gels was determined as the weight of the protein paste before heat treatment

compared to the weight of the gels after cooking. Gels were prepared and heated as described

above (torsion gel testing). After cooking the gel was removed from the tube, lightly dried and

reweighed. The weight of the tube was subtracted from the total initial weight and then

compared to the final weight of the cooked gel. Percent cook loss was determined by:

Cook Loss = (1- [G'el Weight/(hzitiarl Weight Tulbe Weigh }~x 100

Cook loss is in percent, gel weight is the weight of the cooked protein paste after being

removed from the tube, initial weight is the capped tube plus the raw gel stuffed inside, tube

weight is the weight of the tube after the gel has been cooked and removed from the tube.

Heating Rate Gelation Studies

Heating rate gelation studies were conducted at North Carolina State University in the

laboratory of Dr. Tyre Lanier, Raleigh, NC. Torsion tubes, 10 cm in length and 1.8 cm in

diameter were filled with protein paste and subsequently heated from 10oC to a 70oC internal

endpoint, either rapidly by a cylindrical microwave (Riemann and others 2004b), at 20 or









98oC/min to test rapid heating effects, slowly at loC/min by immersion in a programmable water

bath or placed directly in an 700C water bath for 15 minutes. Temperatures were measured by

fiber optic probe (Riemann and others 2004b). Upon reaching 700C, rapidly heated samples were

held for 0 or 20 min prior to rapid cooling by immersion in ice water for sufficient internal

cooling to <100C for ~30 min. Samples were held static in a cylindrical microwave applicator

(length 16 cm, radius 12.5 cm) and heated using power settings calculated previously. After

reaching a final temperature of 700C, this temperature was maintained in gels during the

subsequent holding period by utilizing feedback software (Riemann and others 2004b).

Rheological changes (storage modulus, G') of pastes/gels were non-destructively and

continuously measured as pastes were heated, held and cooled using a 40 mm, 4 degrees slope

cone and plate attachment of a constant stress, small strain rheometer (Stresstech, Rheologica

instruments AB, Lund, Sweden). Oscillation parameters were those used in Riemann and others

(2004). Heating conditions were at either 200C/min, the most rapid heating rate possible for this

apparatus, 0.50C/min, loC/min, 20C/min or 50C/min to an endpoint temperature of 700C

followed by holding for 0 or 20 min prior to cooling at 50C/min to 100C.

Protein Solubility as a Function of pH

The isolate was diluted to 10 mg/ml in DI water with thorough homogenization using a

tissue homognizer (Ultra-Turrax T18, IKA Works Inc. Wilmington, NC). After dilution in

water, 2 ml diluted protein was added to 2 ml buffer with good mixing. The pH range was from

1.5-12 in 0.5 increments. For pH 1.5-6.5, 2 mM citric acid buffer was used and for pH 7.0-12, 2

mM sodium phosphate buffer was used. The buffers were adjusted to the desired pH prior to

protein addition. After protein addition, the mixture was allowed to incubate on ice for 10

minutes. After incubation the protein solution was centrifuged at 3,000 x g for 10 minutes.

After centrifugation, the supernatant was sampled for protein concentration using the biuret









method (Torten and Whitaker 1964). Protein concentration was calculated from a standard curve

based on bovine serum albumin (BSA). Percent solubility was then calculated as a percentage of

an uncentrifuged control by the following equation:

[ protein (mg / ml) sup ernanrrt]/ lr~oteina (mg / ml) total]

Protein Solubility as a Function of Salt Concentration

The isolate was diluted to 5 mg/ml in 20 mM Tris-HCI at pH 7.2 without added NaCl with

thorough homogenization using an Ultra-Turrax T18. After dilution in buffer, 4 ml was taken

and the appropriate amount of NaCl added to each tube to achieve 0 mM, 150 mM, 300 mM 450

mM or 600 mM concentrations. After the appropriate NaCl concentration was achieved, the

samples were centrifuged at 3000 x g for 10 minutes at 50C. After centrifugation, the supernant

was collected in duplicate and soluble protein was determined according to the biuret method

(Torten and Whitaker 1964). The soluble protein was compared to an uncentrifuged control.

Percent soluble protein was calculated as

(proteinz (mg / ml) sup ernanzt) / (rotein (mg / ml) total)

Statistical Analysis

Experimental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on

replicate isolations as discussed above. A replicated (N=2) was determined as acceptable due to

achieving an acceptable power (P=0.80, a=0.05).

One-way independent measures analyses of variance were used to examine the effects of

all methods used except for the analysis of pH solubility and salt solubility which were tested

with 2-way ANOVA analysis. The Kruskal-Wallis ANOVA testing by ranks was used on data

which did not pass normality. Post hoc analysis was conducted only in the presence of

significant population differences. ANOVA statistical comparisons were conducted with










SigmaStat, (Systat Software Inc. San Jose Ca) with a significance level of p<0.05. After

SigmaStat completed the ANOVA analysis, the post hoc analysis recommended by SigmaStat

was used. In most cases this was Tukey's test. Dunn's test was used for all punch test analysis;

force, distance and jelly strength. The Holm-Sikak method was used with the torsion stress,

expressible moisture and ATPase activity. The two-way ANOVA analysis used the Holm-Sidak

pair wise comparisons. The two-way ANOVA tables and the post hoc tests are reported in

appendix A. The heating rate study was analyzed with Graph Pad QuickCalcs online calculator

software (Graph Pad Software Inc. San Diego Ca). Analysis used an unpaired t-test with a

manual bonferoni correction, overall significance of p<0.05 with the individual significant p-

values of p<0.004.







































Centrif ugation
S upe matant
Mostly water, can be reused


Sediment = Protein isolate


Figure 2-1: The process used in acid and alkali pH shift processing.









CHAPTER 3
CHANNEL CATFISH (Ictalunts prnctatus) MUSCLE PROTEIN ISOLATE PERFORMANCE
PROCESSED UNDER DIFFERING ACID-AIDED AND ALKALI-AIDED pH VALUES.

Introduction

The need for an increase in the production of seafood products has become a clear concern

with international seafood shortages proj ected to be seen as early as 2015 (FAO Subcommittee

on Aquaculture, Beijing China 2002). One of the methods which may alleviate some of the

stress currently on the seafood marked is the utilization of byproducts. Byproducts are

considered what is left over from the whole fish after the primary products have been removed.

In catfish these are the fillet and the belly flaps ("nugget"). These byproducts provide an

inexpensive raw material which increases the total utilization, profitability and management of

the fisheries resources.

The production of catfish in the United States has been steadily increasing for the last 35

years (Harvey 2002). In the filleting process the utilization based on live weight is at best 45%,

comprising fillets and nuggets. The byproducts are the frame and cutoffs, these portions of the

fillet/nugget are not acceptable to the consumer. This is a substantial amount of usable material

that, with the appropriate processing, may be used for the production of value-added protein

isolates. A relatively new process developed by Hultin and coworkers (Hultin and Kelleher

1999) utilizes the solubilization properties of specific muscle tissues, primarily myofibrillar

proteins, at extreme low or high pH. This property of myofibril proteins allows for the removal

of membranes and neutral lipids, connective tissue, bones, scales and other non-solublized

components .

The effect of pH-shift processing on muscle proteins has been studied by several workers.

Currently it has been demonstrated that pH-shift processing can modify the physical properties

of the resulting protein isolate. This has been demonstrated in species ranging from cold water









Eish to land animals. The modification of physical properties of pH-shift produced isolates has

resulted in a wide range of effects. Acid (pH 2.5) treatment of myofibrillar proteins showed a

decrease in the storage modulus of cod muscle proteins where as alkali (pH 1 1.0) treatment has

shown an increase in the storage modulus when compared to a surimi control (Kristinsson and

Hultin 2003b). An increase in storage modulus refers to a stiffer gel and might indicate that a

treatment may lead to stronger and better gels. In Atlantic croaker, however, both acid (pH 2.5)

and alkali (pH 11.0) processing showed an increase in the storage modulus as compared to a

surimi control (Kristinsson and Liang 2006). Yongsawatdigul and Park (2004) made rockfish

protein isolates using both acid and alkali pH-shift processing and compared their gelation

properties with washed muscle and whole muscle homogenates. Compared to muscle and

washed muscle, isolates from alkali-aided processing had an increase in gel elasticity and force

needed to penetrate the gel while isolates from acid-aided processing showed a decrease in both

(Yongsawatdigul and Park 2004). It is unknown what the specific effects of solubilizing muscle

proteins at different high or low pHs will have on the functional properties of recovered muscle

proteins.

Muscle gelation is a multi step process with the Einal result being a three dimensional

network. This network may be studied utilizing different methods which provide information on

the various mechanisms involved in its formation and its properties. The modifications,

treatments and additives of a formulated muscle product are a directly influence of the final gel

network formed. The changes to the three dimensional gel structure by utilizing the acid or

alkali solubilizing process have shown changes in gel strength which can be determined using

the Hamann torsion test and the punch test. Small strain oscillatory testing on isolates made with

the pH shift process has shown increases in G' both after heating (~800C) and upon cooling










(~50C) (Park 2005). However, decreases have also been shown with acid processed protein

isolates. Inconsistent results from acid pH processing raise the question of a species/pH

relationship.

Acid and alkali solubilization may be done over a range of pH. The muscle proteins of

interest, mainly actin and myosin, are solubilized under acidic conditions between 2.0-3.0 and

10.5-1 1.5 under basic conditions. This can yield proteins of varying levels of unfolding. It is

hypothesized that utilizing different solubilizing conditions within this pH range will produce

differences in gel forming properties and also differences in the physical properties in the final

gel .

Methods

Raw Material

The raw material used in these studies was fresh catfish fillets obtained 1-3 days post

harvest from a local supplier. Catfish fillets were only purchased which were determined to be

within 3 days of packaging. The catfish fillets were purchased and immediately transported on

ice to the laboratory and processed the same day.

Preparation of Protein Isolates

Protein isolates were prepared according to figure 2-1. Fresh fillets were initially ground

in an Oster heavy duty food grinder (Niles, Ill., U.S.A.) for the preliminary disruption and

collection of the muscle tissue. Following grinding, the comminuted meat was diluted 1:2 (w/v)

with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds.

Following homogenization, the resulting muscle tissue slurry was further diluted to give a final

dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manually stirred with a plastic

spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the

methods described below, using either 2N NaOH or 2N HCI as needed for the pH desired, with









continuous manual mixing. Upon reaching the desired pH, insoluble material was removed by

centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products,

Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at SoC. Following centrifugation, the

soluble middle layer was collected through a kitchen strainer with a mesh size of approximately

0.25 mm to minimize contamination with other separated materials. The soluble material was

readjusted to pH 5.5 as described above. After readjustment the solution was centrifuged to

remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The

precipitated protein was collected by decanting the supernatant containing the unprecipitated

proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH

was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate

was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing

until the moisture content was below 80%. Moisture content was determined using a Cenco

infrared moisture analyzer (CSC Scientific, Fairfax, Va., U. S.A.). Upon completion of manual

dewatering the protein isolation was complete. Preliminary unpublished investigation of protein

isolates in this laboratory found the shelf life of catfish protein isolates to be 5-7 days on ice. All

protein isolates were stored on ice at the precipitation pH and used within 5 days.

Protein Concentration

Protein concentration in the isolates and the subsequent solutions was determined using the

Biuret method, as described by Torten and Whitaker (1964), with of 10% w/v deoxycholic acid

in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any

remaining lipids in the samples. Protein concentration was measured based on a standard curve

based on BSA.









Oscillatory Rheology

For the gelation tests, the isolates were rehydrated to 10% protein and pH adjusted to 7.2

with 20 mM Tris-HCI and HCl/NaOH. For the added NaCl samples, NaCl was added at 2%

w/w. The sample was homogenized at speed 1 using an Ultra-Turrax T18 homogenizer. The

protein paste was then transferred to a peltier plate at 50C attached to an AR2000 advanced

research rheometer (TA Instrument, New Castle, DE) and a head with a flat cross-hatched

polyacrylic surface. The gap was 1000 microns and the head lowered onto the sample using the

controlled speed function provided by the software. The isolates were subjected to heating (5-

800C) and cooling (80-50C), with testing conducted in oscillation mode. The storage modulus

(G'), loss modulus (G") and tan delta (6) was followed as a function of heat and time. All

experiments were done under a controlled strain of 0.01 and controlled frequency of 0. 1 Hz.

Torsion Gel Testing

Protein paste was prepared as above, except at 20% protein concentration. Proteins were

stuffed in cylindrical tubes 10 cm long by 1.8 cm in diameter and cooked at 800C for 20 min in a

water-bath. The interior wall of the tube was coated with a lecithin based spray. The cooked

samples were then removed from the casing after cooling for 30 min on ice, held overnight on

ice, and subsequently cut into 2.54 cm long pieces. These pieces were glued to plastic disks (Gel

Consultants Inc., Raleigh, N.C.) using an instant adhesive, Krazy glue (Toagosel Co., LTD.

Nishi-shinnbashi Minato-Ku, Tokyo). Dumbbell shaped samples were then milled from each

sample to 1 cm diameter in a machine mill (Electro Sales Co., Somerville, Mass., U.S.A.),

wrapped in plastic wrap to prevent moisture loss, and brought to room temperature prior to

torsion testing. For testing, 4 gel specimens were vertically mounted and sheared to the point of

fracture at 2.5 rpm in a modified Brookfield viscometer (Gel Consultants Inc., Raleigh, N.C).

Stress (kPa) and strain dimensionlesss) at fracture were calculated with the manufacturer' s









software for each sample, corresponding to the strength and deformation of the gels, respectively

(Hamann and others 1990).

Punch Test

Punch test samples were prepared by adjusting protein isolates to 20% solids according to

the moisture content and stuffed into plastic tubing, 1.25 inches in diameter. These samples were

cooked for 20 min at 800C, and then cooled rapidly in ice water. Samples were air freighted the

same day they were cooked to the laboratory of Dr. Herbert O. Hultin in Gloucester MA

(University of Massachusetts Marine Station) where punch testing was conducted The samples

were cut to 1.0 inch length and 1.25 inch diameter. The sample was placed on the machine table

and the head adjusted to just above the sample surface. A 5 mm head was used at a speed of 1.0

cm/min with force and distance at failure reported. Punch testing was conducted on a Rheo Tex

Model AP-83 (Sun Science Co., Seattle, Wa).

Fold Test

A gel fold test was conducted according to Nowsad and others (2000). The test was

conducted by slicing 2-mm thick sample disc from the cylindrical gels, mentioned above, and

folding them into halves and quarters. The scales are: 5= no crack when folded into a quarter,

4=no crack when folded into half but crack when folded into quarter, 3=crack when folded into

half, 2=broke and split into halves when folded into half, 1=broke and split prior to being folded

into half.

Gel Water Holding Capacity

Gel water holding capacity was conducted according to Nowsad and others (2000).

Expressible moisture of the gels was determined by compressing a 1.0 g spherical gel slice

between 4 double layers of filter paper at a pressure of 20 Kg/cm2 for 1 min and calculated from









weight before and after the compression. Expressible moisture was determined as the percentage

of original weight after compression.

Cook Loss

Cook loss of gels was determined as the weight of the protein paste before heat treatment

compared to the weight of the gels after cooking. Gels were prepared and heated as described

above (torsion gel testing). After cooking the gel was removed from the tube, lightly dried and

reweighed. The weight of the tube was subtracted from the total initial weight and then

compared to the final weight of the cooked gel. Percent cook loss was determined by:

Clook Loss = (1- [Grel Weight/(hzitial Weight Tube Weigh }~x 100

Cook loss is in percent, gel weight is the weight of the cooked protein paste after being

removed from the tube, initial weight is the capped tube plus the raw gel stuffed inside, tube

weight is the weight of the tube after the gel has been cooked and removed from the tube.

Statistical Analysis

Experimental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on

replicate isolations as discussed above. A replicated (N=2) was determined as acceptable due to

achieving an acceptable power (P=0.80, a=0.05).

One-way independent measures analyses of variance were used to examine the effects of

all methods used. The Kruskal-Wallis ANOVA testing by ranks was used on data which did not

pass normality. Post hoc analysis was conducted only in the presence of significant population

differences. ANOVA statistical comparisons were conducted with SigmaStat, (Systat Software

Inc. San Jose Ca) with a significance level of p<0.05. After SigmaStat completed the ANOVA

analysis, the post hoc analysis recommended by SigmaStat was used. In most cases this was










Tukey's test. Dunn's test was used for all punch test analysis; force, distance and jelly strength.

The Holm-Sikak method was used with the torsion stress and expressible moisture.

Results

Rheological Changes in Protein Isolates During Thermal Gelation

The storage modulus (G') measures the resistance of a material to positive movement of a

probe. Positive movement is the duration of movement during the oscillation of the head which

is moving away from the zero point. G' is used to determine the solid fraction of a material

during testing. In this study G' was followed with temperature to assess the change in the solid-

like characteristics of protein isolates. G' showed a decrease for all isolates without added NaCl

from 50C until 450C, and after 450C, G' began to increase (figure 3-1, 3-4, 3-7, 3-10, 3-13, 3-

16). For isolates made under acidic conditions, G' increased until 600C for pH 2.0 samples

without added NaCl and pH 2.5 samples with and without NaCl and then began to decline until

the final temperature of 80oC was reached (figures 3-1, 3-4, 3-25). The other isolates produced

under acidic conditions, pH 2.0 with added NaCl and pH 3.0, exhibited a decrease in G' until

~720C. Acid isolates with added NaCl showed an initial decrease in G' which ended at ~300C.

Further decrease in G' was then observed at about 70-720C. Isolates produced under basic

conditions with and without added NaCl did not show a second decrease in G' after the initial

decrease ending at 450C.

The G' of gel pastes with no salt added prior to heat treatment (figure 3-43) were not

significantly different (p>0.05) within the acid treated samples. The alkali processed gel paste

treatment pH 11.5 was significantly higher (p<0.05) in G' than pH treatment 10.5, but there were

no other significant differences between the individual alkali treated pH treatments. Between

individual acid and alkali pH treatments, pH 2.5 was significantly lower (p<0.05) in G' than all

alkali pH treatments and pH treatment 2.0 was significantly (p<0.05) lower in G' than pH









treatment 11.5. The control sample was significantly lower (p<0.05) in G' than all alkali pH

treatments but was not significantly different (p>0.05) from any of the acid treatments. The G'

of the gel paste with 2% added salt (figure 3-46) was not significantly different (p>0.05) within

the acid treated samples. The alkali treatment pH 11.5 was significantly (p<0.05) higher in G'

than pH treatments 2.5, 3.0 and 10.5. The control paste was significantly (p<0.05) higher in G'

than pH treatments 2.5, 3.0 and 10.5. No other significant differences (p>0.05) were present

between gel pastes.

Samples treated at low pH had G' values significantly lower (p<0.05) than samples treated

at high pH after heating at 800C and after cooling to 50C (Figure 3-45). This difference was

found for treatment conditions containing NaCl and also for those without added NaC1. The

differences between low and high pH treatment were not the same in the presence or absence of

added salt. Without the addition of salt pH treatments 2.0 and 2.5 were significantly different

(p<0.05) from pH treatment 11.5 with pH treatment 2.5 also being significantly different

(p<0.05) from pH treatment 11.0 when heated to 80oC and when cooled to SoC. With the

addition of salt pH treatments 3.0 and 2.5 were significantly different (p<0.05) from pH

treatment 11.5 after cooling to SoC however after heating to 80oC and before cooling pH

treatment 3.0 was significantly different (p<0.05) from pH treatments 11.0 and 11.5.

The theology studies also provided information about the loss modulus (G") of the protein

isolates as well as the storage modulus. The loss modulus is considered the viscous or liquid like

fraction of a viscoelastic material. The G" of catfish protein isolates in the absence of salt are

shown in figures 3-2 (pH 2.0), 3-5 (pH 2.5), 3-8 (pH 3.0), 3-11 (pH 10.5), 3-14 (pH 11.0) 3-17

(11.5) and 3-20 (whole muscle). The G" of catfish protein isolates treated in the presence of salt

are shown in figures 3-23 (pH 2.0), 3-26 (pH 2.5), 3-29 (pH 3.0), 3-32 (pH 10.5), 3-35 (pH 11.0),









3-38 (pH 11.5) and 3-41 (whole muscle). The changes in G" during both the heating and

cooling phase in acid treated samples without added salt showed a decline in G" during heating

with the minimum at the end of the heating phase, 80oC. During the cooling phase the G"

increased, after decreasing during heating. The G" after cooling was slightly higher than the

starting G" but did not show the increases seen in G'. Alkali treated samples in the absence of

added salt showed a decrease in G" during heating but the decrease during heating in all alkali

samples was characterized by a sharp dip beginning below 40oC and ending just below 60oC.

The increase in G" after the minimum point of the dip did not continue to increase but began to

decrease again. Untreated muscle showed a similar trend to alkali treated samples but showed a

more gradual decline before increasing. After the increase in G" during heating of the untreated

muscle, G" began to decline at 70oC whereas the alkali treated samples after a minor increase,

began to decrease at 60oC for pH treatments 11.0 and 11.5. Samples from the pH 10.5 treatment

however showed a similar trend to the untreated muscle at higher temperatures, beginning to

decrease in G" at 70oC. In the presence of added salt, pH treated samples showed a G" trend

similar to that of G' though the value was lower.

Tan delta is the relationship of G"/G' which provides an analysis of the transition of a

material from a more viscous material to a more elastic material. A perfectly elastic material is

represented as tan delta equal to 0, or stress and strain are perfectly in phase and a perfectly

viscous material is represented as tan delta equal to 90 or stress and strain are perfectly out of

phase (Park 2005). Tan delta was analyzed in all samples by merging and smoothing replicate

rheograms. The tan delta results are shown in figures 3-3 (pH 2.0), 3-6 (pH 2.5), 3-9 (pH 3.0), 3-

12 (pH 10.5), 3-15 (pH 11.0), 3-18 (pH 11.5), 3-21 (whole muscle) for samples treated without

salt. In the absence of salt the tan delta transition showed more of a peak increase in tan delta









prior to the start of the decline in tan delta. The decline in tan delta began at 37oC in samples

subj ected to pH-shift processing. The whole muscle samples did not show a peak increase but a

gradual increase in tan delta leading to a gradual decline in the absence of added salt. Samples

treated with either acid or alkali processing in the presence of added salt is shown in figures 3-24

(pH 2.0), 3-27 (pH 2.5), 3-30 (pH 3.0), 3-33 (pH 10.5), 3-36 (pH 11.0), 3-39 (pH 11.5) and 3-42

(whole muscle). Samples treated with either acid or alkali processing showed a gradual decline

in slope beginning between 19-22oC during heating and did not increase after this decline began.

The tan delta for the control however increased to between 28oC and 42oC with the peak ranging

over SoC from 42-47oC with no change in tan delta then began the decrease, forming a bell

curve. After the decline began at 47oC no increase in tan delta was observed.

Gel Quality of Isolates as Assessed by Torsion Testing

The results of torsion testing are shown in figures 3-49, 3-50. Alkali aided isolate gels

11.0 and 11.5 had significantly higher (p< 0.05) stress and strain values than acid aided isolate

gels or control. Acid and alkali treated isolate gels were significantly higher in stress (p< 0.05)

but not significantly different (p>0.05) in strain from the control. Alkali aided isolate gels

showed significant differences (p< 0.05) within the high pH group, with pH 11 samples showing

significantly higher stress differences (p< 0.05) from pH 10.5 and 11.5, with no significant

differences (p>0.05) between isolates made at pH 10.5 or 11.5. There were no significant

differences (p>0.05) in strain within the alkali aided isolate. Significant differences (p<0.05)

were observed between all pH treatments compared to the control for stress but no differences

were observed in strain values.

Gel Quality of Isolates as Assessed by Punch Testing

Acid and alkali aided isolate gels 2.5, 10.5, 11.0 and 11.5 had a significantly higher (p<

0.05) break force than control samples shown in figures 3-51. The distance required to break all









of the gels did not show significant differences (p>0.05) except pH 3.0 was significantly lower

(p<0.05) than pH 10.5, and the control. The control had a significantly lower (p<0.05) force than

pH treated samples 2.0, 10.5, 11.0 and 11.5. The control was significantly higher (p< 0.05) in

the distance needed to break the gel than gels made with isolate from pH 3.0 treatment. No other

significant differences (p>0.05) were observed between the control and pH treated samples.

Gel Quality of Isolates as Assessed by Expressible Moisture

The results from expressible moisture testing of the samples are shown in figure 3-54. The

results of the study show gels made with acid-aided isolates have higher expressible moisture

than gels made with alkali-aided isolates. Significant differences (p<0.05) were observed

between gels made with alkali treatments 11.0 and 11.5 when compared to the acid treatment at

pH 2.0 and the control. Other individual pH treatments from acid- or alkali-aided isolates did not

show any significant differences (p>0.05).

Gel Quality of Isolates as Assessed Fold Test

The fold test conducted in this study is shown in figure 3-55. These results did not show

any significant differences (p>0.05) between gels from the pH-shift process and the control gels.

All gels made with isolates from the alkali-aided process treatment and the control gels scored a

5, which is the highest score. The gel made with the isolate from the acid-aided process ranged

from 2.5-4, but were not significantly different from the control and the alkali-aided gels.

Gel Quality of Isolates as Assessed by Cook Loss

The cook loss results of this study are shown in figure 3-56. The results of this study did

not show any significant differences (p>0.05) between gels made with isolates from the acid-

aided process as compared to gels made with isolates from the alkali-aided process. Individual

pH treatments within samples treated at low pH or at high pH did not show any significant

differences (p>0.05). Individual pH treatments or grouped pH treatments did not show any









significant differences (p>0.05) as compared to the whole muscle control except pH treatment

11.0 was significantly (p<0.05) lower than the control.

Discussion

The G' or rigidity modulus is the resistance to gel structural movement of the network

which is being formed. G' is derived in oscillatory testing as the fraction of the sigmoid

movement of the head which is positive or negative displacement from 0 as compared to the

fluid fraction of oscillatory measurement which is the return to 0 from positive or negative

movement of the head. As the myofibrillar proteins set into a gel structure the resistance to

movement of the curing system is increased by molecular interactions. The theological testing

of isolate measured non-fracture parameters using micro-strain oscillatory testing. The

resistance to movement, or decreasing fluidity of the gel network is shown as increased G'. The

theological findings of this study show that pH treatments used to produce isolate significantly

influence the final rigidity of the thermally set gel.

The protein paste prior to heat treatment, in both the presence and absence of salt, showed

the same trend as final gel rigidity of the thermally set and cooled gels (figures 3-43 and 3-45 (no

salt, respectively) and 3-46 and 3-48 (added salt, respectively)) except for pH 3.0 treatment. The

pH 3.0 treatment was equal to pH 2.5 treatment prior to heating, but after heating decreased in G'

relative to the other samples. The trends of the gel paste prior to heat treatment may provide an

indication of the relationship of the final gel rigidity of the thermally treated muscle paste with

the pH shift processed catfish muscle used in this study; however, the relative increase [(final G'/

initial G')*100] in gel rigidity from the raw paste to the final cooked gel ranged from 200-

2000%. Due to the consistent trend of the raw paste with the final gel rigidity, standardizing the

start point of theological testing (initial G'- final G') did not affect the final results of the testing

even with the out of trend raw paste seen in pH treatment 3.0.









The acid treated samples had a higher susceptibility to movement whereas alkali treated

samples had a decreased susceptibility or increased structural rigidity (G') when compared to

untreated muscle. These results are inconsistent with results shown for Atlantic croaker which

showed an increase in gel rigidity over muscle without pH-shift processing, for both acid (pH

2.5) and alkali (pH 11.0) treatments (Kristinsson and Liang 2006). Isolates produced at pH 2.0

with added salt increased above that of the untreated catfish muscle. It can be concluded from

the differentiation of gel rigidity as indicated by G' that pH processing at different low or high

pH values allows for the modulation of the gel strength. This modulation of gel strength can

now be tailored as the final product requires.

The theological results of acid aided isolate made from the lowest pH treatment (highest

degree of ionization at the low pH values tested) show gel rigidity increased above that of the

untreated control in the presence of added salt (figure 3-48). When the treatment pH used was to

achieve solubility and separation was less extreme (pH 2.5 and 3), the rigidity of the gel was

equal to or less than the control. These results are similar to those shown by Raghavan and

Kristinsson (2007a), where catfish myosin increased in gel rigidity as pH of acid solubilization

decreased. Previous studies on acid solubilization of muscle proteins from sources other than

catfish have shown acid solubilization increasing gel strength above that of untreated controls

(Kristinsson and Liang 2006; Mireles Dewitt and others 2007). However, the solubilization pH

used in these experiments was not as low as the most extreme pH used here. The increase in gel

rigidity above the control in only the most extreme pH treated sample indicates that a high net

positive charge which influences the level of unfolding is necessary to produce increased gel

rigidity above that of an untreated control is species specific. Studies have demonstrated that

extreme pH may lead to a partially refolded state, often called a molten globule. This modified









structure may be predisposed to promote more protein-protein interactions which can promote

the formation of gels and produced stronger gels via more extensive protein-protein interactions

during thermal processing. Rheological studies on isolated muscle proteins, specifically catfish

myosin, have shown that acid treatment resulted in a gel which increased in G' above untreated

control (Raghavan and Kristinsson 2007a). Increasing gel rigidity of catfish myosin with less

extreme ionization/unfolding than that used here, indicates the contribution of other myofibril

proteins co-precipitated with myosin in the whole protein system (Kristinsson and others 2005b)

after pH adjustment will greatly influence the final rigidity an isolated muscle system is able to

achieve. Thus, when using a whole muscle system it is important to optimize the pH used. The

higher low pH values used to make acid aided catfish protein isolates (in the presence of salt)

resulted in lower G', These results indicate that treatment with less [H+] subj ected to the muscle

proteins produces isolates which do not promote the stronger interactions occurring with the

isolates made with the treatments with the highest level of [H+].

No significant differences (p>0.05) were observed between alkali treated gels in the

presence or absence of added NaC1. Previous studies investigating multiple pH treatments on

tilapia white muscle reported an improvement of gel properties with alkali treatment but

decreased gelling ability of acid treated proteins (Ingadottir 2004). The increase in gel rigidity

with alkali treated samples is consistent with previously published data on pH-shift processed

muscle at alkali pH. The lack of differences in alkali treated catfish samples indicates that the

molecular transitions induced by alkali pH treatment produces protein structures (in the presence

of 2% NaC1) with similar interaction potentials at all alkali pH values tested,, thus resulting in

unfolded structures which have similar gel forming abilities. As muscle gels are formed in the

presence of salt, it may be concluded that alkali solubilized isolate produces a gel network with









the highest resistance to compressibility of the samples tested and all alkali treatments tested

results in gel networks with equivalent non-fracture compressibility.

During the heating phase, all isolates and the control in the absence of added salt

demonstrated a similar decline in G' until about 45oC, which indicates that the molecular

interactions induced by the thermal input during this range were similar for all isolates under the

conditions tested. In the absence of salt, and at the pH tested, there is a significant repulsion

between the muscle proteins at low temperatures, which may be modified as the system is

heated, leading to a more fluid system, hence lower G' as seen here in this study. Above

approximately 45oC, all isolates and control samples with no added salt demonstrated an increase

in G', indicating the development of thermally induced protein-protein interactions. While

alkali-aided isolates made from pH 11 and 11.5 treatments (figures 3-13 and 3-16) showed a

gradual increase in G' until 800C (maximum point of heating), the acid-aided isolates (figures 3-

1, 3-4 and 3-7) peaked at about 600C (pH 2.0 and 2.5) and about 700C (pH 3.0), demonstrating

they had different and weaker interactions than the alkali-aided isolates. The isolate made with

pH 10.5 treatment (figure 3-10) had a similar gelation behavior as the acid-aided isolates,

peaking at about 700C, suggesting it had different protein-protein interactions than the other

alkali-aided treatments.

Gelation behavior of isolates in the presence of salt was different than in the absence of

salt. This is not unexpected as the presence of salt can cause partial solubilization of the proteins

and can also induce protein unfolding. Prior to heating the G' of the samples with salt was lower

than the sampled without added salt. This is most likely due to the fact that salt would screen the

electrostatic repulsive charges between then proteins, and thus lead to a more fluid system, hence

lower G'. Small changes were seen in G' during heating, until between 35-400C for the acid-









aided gels (figures 3-22, 3-25 and 3-28) and about 40-450C for the alkali-aided gels (figure 3-31,

3-34 and 3-37). Unfolding is a prerequisite to thermal gelation of muscle proteins, thus

suggesting that proteins in the acid-aided isolates were more susceptible to heat and presumably

thermally unfolded at a lower temperature than the proteins in the alkali-aided process. Both the

acid- and alkali-aided isolates had a distinctly different gelation behavior on heating in the

presence of salt compared to the control (figure 3-40), which demonstrated an expected dip in G'

between 35 and 500C. This clearly shows that the pH-shift process leads to changes with the

proteins in the isolate which in turn have different gel forming properties.

All isolates, and control, regardless of the presence or absence of salt demonstrated a

significant increase in G' during the cooling phase. The final G' was in all cases substantially

higher than the initial G', but the difference between the two varied greatly between treatments.

The difference in initial and final G' can be an indicator of the ability of a sample to produce a

strong gel. The larger the final G', the more rigid and strong the gel could be. The increase in

initial and final G' for acid-aided gels in the absence of salt ranged from 1.8-3.3 fold, while it

was 5.9-6.4 fold for the alkali-aided gels. Although the control increased its G' 5.5 fold, the final

G' was lower than the G' of alkali-aided gels. In the presence of salt the increase in G' was 3.6-

13 fold for the acid-aided gels but 14-25 fold for the alkali-aided gels. The control had an 8 fold

increase in G'. The much larger increase in G' during the heating phase of gelation exhibited by

alkali processed muscle proteins, clearly indicates that high pH processing results in protein

conformations and supramolecular structures which promote stronger protein-protein interactions

during heating and particularly cooling than acid-aided gels and untreated minced catfish muscle.

The larger increase in G' on cooling for the alkali-aided gels compared to the acid-aided gels

may suggest they lead to increased hydrogen bonding on cooling (Lanier 2000).









Muscle protein gels are characterized from a quality standpoint by color, gel strength and

flexibility. Gel strength is evaluated as resistance to structural breakage or the stress coefficient.

The gel flexibility is the structural elasticity or the strain coefficient. The stress and strain at

fracture of muscle gel may be analyzed in two ways; 1) the rotational stress and strain and 2) the

tangential stress and strain. Torsion testing analyzes the rotational stress and strain by measuring

the force and distance required to twist a cylindrical gel segment until it breaks in half.

Tangential fracture measurement is done using the punch test. The punch test lowers a probe

into a flat cross-section of the gel and measures the force and distance required to puncture the

gel. These two tests are frequently used to determine physical qualities attributed to the quality

of muscle protein products.

Torsion testing is the study of the rotational fracture properties of muscle proteins gels and

was used in this study to compare pH treated and untreated samples using a method widely

accepted for surimi testing. Four primary texture profile properties which have been attributed to

surimi gels by torsion testing (Park 2005), those are mushy, brittle, rubbery, and tough (Park

2005). Mushy is described by having low stress and low strain, with brittle having high stress

and low strain. The other two are high strain descriptors with rubbery having low stress and

tough having high stress. However, the results with catfish protein isolate, as seen in figure 3-57,

do not fall within the parameters of a typical texture profile graph, as the gel stress is too high

except for the control which is considered brittle. As seen in figure 3-57 all treatments resulted

in product that had brittle properties (Park 2005).

The differentiation between acid and alkali treated samples in the way they respond to

rotational fracture indicates alkali treatment forms both a stronger and more elastic gel. The

individual pH treatments did show differences with pH 2.5 having the lowest stress and pH 11.0









the highest. The desired textures of a restructured muscle product are important in the

processing chosen for the production of that product. Kim and others (1996) showed

conventional processing of catfish by-products resulted in a surimi product with acceptable

textural properties for the production of shellfish analogs with the addition of cryoprotectants

and starch. The torsion results of pH-shift processing of catfish muscle when compared to the

results of Kim and others (1996) showed that protein gels (with no added cryoprotectants) made

from alkali processing produced a stronger gel than either acid or no processing. The increased

strength of pH-shift processed gels are however categorized as brittle according to the torsion

texture profile (Park 2005). Acid processing of catfish, however, has led to mixed results. The

acid results reported here for catfish are similar to Mireles Dewitt and others (2007) who showed

that acid treatment resulted in an improvement in gel strength as compared to a ground muscle

control .

The pH-shift process exhibited higher shear stress and lower shear strain than the

processing and formulation additives used in the products tested to develop the torsion texture

profile (Park 2005). The pH-shift processing of catfish muscle proteins compared to croaker,

whiting, rockfish and cod since alkali treatment increased gel strength and elasticity of all

species compared to acid treatment and no treatment. These differences show that the pH used

to produce isolate do in fact provide protein isolates with different rotational physical resistance.

Punch testing, which is a study of tangential fracture properties of muscle protein gels, was

also used in this study to compare pH treatments and untreated samples. This is the other

fracture testing method widely used in the surimi industry. The parameters recorded are the

resistance and distance traveled at breakage. Breakage is when the structure of the gel gives way

and can no longer hold its cohesive form. The results of this study indicate that pH treatment









directly affects the punch test scores as shown in Eigures 3-51, 3-52, and 3-53. Other studies

have shown surimi from carp form a gel with lower break force and distance than acid or alkali

processing (Luo and others 2001a). The effect of changing the punch test scores is that the

surimi quality grade may be based on the jelly score (Park 2005) obtained from punch test

values, the j elly score is the multiplication of force times distance. The j elly score for pH-shift

processed catfish muscle is shown in figure 3-53. The problems with the jelly score are many

(Park 2005), but as the j elly score is the product of gel force and gel deformation the quality of

the protein isolate is not reported accurately. The results of this study show that pH-shift

processing does not change the gel structure as assessed by the punch test as dramatically as

seen in other testing methods, specifically torsion testing.

The fold test is a semi-quantitative test used to determine the flexibility of cooked muscle

protein gels rapidly. Based on the 1-5 or grading scale the fold test allows for a Hyve point

scoring of the gels. The fold test allows for the determination of gels which are considered to be

too brittle. However the fold test does not account for gels which may be too elastic. The fold

test is one of the primary methods used in grading surimi. The lack of significant difference

(p>0.05) between all pH treated samples and the control indicates that catfish muscle proteins

with or without pH shift processing will form acceptable gels under these test conditions. The

alkali results are consistent with pH-shift processing of tilapia (Ingadottir 2004). However, acid

processed tilapia also resulted in fold test scores of 5 which the catfish isolate gels from the acid-

aided process did not. The lack of significant difference (p>0.05) between the acid treatment and

the alkali treatment or the control, but with inconsistent results in acid processed catfish muscle

as compared to the consistent results of acid processed tilapia muscle, indicate that the acid

processing of catfish muscle may lead to a more complex material than the same process did









with tilapia. The production of surimi from catfish, however, showed acceptable results with the

addition of cryoprotectants and starch (Kim and others 1996). This indicates that based on the

fold test, pH-shift processing of catfish muscle, though inconsistent, under the acidic conditions

tested here, provides a gel structure acceptable for the production of a surimi product without the

addition of cryoprotectants or other additives.

The press test investigates water holding capacity of the gel matrix formed after cooking.

The water holding capacity of muscle gels is directly related to the firmness of the gel network

(Kim and others 1996). The results of the study show that alkali treated isolate, pH 11.0 and

11.5, had significantly higher water holding capacity than acid treated isolate, pH 2.0, and the

control. Water holding capacity of pH-shift processed catfish muscle when compared to catfish

surimi indicates that pH-shift processing resulted in decreased water holding capacity compared

to washed catfish surimi with added cryoprotectants which had an expressible moisture of less

than 2 percent (Kim and others 1996). The results ofKim and others (1996) indicate that catfish

mince with or without cryoprotection produced a weak gel with puncture forces less than 500g.

These results are inconsistent with the results found in this study showing that even though there

is a reduction in water holding capacity, the preparation of thermally set gels without

cryoprotection does result in strong gel formation. The stability of pH-shift processed catfish

muscle on frozen storage and the need for cryoprotection has yet to be determined.

Cook loss determines the amount of weight a product may lose during thermal processing.

This weight loss is very important as increased cook loss reduces the final retail weight of the

finished product and is a direct economic loss for the processor. Cook loss is a form of water

holding capacity of muscle gels as the minced muscle product transitions from a protein paste to

a thermally set network. The lack of significant difference (p>0.05) between the pH treated










samples and the control except for pH treatment 11.0 indicates that the water holding capacity of

pH processed isolate during thermal transition is not affected by all pH treatments except pH

11.0. The low cook loss of all samples in the current study (less than 3%) indicated that the

isolates have a better water holding capacity than that reported for acid processed catfish surimi

with added cryoprotectants and phosphates, which are known to aid in water retention (Mireles

Dewitt and others 2007). This means that pH-shift processing of catfish muscle may provide a

protein isolate with comparable or improved properties of phosphates in muscle systems but

without the addition of phosphates.

Conclusions

The results of the gel properties of pH-shift processed catfish muscle indicate that pH

processing results in improved gel strength over conventional processing according to the catfish

results published by Kim and others (1996). These results also indicate that the range of textural

possibilities has not been fully explored. The brittle gels formed, based on the textural schematic

of Park (2000), indicate that optimization of the gel properties of pH-shift processed catfish

muscle is crucial before commercial processing is possible. Increasing the shear strain above

2.25 and decreasing the shear stress below 70 kPa is just the first step in developing a pH-shift

processed catfish muscle into a useable primary product. The most desirable method for

achieving the reduction in shear stress and the increase in shear strain is the addition of water to

the gel paste. The pH-shift processed isolates were tested at 80% moisture compared to 78%

moisture used by Kim and others (1996). This suggests that pH-shift processed isolates may

require a lower protein concentration than conventionally processed catfish muscle. Additional

benefits to the processor and the consumer if pH-shift processing is used include taking

advantage of the different solubilization pH values. pH-shift processing allows the processor to

have greater control over the desired texture for specific applications as seen within the textural










ranges published here. This data also suggests that individual isolates may be combined to

provide an even broader range of textures within a single comminuted product if the appropriate

processing conditions are used.









20000
18000
16000
14000
e~1200 ~Cooling





2000 Heating

0 20 40 60 80 100
Temperatume (oc)
Figure 3-1: The storage modulus (G') of catfish protein isolates produced at pH 2.0 during
heating and cooling as tested by small strain theology. The gel was developed at
10% solids, 2oC/min in the absence of added NaC1. Testing was conducted with
controlled strain, 0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used
with a 40 mm cross-hatched acrylic head set to a gap of 1000 pm. The development
of thermal gelation during heating and cooling shows the physical transitions of
proteins subj ected to pH-shift processing.









I


3500


3000

2500

2000 L Cooling

S1500
1000
heating
500


0 20 40 60 80 100

Temperature (oC)
Figure 3-2: The loss modulus (G") of catfish protein isolates produced at pH 2.0 during heating
and cooling as tested by small strain theology. The gel was developed at 20C/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









0.4

0 5-

0.5




Cooling
0.0 5


0 20 40 60 80 100

Tem pe ratu re (oC)

Figure 3-3: The tan delta (G"/G') of catfish protein isolates produced at pH 2.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









8000
7000
6000

5000 Cooling
e 4000
(33000
2000
1000
Heating

0 20 40 60 80 100

Temperature (oc)
Figure 3-4: The storage modulus (G') of catfish protein isolates produced at pH 2.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.




















eaing


1600
1400
1200
1000
800
600
400
200
0


100


Tem peratu re (o C)


Figure 3-5: The loss modulus (G") of catfish protein isolates produced at pH 2.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









0.4



*0.2 -
0.15


0.15

0.05
0 Cooling

0 20 40 60 80 100

Temperature (oC)

Figure 3-6: The tan delta (G"/G') of catfish protein isolates produced at pH 2.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









45000
40000
35000
30000
25000
a Cooling
20000






0 20 40 60 80 100
Temperature (oC
Figure 3-7: The storage modulus (G') of catfish protein isolates produced at pH 3.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.













Ah /Cool ing






H eating


8000
7000
6000
5000
4000
3000
2000
1000
0


40 60
Temperature (oC)


100


Figure 3-8: The loss modulus (G") of catfish protein isolates produced at pH 3.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.










I IYY-1LYI~ I


0.4


0.35
0.3
0.25 Hetn
*0.2
0.15
0.1
0.05/
0~C'ool ing

0 20 40 60 80 100
Temperature (oC)

Figure 3-9: The tan delta (G"/G') of catfish protein isolates produced at pH 3.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









50000
45000
40000
35000
~ 30000Cooling
e6 25000
b 20000 H~eating
15000
10000
5000 *

0 20 40 60 80 100
Temperature (oc)
Figure 3-10: The storage modulus (G') of catfish protein isolates produced at pH 10.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.









8000
7000
6000
S5000 -ICoo/~n linfg
S4000
(3 3000
2000
1000
H eating g

0 20 40 60 80 100
Temperatu re (o C
Figure 3-11: The loss modulus (G") of catfish protein isolates produced at pH 10.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.










0.4

/Heating


0.15


0.15
0.05
0.0

0 20 40 60 80 100
Temperature (oC)

Figure 3-12: The tan delta (G"/G') of catfish protein isolates produced at pH 10.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









80000
70000
60000

40000
3 0000
Heating

200


0 20 40 60 80 100
Temperature (oc)
Figure 3-13: The storage modulus (G') of catfish protein isolates produced at pH 1 1.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.









10000
9000
8000
7000 oln

S5000
(3 4000
3000
2000
1000
Heating

0 20 40 60 80 100
Temperature (oC)
Figure 3-14: The loss modulus (G") of catfish protein isolates produced at pH 11.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.










I I


0.4


0 35 L~Heating


0.25
*0.2




0.05
Cooling

0 20 40 60 80 100
Temperature (oC

Figure 3-15: The tan delta (G"/G') of catfish protein isolates produced at pH 1 1.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









80000
70000
60000
50000
crs Cooling
S40000
~330000 *
Heating
20000
10000


0 20 40 60 80 100
Temperature (oC)
Figure 3-16: The storage modulus (G') of catfish protein isolates produced at pH 11.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.









10000
9000
8000
7000
ca6000
e 50 Cooling
(3 4000
3000
2000
1000
Heating

0 20 40 60 80 100
Temperature (oq)
Figure 3-17: The loss modulus (G") of catfish protein isolates produced at pH 11.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min in the absence of added NaC1. Testing was conducted with controlled strain,
0.01 at a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-
hatched acrylic head set to a gap of 1000 pm. The development of thermal gelation
during heating and cooling shows the physical transitions of proteins subjected to pH-
shift processing.









0.4
0 35


a0.2


0.05 Cooin
0.




0 20 40 60 80 100
Temperature (oC
Figure 3-18: The tan delta (G"/G') of catfish protein isolates produced at pH 1 1.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min in
the absence of added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.








25000

20000

*& 15000Coln

i3 10000
Heating

5000

0~,

0 20 40 60 80 100
Temperature (oC)
Figure 3-19: The storage modulus (G') of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min in the absence of
added NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1
Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to
a gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.









4000
3500
3000
2500
err Cooling
2000
'3 1500
1@00


0 Heating
0 20 40 60 80 100
Temperature (oC)
Figure 3-20: The loss modulus (G") of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min in the absence of
added NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1
Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to
a gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.









0.3



0 25 Heating


0.05


0.


0 20 40 60 80 100
Temperature (oC
Figure 3-21: The tan delta (G"/G') of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min in the absence of
added NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1
Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to
a gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.









16000
14000
12000

*R10000 1 Cooling
~c8000
6000
4000
2000


0 20 40 60 80 100
Temperature (oC)
Figure 3-22: The storage modulus (G') of catfish protein isolates produced at pH 2.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









2500

2000

Cooling
1500o

63 1000

500 Heating~



0 20 40 60 80 100
Temperatu re (oq)
Figure 3-23: The loss modulus (G") of catfish protein isolates produced at pH 2.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.










I I


0.4


0.35
0.3 Heating
0.25
*0.25
0.15
0.1 ,

0.05 Cooling


0 20 40 60 80 100
Temperature (oC)
Figure 3-24: The tan delta (G"/G') of catfish protein isolates produced at pH 2.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









9000
8000
7000
6000
S5000Coln
4000
3000
Heating

200


0 20 40 60 80 100
Temperature (oc)

Figure 3-25: The storage modulus (G') of catfish protein isolates produced at pH 2.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









1400
1200

1000

800
e. Cooling
600

400 Heating,

200


0 20 40 60 80 100
Temperature (oC)
Figure 3-26: The loss modulus (G") of catfish protein isolates produced at pH 2.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.










0.45

0.35 1 f
0.3 4Heating
a0.25

0 0.25
S0.15
0.15

0.05
0 Cooling

0 20 40 60 80 100
Temperature (oC)
Figure 3-27: The tan delta (G"/G') of catfish protein isolates produced at pH 2.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









I I


3500


203000 III


a 2000

i3 1500
1000
500 ******Heating
50

0 20 40 60 80 100
Temperature (oC)
Figure 3-28: The storage modulus (G') of catfish protein isolates produced at pH 3.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









700

600
500 I

400

S300
200 -c
100
Heating

0 20 40 60 80 100
Temperature (oC)
Figure 3-29: The loss modulus (G") of catfish protein isolates produced at pH 3.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









0.4


Heating

0 01 25


0 .1
0 .0 5Cool ing
0.0

0 20 40 60 80 100
Temperature (oC
Figure 3-30: The tan delta (G"/G') of catfish protein isolates produced at pH 3.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.










50000


40000
Cooling
111' 30 00 0

b3 20000

10000
0etn

0 20 40 60 80 100
Temperature (oC)
Figure 3-31: The storage modulus (G') of catfish protein isolates produced at pH 10.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









9000
8000
7000
6000
5000 oo ing
4000
3000
2000
SHeating
1000

0 20 40 60 80 100
Tem peratu re (oC)
Figure 3-32: The loss modulus (G") of catfish protein isolates produced at pH 10.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









0.45
0.4



0.15


0.15

0.05 Cooling

0 2

Temperatu re4 (oc)0

Figure 3-33: The tan delta (G"/G') of catfish protein isolates produced at pH 10.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.














10000*



15000


500

0 20 40 60 80 100
Tenperature (oC

Figure 3-.34: The storage modulus (G') of catfish protein isolates produced at pH 1 1.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









5000
4500
4000
3500
~r Cooling
2 5 0 03 0

63 2000
1500 Heating

100


0 20 40 60 80 100
Tem peratu re (oC)
Figure 3-35: The loss modulus (G") of catfish protein isolates produced at pH 11.0 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









0.4
0.35
0.3
0.251 ~L~yHeating
a 0.25

S0.2


0.05
0 ~Cooling

0 20 40 60 80 10 0
Temperature (OC

Figure 3-36: The tan delta (G"/G') of catfish protein isolates produced at pH 1 1.0 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









25000


20000 2Co n

15000Coln

(3 10000
Heating

5000



0 20 40 60 80 100
Temperature (oC)
Figure 3-37: The storage modulus (G') of catfish protein isolates produced at pH 11.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









4000
3500
3000
Cool ing
S2500
S2000
(3 1500
H eating
1000
500


0 20 40 60 80 100
Tempe ratu re (oC)
Figure 3-38: The storage modulus (G") of catfish protein isolates produced at pH 1 1.5 during
heating and cooling as tested by small strain theology. The gel was developed at
2oC/min with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at
a frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.










I UY*..r~ I


0.35


0.3

0.25

0 2 Heating




0.05
Cooling

0 20 40 6080 100
Temperature ("C)
Figure 3-39: The tan delta (G"/G') of catfish protein isolates produced at pH 1 1.5 during heating
and cooling as tested by small strain theology. The gel was developed at 2oC/min
with 2% added NaC1. Testing was conducted with controlled strain, 0.01 at a
frequency of 0. 1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched
acrylic head set to a gap of 1000 pm. The development of thermal gelation during
heating and cooling shows the physical transitions of proteins subj ected to pH-shift
processing.









9000
8000

7oao -~, Co olin
6000

4000
3000
Heating

200

0 20 40 60 80 100
Temperature (oC)
Figure 3-40: The storage modulus (G') of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min with 2% added
NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1 Hz.
Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a
gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.









1400

1200
1000

800
Cooling
600
400

200 -Heating

0 20 40 60 80 100
Tem pe ratu re (oC)
Figure 3-41: The loss modulus (G") of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min with 2% added
NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1 Hz.
Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a
gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.









0.35


0025 Heating
a0.25


S0.15

0.1

0.05
Cooling

0 20 40 60 80 100
Temperature (oC

Figure 3-42: The tan delta (G"/G') of untreated catfish muscle during heating and cooling as
tested by small strain theology. The gel was developed at 2oC/min with 2% added
NaC1. Testing was conducted with controlled strain, 0.01 at a frequency of 0. 1 Hz.
Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a
gap of 1000 pm. The development of thermal gelation during heating and cooling
shows the physical transitions of proteins subj ected to pH-shift processing.










16000


14000

12000

10000

S8000
6000

4000

2000

0


2 2.5 3 10.5 11 11.5 control


Treatment


Figure 3-43: The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) prior to heating, in the absence of added NaC1. The significant
differences present between samples (p<0.05) are represented by the letters above the
column. Similar letters represent no significant difference (p>0.05) between
treatments.









16000
14000 C
12000
ABC
10000AB
c-- ABC
8000
(36000 A
4000A
2000 -


2 2.5 3 10.5 11 11.5 control
Treatme nt

Figure 3-44: The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) after heating to 800C, in the absence of added NaC1. The
significant differences present between samples (p<0.05) are represented by the
letters above the column. Similar letters represent no significant difference
(p>0.05) between treatments.













| qBC ABC


'* **********


2 2.5


10.5
Treatment


1 1 1 1.5 co ntrolI


Figure 3-45:


The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) after cooling to 50C, in the absence of added NaC1. The
significant differences present between samples (p<0.05) are represented by the
letters above the column. Similar letters represent no significant difference
(p>0.05) between treatments.


70000

60000
50000

ca 40000

i3 30000
20000
10000

O


ABC


-~f~TA











2000
1800 A
1600
1400__ B
1200B
0 1000 -
A-
600
400 -
200 -


2.0 salt 2.5 salt 3.0 salt 10.5 salt 11.0salt 11.5salt control
salt

Treatment


Figure 3-46: The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) prior to heating, in the presence of 2% added NaC1. The significant differences
present between samples (p<0.05) are represented by the letters above the column. Similar
letters represent no significant difference (p>0.05) between treatments.









-


'


10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0


2 2.5


3 10.5


11.5 control


AB


1 AB
- A
-


Treatment

Figure 3-47: The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) after heating to 800C, in the presence of 2% added NaC1. The
significant differences present between samples (p<0.05) are represented by the
letters above the column. Similar letters represent no significant difference (p>0.05)
between treatments.









40000
35000 A
30000
25000
20000
i315000 AB
10000 -- AhI I I AB

50000 1I I c

2 2.5 3 1 0.5 1 1 1 1.5 co ntrolI
Treatment

Figure 3-48: The storage modulus (G') of catfish protein isolates compared to unprocessed
muscle (control) after cooling to 50C, in the presence of 2% added NaC1. The
significant differences present between samples (p<0.05) are represented by the
letters above the column. Similar letters represent no significant difference (p>0.05)
between treatments.









160

140

120



u, 80
S60

40

20

0


BE
T


AB


C


A


2 2.5 3 10.5 11 11.5 control
Treatme nt

Figure 3-49: The torsion shear stress of catfish protein isolates and unprocessed muscle (control)
heat set gels as tested by torsion shear. The gel was developed with static heating
(80oC for 20 min) and 48 hrs cold setting time, with 2% added NaC1. The significant
differences present between samples (p<0.05) are represented by the letters above the
column. Similar letters represent no significant difference (p>0.05) between
treatments.










B B
AB
AB I I AB
- AB


1.8
1.6
1.4
1.2


0.8
0.6
0.4
0.2
0


2 2 .5 3 1 0.5 1 1 1 1.5 co ntrolI
T reatme nt

Figure 3-50: The torsion shear strain of pH-shift processed catfish protein isolates and
unprocessed muscle (control) heat set gels as tested by torsion shear. The gel was
developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2%
added NaC1. The significant differences present between samples (p<0.05) are
represented by the letters above the column. Similar letters represent no significant
difference (p>0.05) between treatments.











A AA
AB A AB 1-


1000
900
800
700
600
500
400
300
200
100
0


2 2.5 3 10.5 11 11.5 controlI
pH treatme nt
Figure 3-51: The tangential shear stress of pH-shift processed catfish protein isolates and
unprocessed muscle (control) heat set gels as tested by punch testing. The gel was
developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2%
added NaC1. The significant differences present between samples (p<0.05) are
represented by the letters above the column. Similar letters represent no significant
difference (p>0.05) between treatments.












AB

AB A


2.5


Figure 3-52: The tangential shear strain of pH-shift processed catfish protein isolates and
unprocessed muscle (control) heat set gels as tested by punch testing. The gel was
developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2%
added NaC1. The significant differences present between samples (p<0.05) are
represented by the letters above the column. Similar letters represent no significant
difference (p>0.05) between treatments.


10.5
pH treatment


11.5 control











A
A

A A
-
AB AB
- q
-

_ B
-

-


900
800

700
600

500
400

300
200

100
0


2.5


Figure 3-53: The j elly strength of pH-shift processed catfish protein isolates and unprocessed
muscle (control) heat set gels. Jelly strength was calculated from the punch test values
and is tangential shear stress~shear strain.. The gel (with 2% added NaC1) was
developed with static heating (80oC for 20 min) followed by 48 hrs cold setting time.
The significant differences present between samples (p<0.05) are represented by the
letters above the column. Similar letters represent no significant difference (p>0.05)
between treatments.


3 10.5
Sample


11.5 controlI











A AB A


2.5


10.5


1 1 .5 co ntrolI


Treatm ent

Figure 3-54: The expressible moisture of pH-shift processed catfish protein isolates and
unprocessed muscle (control) heat set gels as tested by placing a 3 Kg weight on a gel
slice, 1.8 cm in diameter and 3 mm in thickness. The weight loss is assumed to be
water leakage from the gel matrix and is a determinant of water holding capacity of
the gel matrix. The gel was developed with static heating (80oC for 20 min) and 48
hrs cold setting time, with 2% added NaC1. The significant differences present
between samples (p<0.05) are represented by the letters above the column. Similar
letters represent no significant difference (p>0.05) between treatments.



























2 2.5 3 1 0.5 1 1 1 1.5 co ntrolI
Treatment

Figure 3-55: The shear stress of pH-shift processed catfish protein isolates and unprocessed
muscle (control) heat set gels as tested by folding a 3 mm gel slice, 1.8 cm in
diameter, in half then quarters. The gel score is assessed as 5= no crack when folded
into a quarter, 4=no crack when folded into half but crack when folded into quarter,
3= no crack when folded in half but split when folded into quarter, 2=crack when
folded into half, 1=broke and split into halves. The gel was developed with static
heating (80oC for 20 min) 48 hrs cold setting time, with 2% added NaC1. No
significant differences were present between any of the samples tested.









2.5

2







~0

2 2.5 3 10.5 1 1 1 1 .5 co ntrolI
T treatment
Figure 3-56: The weight loss of pH-shift processed catfish protein isolates and unprocessed
muscle (control) heat set gels with 2% added NaCl developed with static heating
(80oC for 20 min), 48 hrs cold setting (40C), as compared to the weight before
cooking and setting. No significant differences were present between samples
(p>0. 05).


































Figure 3-57: Torsion shear strain vs. shear stress of pH-shift processed catfish muscle and an
untreated control. The texture profie associated with torsion testing is according to
Park (2000). The X in the middle represents the crossing points of the different
textures. The shaded box is the extent which the texture profie was determined. The
points from pH-shift processing outside of the shaded box (all points except the
untreated control) are above the maximum stress value listed in the texture profie.
The pH-shift samples are pH 2.0(*), pH 2.5 (m), pH3.0 (A), pH 10.5 (A), pH 11.0
(0), pH 11.5 (0), untreated control (SK).


- O








Br itle; Tough
"


ii I _I I


.00


130. 00
120. 00
110. 00
100. 00
00.00
80.00
70.00
60.00
50.oo
40.00
30.00
20.00
10.o 0
0.00
1


1.25 1.50 1.75 2.00 2. 25 250 2. 75 3.00


Shear Strain









CHAPTER 4
CHEMICAL PROPERTIES OF ACID AIDED AND ALKALI AIDED PROTEINT ISOLATES
FROM CATFISH (Ictalurus punctatus)

Introduction

Channel catfish (Ictalurus puncta~tus) is one of the fastest growing aquatic products in the

United States. The production and utilization of aquacultured catfish in the United States has

increased over the past 20 years and recently stabilized to a production level of 600,000 pounds

from 1999-2001 (Harvey 2002). The primary product of catfish is the fillet, with the secondary

product being the belly flaps, or the "nugget". The quality and potential utilization of catfish for

other purposes is under on-going investigation with promising results (Kim and others 1996;

Kristinsson and others 2005b; Suvanich and others 2000). The over-harvesting of all aquatic

resources was recently highlighted by Worm and others (2006) with the proj ected depletion of

aquatic resources by 2048. This becomes more of an issue since approximately 40% of the live

weight of many harvested species is discarded. Methods are currently being investigated which

will put more of the total seafood catch into the human consumable market and potentially

diminish or greatly relieve some of the current strain on the fisheries. The present seafood catch

totals approximately 100 million tons (Kristinsson and others 2005b) and can be far better

utilized if new economic technologies are developed. To achieve optimally utilize material

currently being discarded or diverted into other products not intended for human use, it is of the

utmost importance to understand the chemistry of these material so novel processes can be

developed.

The chemical properties of muscle proteins in meat and meat products are known to affect

quality, application and formulation ranging from whole meat to comminuted products (Lanier

2000). The chemistry of muscle proteins directed for human consumption is investigated to

better understand the effect of inherent chemical properties on the functional properties and









quality of proteins. The main functional properties of importance to proteins are their gelation

properties which dictate the difference textural properties you can achieve with muscle protein

products, and also their water holding capacity. Myosin, actin and the complex actomyosin are

known to be the primary contributors of textural properties of muscle gels (Park 2005). The

chemical properties of proteins in muscle vary widely, but are known to affect the utilization of

muscle proteins for food applications, including protein gels. The chemical properties of

proteins include but are not limited to myosin ATPase activity, reactive and total sulfhydryl

groups and surface hydrophobicity (Lanier 2000).

Myosin ATPase activity is used as an indicator of structural changes in myosin and/or

actomyosin integrity during post-harvest handling and processing (Park 2005). These structural

changes have been found to correlate to changes in the gelling properties of muscle proteins.

Yongswawatdigul and Park (2002) state that the gelling differences are due to the oxidation of -

SH groups at the active site on the head portion of the myosin heavy chain.

Modiaication of -SH groups may be monitored by measurement of reactive and total

sulfhydryl groups (Liang and Kristinsson 2005). Protein sulfhydryl groups in gelled systems are

believed to provide structural stability to the gel network by undergoing disulfide interchanges

and -SH oxidation which contributes to disulfide bridges formed during gelation (Visschers and

De Jongh 2005). The presence and availability of -SH groups to participate in forming disulfide

bridging increases protein-protein interactions and thus the integrity of the gel network

(Visschers and De Jongh 2005).

Surface hydrophobicity of proteins in gel networks is known to impart structural integrity

to the gel (Lanier 2000). As the gel is formed, the hydrophobic patches of proteins exposed to

the solvent interact to bury these exposed hydrophobic patches through protein-protein









interactions. Protein surface hydrophobicity and physical properties (i.e. with torsion testing)

have been found to correlate (Lanier 2000). Hydrophobic interactions are believed to occur after

disulfide interchanges as mentioned above during the heat induced gel formation of muscle

proteins (Smyth and others 1998). The hydrophobic interactions which are partly directed by

disulfide links bring proteins into the preliminary three-dimensional network allowing for

hydrogen bonding to occur during the cooling phase of heat induced gels (Smyth and others

1998). Modification of sulfhydryl and/or hydrophobic properties of muscle proteins would be

one way of directing protein gelation as well as investigating the chemical mechanisms

responsible for differences observed in the gel networks formed when implementing new

technologies of muscle protein recovery (Lanier 2000).

Acid-aided and alkali-aided processing is a new technology aimed at utilizing

underutilized products and byproducts of muscle-based systems (Hultin and Kelleher 2000).

Acid aided and alkali aided processing utilizes the solubility properties of muscle proteins,

specifically the myofibrillar proteins. Solubility is imparted to myofibrillar proteins by the

increase or decrease in pH to a point where the proteins are highly ionized resulting in their

solubility. Solubility of myofibril proteins is important because it allows for the separation and

removal of unwanted materials such as collagen and fat. These unwanted materials are not easily

separated or removed in other recovery processes, such as surimi. Acid-aided and alkali-aided

processing of channel catfish has been shown to provide an improved protein isolate by reducing

the lipid and heme content. The reduction of lipid and heme content increases whiteness and

increases the oxidative stability of the isolate (Kristinsson and others 2005b). Acid aided and

alkali aided processing has been shown to alter the structural states of muscle proteins in cod and

catfish myosin (Davenport and Kristinsson. 2003; Kristinsson and Hultin 2003a; Raghavan and









Kristinsson 2007a, 2007b) and whiting and rockfish protein isolate made from whole ground

muscle (Choi and Park 2002; Yongsawatdigul and Park 2004). However, the structural stability

of catfish muscle proteins as a whole when subjected to the acid aided or alkali aided processing

has not yet been investigated. We hypothesize that acid-aided and alkali-aided processing will

structurally modify channel catfish muscle proteins. The goal of this study is to characterize the

effect of acid and alkali processing on some key structural properties of catfish muscle protein.

Methods

Raw Material

The raw material used in these studies was fresh catfish fillets obtained 1-3 days post

harvest from a local supplier. Catfish fillets were only purchased which were determined to be

within 3 days of packaging. The catfish fillets were purchased and immediately transported on

ice to the laboratory and processed the same day.

Preparation of Protein Isolates

Protein isolates were prepared according to figure 2-1. Fresh fillets were initially ground

in an Oster heavy duty food grinder (Niles, Ill., U.S.A.) for the preliminary disruption and

collection of the muscle tissue. Following grinding, the comminuted meat was diluted 1:2 (w/v)

with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds.

Following homogenization, the resulting muscle tissue slurry was further diluted to give a final

dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manually stirred with a plastic

spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the

methods described below, using either 2N NaOH or 2N HCI as needed for the pH desired, with

continuous manual mixing. Upon reaching the desired pH, insoluble material was removed by

centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products,

Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at SoC. Following centrifugation, the









soluble middle layer was collected through a kitchen strainer with a mesh size of approximately

0.25 mm to minimize contamination with other separated materials. The soluble material was

readjusted to pH 5.5 as described above. After readjustment the solution was centrifuged to

remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The

precipitated protein was collected by decanting the supernatant containing the unprecipitated

proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH

was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate

was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing

until the moisture content was below 80%. Moisture content was determined using a Cenco

infrared moisture analyzer (CSC Scientific, Fairfax, Va., U. S.A.). Upon completion of manual

dewatering the protein isolation was complete. Preliminary unpublished investigation of protein

isolates in this laboratory found the shelf life of catfish protein isolates to be 5-7 days on ice. All

protein isolates were stored on ice at the precipitation pH and used within 5 days.

Protein Concentration

Protein concentration in the isolates and the subsequent solutions was determined using the

Biuret method, as described by Torten and Whitaker (1964), with of 10% w/v deoxycholic acid

in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any

remaining lipids in the samples. Protein concentration was measured based on a standard curve

based on BSA.

Protein Surface Hydrophobicity

Protein surface hydrophobicity was conducted according to Liang and Kristinsson (2005).

To measure surface hydrophobicity the isolate was diluted to give a stock solution of 10 mg/ml

in a 20 mM tris-HCI buffer, 0.6 M NaC1, pH 7.2. The stock solution was serially diluted to

obtain a concentration curve. Increasing volume (100 CIl, 200 CIl, 300 CIl, 400 Cll and 500 Cll) of









the stock solution were added to Tris-HCI buffer to a final volume of 4.0 ml. Then, 10 Cll of 6-

propionyl-2-(dimethylamino) naphthalene (PRODAN) (11.35 Clg/ml in methanol) was added and

the samples mixed for ~15 seconds. After mixing the samples were then incubated for 15 min in

the dark. All sample preparations and incubations were performed on ice in disposable test tubes.

After incubation, the sample was transferred to a fluorescence cuvette and the fluorescence

emission intensity scanned between 380-560 nm with excitation at 365 nm in a Perkin Elmer LS

45 Luminescence Spectrophotometer (Norwalk, CT). As the isolate is a collection of proteins,

the fluorescence peak of the samples is a relatively flat and broad peak between 430-460 nm.

The maximal fluorescence of the protein isolate was taken and used as the wavelength for

analysis. The surface hydrophobicity was calculated as the slope of the net fluorescence versus

protein concentration (mg/ml) of the samples.

Reactive Sulfhydryl Groups

Reactive sulfhydryl groups were determined according to Kim and others (2003). The

isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer

(M133/1281-0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with

600 mM NaCl to 250 Clg/ml. After dilution, 80 Cll of 10 mM 5,5' -dithiobis(2-nitrobenzoic acid)

(DTNB) was added to the sample. This mixture was then incubated for 1 hr on ice. After

incubation the sample was read at 420 nm using an Agilent 8453 diode array UV-visible

spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany).

Micromolar determination of SH groups per gram in the sample were done using the following

equation:

SH/ g = [arbsorbanrce x d~ilurtion factor x 100]/[1 3600(mlol/cmz)x sampleconcentration(mg/mill)









Total Sulfhydryl Groups

Total sulfhydryl groups were determined according to Kim and others (2003) The isolate

was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-

0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with 600 mM

NaCl to 250 Clg/ml. After dilution, 0.5 ml of diluted isolate was mixed with 2.5 ml urea buffer.

The urea buffer contained 8 M urea, 0.2 M Tris-HC1, 2% SDS, 10 mM EDTA and was adjusted

to pH 8.5. After mixing with urea, 50 Cll of 10 mM DTNB was added to the sample. The

mixture was incubated in a water bath for 15 minutes at 400C. After incubation the sample was

read at 420 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent

Technologies Deutschland GmbH, Waldbronn, Germany). Total sulfhydryl groups were

calculated using the following equation.

SH/ g = absorbancee x dilution factor x 1 00 1/[1 3600(mol/cm) x sample concentration (mg/ml)l

Myosin ATPase Activity Assay

Myosin ATPase activity was determined by the method of Perry (1955). The buffers to

initiate the enzymatic hydrolysis of ATP by myosin were prepared prior to sample preparation.

The reaction buffers used in this assay were 0.2 M glycine-NaOH buffer (pH 9.0), 0.1 M calcium

chloride, 0.05 M ATP, sodium salt (pH 6.8) and 15% trichloroacetic acid (TCA). Liberation of

inorganic phosphate was monitored in this reaction as the determinant of enzymatic function.

For the estimation of inorganic phosphate the following buffers were used: 12% TCA, 10%

(w/v) ammonium molybdate stock solution in 10N sulfuric acid. This ammonium molybdate

stock was used in the preparation of the ferrous sulfate ammonium molybdate reagent which

was made fresh the day of analysis. For analysis of myosin ATPase activity the isolate was

diluted to 1 mg/ml in Tris-HCI buffer containing 600 mM NaCl pH 7.2 and homogenized

thoroughly. This was kept on ice until needed. In separate tubes for each reaction, prior to









isolate addition, the reaction buffer was prepared by adding 1.3 ml glycine-NaOH buffer, 0.2 ml

calcium chloride, 0.3 ml ATP. This mixture was then incubated at 25oC for 5 min to allow the

temperature to equilibrate. After temperature equilibration, 0.2 ml of the individual isolate

solution was added to each reaction tube with proper mixing and allowed to incubate at 25oC for

5 min. After 5 min, 1 ml of 15% TCA was added to each reaction tube to stop the reaction. This

was centrifuged for 10 minutes at 25,000 x g to precipitate the proteins from solution and leaving

liberated phosphate from ATP in solution. Next 0.5 ml of supernatant was added to 3.2 ml of 12

% TCA with good mixing and left to stand at 25oC for 10 minutes. This solution (3.0 ml) of this

solution was removed and mixed with 2 ml of the ferrous sulfate ammonium molybdate

reagent and incubated at room temperature for 1 min. After 1 min of incubation the solution was

transferred to a cuvette and read at 363 nm using an Agilent 8453 diode array UV-visible

spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The

color of this reaction intensifies with time so only one reaction was done at a time to standardize

the incubation time of all samples to 1 min. The results of myosin ATPase activity were assayed

as relative results to the control. The control absorbance was set to 100% and the percent

activity of the isolates was calculated from the absorbance of the samples relative to the control.

activity = pH treatedsample/lcontrolsample

Protein Solubility as a Function of pH

The isolate was diluted to 10 mg/ml in DI water with thorough homogenization using a

tissue homognizer (Ultra-Turrax T18, IKA Works Inc. Wilmington, NC). After dilution in

water, 2 ml diluted protein was added to 2 ml buffer with good mixing. The pH range was from

1.5-12 in 0.5 increments. For pH 1.5-6.5, 2 mM citric acid buffer was used and for pH 7.0-12, 2

mM sodium phosphate buffer was used. The buffers were adjusted to the desired pH prior to









protein addition. After protein addition, the mixture was allowed to incubate on ice for 10

minutes. After incubation the protein solution was centrifuged at 3,000 x g for 10 minutes.

After centrifugation, the supernatant was sampled for protein concentration using the biuret

method (Torten and Whitaker 1964). Protein concentration was calculated from a standard curve

based on bovine serum albumin (BSA). Percent solubility was then calculated as a percentage of

an uncentrifuged control by the following equation:

[ protein (mg / ml) sup ernanlt]/r~otein (mg / ml) total]

Protein Solubility as a Function of Salt Concentration

The isolate was diluted to 5 mg/ml in 20 mM Tris-HCI at pH 7.2 without added NaCl with

thorough homogenization using an Ultra-Turrax T18. After dilution in buffer, 4 ml was taken

and the appropriate amount of NaCl added to each tube to achieve 0 mM, 150 mM, 300 mM 450

mM or 600 mM concentrations. After the appropriate NaCl concentration was achieved, the

samples were centrifuged at 3000 x g for 10 minutes at 50C. After centrifugation, the supernant

was collected in duplicate and soluble protein was determined according to the biuret method

(Torten and Whitaker 1964). The soluble protein was compared to an uncentrifuged control.

Percent soluble protein was calculated as

proteinsz mg / ml) sup ernant) proteinin (mg / ml) total)

Statistical Analysis

Experimental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on

replicate isolations as discussed above. A replicated (N=2) was determined as acceptable due to

achieving an acceptable power (P=0.80, a=0.05).

One-way independent measures analyses of variance were used to examine the effects of

all methods used except for the analysis of pH solubility and salt solubility which were tested









with 2-way ANOVA analysis. The Kruskal-Wallis ANOVA testing by ranks was used on data

which did not pass normality. Post hoc analysis was conducted only in the presence of

significant population differences. ANOVA statistical comparisons were conducted with

SigmaStat, (Systat Software Inc. San Jose Ca) with a significance level of p<0.05. After

SigmaStat completed the ANOVA analysis, the post hoc analysis recommended by SigmaStat

was used. In most cases this was Tukey's test. The Holm-Sikak method was used with ATPase

activity. The two-way ANOVA analysis used the Holm-Sidak pair wise comparisons. The two-

way ANOVA tables and the post hoc tests are reported in appendix A.

Results

Myosin ATPase

Myosin ATPase activity was reduced by pH-shift processing (Figure 4-1). This suggests

that the pH treatment leads to changes in the myosin recovered with the pH-shift isolates.

Myosin ATPase activity was significantly reduced (p<0.05) by pH treatments 2.0, 2.5, 3.0 and

10.5. No significant differences (p>0.05) were seen between any of the pH-shift isolates. The

isolates retained from 3 5% to 61% myosin ATPase activity of the raw material.

Surface Hydrophobicity

Surface hydrophobicity of the proteins in the isolates was not significantly (p>0.05)

reduced compared to the proteins in the raw material (Figure 4-2). All isolate treatments had

lower surface hydrophobicities but no main effect was determined. As with the myosin ATPase

results, there were no significant differences (p>0.05) between any of the acid or alkali

treatments.

Total Sulfhydryl Groups

When total sulfhydryls were analyzed no significant differences (p>0.05) were seen

between any of the acid or alkali treatments within groups or between groups (figure 4-3). All










the pH treatments were not significantly different (p>0.05) from the control as individual

treatments or as a population.

Reactive Sulfhydryl Groups

There were no significant differences (p>0.05) in surface sulfhydryl groups between any of

the acid or alkali treatments within groups or between groups (Higure 4-4). No significant

differences were present between any of the pH-shift treated isolates and the control. No

significant differences were present between any individual treatments or within the population.

Solubility of Proteins at Different Salt Levels

The results of the salt solubility study are shown in Eigure 4-5. The isolates and untreated

control were compared with the Holmes-Sidak multiple pair wise comparison with a p<0.05.

The pair wise comparison of the salt solubility is shown in appendix A. Significant differences

were present between all comparisons made, both within pH treatments and within salt

concentrations.

Solubility of Proteins at Different pH Values

The results of pH solubility of isolates and untreated catfish muscle are shown in Eigure 4-

6. The isolates and untreated control were compared with the Holmes-Sidak multiple pair wise

comparison with a p<0.05. The pair wise comparison of the pH solubility is shown in appendix

A. The isolates showed significant differences (p<0.05) at extreme pH values but no significant

differences were present between pH 4 to pH 7.5.

Discussion

The globular head of myosin has an enzymatic function where ATP is hydrolyzed to ADP

and phosphate. The purpose of the enzymatic function is to break the myosin/actin bridge which

is important for the muscle contraction/relaxation cycle (Berne and others 2004). Myosin

ATPase activity has been reported as a good indicator of protein quality and quality of processed









fish muscle food products, such as surimi (Park, 2005). The results here for catfish show that

there were no significant differences (p>0.05) in myosin ATPase activity between the isolate

treatments. The isolate treatments had however significantly (p<0.05) lower ATPase activity

than the control (Figure 4-1). Isolate treatments did not completely inactivate myosin ATPase

activity but did reduce the activity, suggesting high and low pH treatments the proteins were

subj ected to modified the structure of the myosin catalytic site. The change in myosin ATPase

activity has been attributed to a shifting of the sulfhydryl groups at the myosin active site

(Yongsawatdigul and Park 2004). The reduction in myosin ATPase activity was highly variable

as indicated from a large standard deviation (Figure 4-1). In the pH 10.5 treatment group, the

myosin ATPase activity ranged from 0-62%. This range may be due to the inconsistent exposure

of the myosin active site to the solvent at high pH. These inconsistencies were present in all

samples with some varying more than others. The lack of significant difference (p>0.05) in the

myosin ATPase activity (figure 4-1) may be due to the variability of the pH treated systems and

differentiation between acid and alkali treatments of muscle proteins was not possible. Isolate

production with either acid or alkali may or may not go through the same structural changes,

however, the product is similar in that the acid or alkali treatments produce a reduction of

ATPase activity which is not significantly different (p>0.05) among the treatment groups. This

means that though there is a reduction in the ATPase activity when acid or alkali processing is

used there is not a complete inactivation of myosin ATPase activity.

Surface hydrophobicity measurement is an analysis of the hydrophobic patches on the

surface of a protein that bind a fluorescent probe. In this experiment, PRODAN was used as the

fluorescent probe. Figure 4-2 shows no significant difference (p>0.05) in surface hydrophobicity

between treatment groups. However, the control was higher in surface hydrophobicity than all









pH treatmented samples though no main effect was determined. Previous studies have shown

that pH-shift processing increased the surface hydrophobicity of catfish myosin (Kristinsson and

Hultin 2003a; Raghavan and Kristinsson 2007a, 2007b). The surface hydrophobicity of isolated

catfish myosin shows pH treatment 2.5 having the greatest surface hydrophobicity followed by

pH treatment 11.0 and then followed by untreated control (Davenport and Kristinsson 2003).The

reduction of surface hydrophobic patches during acid and alkali treatment could be the result of

structural modification during the pH readjustment to 5.5, burying the hydrophobic patches

previously on the surface of the protein. However this reduction of surface hydrophobicity in the

whole isolate system could also be due to micro-aggregation preventing the exposure of

hydrophobic patches on the surface of the proteins.

Total sulfhydryls are the total number of -SH groups exposed after treatment in 8 M urea.

Reactive sulfhydryls are the -SH groups of the protein which are exposed to the solvent (Tris

buffer with 0.6M NaC1) and are able to react with DTNB. The difference between total

sulfhydryls and reactive sulfhydryls is the molar content of sulfhydryl groups which are buried

within the protein structure of the system. There were no significant differences (p>0.05)

between total sulfhydryls in all treatment groups and the control. Figure 4-4 shows no -SH

groups were altered during acid or alkali processing. The total sulfhydryl groups present in the

samples, which may participate in gel formation, remained the same. Figure 4-3 depicts the

reactive sulfhydryls measured in the control and all treatments. No significant differences

(p>0.05) were observed between any of the treatment groups or between any of the treatment

groups and the control. Due to a lack of significant differences (p>0.05) in total sulfhydryl and

reactive sulfhydryl groups it may be concluded that sulfhydryl content was not significantly

(p>0.05) linked to changes in gel strength (see gelation data in Chapter 3). However, structural









susceptibility during heating denaturation to expose sulfhydryl groups during thermal treatment

was not assayed. Thus the molar content of disulfide bonds in the thermally set gel is currently

unknown.

Salt solubility of muscle proteins is one way to assess whether functionality has been

modified by pH-shift processing. Muscle proteins, myofibrillar protein in particular, are highly

sensitive to changes in ionic strength and type of ions in their environment. The ability of the

proteins in the isolate to enter into solution at varying concentrations of salt was tested at pH 7.0

(Figure 4-5). Myofibrillar proteins are the contractile proteins of muscle, those proteins which

are salt soluble, or more specifically go into solution above an ionic strength of ~0.3. Figure 4-5

shows a reduction in solubility of the protein isolates in the presence of high salt concentrations.

This reduction may be due to structural changes and/or micro-aggregation which prevent the

continued solubility of these proteins under conditions which normally would solublize

myofibrillar proteins. The reduction in salt solubility of isolates follows the same trend as the

reduction of surface hydrophobicity. These tests provided results when compared to previous

results on cod muscle protein isolates and cod myosin where surface hydrophobicity was

increased while solubility was decreased (Kristinsson and Hultin 2003b) indicating a species

effect of pH-shift processing. These results indicate that in catfish isolates, the protein-protein

interactions are changed in such a way to prevent ionization of the protein surface thus resulting

in a reduction of solubility. As stated above, the reduction in surface hydrophobicity may be due

to microaggregation of misfolded proteins, thus leaving little exposed hydrophobic surface.

Increased salt will solubilize muscle proteins, but it is possible the interactions between these

microaggregates were too strong for the salt to overcome, thus leading to only a small increase in

solubility. Due to the similarity of results observed between isolated catfish myosin (Raghavan










and Kristinsson 2007a) and whole muscle isolate, the same molecular mechanism is most likely

responsible for these differences.

The modification of catfish muscle proteins by pH-shift processing resulted in isolates with

different pH solubility profies. The increased solubility of the control in the pH range 3.5-10 is

most likely due to a higher amount of sarcoplasmic proteins compared to isolates. Previous work

on pH-shift processing of catfish muscle (Kristinsson and others 2005b) has shown an increased

retention of sarcoplasmic proteins when catfish is processed at pH 2.5 as compared to alkali

processing at pH 11.0. This suggests that sarcoplasmic proteins which are largely soluble in the

control co-aggregate with the myofibrillar proteins and thus become largely insoluble, explaining

the different pH profile seen in the pH 3.5-10 range. The low solubility in this range would

indicate that the protein isolates are more prone to aggregation than untreated control. This is

likely due to the fact that the isolates are composed of only partially refolded protein structures

which favor interactions with each other more than the solvent. It is also interesting to note that

all isolates expect the pH 10.5 treatment, became soluble at a lower pH than the control,

suggesting more protonation was needed to break the interactions between them. The pH 10.5

treatment may have been milder than the other treatment, thus giving isolates with weaker

protein-protein interactions. It is also interesting to note that contrary to the results seen at low

pH the isolate solubilized at lower high pH values than the control, indicating that they were

more sensitive to increases in negative charges than positive charges. These data therefore

suggest the pH-shift process modified the protein structure and their interactions compared to

control .

Conclusions

No significant differences (p>0.05) were seen between total or reactive sulfhydryl groups

of catfish protein isolates compared to control. No significant differences (p>0.05) were seen









between the surface hydrophobicity or myosin ATPase activity of the isolates but all isolate

samples showed reduction in both compared to control. Significant differences (p<0.05) were

observed in salt- and pH-dependent solubility of isolates compared to control. These results

indicate that, although significant differences are not detected (p>0.05) in some of the commonly

used tests to describe the chemical nature of protein products, isolates subj ected to different pH-

shift processing conditions do in fact have different properties and pH-shift processed muscle

proteins may have chemical modifications which are different from traditional muscle systems.

The chemical modification of catfish muscle proteins by pH-shift processing may also lead to

structural and functional modification of catfish muscle proteins. Further studies are needed to

determine whether the structural and functional effects when using this new technology are

dictated by any chemical changes to specific amino acids during processing. With further study

and characterization the formulation of custom protein isolates to provide application specific

utilization may be possible.










100
90
AB AB
S80
> 70 -
.t A A A A B
60

< 50 .
40 --

*g 30
20
10
2 2.5 3 10.5 11 11.5 con


Figure 4-1: Myosin ATPase activity of catfish muscle isolates compared to a control sample.
The control was normalized to 100% activity and the percentage of activity was
calculated relative to the control. The significant differences present between
samples (p<0.05) are represented by the letters above the column. Similar letters
represent no significant difference (p>0.05) between treatments.









1400

1200

1000

0- 800

600





20


Figure 4-2: The surface hydrophobicity of catfish muscle isolates and untreated control.
Hydrophobicity was assayed by developing a protein concentration curve and
measuring the maximal fluorescence response of the fluorescent probe PRODAN at
~400 nm. A main effect was not determined for the sample population (p>0.05).









































T


I


S11.5 I


10.5 || I


0.07

0.06

0.05

0.04

0.03

0.02


0.02 ::: 2.5 I ~3 1 q.5 II 1 1 ~1 1 .5 llcon


Figure 4-3: Total sulfhydryl content of catfish protein isolates compared to untreated control.
Total sulfhydryl content was assayed by the reactivity of catfish proteins to DTNB
after being treated for 10 min in 8M urea at 400C. No significant differences
(p>0.05) were present between any of the samples tested, thus it is concluded that pH-
shift processing did not affect total sulfhydryl content.


-


-


-


-


0.014


0.012


.50.01


a. 0.008

S0.006


-s 0.004


11


2


con:


0.002


0


Figure 4-4: Reactive sulfhydryl content of catfish protein isolates compared to untreated control.
Reactive sulfhydryl content was assayed by the reactivity of catfish muscle proteins
to DTNB in T-HCI buffer. No significant differences (p>0.05) were present between
any of the samples tested, thus it is concluded that pH-shift processing did not affect
reactive sulfhydryl content.












- p 2.0
-m pH 2.5

- -- pH 3.0
-x- pH 10.5
-a- pH 1 1.0
-* pH 1 1.5
+ control


100
90
80

7 0
60 s



30


20


OmM


150mM


300mM
mM NaCI


450mM


600mM


Figure 4-5: Solubility of catfish protein isolates and untreated control in varying concentrations
of sodium chloride at pH 7.2 in 25 mM tris-HCI buffer. The percent solubility was
determined by dividing protein concentration in the supernatant after centrifugation
by the protein concentration in an uncentrifuged sample. Significant differences
compared within pH treatments and salt concentrations are tabulated and reported in
appendix A.





Figure 4-6: The pH solubility profile of catfish protein acid-aided isolates and untreated control
from pH 1.5-12 with the non-centrifuged samples reported as the right most point on
the graph. Solubility was calculated as explained in Fig. 4-5. Significant differences
present between samples are shown in appendix A.


+ pH 2.0
-c- pH 2.5
+ pH3.0
-1 Control


100

,80

~60

"a 40

20

0


1.5 2.5 3.5


4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 NC
pH

































Figure 4-7: The pH solubility profile of catfish protein acid-aided isolates and untreated control
from pH 1.5-12 with the non-centrifuged samples reported as the right most point on
the graph. Solubility was calculated as explained in Fig. 4-5. Significant differences
present between samples are shown in appendix A.


-M- pH10.5

I -a- pH 11.0
SpH 11.5
SControl


100


80


,60





20


0


rU
in in


C~3
in


P U1
in in in


03
in in









CHAPTER 5
THE EFFECT OF pH-SHIFT PROCESSING ON THE STRUCTURAL AND THERMAL
PROPERTIES OF CATFISH (Ictalunts prnctatus) PROTEIN ISOLATES

Introduction

The structure function relationship of muscle proteins is important to the application and

performance of muscle based food products. The modification of protein structure from the

native state is critical for meat functionality. The native structures of muscle proteins, though

crucial for the function of the muscle proteins in the living system, do not exhibit functional

characteristics associated with processed meat. The change in secondary structures are related to

the functionality of muscle proteins, however the intermediary transition of secondary structures

from native to denatured are considered to bear a more direct relationship to muscle protein

functionality (Xiong 1997). Muscle based products are first influenced by the growth conditions,

slaughter method and muscle type of the animal The production of functional comminuted meat

products is further dictated by the processing conditions and chemical additives used. The

production of comminuted meat products frequently includes the addition of salts, primarily

sodium chloride. Addition of salt induces structural changes of muscle proteins due to the

electrostatic interaction between proteins and the sodium and chloride ions which results in

muscle fiber swelling (Xiong 1997). The swelling of muscle fibers allows for increased water

retention of the meat system, and thus modifies the functional properties and ultimately the

perception of the product. These changes are directed by the state of the intermediate structural

characteristics of the muscle proteins. The change in intermediate structures can thus be dictated

by processing conditions and chemical additives resulting in the functional properties of muscle

proteins being determined by external factors (Xiong 1997).









The use of acid or alkali processing has been shown to change the structural conformations

of isolated muscle proteins, specifically myosin (Kristinsson and Hultin 2003a; Raghavan and

Kristinsson 2007a, 2007b), resulting in molten globular or stable intermediate protein structures.

In this study it is hypothesized that the utilization of acid-aided and alkali-aided extraction

method on a warm water species (catfish) will lead to unique protein structural properties that are

pH dependent. This hypothesis was investigated by determining the effect of pH shift processing

at different pH on the structure and stability of catfish protein isolates produced at low and high

pH treatments.

Methods

Raw Material

The raw material used in these studies was fresh catfish fillets obtained 1-3 days post

harvest from a local supplier. Catfish fillets were only purchased which were determined to be

within 3 days of packaging. The catfish fillets were purchased and immediately transported on

ice to the laboratory and processed the same day.

Preparation of Protein Isolates

Protein isolates were prepared according to figure 2-1. Fresh fillets were initially ground

in an Oster heavy duty food grinder (Niles, Ill., U.S.A.) for the preliminary disruption and

collection of the muscle tissue. Following grinding, the comminuted meat was diluted 1:2 (w/v)

with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds.

Following homogenization, the resulting muscle tissue slurry was further diluted to give a final

dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manually stirred with a plastic

spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the

methods described below, using either 2N NaOH or 2N HCI as needed for the pH desired, with

continuous manual mixing. Upon reaching the desired pH, insoluble material was removed by









centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products,

Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at SoC. Following centrifugation, the

soluble middle layer was collected through a kitchen strainer with a mesh size of approximately

0.25 mm to minimize contamination with other separated materials. The soluble material was

readjusted to pH 5.5 as described above. After readjustment the solution was centrifuged to

remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The

precipitated protein was collected by decanting the supernatant containing the unprecipitated

proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH

was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate

was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing

until the moisture content was below 80%. Moisture content was determined using a Cenco

infrared moisture analyzer (CSC Scientific, Fairfax, Va., U. S.A.). Upon completion of manual

dewatering the protein isolation was complete. Preliminary unpublished investigation of protein

isolates in this laboratory found the shelf life of catfish protein isolates to be 5-7 days on ice. All

protein isolates were stored on ice at the precipitation pH and used within 5 days.

Protein Concentration

Protein concentration in the isolates and the subsequent solutions was determined using the

Biuret method, as described by Torten and Whitaker (1964), with of 10% w/v deoxycholic acid

in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any

remaining lipids in the samples. Protein concentration was measured based on a standard curve

based on BSA.

Circular Dichroism (CD)

CD was done according to Kristinsson and Hultin (2003a). The isolate was diluted and

homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec









Products Inc., Bartlesville, OK) in 20 mM Tris-HCI buffer, pH 7.2 with 600 mM NaCl to a

concentration of 10 mg/ml. This protein stock solution of 10 mg/ml was prepared and diluted to

2 mg/ml 1 hr before analysis and held on ice. For analysis of secondary structure the sample was

scanned from 260-200 nm in a 0.1 cm quartz cuvette, 0.2 nm resolution scanned at 50 nm/min on

a Jasco J-500C circular dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room

temperature. The time duration of the scan was no more than 10 minutes. Differences in total

alpha helix beta structure and random coil structures present were determined from

DICHROWEB using the K2D analysis program (Lobley et. al., 2002).

Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HC1)

Catfish protein isolate and the control, untreated ground catfish muscle, were treated with

Gu-HCI over the range of 0-6M in 0.5 M increments. For assessment of denaturation, 222 nm

was used as an indicator wavelength of alpha helical content, scanning from 220-225 nm in a

0.10 cm quartz cuvette, 0.2 nm resolution scanned at 50nm/min on a Jasco J-500C circular

dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The protein

concentration was 1 mg/ml. Samples were allowed to incubate for 5 minutes on ice prior to

reading.

Differential Scanning Calorimetry (DSC)

DSC was conducted according to Fukushima and others (2003) on a MicroCal DSC

(MicroCal, LLC, North Hampton, MA). Isolate were diluted in sample buffer (20 mM tris-HC1,

pH 7.2 with 600 mM NaC1) to a protein concentration of 10 mg/ml and homogenized for 1

minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec Products Inc.,

Bartlesville, OK). The sample was degassed under vacuum for 5 min at SoC. After degassing,

0.6 ml was loaded into the sample cell, the reference cell contained sample buffer. The sample

was then linearly heated at loC/min from SoC-80oC. Analysis of the data was conducted on the









software provided by the manufacturer, Origin Pro 7.5, for determination of both exothermic and

endothermic events.

Susceptibility of Proteins to Transglutaminase-Induced Cross Linking

The susceptibility of pH-shift processed catfish muscle to protein cross linking was

assayed using the commercial transglutaminase (TGase) Activia TI from Ajinomoto LLC

(Ajinomoto Food Ingredients LLC Chicago II). 0.2% TGase (w/w) was added to isolate diluted

to 10% solids in 20mM Tris-HCI buffer, 600mM NaC1. TGase activity was assayed by using an

AR2000 advanced research rheometer (TA Instrument, New Castle, DE) with a head with a flat

cross-hatched polyacrylic surface and thermally controlled plate. The gap was 1000 microns and

the head lowered onto the sample using the controlled speed function provided by the software.

The samples were tested in oscillatory mode under controlled frequency at 0.1 Hz, and strain

controlled at 0.01. TGase activity was monitored by the increase in G' over 1 hour at 30oC.

Activity was compared against samples with no TGase added. G' was plotted against time and

the slope of the line from linear regression was used to calculate the TGase activity. The activity

coefficient was calculated as the ratio of treated to untreated samples.

Statistical Analysis

Experimental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on

replicate isolations as discussed above. A replicated (N=2) was determined as acceptable due to

achieving an acceptable power (P=0.80, a=0.05).

One-way independent measures analyses of variance were used to examine the effects of

all methods. The Kruskal-Wallis ANOVA testing by ranks was used on data which did not pass

normality. Post hoc analysis was conducted only in the presence of significant population

differences. ANOVA statistical comparisons were conducted with SigmaStat, (Systat Software









Inc. San Jose Ca) with a significance level of p<0.05. After SigmaStat completed the ANOVA

analysis, the post hoc analysis recommended by SigmaStat was Tukey's test.

Results

Circular Dichroism (CD)

pH-shift processing led to changes in secondary structure of protein isolates as seen by the

CD results (figures 5-1, 5-2, 5-3). The order of secondary structure was found to be

control>alkali>acid. Acid treated protein isolates had lower relative a-helix content than the

control and higher relative beta structure content than the control though no main effect was

observed. Alkali treated protein isolates also showed relatively lower a-helical content and

higher beta structure content than the control though no main effect was observed. There were

no significant differences (p>0.05) in secondary structure between acid and alkali treated protein

isolates. No significant difference (p>0.05) was observed in random coil content among any

treatment groups as compared with each other or with the control.

Guanidine Hydrochloride Denaturation (Gu-HC1)

Subj ecting protein samples to increasing concentration of Gu-HCI gave unexpected results

(figure 5-4 and 5-5). Increasing Gu-HCI to 0.5 M led to substantial protein denaturation of the

control samples (70%), some additional denaturation of alkali-aided isolate samples, while acid-

aided isolate was less affected. Isolates made with the pH 11.5 treatment were most affected and

had fully unfolded in 0.5 M Gu-HC1. All samples demonstrated a refolding behavior in 1 M Gu-

HC1, with all isolates (except the one made with the pH 3 treatment) and control having higher

level of structure than untreated control at 0 M Gu-HC1. At 1.5 M Gu-HC1, control samples

showed some unfolding, while acid-aided isolate samples continued to refold (figure 5-4).

Alkali-aided samples were little affected going from 1 to 1.5 M Gu-HCI (figure 5-5). Going from

1.5 M to 2 M Gu-HCI led to almost complete unfolding of all samples.










Micro-Differential Scanning Calorimetry (DSC)

The thermal events observed for all pH-shift processed samples are shown in figures 5-6,

5-7 and table 5-1. The event boxes in blue (table 5-1) are endothermic events and the event

boxes in red are exothermic events. The numbers listed are first the temperature and then the

specific heat, normalized from an internally derived baseline using Origin software. The table

boxes are grouped on a per-degree basis with samples grouped together in one row if the thermal

events happen within one degree of each other.

The first event occurred at 11.8 +/- 0.090C. The event was endothermic in all samples

except pH treatment 11.5 was exothermic. The next event occurred at 20.8-21.740C in pH

treatment 10.5 and the control. Following this event a single exothermic event occurred in pH

treatment 10.5 at 23.730C. Exothermic events occurred in pH treatments 2.0, 2.5, 10.5, 11.0 and

1 1.5 between 28.27-29.3 50C. The next event was an endothermic event in all the acid treated

samples over the range of 34.79-3 5.260C. The alkali treated samples had an event at 36.83-

37.310C. The control, however, had an exothermic event at 36.560C. The pH treatments 2.5 and

3.0, had an endothermic event at 39.650C and 39.820C, respectively. The pH-treatments, 2.0,

10.5, 11.0, 11.5, had an exothermic event between 41.21-42. 1000. The control sample had an

endothermic event at 43.470C. An endothermic event was recorded among all the pH-treated

samples occurring between 44.52-45.670C The pH-treatments 2.0, 3.0, 10.5, 11.0, 11.5 had an

endothermic event ranging from 47.69-48.340C. The control had an exothermic event at

47.20oC. In pH treatment samples 2.0, 3.0, 10,5, 11.0, 11.5 and the control, an endothermic

event was recorded, while pH-treatment 2.5 had an exothermic event between 50.04-51.750C.

An exothermic event in pH-treatment 2.5 occurred at 55.710C. The pH-treatments 11.0 and 2.0

had exothermic events at 56.830C and 57.170C respectively. An exothermic event occurred in

pH treated samples 2.5, 10.5, 11.5 and the control between 57.82-58.810C. The pH-treatments










3.0 and 11.5 had an endothermic event at 60.390C and 59.610C respectively. In pH treatment

samples 2.0 and 2.5 an endothermic event occurred at 62.600C and 63.160C, respectively. The

pH-treatments 3.0 and 10.5 had exothermic events occur at 63.630C and 63.810C respectively.

An endothermic event occurred in the control at 64.970C. Exothermic events occurred between

66.49-67.380C in pH-treatments 2.0, 3.0, 10.5 and 11.5. Exothermic events occurred in pH-

treatment 2.5 and the control at 68.420C and 69.360C respectively. Endothermic events at

71.180C occurred in pH-treatments 2.0 and 10.5. The pH-treatment 2.0 had an exothermic event

occurring at 72.880C. The last events ranged from 74.18-76.390C. Endothermic events occurred

in pH-treatments 2.0, 2.5 and the control at 74.180C, 75.950C and 75.120C, respectively. The

pH-treatments 10.5 and 11.0 had exothermic events occurring at 74.830C and 76.390C,

respectively .

Susceptibility of Proteins to Cross-linking by Transglutaminase

Susceptibility of protein isolates and control samples to transglutaminase (TGase) induced

cross-linking was measured by following increases in G' of the system as a function of time and

calculating the difference between final G' of the untreated and treated sample (i.e. susceptibility

ratio). Figures 5-9 to 5-15 show that G' for all TGase containing samples, except control and

isolates made with the pH 2.0 treatment, increased over untreated samples as time progressed

however no main effect was determined. All samples however had a higher final G' for TGase

samples after 1 h compared to untreated samples, resulting in a positive susceptibility ratio for all

samples (figure 5-8). No significant differences (p>0.05) were seen in the susceptibility ratio

(figure 5-8) between the acid treated samples or the alkali treated samples. No significant

differences in the susceptibility ratios (p>0.05) were found between acid treatment and alkali

treatments compared to the control.









Discussion

Circular dichroism was used as a comparative method to study the average change in the

structural properties of all proteins collected during pH-shift processing. The differences

observed between the average structural content of pH-shift treated samples and control samples

indicate a structural transition from alpha helix to beta structure as denaturation increases. The

native secondary structural content of muscle proteins followed the order of control>alkali>acid.

The increase in beta structure content indicates that extreme pH used during pH shift processing

induced protein unfolding at the high or low pH used to extract the proteins. After adjustment of

the protein system from high or low pH to pH 5.5, refolding occurs resulting in an increase in the

beta structure content of protein isolates. Previous studies have shown that during the

denaturation process of proteins the beta structure secondary structure is more structurally stable

than the alpha helix secondary structure and alpha helix structures may transition to beta

structures before completely unfolding into a random coil (Drummy and others 2005). The

structural changes induced by pH-shift processing may be species specific because, in contrast to

the present study, results shown for pH adjusted cod myosin with pH-shift processing showed a

minimal effect on the secondary structure of refolded myosin (Kristinsson and Hultin 2003a).

However, similar to the catfish used in the present study, it was reported that acid treatment

resulted in the reduction of secondary structure with isolated catfish myosin (Raghavan and

Kristinsson 2007a). This indicates that the structural trends observed when testing the whole

protein isolates system are specific to fish species and similar to the results obtained for isolated

catfish myosin. However the differences observed between catfish myosin and cod myosin

further indicate that the unfolding and refolding parameters need to determined across species

and different fish may possibly impart different gel properties to the isolated protein. Though the

refolding properties of catfish myosin (Raghavan and Kristinsson 2007a) showed similarities









with the results attained from the present protein isolates those results applied best to the pH 2.5

treatment. The results show that catfish myosin treated at pH 2.0 showed a larger decrease in

secondary structure than treatment at pH 2.5 (Raghavan and Kristinsson, 2007a). These results

are different from the effect of pH 2.0 treatment on protein isolates (figures 5-1 and 5-2) which

showed that this pH did not significantly decrease (p>0.05) in secondary structure and in

particular the alpha helix content. This means that similarities exist between the isolated

individual proteins and the whole muscle system; however, the structure of protein isolates has

unique properties that are not dictated exclusively by myosin.

Gu-HCI treatment is used for investigating the susceptibility of proteins to chemical

denaturation. Gu-HCI as denaturant can be used to establish the chemical denaturation kinetics

of a protein system. Normally one would expect a somewhat sigmoidal denaturation curve at

increasing levels of Gu-HC1, but this was not seen here for the catfish proteins. Gu-HCI induced

a small change in protein structure at 0.5 M for acid-aided isolates, while alkali-aided isolates

were further denatured. Control became highly denatured at this low level of Gu-HC1. A further

increase in Gu-HCI led to refolding (more negative response at 222 nm), followed by more

unfolding (less negative response at 222 nm) as Gu-HCI increased. This data therefore does not

allow for a two-state kinetic model to be established. Structural events occurring in protein

folding after pH-shift processing and apparent increases in folding of protein isolates with

increased Gu-HCI indicate molten globular structures are formed. However these molten

globules are not unique to the isolates as the control also demonstrated apparent refolding

behavior. Fan and others (1996) showed an increase in chicken liver dihydrofolate reductase

activity in low concentrations of Gu-HCI with no significant change in secondary structure

according to circular dichroism results of secondary structural analysis (Fan and others 1996).









The results of Fan and others (1996) and those shown here support the presence of a range of

conformational isomers with similar low energy states (Kumar and others 2000) resulting in

structures which have modified functional properties.

The DSC results from this study show that the thermal transitions of pH-shift processed

catfish muscle differ not only from the control but are also dependent on the pH used during

processing. The first temperature range which an event was recorded at was 110C. This event

occurred in all samples however pH treatment 11.5 showed an exothermic event at this low

temperature compared to all other samples showing endothermic events.

The next thermal event was recorded between 20.8-21.74oC in pH treatment 10.5 and the

control with pH treatment 10.5 having another small event closely following at 23.73oC. Major

distinguishing events occurred from 34.79-35.26oC in the acid treated samples and from 36.83-

37.31oC in alkali treated samples. In these temperature ranges endothermic events were recorded

for pH-treated samples. At 36.56oC, within the alkali treated range, the control had an

exothermic event. Catfish muscle subjected to pH-shift processing resulted in an endothermic

event occurring rather than an exothermic event. Acid processing of catfish muscle further

changed the thermal sensitivity of the isolated proteins reducing the temperature, 34.79-3 5.26oC,

at which this reaction occurred. The change in the thermal response of acid treated catfish

muscle may be due to the proteins co-precipitating with myosin as acid treated isolated myosin

shows the first thermal event at 37oC (Raghavan and Kristinsson 2007a, 2007b).

The next series of events occurred between 39.65-48.34oC. The event leading this off was

catfish muscle processed at pH 2.5 and 3.0 which had exothermic events occurring at 39.65oC

and 39.82oC respectively whereas the other pH shift processed samples had endothermic events

occurring between 41.22-42.1oC, with the control event occurring at 43.47oC. Catfish muscle









subj ected to pH-shift processing exhibited a lower temperature range of thermal events, (39.65-

42. 1oC), than the control sample, (43.47oC). The control peak can be associated with the second

transition of whole myosin peaks as seen for catfish myosin at pH 7.3 which exhibited a second

transition peak at 44.90C (Raghavan and Kristinsson 2007b). The control at 43.47oC and 47.2oC

showed very large exothermic peaks as seen in Eigure 5-6, the 4. These higher temperature peaks

however did not correlate with the two myosin peaks shown by Raghavan and Kristinsson

(2007b) for isolated catfish myosin which occurred at 37oC and 44.90C. The first large peak at

43.47oC was shifted from the pH treated samples as seen in table 5-1. The second large peak

was consistent with the pH-treated samples except for pH treatment 2.5. However within these

two large peaks seen in the control all the pH shift processed samples had an endothermic event

between 44.52-45.66oC. The range observed here would fall within an expected range with the

second myosin peak shown by Raghavan and Kristinsson (2007b) with catfish myosin. Over the

39.65-42. 1oC temperature range Xiong (1997) reported the first transition peak of chicken breast

myosin at 40oC. The results of this study and those of Raghavan and Kristinsson (2007b) do not

indicate that catfish myosin has the first transition at 40oC but that an additional peak is observed

either by a co-precipitated protein or due to the structural modification of catfish myosin during

the precipitation process in the presence of other muscle proteins.

Following these series of events, between 50.04-51.02oC an endothermic event occurred in

all samples. This indicates that though pH-shift processing alters the structure and thermal

response of muscle proteins, there is this one temperature range which is not affected by pH-shift

processing and some part of myosin or another co-precipitated protein. The thermal events

occurring within this temperature region have been associated with the transitions of myosin S-2

subfragment (51oC) in rabbit and chicken breast, the light meromyosin chains in chicken breast









(52oC) and the myosin light chains in rabbit (51oC) (Xiong 1997). The thermal transitions above

the 50-5 1oC range show different patterns of thermal reactivity. These differences in the thermal

events, of individual pH treatments and the control, may provide insight into the differences

observed in other properties of pH-shift processed muscle proteins.

Transglutaminase (TGase) is an enzyme which promotes the formation of covalent bonds

in muscle proteins. This bond is based on the cross linking of lysine and glutamine to form the

dipeptide e-(y-glutamyl)1ysine (Perez-Mateos and Lanier 2007). The formation of covalent cross

links in muscle proteins in the absence of TGase are primarily by the formation of disulfide

bonds (Stone and Stanley 1992).

The formation of covalent cross links in muscle proteins promotes the formation of

stronger heat set gels at temperatures below the point where muscle proteins form irreversible

gels, normally between 25-500C. This increase in gel strength at low temperature is a setting

effect referred to as suwari setting. The use of TGase in muscle systems has been employed to

formulate muscle products from smaller pieces of meat such as scallops, steaks, shrimp and ham

(Ajinomoto 2007). The use of TGase in surimi processing has been employed for the

improvement of lower grade surimi or the mixture of lower grade surimi with higher grade

surimi to form the gel strengths needed for appropriate product formulation.

The susceptibility of muscle products to TGase activity is based on the availability of both

lysine and glutamine on the surface of the proteins to interact with the active site of TGase to

form the covalent cross link. The protein motifs which facilitate TGase activity have not been

characterized; however, TGase activity in muscle systems has been directly correlated with the

decrease in myosin heavy chain (Perez-Mateos and Lanier 2007).









The results of this study indicate that pH treatment of catfish muscle proteins may promote

interactions of the isolated proteins with TGase for all alkaline-aided isolates but not all acid-

aided isolates. Thus these results indicate that structural modifications which occur with pH

treatment result in the interactions of isolated proteins with TGase appear greater than those

without pH treatment under most treatment conditions. Perez-Mateos and Lanier (2007) have

indicated that myosin heavy chain is reduced when muscle is treated with TGase, suggesting

cross-linking between myosin. The structural modifications during acid processing were more

pH specific with respect to TGase treatment as treatment at pH 2.0 was not significantly different

from the control.

These results, when compared with the structural results indicate that the specific changes

of muscle proteins isolated at pH 2.0 are not identifiable from other acid treatments by CD but

result in surface modification of the proteins as shown by TGase treatment. The high variability

of pH-treatment 10.5 indicates that the refolded surface structure at this pH is not as consistent as

the refolding of proteins treated under more extreme alkali conditions. The use of pH-shift

processing is also species specific as the results of this study are in contrast to the results

reported (Perez-Mateos and Lanier 2007) which found no increase in gel strength by TGase

treatment to acid solubilization of protein isolates made from Atlantic Menhaden. The reactivity

of muscle proteins with TGase indicates the accessibility of specific amino acids to TGase.

Changes in the accessibility of specific amino acids indicate structural changes in the proteins

directly affect the surface structure of proteins. The characterization of the specific location and

proteins involved in the interaction with TGase may lead to further understanding of the protein

motifs responsible for enzyme induced cross-linking and increase the understanding of pH-shift

processing at specific sites within a protein or protein mixtures.









Conclusions

This study demonstrated that pH shift processing decreases the alpha helical content of

protein isolates, increases the structural flexibility and modified the temperatures and types of

thermal events catfish muscle proteins underwent. These results support our hypothesis that pH-

shift processing will affect the structure and thermal reactivity of catfish muscle proteins and

those effects were unique to the pH used during processing. It may be concluded that pH-shift

processing affects catfish muscle protein secondary structure. This modification of secondary

structure may lead to the modification of the thermal reactivity and the susceptibility of protein

isolates to enzymatic modification and ultimately the modification of the thermal gelation

properties of catfish muscle proteins. Additionally the use of enzyme treatment, specifically

TGase, may provide an effective method of analysis for determining the modification of the

surface structure of muscle proteins and provide insight to the mechanisms involved in the

interaction and gelation of a highly complex material.
















1.5
1
0.5
0
-0.5
-1
-1.5
-2

-2.5
-3
-3.5
200


210 220


Figure 5-1: Circular dichroism of catfish protein isolate made with acid processing and untreated
catfish muscle recorded from 200-260nm. Delta epsilon units reported were obtained
from molar ellipiticity. Samples were scanned in 25 mM Tris-HCI and 600 mM

NaC1, pH 7.2. The treatment pH is shown at the top.


0.5'
0

-0.5

-1
-1.5

-2
-2.
-3


-3.5
200


210 220


Figure 5-2: Circular dichroism of catfish protein isolate made with alkali processing and
untreated catfish muscle recorded from 200-260nm. Delta epsilon units reported
were obtained from molar ellipiticity. Samples were scanned in 25 mM Tris-HCI and
600 mM NaC1, pH 7.2. The treatment pH is shown at the top.











0 %alpha helix n % beta sheet


n % random coil


60.00


50.00

c40 00 --

3 30.00

A 20.00

10.00 -

0.00
2.0 2.5 3.0 10.5 11 .0 1 1.5 co ntrolI
pH Treatment


Figure 5-3: Level of apparent a-helix, P-structureand random coil in catfish protein isolates and
untreated control. Percent structure was calculated from Dichroweb using the K2D
model. The K2D model did not require a protein reference set and provided a
secondary structural model without requiring data points below 200 nm. No
significant differences (p>0.05) were present between any of the samples for all
structures.











1.1

1 *

0.8

c 0.7
0.6
2.0
S0.5
0.4 2.5




0.2
-0.3

-i0.4




-0.5
[GuHCI]

Figure 5-4: Effect of Gu-HCI (0-2 M) on apparent structure of low pH processed catfish protein
isolates and untreated control. GuHCI treatment used 222 nm as the indicator
wavelength of alpha helix content. The change in structural content was determined
by whole muscle control in 0 M GuHCI having 100% native or 0% denatured
structure. 100% denatured structure was calculated from the response at 222 nm of
the control in 6 M GuHC1. The two state denaturation model was then used to
calculate the fraction of denaturation of each sample.










1.1

1.

0.8
c 0.7

*= 0.. 6
3 10.5
0a / 11.0
o 11.5
o0.2 a control
Pi 0.1

o
O -0.1 0 0.5 1; 1. 2

-023

-0.4
-0.5
[GuHCI]

Figure 5-5: Effect of Gu-HCI (0-2M) on apparent structure of high pH processed catfish protein
isolates and untreated control. GuHCI treatment used 222 nm as the indicator
wavelength of structural content. The change in structural content was determined by
whole muscle control in 0 M GuHCI having 100% native or 0% denatured structure.
100% denatured structure was calculated from the response at 222 nm of the control
in 6 M GuHC1. The two state denaturation model was then used to calculate the
fraction of denaturation of each sample.























-0.1

-0.2

Temperature OC

Figure 5-6: DSC thermograms of low pH treated catfish protein isolates and untreated control.
The thermograms represented here are the average of 4 replicate scans from two
separate isolations. The curves were smoothed together by 3 point averaging.
Average smoothing was repeated 4 times to obtain the smoothed graph. The black
line is the untreated control.


Temperature oC


Figure 5-7: DSC thermograms of high pH treated catfish protein isolates and untreated control.
The thermograms represented here are the average of 4 replicate scans from two
separate isolations. The curves were smoothed together by 3 point averaging.
Average smoothing was repeated 4 times to obtain the smoothed graph. The black
line is the untreated control.





o 4.5
m 4.0


S3.0


S2.0


1.5
-a0


11.0


Figure 5-8:The susceptibility of catfish protein isolates and untreated control to enzymatic
treatment with transglutaminase (TGase). Measurement of TGase activity was the
relative increase in the storage modulus between 0.02% TGase treated and untreated
incubated at 300C for 1 hr. A main effect was not determined for the sample
population (p>0.05).


i~F


10.5
isolation pH


11.5 control










* TGase
Untreated


20

15

10

5

0-


1000


2000
Time (s)


3000


4000


Figure 5-9: Compiled and smoothed rheogram of pH treatment 2.0 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.


120

100
-80

o 60

S40
e 20
o


1000 2000 3000
Time (s)


4000


Figure 5-10: Compiled and smoothed rheogram of pH treatment 2.5 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.






















Time (s)

Figure 5-11: Compiled and smoothed rheogram of pH treatment 3.0 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.


_ _


1


__


140
120
100
80
60


40


*TGase
Untreated


1000


2000


3000


4000


O 1000 2000 3000
Time (s)


4000


Figure 5-12: Compiled and smoothed rheogram of pH treatment 10.5 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.






















0 1000 2000 3000
Time (s)


4000


Figure 5-13: Compiled and smoothed rheogram of pH treatment 1 1.0 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.


S50
- 40

o 30

e 20

o
3j0


0 1000 2000 3000
Time (s)


4000


Figure 5-14: Compiled and smoothed rheogram of pH treatment 1 1.5 with (black) and without
(gray) TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-
HCI buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour.
Rheograms were combined and smoothed using TA theology advantage data analysis
software.











(3 TGase
S10
u, Untreated

o 6




cr> 0 -
0 1000 2000 3000 4000
Time (s)

Figure 5-15: Compiled and smoothed rheogram of the control with (black) and without (gray)
TGase treatment. Protein pastes were prepared at 10% solids in 20 mM tris-HCI
buffer with 0.6 M NaC1, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms
were combined and smoothed using TA theology advantage data analysis software.











Table 5-1: Thermal events of catfish protein isolates and untreated control as recorded by DSC. The smoothed transposed data was
used to generate this peak table. The columns are separated into samples and the rows are thermal events. Thermal events
are paired to -loC. Blue represents an endothermic event, orange represents an exothermic and yellow denotes no event
was present.


pH 2.0 pH 2.5 pH 3.0 pH 10.5 pH 11.0 pH 11.5 Control
T(oC) CP T(oC) CP T(oC) CP T(oC) CP T(oC) CP T(oC) CP T(C) CP
11.79 0.1044 11.82 0.0476 11.80 0.2234 11.84 0.0386 11.76 0.2021 11.79 0.0622




34.80 0.0852 35.26 0.0722 34.79 0.0845
37.31 0.0803 37.25 0.1111 36.83 0.0908
39.65 0.0076 39.82 0.0010

43.47 0.3714
45.19 0.0563 44.52 0.1175 45.64 0.0838 45.42 0.0985 45.34 0.0719 45.67 0.0398





60.39 -0.0284 59.61 0.0392

64.97 0.0109



71.18 0.0020 71.18 0.0001









CHAPTER 6
THE EFFECT OF HEATING RATE ON pH-SHIFT PROCESSED CATFISH (Ictalitrus
princtatus) MUSCLE PROTEINS

Introduction

The functional characteristics of muscle proteins are determined by the structural

conformation of the proteins in raw muscle and the intermediate structures of proteins during the

transition from the native to denatured state (Xiong 1997). One of the ways to dictate the

structural transition of muscle proteins from the native state to the non-native state is by the

application of heat (Phillips and others 1994). Temperatures above the denaturation temperature

are usually required to achieve the structural intermediates necessary which result in the

formation of a gel network of proteins (Phillips and others 1994). Muscle proteins denature over

a wide temperature range. Thermal transitions points of isolated myosin are in the range of 40oC-

67oC (Xiong 1997). Muscle proteins show increases in gel strength when subjected to thermal

treatment., However, at medium temperatures, or about 35-55oC, muscle proteins have exhibited

weakening in gel strength which is thought to be due to the disassociation of the helix-coil of

myosin. Above this temperature range strengthening of protein-protein interactions occur, and

gel strength increases again, This dissociation is thought to disrupt the gel network as it is

forming but further increases in temperature result in the reformation of the gel network resulting

in the final set form (Xiong 1997). Thus certain structural and conformational changes of muscle

proteins during thermal processing are required to achieve desirable functionalities (Xiong

1997). The modification of thermal processing by altering the heating rate has been shown to

affect the functional properties of comminuted muscle protein products (Riemann and others

2004a). For example, slower heating rates allows progressive, sequential unfolding of proteins

leading to a finer texture and increased water holding capacity of muscle proteins (Xiong 1997).









The obj ective of this study was to investigate the effect of various heating rates on the gel

forming ability of protein isolates made from acid and alkali processed catfish protein isolates.

Methods

Heating rate gelation studies were conducted at North Carolina State University (Raleigh,

NC) in the laboratory of Dr. Tyre Lanier.

Production of Protein Isolates

Fresh channel catfish was obtained for the study from a local supplier. Protein isolates

were produced as outlined in Figure 2-1: pH 2.5 for the acid-aided process and pH 11.0 for the

alkali-aided process. Precipitation following filtration was preformed at pH 5.5. Removal of

insoluble materials was accomplished by screening the material 3 times through progressively

tighter mesh. After precipitation of the clarified muscle protein homogenates chiffon cloth was

used for dewatering protein paste to less than 78% moisture.

Protein Composition

Protein isolates were assayed for moisture in a drying oven for 24hrs. Protein content was

assumed to be 97% dry weight (Kristinsson and others 2005b).

Torsion

Torsion tubes, 10 cm in length and 1.8 cm in diameter were filled with protein paste at

78% moisture and subsequently heated from 100C to a 700C internal endpoint, either rapidly by

a cylindrical microwave (Riemann and others 2004b), at 20 or 980C/min to test rapid heating

effects, slowly at loC/min by immersion in a programmable water bath or placed directly in an

700C water bath for 15 minutes. Temperatures were measured by a fiber optic probe (Riemann

and others 2004b). Upon reaching 700C, rapidly heated samples were held for 0 or 20 min prior

to rapid cooling by immersion in ice water for sufficient internal cooling to <100C for ~30 min.

Samples were held static in a cylindrical microwave applicator (length 16 cm, radius 12.5 cm)









and heated using power settings calculated previously. After reaching a final temperature of

700C, this temperature was maintained in gels during the subsequent holding period by utilizing

feedback software (Riemann and others 2004b).

Rheology

Rheological changes (storage modulus, G') of pastes/gels at 78% moisture were non-

destructively and continuously measured as pastes were heated, held and cooled using a 40 mm,

4 degrees slope cone and plate attachment of a constant stress, small strain rheometer (Stresstech,

Rheologica instruments AB, Lund, Sweden). Oscillation parameters were those used in

Riemann et al 2004. Heating conditions were at either 200C/min, the most rapid heating rate

possible for this apparatus, 0.50C/min, loC/min, 20C/min or 50C/min to an endpoint temperature

of 700C followed by holding for 0 or 20 min prior to cooling at 50C/min to 100C.

Statistical Analysis

Preliminary investigation of the affect of heating rate on pH-shift processed catfish protein

isolates was conducted on a single isolation of catfish fillets. The fillets used in this study were

obtained from two lots of fillets shipped to a local supplier. Torsion studies were conducted on

four to six cooked gels. Each gel was sampled 2 to 4 times. Rheological studies were conducted

in duplicate.

The heating rate study was analyzed with Graph Pad QuickCalcs online calculator software

(Graph Pad Software Inc. San Diego Ca). Analysis used an unpaired t-test with a manual

bonferoni correction, overall significance of p<0.05 with the individual significant p-values of

p<0.004.











Torsion

The torsion stress and strain results were obtained by thermally treating catfish muscle

isolated at both acid (2.5) and alkali (11.0) pH by two heating methods: microwave heating at a

heating rate of 200C per minute with (20/20) and without (20/0) a 20 minute hold, a heating rate

of 980C per minute with a 20 minute hold (98/20) and water bath heating rates of 0.50C (0.5

water), 1.00C (1 water), and 100C (10 water) per minute. Rapid heating provided total process

times of 3 23 and 21 min, respectively, for 200C with and without 20 minute holding and 980C

per minute with a 20 minute hold. The stress and strain are shown in figures 6-1 and 6-2,

respectively .

The stress results showed that alkali treatments were significantly higher (p<0.05) than

acid treatment. Alkali processing showed an order of 20/20>1 water>0.5 water>98/20>10

water>20/0. Significant differences were not present (p>0.05) between alkali treated samples

20/0 and 10 water. Significant differences were not present (p>0.05) 0.5 water and1 water when

compared to 20/20 and 98/20 however significant differences were present between 98/20 and

20/20. Acid processing showed an order of 0.5 water>1 water>98/20>20/20>10 water>20/0.

Significant differences were not present (p>0.05) between acid treated samples 20/0, 20/20 and

10 water. Significant differences were not present (p>0.05) between acid treated samples 98/20

and 1 water. Acid treatment 20/20 was not significantly different (p>0.05) from 1 water

however it wsa significantly different (p<0.05)from 98/20. Significant differences were present

(p<0.05) between the acid treated sample 0.5 water and all other acid treated samples.

The strain results showed that alkali treated catfish protein was significantly higher

(p<0.05) than acid treatment 10 water. The strain results showed alkali treated catfish protein

20/0 had significantly higher (p<0.05) strain values than acid treated catfish protein 20/20, 98/20,


Results









0.5 water and 10 water. The alkali treated isolate showed an order of 20/20>0.5 water>20/0>1

water>98/20>10 water. Acid treatment showed an order of 1 water=20/20>98/20>0.5 water>10

water>20/0.

Rheology

The rheograms shown in Eigures 6-4 6-10 show the gel formation process of varied heat

treatments. During the heating phase all rapidly heated samples, both acid and alkali, followed

almost exactly the same heating pattern (Higure 6-10) with differentiation between the gels

occurring at either the onset of holding or cooling, depending on the treatment. The acid sample,

though not significant (p>0.05), did not reach as high a G' during high temperature holding as

the alkali treatment. Acid treated catfish muscle not held at high temperature exhibited a

reduction in G' on cooling at 32oC. No decrease on cooling was observed for the acid sample

held at high temperature or the alkali treated samples. The acid and alkali samples heated at

0.5oC per minute did not follow the same gelation pattern on heating or cooling after heating

~30oC (figure 6-6, 6-9). During the heating phase of the samples heated at 0.5oC the G'

decreased until ~30oC. After this decrease the G' began to increase in both the acid and alkali

treated samples. The alkali treated sample increased at a greater rate than the acid treated sample

and did not exhibit a maj or decline in G' after it began to increase at 30oC. The acid treated

sample increased in G' until reaching the maximum G' of the heating and cooling regime at

~42oC. Above ~42oC the acid treated sample decreased in G' until the start of the cooling phase

at 70oC.

The theology results shown figure 6-3 of this study show that prior to heating there were

no significant differences (p>0.05) between any of the samples treated under acid or alkali

conditions. After heating at 700C prior to either holding or cooling, the G' of alkali isolate

heated at 200C per minute with no hold was significantly higher (p<0.05) than acid treated









isolate heated under the same conditions. After holding at 700C no significant differences

(p>0.05) were shown between any of the samples, before holding or after holding. No

significant differences (p>0.05) were observed based on heating regimen within acid or alkali

treated samples. When comparing acid treatment to alkali treatment heating at 200C per minute

with or without holding alkali treated protein isolates had a significantly higher (p<0.05) G',

however, acid treated isolate held for 20 minutes at 700C did not show significant differences

(p>0.05) between any of the acid treated or alkali treated samples.

Discussion

Structural shifting from the native state to intermediate states results in modifications of

muscle protein functionality. The transition of muscle proteins from native to intermediate states

may be induced by the heating method applied to a muscle protein paste Heating rate has

shown to affect the functional properties of muscle proteins.

Isolates heated at a high rate with holding at high temperature, had a significantly higher

(p<0.05) stress than rapidly heated samples with no holding at high temperature. The increase

gel strength of isolates treated held at high temperature may allow the requisite time for muscle

proteins to unfold into a conformation of higher functionality. Previous studies on high

temperature holding with rapid heating rates showed muscle proteins improving in gel strength

during short high temperature holding times. These results follow previously published studies

showing that rapid heating rates with no holding produce gels with lower strength than those

cooked at slower rates (Riemann and others 2004). The application of high temperature holding

for alkali treated catfish muscle proteins followed the results reported by Riemann and others

(2004) showing an increase in gel strength with increased high temperature holding on rapidly

heated comminuted muscle pastes (Riemann and others 2004). The acid processed catfish

isolate, however, did not follow this same trend with high temperature holding of rapidly heated









gels increasing gel strength. Heating at 20oC/min with or without holding did not significantly

affect (p>0.05) the gel strength of acid treated catfish muscle. However the very high heating

rate of 98oC/min with 20 min holding resulted in significant increases (p<0.05) in gel strength.

The increased gel strength of rapidly heated gels were significantly lower than gels cooked at the

slowest heating rate of 0.5oC/min (p<0.05) but were not significantly lower than gels cooked at

loC/min heating rate (p>0.05).

The theological results from this study indicate that during rapid heating, the high heating

rate has the predominating effect on the mechanism of gelation of pH-shift processed muscle as

seen by the similar patterns of gel formation in Eigure 6-10. The modification of catfish muscle

proteins by pH-shift processing changing the gel rigidity on cooling, indicate that the modified

protein structures alter the hydrogen bond formation associated with the cooling phase of

gelation (Lanier 2000). The effect of high temperature holding on the gel structure after cooling

may be due to increased covalent linkages formed at high temperature (Lanier 2000) or the

application of enough energy into the muscle protein matrix to allow for a more complete

denaturation of muscle proteins resulting in a more favorable structural rearrangement (Xiong

1997) which promotes the formation of hydrogen bonds (Lanier 2000).

The proposed equivalence point calculation (Riemann and others 2004a) for the

determination of functional performance of heated muscle pastes does not apply to acid

processed catfish muscle as the equivalent points in the heated products did not result in

equivalent improvements in gel strength. The lack of improvement with the equivalent point

method as proposed may be due to the modification of the protein structure during acid

processing, which resulted in larger decreases in alpha helical structure and thus reduced the









overall reactive potential of acid produced catfish isolate as shown in chapter 3 with the

reduction in gel strength of catfish muscle processed at pH 2.5.

Conclusions

These results show that acid and alkali processing form gels with different gel strengths at

fracture regardless of the rate heat is applied. However during rapid heating the effect on gel

formation, as shown by the theology results, indicates that the predominant effect on gel

formation is the rate heat is applied. Using the equivalent point method, rapid heating with

extended high temperature holding for equal thermal input to the system, for the production and

analysis of comminuted muscle products produced with alkali processing was shown to be

effective whereas the structural modification of acid processing may not allow for the use of

rapid heating to produce thermally set gels. The further modification of gel properties and the

functional performance of muscle proteins subj ected to pH-shift processing by modulating the

rate of heat application as shown here may increase the future utilization and application of pH-

shift processed protein isolates by expanding the range of textural properties achievable and

reducing the required cook time for high quality comminuted muscle products.










100
B C BC BC A
90 -E
80 A
70 -
ca F EG
60 -
r D DG E I

40 -
(n 30 -
20 -

10

20/min no 20/min 98/min 20 water water water
hold m icro hold 20 min hold .5/min 1/min 10o/min
min micro

Figure 6-1: Stress response of acid-aided (pH 2.5) in dark grey and alkali-aided (1 1.0) in light
grey catfish protein isolates (pH 11.0) recorded on the Torsion Gelometer. The
microwaved samples were heated at 20oC/min with no high temperature holding
(20/min no hold micro) or with a 20 minute high temperature hold time (20/min hold
20 min micro) and at 98oC/min with a 20 minute hold (98/min 20 min hold) for both
acid- and alkali-aided isolates. The lower heating rates were achieved in a water bath.
The 0.5oC/min (water .5/min) and loC/min (water 1/min) heating rates were done
using a computer controlled water bath. The 10oC/min (water 10/min) heating rate
was achieved by preheating a water bath to 70oC and placing the torsion tubes
directly into the hot water bath for 15 minutes. Gel diameter was I cm and the
machine rotated the gel at 2.5 RPM. The significant differences present between
samples (p<0.05) are represented by the letters above the column. Similar letters
represent no significant difference (p>0.05) between treatments.










2.5


2

1.5


1

0.5


0


20/min no 20/min 98/min 20 water water water
hold micro hold 20 min hold .5/min 1/mi n 1 0/min
min micro

Figure 6-2: Strain response of acid-aided (pH 2.5) in dark grey and alkali-aided (1 1.0) in light
grey catfish protein isolates (pH 11.0) recorded on the Torsion Gelometer. The
microwaved samples were heated at 20oC/min with no high temperature holding
(20/min no hold micro) or with a 20 minute high temperature hold time (20/min hold
20 min micro) and at 98oC/min with a 20 minute hold (98/min 20 min hold) for both
acid- and alkali-aided isolates. The lower heating rates were achieved in a water bath.
The 0.5oC/min (water .5/min) and loC/min (water 1/min) heating rates were done
using a computer controlled water bath. The 10oC/min (water 10/min) heating rate
was achieved by preheating a water bath to 70oC and placing the torsion tubes
directly into the hot water bath for 15 minutes. Gel diameter was I cm and the
machine rotated the gel at 2.5 RPM. The significant differences present between
samples (p<0.05) are represented by the letters above the column. Similar letters
represent no significant difference (p>0.05) between treatments.










120000


S100000

180000

o 60000 1

S 40000

S 20000




Fiur -3 Relogia epneo cdadd(H25n alal aie ~catis prtiioae
(p 10 eoddo T ontrle srsrhoer.Tefstdhdcoluni




Figur 20/20) Rhoogc r witout e (aci 20/0, ed(H .) alkali 200 2 ints odin atfs 70oC n eaing ioate
0p I.5oC/mn(cird 0.5 alkl 0.5) cThoed sigiatrs difermenes. (p<.05 are denoted byum i
letaters aboeth olumn. Theo lt a thersal anbrepresent. the sigifcant differelunces

(p<0.05 between sample after heating to 70 t10oC/i. The ltters cand drepresents the



significant differences (p<0.05) between samples after heating and cooling to SoC but
before holding or cooling. No other significant differences were present between the
samples.









70000


60000 L
50000
.~ Cooling
S40000

S30000 **
2 0 0 0Heating**..
v 10000 +


0 10 20 30 40 50 60 70 80

Temperature oC

Figure 6-4: Rheogram of alkali treated catfish muscle heated at 20oC/minute with no high
temperature holding. Duplicate samples at 78% moisture were heated in the presence
of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled
stress of 100 pa at a frequency of 0. 1 Hz.









80000


70000
S60000
-50000
o 40000
Q~30000

S20000
10000
0


0 10 20 30 40 50 60 70 80

Temperature oC


Figure 6-5: Rheogram of alkali treated catfish muscle heated at 20oC/minute with 20 minutes
high temperature holding. Duplicate samples at 78% moisture were heated in the
presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a
controlled stress of 100 pa at a frequency of 0. 1 Hz.


80000
70000
2 60000
S50000
o 40000
Q~30000

S20000
10000


0 10 20 30 40 50 60 70 80

Temperature oC

Figure 6-6: Rheogram of alkali treated catfish muscle heated at 0.5oC/minute with no high
temperature holding. Duplicate samples at 78% moisture were heated in the presence
of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled
stress of 100 pa at a frequency of 0. 1 Hz.










30000

25000

20000

15000

10000

5000


+o o
o o


0 10 20 30 40 50 60 70 E


Temperature oC

Figure 6-7: Rheogram of acid treated catfish muscle heated at 20oC/minute with no high
temperature holding. Duplicate samples at 78% moisture were heated in the presence
of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled
stress of 100 pa at a frequency of 0. 1 Hz.


50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0


Lbuu
a







O O


0 10 20 30 40 50 60 70 E


Temperature oC

Figure 6-8: Rheogram of acid treated catfish muscle heated at 20oC/minute with 20 minutes high
temperature holding. Duplicate samples at 78% moisture were heated in the presence
of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled
stress of 100 pa at a frequency of 0. 1 Hz.









18000
16000
i3 14000


*0 10000
E 8000

S6000
B 4000
2000


0 10 20 30 40 50 60 70 80

Temperature oC

Figure 6-9: Rheogram of acid treated catfish muscle heated at 0.5oC/minute with no high
temperature holding. Duplicate samples at 78% moisture were heated in the presence
of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled
stress of 100 pa at a frequency of 0. 1 Hz.









80000


70000
60000 t
S50000 1*44,






100

0 10 20 30 40 50 60 70 80

Temperature oC

Figure 6-10: Rheograms of acid and alkali treated catfish muscle heated at 20oC/minute with
and without 20 minutes high temperature holding. Duplicate samples at 78%
moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged.
Acid treated samples heated at 20oC with no hold (0), acid treated samples heated at
20oC with a 20 minute high temperature hold (x). Alkali treated samples heated at
20oC with no hold (A), alkali treated samples heated at 20oC with a 20 minute high
temperature hold (0). The sample was tested under a controlled stress of 100 pa at a
frequency of 0. 1 Hz.









CHAPTER 7
GENERAL DISCUSSION

The results of these studies show that pH-shift processing leads to structural modification

of catfish muscle proteins. The structural modification of catfish muscle proteins appears to be

responsible for the modification in solubility of catfish muscle proteins but does not lead to

significant differences in the chemical properties tested. The structural modification of catfish

muscle proteins by pH-shift processing was found to lead to the change in the thermal and

enzymatic sensitivity of catfish muscle proteins ultimately leading to the modifications observed

in the physical properties of thermally treated catfish muscle proteins. By using pH-treatment on

catfish muscle proteins, different functional properties are exhibited based on the type of pH-

treatment used. The modification of these functional properties and the use of one or more of the

pH-treatments studied provide a protein product with a wide variety of physical properties

possibly leading to the custom formulation of muscle proteins products. The custom formulation

of muscle products may be accomplished by the utilization of one or more pH-processing

techniques studied here. For example using one pH treatment a soft gel could be formed, but a

hard elastic gel with another pH treatment. The results of this study indicate that further

investigation is needed to fully understand the mechanisms responsible for the changes in the

physical properties observed, for example studying other chemical properties of muscle proteins

not investigated here and the effect of multiple proteins on the refolding of mechanisms of

muscle proteins.

Chemical analysis of pH-shift processed catfish muscle proteins showed no differences

between reactive or total sulfhydryl groups. These results therefore show that pH-shift

processing does not affect the sulfhydryl content of catfish muscle prior to thermal treatment.

The change in surface hydrophobic groups did not show any significant difference (p>0.05) in









the type of pH-shift processing used, however untreated catfish muscle proteins indicated higher

surface hydrophobicity than pH-shift processed catfish muscle. The lower surface

hydrophobicity for isolates was unexpected, as a higher hydrophobicity would have been

expected since proteins were only partially refolded. The most plausible explanation is that

microaggregates of proteins formed when the bulk of hydrophobic groups were buried from the

solvent. The decrease in myosin ATPase activity by pH-shift processing indicates that the active

site on the myosin head was modified. Reduction in myosin ATPase has been attributed to

modification in the SH group in the active site of the myosin head as previously concluded

(Yongswawatdigul and Park 2002). Since no significant difference was seen in the reactive and

total sulfhydryl groups the reduction in myosin ATPase activity may be due to a structural shift

at the active site of the myosin head as no conclusive data was shown on the modification of SH

groups. The change in solubility of catfish muscle proteins by pH-shift processing shows that

pH-shift processing does reduce the solubility in both sodium chloride and modify the pH

solubility profile of muscle proteins. The reduction in solubility, especially for low pH

treatments, indicates that though sarcoplasmic proteins are retained during low pH treatment

(Kristinsson and others 2005b) the solubility of sarcoplasmic proteins is reduced by low pH

treatment. High pH treatment also retains sarcoplasmic proteins, but to a lesser degree than low

pH treatment (Kristinsson and others 2005b) however the solubility results shown here indicate

that high pH treatment does not reduce the solubility of sarcoplasmic proteins as drastically as

low pH treatment. The reduction in solubility of the isolates is supported by the structural data

which show only partial refolding after pH-treatment and thus presumably structures more prone

to protein-protein interactions.









The theological properties of catfish muscle proteins show that pH-shift processing

reduces the temperature at which the phase change from a more viscous state to a gel like

material occurs. The reduction in temperature as shown by the phase angle to the onset of the

thermal transition of pH-shift processed catfish muscle proteins does not correlate with an

increase in gel rigidity but the results as shown in the storage modulus indicate that with the

correct pH salt combination increases in gel rigidity are possible. The range of differences seen

within all the pH treated samples provides a textural schematic of what pH shift processing can

do for muscle proteins.

The structural changes induced by pH-shift processing suggest that a molten globular form

is induced after pH-shift processing. The modification of the protein structure into a molten

globular form is presumed to be responsible for the chemical and physical changes seen in pH-

shift processed catfish muscle. The changes in structure also cause the muscle proteins to be

more flexible as treatment with low concentrations of guanidine hydrochloride induced an

increased response at 222 nm which may indicate the reformation of alpha helix structure in pH-

shift processed muscle proteins. The structural changes due to pH-shift processing caused a

change in thermal sensitivity as seen by the DSC data. The shifting of thermal events in both

type and temperature show the important effect of pH-shift processing on muscle proteins as

thermal treatment is the most common processing treatment to muscle proteins. This molten

globular form of the muscle proteins after pH-shift processing is attributed to the changes seen in

the chemical and functional properties of the protein isolates. The modifications of thermal

events show that the reactivity of catfish muscle proteins to thermal treatment undergoes

different transitions and reactions at different temperatures. The change in structure of catfish

muscle proteins also increased the susceptibility to enzymatic cross-linking. The increase in










enzymatic sensitivity indicates that pH-shift processing changes the structure of the muscle

proteins to promote interaction with transglutaminase.

The change in the thermal treatment regime of pH-shift processed catfish muscle showed

that pH-shift processed catfish muscle can be modified functionally by the thermal treatment

regime. Further investigation into the cooking methods used in pH-shift processed proteins may

lead to an increased range of textural properties and reduced processing times. The reduced

processing times may lower energy requirements and reduce the cost of processing.

This new understanding of pH-shift processing allows the production of meat based

products designed with specific physical properties. This is possible due to the range of gel

textures generated by the different pH processing parameters. In addition the combination of

gels produced with multiple isolates may provide a variety of textures in a single product which

is more closely related to meat and fillets. While the studies presented here show that pH-shift

processed catfish muscle proteins undergo structural changes which lead to changes in the

physical properties, future research is needed to determine the mechanisms for transforming pH-

shift produced isolates into manufactured fillets.










APPENDIX
STATISTICAL TABLES


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis
Cook Loss
Group N Missing Median
CLpH2 4 0 1.611
CLpH2.5 4 0 2.251
CLpH3 4 0 0.942
CLpH10.5 4 0 1.242
CLpH11 4 0 1.036
CLpH11.5 4 0 1.765
CL control 4 0 1.766
H = 7.621 with 6 degrees of freedom.

One Way Analysis of Variance


of Variance on Ranks


25%
1.331
2.072
0.189
0.971
0.952
1.158
1.301
(P = 0.267)


75%
1.890
2.430
1.695
1.512
1.119
2.372
2.030


Press Test
Group Name N Missing
PLpH2 4 0
PLpH2.5 4 0
PLpH3 4 0
PLpH10.5 4 0
PLpH11 4 0
PLpH11.5 4 0
PLeontrol 4 0
Source of Variation DF
Between Groups 6
Residual 21
Total 27


Mean
9.802
9.101
6.949
8.347
6.170
6.271
10.187
SS
66.830
43.830
110.660


Std Dev
1.032
2.149
1.242
1.798
1.183
1.481
0.745
MS
11.138
2.087


SEM
0.516
1.075
0.621
0.899
0.592
0.741
0.373
F
5.337


P
0.002


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis
Fold Test
Group N Missing Median
FpH2 4 0 3.750
FpH2.5 4 0 2.500
fpH3 4 0 4.000
fpH10.5 4 0 5.000
FpH11 4 0 5.000
FpH11.5 4 0 5.000
Control 4 0 5.000


of Variance on Ranks


25%
3.000
2.000
3.000
5.000
5.000
5.000
5.000


75%
4.750
3.000
5.000
5.000
5.000
5.000
5.000


H = 20.016 with 6 degrees of freedom. (P = 0.003)











One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Transglutaminase treatment
Group N Missing Median 25% 75%
TGASE2 4 0 1.707 0.716 2.635
TGASE2.5 4 0 2.306 2.191 2.608
TGASE'3 4 0 2.598 1.410 3.369
TGASE10.5 4 0 3.783 1.444 10.555
TGASEl l 4 0 4.031 2.899 4.290
TGASE11.5 4 0 2.863 2.526 3.346
TGASECONTROL 4 0 1.049 1.016 1.112
H = 1 1.121 with 6 degrees of freedom. (P = 0.085)

One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Rheology with salt, 80oC
Group N Missing Median 25% 75%
REHWS80CG'2 4 0 3103.000 2179.500 4052.000
REHWS80CG'2.5 4 0 2151.500 2042.000 2323.000
REHWS80CG'3 4 0 1232.000 1071.000 1393.000
REHWS80CG'10.5 4 0 4516.500 4194.500 4838.000
REHW S 80CG'll 4 0 7151.500 4971.500 10221.000
REHWS80CG'll.5 4 0 4815.500 4468.500 5874.000
REHWS80CG' CONTROL 4 0 2224.500 2122.500 2320.000
H = 23.488 with 6 degrees of freedom. (P = <0.001)

One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Rheology without salt, 80oC
Group N Missing Median 25% 75%
REHNS80CG'2 4 0 3900.500 2296.000 5560.500
REHNS80CG'2.5 4 0 1522.500 1365.500 1750.000
REHNS80CG'3 4 0 8551.000 7724.500 10739.000
REHNS80CG'10.5 4 0 8353.000 8053.500 9306.500
REHNS80CG'll1 4 0 13270.000 9532.000 16600.000
REHNS80CG'll.5 4 0 14050.000 13055.000 15400.000
REHNS80CG' CONTROL 4 0 6379.500 5375.500 7099.000
H = 23.675 with 6 degrees of freedom. (P = <0.001)

One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Rheology with salt after cooling to SoC
Group N Missing Median 25% 75%
REHSALTG'2 4 0 10487.500 6824.500 15360.000
REHSALTG'2.5 4 0 6843.500 6458.500 7463 .000










REHSALTG'3 4 0 3018.500 2532.000
REHSALTG' 109.5 4 0 14825.000 13920.000
REHSALTG'll1 4 0 14190.000 11350.000
REHSALTG'll.5 4 0 19810.000 18385.000
REHSALTG' CONTROL 4 0 7688.000
H = 22.379 with 6 degrees of freedom. (P = 0.001)


3505.000
15545.000
17260.000
24010.000
7521.500


8113.000


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Rheology without salt after cooling to SoC
Group N Missing Median 25% 75%
REHG'2 4 0 12315.000 8375.500 19625.000
REHG'2.5 4 0 5190.500 3929.000 6506.500
REHG'3 4 0 35500.000 29775.000 44675.000
REHG' 109.5 4 0 31550.000 30275.000 35610.000
REHG'll1 4 0 51540.000 37565.000 63940.000
REHG'll1.5 4 0 57295.000 55460.000 63645.000
REHG' CONTROL 4 0 21855.000 18650.000 24095.000
H = 24.214 with 6 degrees of freedom. (P = <0.001)

One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Torsion strain
Group N Missing Median
TORSTRAIN2 8 0 1.325
TORSTRAIN2.5 8 0 1.175
TORSTRAIN3 8 0 1.085
TORSTRAIN10.5 8 0 1.395
TORSTRAIN11 8 0 1.560
TORSTRAIN11.5 8 0 1.575
TORSTRAIN CONTROL 8 0
H = 23.804 with 6 degrees of freedom. (P


25%
1.255
1.140
0.970
1.020
1.380
1.525
1.250
<0.001)


75%
1.410
1.295
1.150
1.605
1.715
1.660
1.025 1.435


One Way Analysis of Variance
Torsion stress


Group Name N Mi
TORSTRESS2 8
TORSTRESS2.5 8
TORSTRESS3 8
TORSTRESS10.5 8
TORSTRESS11 8
TORSTRESS11.5 8
TORSTRESS CONTROL
Source of Variation DF
Between Groups 6
Residual 49


ssing Mean
0 84.790
0 73.833
0 89.440
0 99.359
0 127.396
0 108.529
8 0
SS


Std Dev
11.315
5.836
18.116
14.489
18.656
13.181
41.960
MS


SEM
4.001
2.063
6.405
5.123
6.596
4.660
4.667
F


1.650
P


35383.363 5897.227 33.189 <0.001
8706.538 177.684










Total


55 44089.901


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Punch Test Jelly Strength
Group N Missing
PTJS2 9 0
PTJS2.5 9 0
PTJS3 9 0
PTJS10.5 7 0
PTJS11 6 0
PTJS11.5 7 0
PTJS CONTROL 9
H = 32.504 with 6 degrees (


Median
6292.500
4636.400
3872.600
6374.400
6153.500
7174.400
0
of freedom.


25% 75%
3196.050 8430.250
4177.200 5239.200
3259.250 4764.600
4860.575 7720.000
5349.600 7173.200
6230.325 7675.150
1728.000 1546.425
(P = <0.001)


2127.375


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Punch Test Distance
Group N Missing
PTD2 9 0
PTD2.5 9 0
PTD3 9 0
PTD10.5 7 0
PTD11 6 0
PTD11.5 7 0
PTD CONTROL 9


Median
7.600
6.700
6.700
8.300
8.000
7.900
0


25%
7.150
6.500
6.350
7.975
7.700
7.600
9.100
(P = 0.004)


75%
12.925
8.600
7.125
9.325
8.500
8.950
7.175 10.250


H = 19.340 with 6 degrees of freedom.


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Punch Test Force
Group N Missing
PTF2 9 0
PTF2.5 9 0
PTF3 9 0
PTF10.5 7 0
PTF11 6 0
PTF11.5 7 0
PTF CONTROL 9


Median
491.000
618.000
578.000
768.000
791.500
876.000
0


25%
455.000
605.000
513.750
576.000
701.000
608.250
192.000


75%
819.500
688.250
661.750
877.500
853.000
938.500
186.750


222.750


H = 30.333 with 6 degrees of freedom. (P = <0.001)











One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Reactive Sulfhydryl Groups
Group N Missing
RSG2 4 0
RSG2.5 4 0
RSG3 4 0
RSG10.5 4 0
RSG11 4 0
RSG11.5 4 0
RSG CONTROL 4


Median
0.00973
0.0104
0.00927
0.00858
0.0109
0.0101
0


25%
0.00584
0.00784
0.00555
0.00857
0.00784
0.00830
0.00706
(P = 0.493)


75%
0.0139
0.0126
0.0128
0.00872
0.0143
0.0119
0.00677


0.00738


H = 5.409 with 6 degrees of freedom.


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis
Total Sulfhydryl Groups


of Variance on Ranks


Group N M
TSGpH2 4
TSGpH2.5 4
TSGpH3 4
TSGpH10.54
TSGpH11 4
TSGpH11.54
TSGcontrol 4
H = 11.209 with 6


missingg
0
0
0
0
0
0
0


Median
0.0313
0.0363
0.0322
0.0293
0.0312
0.0344
1.766


25% 75%
0.0303 0.0335
0.0294 0.0388
0.0318 0.0333
0.0224 0.0356
0.0301 0.0345
0.0234 0.0456
1.301 2.030
(P = 0.082)


degrees of freedom.


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis
Surface Hydrophobicity


of Variance on Ranks

25% 75%
413.460 632.420
387.580 529.155
435.430 531.250
410.955 509.775
361.400 456.695
388.550 474.115
989.180 1274.500
(P = 0.191)


Group N
SHpH2 4
SHpH2.5 4
SHpH3 4
SHpH10.5 4
SHpH11 4
SHpH11.5 4
SHcontrol 2


Missing
0
0
0


Median
534.735
452.370
495.025
472.125
398.455
431.795
1131.840


H = 8.701 with 6 degrees of freedom.











One Way Analysis of Variance


ATPase activity
Group Name N
ATPpH2 4
ATPpH2.5 4
ATPpH3 4
ATPpH 10.5 4
ATPpH11 4
ATPpH 11.5 4
ATPcontrol 4
Source of Variati
Between Groups
Residual
Total


Missing
0
0
0
0
0
0
0
on DF
6
21
27


Mean Std Dev SEM


51.014
51.858
35.211
36.729
61.346
59.045
100.000
SS
11271.662
8377.625
19649.287


22.191
17.213
16.573
28.726
19.634
22.770
0.000
MS
1878.61(
398.935


11.095
8.606
8.286
14.363
9.817
11.385
0.000
F
04.709


P
0.003














0.538


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks
Group N Missing Median 25% 75%
CDRAN2 5 0 0.480 0.480 0.495
CDRAN2.5 5 0 0.480 0.480 0.515
CDRAN3 5 0 0.480 0.480 0.502
CDRAN 10.5 5 0 0.490 0.480 0.560
CDRAN11 5 0 0.500 0.487 0.528
CDRAN11.5 5 0 0.490 0.480 0.520
CDRAN CONTROL 5 0 0.510 0.338
H = 2.266 with 6 degrees of freedom. (P = 0.894)


One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on
Group N Missing Median 25%
CDA2 5 0 0.0800 0.0725
CDA2.5 5 0 0.0800 0.0650
CDA3 5 0 0.0800 0.0800
CDA 10.5 5 0 0.150 0.108
CDAll1 5 0 0.210 0.150
CDAll1.5 5 0 0.110 0.0975
CDA CONTROL 5 0 0.310
H = 12.623 with 6 degrees of freedom. (P = 0.049)


Ranks
75%
0.220
0.255
0.250
0.340
0.320
0.302
0.292 0.563











One Way Analysis of Variance
Kruskal-Wallis One Way Analysis of Variance on Ranks


Group N Missing Median
CDB2 5 0 0.430
CDB2.5 5 0 0.440
CDB3 5 0 0.430
CDB 10.5 5 0 0.370
CDB 11 5 0 0.290
CDB 11.5 5 0 0.400
CDB CONTROL 5 0
H = 12.285 with 6 degrees of freedom.


25%
0.285
0.232
0.248
0.1000
0.153
0.178
0.130
(P = 0.056)


75%
0.448
0.455
0.440
0.412
0.368
0.412
0.0950


0.183


Two Way ANOVA of pH Solubility data

Two Way Analysis of Variance
General Linear Model
Dependent Variable: Solubility
Source of Variation DF SS
Sample pH 22 796410.028
Treatment pH 6 15843.955
Sample pH x Treatment pH132 32574.525
Residual 479 36729.665
Total 639 883137.566


MS
36200.456
2640.659
246.777
76.680
1382.062


F
472.098
34.437
3.218


P
<0.001
<0.001
<0.001


Main effects cannot be properly interpreted if significant interaction is determined. This is
because the size of a factor's effect depends upon the level of the other factor.

The effect of different levels of Sample pH depends on what level of Treatment pH is present.
There is a statistically significant interaction between Sample pH and Treatment pH. (P =
<0.001)


Power of performed test with alpha
Power of performed test with alpha
Power of performed test with alpha


0.0500: for Sample pH : 1.000
0.0500: for Treatment pH : 1.000
0.0500: for Sample pH x Treatment pH : 1.000


All Pairwise Multiple Comparison Procedures (Holm-Sidak method):
Overall significance level = 0.05
Comparisons for factor: Treatment pH within 1.5: No Significant Differences


Comparisons for factor: Treatment pH within :
Comparison Diff of Means t
10.500 vs. 11.500 31.839 5.142
3.000 vs. 11.500 31.523 5.091
11.000 vs. 11.500 24.463 3.951
2.500 vs. 11.500 23.433 3.784


Unadjusted P Critical Level Significant?
0.000 0.002 Yes
0.000 0.003 Yes
0.000 0.003 Yes
0.000 0.003 Yes











Comparisons for factor: Treatment pH within 2.5


Comparison
con vs. 2.000
con vs. 11.500
con vs. 2.500
con vs. 11.000
con vs. 3.000
con vs. 10.500
10.500 vs. 2.000
10.500 vs. 11.500


Diff of Means
57.028
53.758
53.306
49.815
44.938
35.612
21.416
18.146


t
9.210
8.682
8.609
8.045
7.258
5.751
3.459
2.931


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.004


Critical Level
0.002
0.003
0.003
0.003
0.003
0.003
0.003
0.004


Critical Level
0.002
0.003
0.003
0.003
0.003
0.003


Critical Level
0.002
0.003
0.003
0.003
0.003


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Treatment pH within 3
Comparison Diff of Means t Unadjusted P
con vs. 11.500 57.549 9.294 0.000
con vs. 3.000 57.127 9.226 0.000
con vs. 2.500 56.663 9.151 0.000
con vs. 2.000 56.613 9.143 0.000
con vs. 11.000 54.283 8.767 0.000
con vs. 10.500 49.738 8.033 0.000

Comparisons for factor: Treatment pH within 3.5
Comparison Diff of Means t Unadjusted P
con vs. 11.500 24.689 3.987 0.000
con vs. 3.000 23.942 3.867 0.000
con vs. 2.000 23.548 3.803 0.000
con vs. 11.000 20.575 3.323 0.001
con vs. 2.500 20.255 3.271 0.001


Comparisons for factor: Treatment pH within 4: No Significant Differences

Comparisons for factor: Treatment pH within 4.5: No Significant Differences

Comparisons for factor: Treatment pH within 5: No Significant Differences

Comparisons for factor: Treatment pH within 5.5: No Significant Differences

Comparisons for factor: Treatment pH within 6: No Significant Differences

Comparisons for factor: Treatment pH within 6.5: No Significant Differences

Comparisons for factor: Treatment pH within 7: No Significant Differences

Comparisons for factor: Treatment pH within 7.5: No Significant Differences












Critical Level
0.002
0.003


Critical Level
0.002
0.003


Critical Level
0.002
0.003


Critical Level
0.002
0.003


Critical Level
0.002


Critical Level
0.002
0.003
0.003
0.003
0.003
0.003
0.003
0.004
0.004


Critical Level
0.002
0.003
0.003
0.003
0.003
0.003
0.003


Significant?
Yes
Yes



Significant?
Yes
Yes



Significant?
Yes
Yes



Significant?
Yes
Yes



Significant?
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Treatment pH within 8
Comparison Diff of Means t Unadjusted P
con vs. 2.000 20.053 3.239 0.001
con vs. 2.500 18.845 3.043 0.002

Comparisons for factor: Treatment pH within 8.5
Comparison Diff of Means t Unadjusted P
con vs. 2.000 19.850 3.206 0.001
con vs. 2.500 19.338 3.123 0.002

Comparisons for factor: Treatment pH within 9
Comparison Diff of Means t Unadjusted P
con vs. 2.000 19.903 3.214 0.001
con vs. 2.500 19.273 3.113 0.002

Comparisons for factor: Treatment pH within 9.5
Comparison Diff of Means t Unadjusted P
con vs. 2.000 22.572 3.645 0.000
con vs. 2.500 19.634 3.171 0.002

Comparisons for factor: Treatment pH within 10
Comparison Diff of Means t Unadjusted P
con vs. 2.000 20.492 3.309 0.001

Comparisons for factor: Treatment pH within 10.5


Comparison
10.500 vs. 2.000
10.500 vs. 2.500
11.000 vs. 2.000
11.000 vs. 2.500
10.500 vs. 3.000
con vs. 2.000
10.500 vs. 11.500
11.000 vs. 3.000
con vs. 2.500


Diff of Means
26.817
24.607
24.195
21.985
21.658
21.241
19.350
19.036
19.031


Unadjusted P
0.000
0.000
0.000
0.000
0.001
0.001
0.002
0.002
0.002


t
4.331
3.974
3.907
3.551
3.498
3.430
3.125
3.074
3.073


Comparisons for factor: Treatment pH within 11


Comparison
3.000 vs. con
10.500 vs. con
11.500 vs. con
3.000 vs. 2.000
11.000 vs. con
2.500 vs. con
10.500 vs. 2.000


Diff of Means
44.686
38.914
32.067
27.165
27.067
26.711
21.393


t
7.217
6.285
5.179
4.387
4.371
4.314
3.455


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.001











Comparisons for factor: Treatment pH within 11.5
Comparison Diff of Means t Unadjusted P Critical Level
3.000 vs. 11.000 23.241 3.753 0.000 0.002

Comparisons for factor: Treatment pH within 12
Comparison Diff of Means t Unadjusted P Critical Level
10.500 vs. 11.000 26.405 4.264 0.000 0.002
2.000 vs. 11.000 23.213 3.749 0.000 0.003
3.000 vs. 11.000 21.632 3.494 0.001 0.003
con vs. 11.000 21.485 3.470 0.001 0.003
Comparisons for factor: Treatment pH within NC: No Significant Differences


Significant?
Yes



Significant?
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within 2


Comparison
NC vs. 5.500
NC vs. 5.000
NC vs. 6.500
NC vs. 6.000
NC vs. 4.500
12.000 vs. 5.500
12.000 vs. 5.000
12.000 vs. 6.500
NC vs. 4.000
12.000 vs. 6.000
12.000 vs. 4.500
NC vs. 7.000
NC vs. 3.500
12.000 vs. 4.000
11.500 vs. 5.500
NC vs. 7.500
11.500 vs. 5.000
NC vs. 8.000
11.500 vs. 6.500
11.500 vs. 6.000
12.000 vs. 7.000
12.000 vs. 3.500
11.500 vs. 4.500
12.000 vs. 7.500
NC vs. 3.000
NC vs. 8.500
12.000 vs. 8.000
11.500 vs. 4.000
NC vs. 10.000
NC vs. 9.000
NC vs. 9.500


Diff of Means
101.286
100.893
100.387
99.892
99.343
98.441
98.048
97.543
97.352
97.047
96.499
94.696
94.664
94.507
93.528
93.296
93.134
92.704
92.629
92.133
91.852
91.819
91.585
90.451
90.370
89.956
89.860
89.593
89.377
89.227
88.232


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Critical Level
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


t
16.358
16.294
16.213
16.133
16.044
15.898
15.835
15.753
15.722
15.673
15.585
15.294
15.288
15.263
15.105
15.067
15.041
14.972
14.960
14.880
14.834
14.829
14.791
14.608
14.595
14.528
14.512
14.469
14.434
14.410
14.250










12.000 vs. 3.000 87.525 14.135 0.000 0.000 Yes
12.000 vs. 8.500 87.111 14.069 0.000 0.000 Yes
11.5 00 vs. 7.000 86.938 14.041 0.000 0.000 Yes
11.5 00 vs. 3.500 86.905 14.035 0.000 0.000 Yes
12.000 vs. 10.000 86.532 13.975 0.000 0.000 Yes
12.000 vs. 9.000 86.383 13.951 0.000 0.000 Yes
11.5 00 vs. 7.500 85.537 13.814 0.000 0.000 Yes
12.000 vs. 9.500 85.387 13.790 0.000 0.000 Yes
1.500 vs. 5.500 85.196 13.759 0.000 0.000 Yes
2.000 vs. 5.500 85.133 13.749 0.000 0.000 Yes
11.5 00 vs. 8.000 84.946 13.719 0.000 0.000 Yes
1.500 vs. 5.000 84.803 13.696 0.000 0.000 Yes
2.000 vs. 5.000 84.740 13.686 0.000 0.000 Yes
1.500 vs. 6.500 84.297 13.614 0.000 0.000 Yes
2.000 vs. 6.500 84.235 13.604 0.000 0.000 Yes
NC vs. 10.500 83.864 13.544 0.000 0.000 Yes
1.500 vs. 6.000 83.802 13.534 0.000 0.000 Yes
2.000 vs. 6.000 83.739 13.524 0.000 0.000 Yes
1.500 vs. 4.500 83.253 13.445 0.000 0.000 Yes
2.000 vs. 4.500 83.191 13.435 0.000 0.000 Yes
NC vs. 2.500 83.119 13.424 0.000 0.000 Yes
11.5 00 vs. 3.000 82.611 13.342 0.000 0.000 Yes
11.5 00 vs. 8.500 82.197 13.275 0.000 0.000 Yes
11.5 00 vs. 10.000 81.619 13.181 0.000 0.000 Yes
11.5 00 vs. 9.000 81.469 13.157 0.000 0.000 Yes
1.500 vs. 4.000 81.262 13.124 0.000 0.000 Yes
2.000 vs. 4.000 81.199 13.114 0.000 0.000 Yes
12.000 vs. 10.500 81.019 13.085 0.000 0.000 Yes
11.5 00 vs. 9.500 80.474 12.997 0.000 0.000 Yes
12.000 vs. 2.500 80.274 12.964 0.000 0.000 Yes
11.000 vs. 5.500 80.186 12.950 0.000 0.000 Yes
11.000 vs. 5.000 79.793 12.887 0.000 0.000 Yes
11.000 vs. 6.500 79.287 12.805 0.000 0.000 Yes
11.000 vs. 6.000 78.792 12.725 0.000 0.000 Yes
1.500 vs. 7.000 78.606 12.695 0.000 0.000 Yes
1.500 vs. 3.500 78.574 12.690 0.000 0.000 Yes
2.000 vs. 7.000 78.544 12.685 0.000 0.000 Yes
2.000 vs. 3.500 78.511 12.680 0.000 0.000 Yes
11.000 vs. 4.500 78.243 12.636 0.000 0.000 Yes
1.500 vs. 7.500 77.206 12.469 0.000 0.000 Yes
2.000 vs. 7.500 77.143 12.459 0.000 0.000 Yes
1.500 vs. 8.000 76.615 12.373 0.000 0.000 Yes
2.000 vs. 8.000 76.552 12.363 0.000 0.000 Yes
11.000 vs. 4.000 76.252 12.315 0.000 0.000 Yes
11.5 00 vs. 10.500 76.105 12.291 0.000 0.000 Yes
11.5 00 vs. 2.500 75.360 12.171 0.000 0.000 Yes










1.500 vs. 3.000
2.000 vs. 3.000
1.500 vs. 8.500
2.000 vs. 8.500
11.000 vs. 7.000
11.000 vs. 3.500
1.500 vs. 10.000
2.000 vs. 10.000
1.500 vs. 9.000
2.000 vs. 9.000
11.000 vs. 7.500
1.500 vs. 9.500
2.000 vs. 9.500
11.000 vs. 8.000
11.000 vs. 3.000
11.000 vs. 8.500
11.000 vs. 10.000
11.000 vs. 9.000
1.500 vs. 10.500
2.000 vs. 10.500
11.000 vs. 9.500
1.500 vs. 2.500
2.000 vs. 2.500
11.000 vs. 10.500
11.000 vs. 2.500


74.280
74.217
73.866
73.803
73.596
73.564
73.287
73.224
73.138
73.075
72.196
72.142
72.079
71.605
69.270
68.856
68.277
68.128
67.774
67.711
67.132
67.029
66.966
62.764
62.019


11.996
11.986
11.929
11.919
11.886
11.881
11.836
11.826
11.812
11.802
11.660
11.651
11.641
11.564
11.187
11.120
11.027
11.003
10.945
10.935
10.842
10.825
10.815
10.136
10.016


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within 2.5


Comparison
NC vs. 4.500
NC vs. 5.000
NC vs. 6.500
NC vs. 6.000
NC vs. 5.500
NC vs. 4.000
NC vs. 7.000
NC vs. 7.500
2.000 vs. 4.500
2.000 vs. 5.000
2.000 vs. 6.500
NC vs. 8.000
NC vs. 3.500
2.000 vs. 6.000
NC vs. 3.000
NC vs. 8.500
2.000 vs. 5.500
11.000 vs. 4.500


Diff of Means
100.668
100.529
100.132
99.828
97.908
96.841
95.503
93.288
92.150
92.010
91.614
91.497
91.370
91.310
90.419
89.444
89.390
88.759


t
16.258
16.235
16.171
16.122
15.812
15.640
15.424
15.066
14.882
14.860
14.796
14.777
14.756
14.747
14.603
14.445
14.437
14.335


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Critical Level
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes










11.5 00 vs. 4.500 88.737 14.331 0.000 0.000 Yes
1.500 vs. 4.500 88.725 14.329 0.000 0.000 Yes
11.000 vs. 5.000 88.619 14.312 0.000 0.000 Yes
11.5 00 vs. 5.000 88.597 14.309 0.000 0.000 Yes
NC vs. 9.000 88.597 14.309 0.000 0.000 Yes
1.500 vs. 5.000 88.586 14.307 0.000 0.000 Yes
2.000 vs. 4.000 88.323 14.264 0.000 0.000 Yes
11.000 vs. 6.500 88.223 14.248 0.000 0.000 Yes
11.5 00 vs. 6.500 88.201 14.245 0.000 0.000 Yes
1.500 vs. 6.500 88.189 14.243 0.000 0.000 Yes
11.000 vs. 6.000 87.919 14.199 0.000 0.000 Yes
11.5 00 vs. 6.000 87.897 14.195 0.000 0.000 Yes
1.500 vs. 6.000 87.886 14.194 0.000 0.000 Yes
2.000 vs. 7.000 86.985 14.048 0.000 0.000 Yes
12.000 vs. 4.500 86.669 13.997 0.000 0.000 Yes
12.000 vs. 5.000 86.529 13.975 0.000 0.000 Yes
NC vs. 10.000 86.409 13.955 0.000 0.000 Yes
12.000 vs. 6.500 86.133 13.911 0.000 0.000 Yes
11.000 vs. 5.500 85.999 13.889 0.000 0.000 Yes
11.5 00 vs. 5.500 85.977 13.885 0.000 0.000 Yes
1.500 vs. 5.500 85.965 13.883 0.000 0.000 Yes
12.000 vs. 6.000 85.829 13.861 0.000 0.000 Yes
NC vs. 9.500 85.294 13.775 0.000 0.000 Yes
11.000 vs. 4.000 84.932 13.717 0.000 0.000 Yes
11.5 00 vs. 4.000 84.910 13.713 0.000 0.000 Yes
1.500 vs. 4.000 84.899 13.711 0.000 0.000 Yes
2.000 vs. 7.500 84.770 13.690 0.000 0.000 Yes
12.000 vs. 5.500 83.909 13.551 0.000 0.000 Yes
11.000 vs. 7.000 83.594 13.500 0.000 0.000 Yes
11.5 00 vs. 7.000 83.572 13.497 0.000 0.000 Yes
1.500 vs. 7.000 83.560 13.495 0.000 0.000 Yes
2.000 vs. 8.000 82.979 13.401 0.000 0.000 Yes
2.000 vs. 3.500 82.852 13.381 0.000 0.000 Yes
12.000 vs. 4.000 82.842 13.379 0.000 0.000 Yes
2.000 vs. 3.000 81.901 13.227 0.000 0.000 Yes
NC vs. 10.500 81.654 13.187 0.000 0.000 Yes
12.000 vs. 7.000 81.504 13.163 0.000 0.000 Yes
11.000 vs. 7.500 81.379 13.143 0.000 0.000 Yes
11.5 00 vs. 7.500 81.357 13.139 0.000 0.000 Yes
1.500 vs. 7.500 81.345 13.137 0.000 0.000 Yes
2.000 vs. 8.500 80.925 13.069 0.000 0.000 Yes
2.000 vs. 9.000 80.079 12.933 0.000 0.000 Yes
11.000 vs. 8.000 79.588 12.853 0.000 0.000 Yes
11.5 00 vs. 8.000 79.566 12.850 0.000 0.000 Yes
1.500 vs. 8.000 79.554 12.848 0.000 0.000 Yes
11.000 vs. 3.500 79.461 12.833 0.000 0.000 Yes










11.500 vs. 3.500
1.500 vs. 3.500
NC vs. 2.500
12.000 vs. 7.500
11.000 vs. 3.000
11.500 vs. 3.000
1.500 vs. 3.000
2.000 vs. 10.000
11.000 vs. 8.500
11.500 vs. 8.500
1.500 vs. 8.500
12.000 vs. 8.000
12.000 vs. 3.500
2.000 vs. 9.500
11.000 vs. 9.000
11.500 vs. 9.000
1.500 vs. 9.000
12.000 vs. 3.000
12.000 vs. 8.500
12.000 vs. 9.000
11.000 vs. 10.000
11.500 vs. 10.000
1.500 vs. 10.000
11.000 vs. 9.500
11.500 vs. 9.500
1.500 vs. 9.500
2.000 vs. 10.500
12.000 vs. 10.000
12.000 vs. 9.500
2.000 vs. 2.500
11.000 vs. 10.500
11.500 vs. 10.500
1.500 vs. 10.500
12.000 vs. 10.500
11.000 vs. 2.500
11.500 vs. 2.500
1.500 vs. 2.500
12.000 vs. 2.500


79.439
79.427
79.397
79.289
78.510
78.488
78.476
77.890
77.534
77.512
77.501
77.498
77.371
76.776
76.688
76.666
76.654
76.420
75.444
74.598
74.499
74.477
74.466
73.385
73.363
73.351
73.136
72.409
71.295
70.878
69.745
69.723
69.711
67.655
67.488
67.465
67.454
65.398


12.829
12.828
12.823
12.805
12.679
12.676
12.674
12.579
12.522
12.518
12.516
12.516
12.496
12.399
12.385
12.382
12.380
12.342
12.184
12.048
12.032
12.028
12.026
11.852
11.848
11.846
11.811
11.694
11.514
11.447
11.264
11.260
11.258
10.926
10.899
10.896
10.894
10.562


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within 3
Comparison Diff of Means t
11.000 vs. 6.500 105.648 17.062
11.000 vs. 4.000 105.532 17.044
11.000 vs. 4.500 105.332 17.011
11.000 vs. 5.500 105.046 16.965
11.000 vs. 5.000 104.667 16.904


Unadjusted P
0.000
0.000
0.000
0.000
0.000


Critical Level
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes










11.000 vs. 6.000 104.523 16.881 0.000 0.000 Yes
11.5 00 vs. 6.500 101.939 16.463 0.000 0.000 Yes
11.5 00 vs. 4.000 101.823 16.444 0.000 0.000 Yes
11.5 00 vs. 4.500 101.622 16.412 0.000 0.000 Yes
11.5 00 vs. 5.500 101.336 16.366 0.000 0.000 Yes
11.000 vs. 3.500 101.122 16.331 0.000 0.000 Yes
11.000 vs. 7.000 100.984 16.309 0.000 0.000 Yes
11.5 00 vs. 5.000 100.958 16.305 0.000 0.000 Yes
11.5 00 vs. 6.000 100.814 16.282 0.000 0.000 Yes
NC vs. 6.500 99.583 16.083 0.000 0.000 Yes
NC vs. 4.000 99.467 16.064 0.000 0.000 Yes
NC vs. 4.500 99.267 16.032 0.000 0.000 Yes
2.000 vs. 6.500 99.155 16.014 0.000 0.000 Yes
11.000 vs. 7.500 99.148 16.012 0.000 0.000 Yes
2.000 vs. 4.000 99.039 15.995 0.000 0.000 Yes
NC vs. 5.500 98.981 15.985 0.000 0.000 Yes
2.000 vs. 4.500 98.838 15.962 0.000 0.000 Yes
NC vs. 5.000 98.602 15.924 0.000 0.000 Yes
2.000 vs. 5.500 98.552 15.916 0.000 0.000 Yes
NC vs. 6.000 98.458 15.901 0.000 0.000 Yes
2.000 vs. 5.000 98.174 15.855 0.000 0.000 Yes
2.000 vs. 6.000 98.030 15.832 0.000 0.000 Yes
11.5 00 vs. 3.500 97.413 15.732 0.000 0.000 Yes
11.5 00 vs. 7.000 97.275 15.710 0.000 0.000 Yes
11.000 vs. 3.000 96.948 15.657 0.000 0.000 Yes
11.000 vs. 8.000 95.706 15.457 0.000 0.000 Yes
11.5 00 vs. 7.500 95.438 15.413 0.000 0.000 Yes
12.000 vs. 6.500 95.157 15.368 0.000 0.000 Yes
NC vs. 3.500 95.057 15.352 0.000 0.000 Yes
12.000 vs. 4.000 95.041 15.349 0.000 0.000 Yes
NC vs. 7.000 94.919 15.329 0.000 0.000 Yes
12.000 vs. 4.500 94.841 15.317 0.000 0.000 Yes
2.000 vs. 3.500 94.629 15.283 0.000 0.000 Yes
12.000 vs. 5.500 94.555 15.271 0.000 0.000 Yes
2.000 vs. 7.000 94.490 15.260 0.000 0.000 Yes
12.000 vs. 5.000 94.176 15.210 0.000 0.000 Yes
12.000 vs. 6.000 94.032 15.186 0.000 0.000 Yes
11.5 00 vs. 3.000 93.239 15.058 0.000 0.000 Yes
11.000 vs. 8.500 93.210 15.053 0.000 0.000 Yes
NC vs. 7.500 93.083 15.033 0.000 0.000 Yes
1.500 vs. 6.500 92.943 15.010 0.000 0.000 Yes
1.500 vs. 4.000 92.827 14.992 0.000 0.000 Yes
2.000 vs. 7.500 92.654 14.964 0.000 0.000 Yes
1.500 vs. 4.500 92.627 14.959 0.000 0.000 Yes
1.500 vs. 5.500 92.341 14.913 0.000 0.000 Yes
11.5 00 vs. 8.000 91.997 14.858 0.000 0.000 Yes










1.500 vs. 5.000 91.963 14.852 0.000 0.000 Yes
1.500 vs. 6.000 91.818 14.829 0.000 0.000 Yes
11.000 vs. 9.000 91.790 14.824 0.000 0.000 Yes
11.000 vs. 10.000 90.920 14.684 0.000 0.000 Yes
NC vs. 3.000 90.883 14.678 0.000 0.000 Yes
12.000 vs. 3.500 90.631 14.637 0.000 0.000 Yes
12.000 vs. 7.000 90.493 14.615 0.000 0.000 Yes
2.000 vs. 3.000 90.455 14.609 0.000 0.000 Yes
11.000 vs. 9.500 90.202 14.568 0.000 0.000 Yes
NC vs. 8.000 89.641 14.477 0.000 0.000 Yes
11.5 00 vs. 8.500 89.500 14.454 0.000 0.000 Yes
2.000 vs. 8.000 89.212 14.408 0.000 0.000 Yes
12.000 vs. 7.500 88.657 14.318 0.000 0.000 Yes
1.500 vs. 3.500 88.418 14.280 0.000 0.000 Yes
1.500 vs. 7.000 88.279 14.257 0.000 0.000 Yes
11.5 00 vs. 9.000 88.080 14.225 0.000 0.000 Yes
11.5 00 vs. 10.000 87.211 14.085 0.000 0.000 Yes
NC vs. 8.500 87.145 14.074 0.000 0.000 Yes
2.000 vs. 8.500 86.716 14.005 0.000 0.000 Yes
11.5 00 vs. 9.500 86.493 13.969 0.000 0.000 Yes
12.000 vs. 3.000 86.457 13.963 0.000 0.000 Yes
1.500 vs. 7.500 86.443 13.961 0.000 0.000 Yes
NC vs. 9.000 85.725 13.845 0.000 0.000 Yes
2.000 vs. 9.000 85.296 13.775 0.000 0.000 Yes
12.000 vs. 8.000 85.215 13.762 0.000 0.000 Yes
NC vs. 10.000 84.855 13.704 0.000 0.000 Yes
11.000 vs. 10.500 84.770 13.690 0.000 0.000 Yes
2.000 vs. 10.000 84.427 13.635 0.000 0.000 Yes
1.500 vs. 3.000 84.244 13.605 0.000 0.000 Yes
NC vs. 9.500 84.137 13.588 0.000 0.000 Yes
2.000 vs. 9.500 83.709 13.519 0.000 0.000 Yes
1.500 vs. 8.000 83.001 13.405 0.000 0.000 Yes
12.000 vs. 8.500 82.719 13.359 0.000 0.000 Yes
12.000 vs. 9.000 81.299 13.130 0.000 0.000 Yes
11.5 00 vs. 10.500 81.061 13.091 0.000 0.000 Yes
1.500 vs. 8.500 80.505 13.002 0.000 0.000 Yes
12.000 vs. 10.000 80.429 12.989 0.000 0.000 Yes
12.000 vs. 9.500 79.711 12.873 0.000 0.000 Yes
1.500 vs. 9.000 79.085 12.772 0.000 0.000 Yes
NC vs. 10.500 78.705 12.711 0.000 0.000 Yes
2.000 vs. 10.500 78.276 12.642 0.000 0.000 Yes
1.500 vs. 10.000 78.216 12.632 0.000 0.000 Yes
1.500 vs. 9.500 77.498 12.516 0.000 0.000 Yes
11.000 vs. 2.500 77.093 12.451 0.000 0.000 Yes
12.000 vs. 10.500 74.279 11.996 0.000 0.000 Yes
11.5 00 vs. 2.500 73.384 11.852 0.000 0.000 Yes










1.500 vs. 10.500
NC vs. 2.500
2.000 vs. 2.500
12.000 vs. 2.500
1.500 vs. 2.500
2.500 vs. 6.500
2.500 vs. 4.000
2.500 vs. 4.500
2.500 vs. 5.500
2.500 vs. 5.000
2.500 vs. 6.000
2.500 vs. 3.500
2.500 vs. 7.000


72.065
71.028
70.600
66.602
64.389
28.555
28.439
28.238
27.952
27.574
27.430
24.029
23.890


11.639
11.471
11.402
10.756
10.399
4.612
4.593
4.560
4.514
4.453
4.430
3.881
3.858


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within 10.5


Comparison
12.000 vs. 6.000
11.000 vs. 6.000
12.000 vs. 5.500
11.000 vs. 5.500
NC vs. 6.000
NC vs. 5.500
2.000 vs. 6.000
2.000 vs. 5.500
12.000 vs. 5.000
11.000 vs. 5.000
NC vs. 5.000
2.000 vs. 5.000
12.000 vs. 4.500
11.000 vs. 4.500
NC vs. 4.500
2.000 vs. 4.500
12.000 vs. 6.500
11.000 vs. 6.500
NC vs. 6.500
2.000 vs. 6.500
12.000 vs. 4.000
11.000 vs. 4.000
NC vs. 4.000
2.000 vs. 4.000
12.000 vs. 7.000
11.000 vs. 7.000
NC vs. 7.000
2.000 vs. 7.000
11.500 vs. 6.000
11.500 vs. 5.500


Diff of Means
99.546
99.493
99.449
99.396
99.200
99.103
99.087
98.990
97.840
97.787
97.493
97.381
96.262
96.209
95.915
95.802
95.542
95.489
95.195
95.083
93.501
93.448
93.154
93.041
91.919
91.866
91.572
91.459
91.095
90.998


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Critical Level
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


t
16.077
16.068
16.061
16.053
16.021
16.005
16.003
15.987
15.801
15.793
15.745
15.727
15.546
15.538
15.490
15.472
15.430
15.421
15.374
15.356
15.100
15.092
15.044
15.026
14.845
14.836
14.789
14.771
14.712
14.696










12.000 vs. 7.500 90.445 14.607 0.000 0.000 Yes
11.000 vs. 7.500 90.391 14.598 0.000 0.000 Yes
NC vs. 7.500 90.098 14.551 0.000 0.000 Yes
2.000 vs. 7.500 89.985 14.533 0.000 0.000 Yes
11.5 00 vs. 5.000 89.389 14.436 0.000 0.000 Yes
11.5 00 vs. 4.500 87.810 14.181 0.000 0.000 Yes
12.000 vs. 3.500 87.760 14.173 0.000 0.000 Yes
11.000 vs. 3.500 87.707 14.165 0.000 0.000 Yes
NC vs. 3.500 87.413 14.117 0.000 0.000 Yes
2.000 vs. 3.500 87.300 14.099 0.000 0.000 Yes
11.5 00 vs. 6.500 87.091 14.065 0.000 0.000 Yes
1.500 vs. 6.000 86.172 13.917 0.000 0.000 Yes
1.500 vs. 5.500 86.075 13.901 0.000 0.000 Yes
11.5 00 vs. 4.000 85.049 13.736 0.000 0.000 Yes
1.500 vs. 5.000 84.465 13.641 0.000 0.000 Yes
12.000 vs. 3.000 83.841 13.540 0.000 0.000 Yes
11.000 vs. 3.000 83.788 13.532 0.000 0.000 Yes
NC vs. 3.000 83.494 13.484 0.000 0.000 Yes
11.5 00 vs. 7.000 83.468 13.480 0.000 0.000 Yes
2.000 vs. 3.000 83.381 13.466 0.000 0.000 Yes
1.500 vs. 4.500 82.887 13.386 0.000 0.000 Yes
1.500 vs. 6.500 82.167 13.270 0.000 0.000 Yes
11.5 00 vs. 7.500 81.993 13.242 0.000 0.000 Yes
1.500 vs. 4.000 80.126 12.940 0.000 0.000 Yes
11.5 00 vs. 3.500 79.308 12.808 0.000 0.000 Yes
1.500 vs. 7.000 78.544 12.685 0.000 0.000 Yes
12.000 vs. 9.000 77.209 12.469 0.000 0.000 Yes
11.000 vs. 9.000 77.156 12.461 0.000 0.000 Yes
1.500 vs. 7.500 77.070 12.447 0.000 0.000 Yes
NC vs. 9.000 76.862 12.413 0.000 0.000 Yes
2.000 vs. 9.000 76.749 12.395 0.000 0.000 Yes
12.000 vs. 8.500 76.447 12.346 0.000 0.000 Yes
11.000 vs. 8.500 76.394 12.338 0.000 0.000 Yes
NC vs. 8.500 76.100 12.290 0.000 0.000 Yes
2.000 vs. 8.500 75.988 12.272 0.000 0.000 Yes
12.000 vs. 10.000 75.645 12.217 0.000 0.000 Yes
11.000 vs. 10.000 75.592 12.208 0.000 0.000 Yes
11.5 00 vs. 3.000 75.389 12.175 0.000 0.000 Yes
NC vs. 10.000 75.298 12.161 0.000 0.000 Yes
2.000 vs. 10.000 75.186 12.143 0.000 0.000 Yes
1.500 vs. 3.500 74.385 12.013 0.000 0.000 Yes
12.000 vs. 8.000 88.031 11.608 0.000 0.000 Yes
11.000 vs. 8.000 87.977 11.601 0.000 0.000 Yes
NC vs. 8.000 87.684 11.562 0.000 0.000 Yes
2.000 vs. 8.000 87.571 11.548 0.000 0.000 Yes
12.000 vs. 9.500 71.218 11.502 0.000 0.000 Yes










11.000 vs. 9.500 71.165 11.493 0.000 0.000 Yes
NC vs. 9.500 70.871 11.446 0.000 0.000 Yes
2.000 vs. 9.500 70.758 11.428 0.000 0.000 Yes
1.500 vs. 3.000 70.466 11.380 0.000 0.000 Yes
11.5 00 vs. 9.000 68.757 11.104 0.000 0.000 Yes
11.5 00 vs. 8.500 67.996 10.981 0.000 0.000 Yes
11.5 00 vs. 10.000 67.194 10.852 0.000 0.000 Yes
11.5 00 vs. 8.000 79.579 10.494 0.000 0.000 Yes
1.500 vs. 9.000 63.834 10.309 0.000 0.000 Yes
1.500 vs. 8.500 63.072 10.186 0.000 0.000 Yes
11.5 00 vs. 9.500 62.766 10.137 0.000 0.000 Yes
1.500 vs. 10.000 62.270 10.057 0.000 0.000 Yes
12.000 vs. 2.500 62.049 10.021 0.000 0.000 Yes
11.000 vs. 2.500 61.996 10.012 0.000 0.000 Yes
NC vs. 2.500 61.702 9.965 0.000 0.000 Yes
2.000 vs. 2.500 61.590 9.947 0.000 0.000 Yes
1.500 vs. 8.000 74.656 9.844 0.000 0.000 Yes
1.500 vs. 9.500 57.843 9.342 0.000 0.000 Yes
12.000 vs. 10.500 57.394 9.269 0.000 0.000 Yes
11.000 vs. 10.500 57.341 9.261 0.000 0.000 Yes
NC vs. 10.500 57.047 9.213 0.000 0.000 Yes
2.000 vs. 10.500 56.934 9.195 0.000 0.000 Yes
11.5 00 vs. 2.500 53.598 8.656 0.000 0.000 Yes
11.5 00 vs. 10.500 48.942 7.904 0.000 0.000 Yes
1.500 vs. 2.500 48.674 7.861 0.000 0.000 Yes
1.500 vs. 10.500 44.019 7.109 0.000 0.000 Yes
10.500 vs. 6.000 42.153 6.808 0.000 0.000 Yes
10.500 vs. 5.500 42.056 6.792 0.000 0.000 Yes
10.500 vs. 5.000 40.446 6.532 0.000 0.000 Yes
10.500 vs. 4.500 38.868 6.277 0.000 0.000 Yes
10.500 vs. 6.500 38.148 6.161 0.000 0.000 Yes
2.500 vs. 6.000 37.497 6.056 0.000 0.000 Yes
2.500 vs. 5.500 37.400 6.040 0.000 0.000 Yes
10.500 vs. 4.000 36.107 5.831 0.000 0.000 Yes
2.500 vs. 5.000 35.791 5.780 0.000 0.000 Yes
10.500 vs. 7.000 34.525 5.576 0.000 0.000 Yes
2.500 vs. 4.500 34.213 5.525 0.000 0.000 Yes
2.500 vs. 6.500 33.493 5.409 0.000 0.000 Yes
10.500 vs. 7.500 33.051 5.338 0.000 0.000 Yes
2.500 vs. 4.000 31.452 5.079 0.000 0.000 Yes
10.500 vs. 3.500 30.366 4.904 0.000 0.000 Yes
2.500 vs. 7.000 29.870 4.824 0.000 0.000 Yes
2.500 vs. 7.500 28.395 4.586 0.000 0.000 Yes
9.500 vs. 6.000 28.329 4.575 0.000 0.000 Yes
9.500 vs. 5.500 28.232 4.559 0.000 0.000 Yes
9.500 vs. 5.000 26.622 4.300 0.000 0.000 Yes










10.500 vs. 3.000
2.500 vs. 3.500
9.500 vs. 4.500
10.500 vs. 8.000
9.500 vs. 6.500
10.000 vs. 6.000
10.000 vs. 5.500
8.500 vs. 6.000
8.500 vs. 5.500
9.000 vs. 6.000
9.500 vs. 4.000
9.000 vs. 5.500
10.000 vs. 5.000


26.447
25.711
25.044
30.637
24.324
23.901
23.804
23.099
23.002
22.338
22.283
22.241
22.195


4.271
4.152
4.045
4.040
3.928
3.860
3.844
3.731
3.715
3.608
3.599
3.592
3.585


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within 11


Comparison
NC vs. 5.000
NC vs. 6.000
NC vs. 6.500
NC vs. 5.500
NC vs. 4.000
NC vs. 4.500
NC vs. 7.000
NC vs. 3.500
1.500 vs. 5.000
1.500 vs. 6.000
2.000 vs. 5.000
2.000 vs. 6.000
1.500 vs. 6.500
NC vs. 7.500
1.500 vs. 5.500
2.000 vs. 6.500
2.000 vs. 5.500
1.500 vs. 4.000
2.000 vs. 4.000
NC vs. 3.000
1.500 vs. 4.500
2.000 vs. 4.500
NC vs. 8.000
11.000 vs. 5.000
1.500 vs. 7.000
11.000 vs. 6.000
2.000 vs. 7.000
11.000 vs. 6.500
11.000 vs. 5.500
1.500 vs. 3.500


Diff of Means
97.821
97.578
96.964
96.675
95.928
94.476
93.276
91.691
90.686
90.443
90.333
90.090
89.829
89.585
89.540
89.476
89.187
88.793
88.441
88.039
87.341
86.988
86.317
86.268
86.141
86.025
85.788
85.411
85.122
84.555


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Critical Level
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


t
15.798
15.759
15.660
15.613
15.493
15.258
15.064
14.808
14.646
14.607
14.589
14.550
14.507
14.468
14.461
14.451
14.404
14.340
14.283
14.218
14.106
14.049
13.940
13.932
13.912
13.893
13.855
13.794
13.747
13.656










11.000 vs. 4.000 84.375 13.627 0.000 0.000 Yes
2.000 vs. 3.500 84.203 13.599 0.000 0.000 Yes
11.000 vs. 4.500 82.923 13.392 0.000 0.000 Yes
1.500 vs. 7.500 82.450 13.316 0.000 0.000 Yes
NC vs. 9.000 82.287 13.289 0.000 0.000 Yes
2.000 vs. 7.500 82.097 13.259 0.000 0.000 Yes
11.000 vs. 7.000 81.723 13.198 0.000 0.000 Yes
NC vs. 8.500 81.163 13.108 0.000 0.000 Yes
1.500 vs. 3.000 80.904 13.066 0.000 0.000 Yes
NC vs. 10.000 80.674 13.029 0.000 0.000 Yes
2.000 vs. 3.000 80.551 13.009 0.000 0.000 Yes
11.000 vs. 3.500 80.137 12.942 0.000 0.000 Yes
1.500 vs. 8.000 79.182 12.788 0.000 0.000 Yes
2.000 vs. 8.000 78.829 12.731 0.000 0.000 Yes
11.000 vs. 7.500 78.031 12.602 0.000 0.000 Yes
11.5 00 vs. 5.000 76.935 12.425 0.000 0.000 Yes
11.5 00 vs. 6.000 76.693 12.386 0.000 0.000 Yes
11.000 vs. 3.000 76.486 12.352 0.000 0.000 Yes
11.5 00 vs. 6.500 76.079 12.287 0.000 0.000 Yes
NC vs. 2.500 75.905 12.259 0.000 0.000 Yes
11.5 00 vs. 5.500 75.790 12.240 0.000 0.000 Yes
NC vs. 9.500 75.254 12.154 0.000 0.000 Yes
1.500 vs. 9.000 75.151 12.137 0.000 0.000 Yes
11.5 00 vs. 4.000 75.043 12.119 0.000 0.000 Yes
2.000 vs. 9.000 74.799 12.080 0.000 0.000 Yes
11.000 vs. 8.000 74.764 12.074 0.000 0.000 Yes
1.500 vs. 8.500 74.028 11.956 0.000 0.000 Yes
2.000 vs. 8.500 73.676 11.899 0.000 0.000 Yes
11.5 00 vs. 4.500 73.591 11.885 0.000 0.000 Yes
1.500 vs. 10.000 73.539 11.877 0.000 0.000 Yes
2.000 vs. 10.000 73.186 11.820 0.000 0.000 Yes
11.5 00 vs. 7.000 72.391 11.691 0.000 0.000 Yes
12.000 vs. 5.000 71.763 11.590 0.000 0.000 Yes
12.000 vs. 6.000 71.520 11.551 0.000 0.000 Yes
12.000 vs. 6.500 70.907 11.451 0.000 0.000 Yes
11.5 00 vs. 3.500 70.805 11.435 0.000 0.000 Yes
11.000 vs. 9.000 70.733 11.423 0.000 0.000 Yes
12.000 vs. 5.500 70.618 11.405 0.000 0.000 Yes
12.000 vs. 4.000 69.871 11.284 0.000 0.000 Yes
11.000 vs. 8.500 69.610 11.242 0.000 0.000 Yes
11.000 vs. 10.000 69.120 11.163 0.000 0.000 Yes
1.500 vs. 2.500 68.770 11.106 0.000 0.000 Yes
11.5 00 vs. 7.500 68.699 11.095 0.000 0.000 Yes
12.000 vs. 4.500 68.418 11.050 0.000 0.000 Yes
2.000 vs. 2.500 68.417 11.049 0.000 0.000 Yes
1.500 vs. 9.500 68.119 11.001 0.000 0.000 Yes










2.000 vs. 9.500
12.000 vs. 7.000
11.500 vs. 3.000
12.000 vs. 3.500
11.500 vs. 8.000
11.000 vs. 2.500
11.000 vs. 9.500
12.000 vs. 7.500
12.000 vs. 3.000
11.500 vs. 9.000
11.500 vs. 8.500
12.000 vs. 8.000
11.500 vs. 10.000
NC vs. 10.500
12.000 vs. 9.000
12.000 vs. 8.500
11.500 vs. 2.500
12.000 vs. 10.000
11.500 vs. 9.500
1.500 vs. 10.500
2.000 vs. 10.500
12.000 vs. 2.500
12.000 vs. 9.500
11.000 vs. 10.500
11.500 vs. 10.500
10.500 vs. 5.000
10.500 vs. 6.000
10.500 vs. 6.500
10.500 vs. 5.500
10.500 vs. 4.000
10.500 vs. 4.500
12.000 vs. 10.500
10.500 vs. 7.000
10.500 vs. 3.500
10.500 vs. 7.500
10.500 vs. 3.000
10.500 vs. 8.000
NC vs. 12.000
10.500 vs. 9.000
9.500 vs. 5.000
9.500 vs. 6.000


67.766
67.218
67.154
65.633
65.432
64.351
63.701
63.527
61.981
61.401
60.278
60.259
59.788
59.669
56.229
55.106
55.019
54.616
54.369
52.534
52.181
49.847
49.196
48.115
38.783
38.152
37.909
37.295
37.006
36.260
34.807
33.611
33.607
32.022
29.916
28.370
26.648
26.058
22.618
22.567
22.324


10.944
10.856
10.845
10.600
10.567
10.393
10.288
10.260
10.010
9.916
9.735
9.732
9.656
9.637
9.081
8.900
8.886
8.821
8.781
8.484
8.427
8.050
7.945
7.771
6.264
6.162
6.122
6.023
5.977
5.856
5.621
5.428
5.428
5.172
4.831
4.582
4.304
4.208
3.653
3.645
3.605


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Sample pH within
Comparison Diff of Means t
NC vs. 5.500 100.248 16.190
NC vs. 5.000 99.849 16.126


11.5
Unadjusted P
0.000
0.000


Critical Level
0.000
0.000


Significant?
Yes
Yes










NC vs. 6.000 99.816 16.120 0.000 0.000 Yes
NC vs. 4.500 99.122 16.008 0.000 0.000 Yes
NC vs. 6.500 97.396 15.730 0.000 0.000 Yes
11.5 00 vs. 5.500 96.012 15.506 0.000 0.000 Yes
NC vs. 3.500 95.804 15.472 0.000 0.000 Yes
11.5 00 vs. 5.000 95.614 15.442 0.000 0.000 Yes
11.5 00 vs. 6.000 95.580 15.436 0.000 0.000 Yes
11.5 00 vs. 4.500 94.887 15.324 0.000 0.000 Yes
NC vs. 4.000 94.510 15.263 0.000 0.000 Yes
11.000 vs. 5.500 93.694 15.132 0.000 0.000 Yes
11.000 vs. 5.000 93.296 15.067 0.000 0.000 Yes
11.000 vs. 6.000 93.262 15.062 0.000 0.000 Yes
11.5 00 vs. 6.500 93.160 15.045 0.000 0.000 Yes
11.000 vs. 4.500 92.569 14.950 0.000 0.000 Yes
11.5 00 vs. 3.500 91.568 14.788 0.000 0.000 Yes
NC vs. 3.000 91.306 14.746 0.000 0.000 Yes
NC vs. 7.500 91.246 14.736 0.000 0.000 Yes
11.000 vs. 6.500 90.842 14.671 0.000 0.000 Yes
11.5 00 vs. 4.000 90.274 14.579 0.000 0.000 Yes
11.000 vs. 3.500 89.250 14.414 0.000 0.000 Yes
12.000 vs. 5.500 88.361 14.270 0.000 0.000 Yes
12.000 vs. 5.000 87.963 14.206 0.000 0.000 Yes
11.000 vs. 4.000 87.956 14.205 0.000 0.000 Yes
12.000 vs. 6.000 87.929 14.201 0.000 0.000 Yes
NC vs. 8.000 87.329 14.104 0.000 0.000 Yes
12.000 vs. 4.500 87.236 14.089 0.000 0.000 Yes
11.5 00 vs. 3.000 87.070 14.062 0.000 0.000 Yes
11.5 00 vs. 7.500 87.010 14.052 0.000 0.000 Yes
12.000 vs. 6.500 85.510 13.810 0.000 0.000 Yes
11.000 vs. 3.000 84.752 13.688 0.000 0.000 Yes
11.000 vs. 7.500 84.692 13.678 0.000 0.000 Yes
NC vs. 8.500 84.336 13.620 0.000 0.000 Yes
12.000 vs. 3.500 83.918 13.553 0.000 0.000 Yes
NC vs. 9.000 83.518 13.488 0.000 0.000 Yes
11.5 00 vs. 8.000 83.093 13.420 0.000 0.000 Yes
NC vs. 10.000 82.628 13.344 0.000 0.000 Yes
12.000 vs. 4.000 82.624 13.344 0.000 0.000 Yes
1.500 vs. 5.500 80.914 13.068 0.000 0.000 Yes
11.000 vs. 8.000 80.775 13.045 0.000 0.000 Yes
1.500 vs. 5.000 80.516 13.003 0.000 0.000 Yes
1.500 vs. 6.000 80.482 12.998 0.000 0.000 Yes
11.5 00 vs. 8.500 80.100 12.936 0.000 0.000 Yes
NC vs. 2.500 79.848 12.896 0.000 0.000 Yes
1.500 vs. 4.500 79.789 12.886 0.000 0.000 Yes
12.000 vs. 3.000 79.419 12.826 0.000 0.000 Yes
12.000 vs. 7.500 79.359 12.817 0.000 0.000 Yes










11.5 00 vs. 9.000 79.282 12.804 0.000 0.000 Yes
11.5 00 vs. 10.000 78.392 12.660 0.000 0.000 Yes
1.500 vs. 6.500 78.063 12.607 0.000 0.000 Yes
11.000 vs. 8.500 77.783 12.562 0.000 0.000 Yes
11.000 vs. 9.000 76.964 12.430 0.000 0.000 Yes
NC vs. 9.500 76.623 12.375 0.000 0.000 Yes
1.500 vs. 3.500 76.471 12.350 0.000 0.000 Yes
NC vs. 10.500 76.397 12.338 0.000 0.000 Yes
11.000 vs. 10.000 76.074 12.286 0.000 0.000 Yes
11.5 00 vs. 2.500 75.612 12.211 0.000 0.000 Yes
12.000 vs. 8.000 75.442 12.184 0.000 0.000 Yes
1.500 vs. 4.000 75.177 12.141 0.000 0.000 Yes
NC vs. 7.000 92.065 12.140 0.000 0.000 Yes
11.000 vs. 2.500 73.295 11.837 0.000 0.000 Yes
12.000 vs. 8.500 72.450 11.701 0.000 0.000 Yes
11.5 00 vs. 9.500 72.388 11.691 0.000 0.000 Yes
11.5 00 vs. 10.500 72.161 11.654 0.000 0.000 Yes
1.500 vs. 3.000 71.972 11.624 0.000 0.000 Yes
1.500 vs. 7.500 71.912 11.614 0.000 0.000 Yes
11.5 00 vs. 7.000 87.829 11.582 0.000 0.000 Yes
12.000 vs. 9.000 71.631 11.569 0.000 0.000 Yes
12.000 vs. 10.000 70.742 11.425 0.000 0.000 Yes
11.000 vs. 9.500 70.070 11.316 0.000 0.000 Yes
11.000 vs. 10.500 69.843 11.280 0.000 0.000 Yes
11.000 vs. 7.000 85.511 11.276 0.000 0.000 Yes
2.000 vs. 5.500 68.296 11.030 0.000 0.000 Yes
1.500 vs. 8.000 67.995 10.981 0.000 0.000 Yes
12.000 vs. 2.500 67.962 10.976 0.000 0.000 Yes
2.000 vs. 5.000 67.898 10.966 0.000 0.000 Yes
2.000 vs. 6.000 67.865 10.960 0.000 0.000 Yes
2.000 vs. 4.500 67.171 10.848 0.000 0.000 Yes
12.000 vs. 7.000 80.178 10.573 0.000 0.000 Yes
2.000 vs. 6.500 65.445 10.569 0.000 0.000 Yes
1.500 vs. 8.500 65.003 10.498 0.000 0.000 Yes
12.000 vs. 9.500 64.737 10.455 0.000 0.000 Yes
12.000 vs. 10.500 64.510 10.418 0.000 0.000 Yes
1.500 vs. 9.000 64.184 10.366 0.000 0.000 Yes
2.000 vs. 3.500 63.853 10.312 0.000 0.000 Yes
1.500 vs. 10.000 63.295 10.222 0.000 0.000 Yes
2.000 vs. 4.000 62.559 10.103 0.000 0.000 Yes
1.500 vs. 2.500 60.515 9.773 0.000 0.000 Yes
1.500 vs. 7.000 72.731 9.591 0.000 0.000 Yes
2.000 vs. 3.000 59.354 9.586 0.000 0.000 Yes
2.000 vs. 7.500 59.294 9.576 0.000 0.000 Yes
1.500 vs. 9.500 57.290 9.252 0.000 0.000 Yes
1.500 vs. 10.500 57.063 9.216 0.000 0.000 Yes










2.000 vs. 8.000
2.000 vs. 8.500
2.000 vs. 9.000
2.000 vs. 10.000
2.000 vs. 7.000
2.000 vs. 2.500
2.000 vs. 9.500
2.000 vs. 10.500
NC vs. 2.000
11.500 vs. 2.000
11.000 vs. 2.000
10.500 vs. 5.500
9.500 vs. 5.500
10.500 vs. 5.000
10.500 vs. 6.000
9.500 vs. 5.000
9.500 vs. 6.000
10.500 vs. 4.500
9.500 vs. 4.500

Comparisons for
Comparison
NC vs. 5.000
NC vs. 4.500
NC vs. 5.500
NC vs. 6.000
NC vs. 4.000
12.000 vs. 5.000
NC vs. 6.500
12.000 vs. 4.500
12.000 vs. 5.500
12.000 vs. 6.000
12.000 vs. 4.000
NC vs. 7.000
11.500 vs. 5.000
12.000 vs. 6.500
11.500 vs. 4.500
NC vs. 7.500
11.500 vs. 5.500
11.500 vs. 6.000
11.500 vs. 4.000
12.000 vs. 7.000
11.500 vs. 6.500
12.000 vs. 7.500
NC vs. 8.000
2.000 vs. 5.000


55.377
52.385
51.567
50.677
60.113
47.897
44.672
44.445
31.951
27.716
25.398
23.851
23.624
23.453
23.419
23.226
23.192
22.726
22.499


8.943
8.460
8.328
8.184
7.927
7.735
7.215
7.178
5.160
4.476
4.102
3.852
3.815
3.788
3.782
3.751
3.746
3.670
3.634


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


factor: Sample pH within con
Diff of Means t Unadjusted P
87.813 14.182 0.000
86.677 13.998 0.000
85.814 13.859 0.000
84.784 13.693 0.000
84.722 13.683 0.000
83.240 13.443 0.000
82.837 13.378 0.000
82.104 13.260 0.000
81.241 13.121 0.000
80.211 12.954 0.000
80.149 12.944 0.000
79.483 12.837 0.000
79.070 12.770 0.000
78.264 12.640 0.000
77.933 12.586 0.000
77.915 12.583 0.000
77.071 12.447 0.000
76.040 12.281 0.000
75.978 12.271 0.000
74.911 12.098 0.000
74.093 11.966 0.000
73.342 11.845 0.000
72.652 11.733 0.000
72.059 11.638 0.000


Critical Level
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes










NC vs. 3.500 71.116 11.485 0.000 0.000 Yes
2.000 vs. 4.500 70.922 11.454 0.000 0.000 Yes
1.500 vs. 5.000 70.827 11.439 0.000 0.000 Yes
11.5 00 vs. 7.000 70.740 11.425 0.000 0.000 Yes
NC vs. 8.500 70.105 11.322 0.000 0.000 Yes
2.000 vs. 5.500 70.060 11.315 0.000 0.000 Yes
1.500 vs. 4.500 69.691 11.255 0.000 0.000 Yes
NC vs. 9.000 69.324 11.196 0.000 0.000 Yes
11.5 00 vs. 7.500 69.172 11.171 0.000 0.000 Yes
2.000 vs. 6.000 69.029 11.148 0.000 0.000 Yes
2.000 vs. 4.000 68.968 11.138 0.000 0.000 Yes
NC vs. 10.000 68.885 11.125 0.000 0.000 Yes
1.500 vs. 5.500 68.828 11.116 0.000 0.000 Yes
12.000 vs. 8.000 68.079 10.995 0.000 0.000 Yes
1.500 vs. 6.000 67.798 10.949 0.000 0.000 Yes
1.500 vs. 4.000 67.736 10.939 0.000 0.000 Yes
2.000 vs. 6.500 67.082 10.834 0.000 0.000 Yes
12.000 vs. 3.500 66.543 10.747 0.000 0.000 Yes
1.500 vs. 6.500 65.851 10.635 0.000 0.000 Yes
NC vs. 9.500 65.660 10.604 0.000 0.000 Yes
12.000 vs. 8.500 65.533 10.584 0.000 0.000 Yes
12.000 vs. 9.000 64.751 10.457 0.000 0.000 Yes
12.000 vs. 10.000 64.312 10.387 0.000 0.000 Yes
11.5 00 vs. 8.000 63.908 10.321 0.000 0.000 Yes
2.000 vs. 7.000 63.729 10.292 0.000 0.000 Yes
NC vs. 10.500 62.623 10.114 0.000 0.000 Yes
1.500 vs. 7.000 62.498 10.093 0.000 0.000 Yes
11.5 00 vs. 3.500 62.372 10.073 0.000 0.000 Yes
2.000 vs. 7.500 62.161 10.039 0.000 0.000 Yes
2.500 vs. 5.000 61.723 9.968 0.000 0.000 Yes
11.5 00 vs. 8.500 61.362 9.910 0.000 0.000 Yes
12.000 vs. 9.500 61.087 9.866 0.000 0.000 Yes
1.500 vs. 7.500 60.929 9.840 0.000 0.000 Yes
2.500 vs. 4.500 60.586 9.785 0.000 0.000 Yes
11.5 00 vs. 9.000 60.581 9.784 0.000 0.000 Yes
11.5 00 vs. 10.000 60.142 9.713 0.000 0.000 Yes
2.500 vs. 5.500 59.724 9.645 0.000 0.000 Yes
2.500 vs. 6.000 58.693 9.479 0.000 0.000 Yes
2.500 vs. 4.000 58.632 9.469 0.000 0.000 Yes
12.000 vs. 10.500 58.050 9.375 0.000 0.000 Yes
11.5 00 vs. 9.500 56.917 9.192 0.000 0.000 Yes
2.000 vs. 8.000 56.897 9.189 0.000 0.000 Yes
2.500 vs. 6.500 56.746 9.165 0.000 0.000 Yes
1.500 vs. 8.000 55.666 8.990 0.000 0.000 Yes
2.000 vs. 3.500 55.361 8.941 0.000 0.000 Yes
2.000 vs. 8.500 54.351 8.778 0.000 0.000 Yes










1.500 vs. 3.500 54.130 8.742 0.000 0.000 Yes
3.000 vs. 5.000 54.057 8.730 0.000 0.000 Yes
11.5 00 vs. 10.500 53.880 8.702 0.000 0.000 Yes
2.000 vs. 9.000 53.570 8.652 0.000 0.000 Yes
2.500 vs. 7.000 53.393 8.623 0.000 0.000 Yes
2.000 vs. 10.000 53.131 8.581 0.000 0.000 Yes
1.500 vs. 8.500 53.120 8.579 0.000 0.000 Yes
3.000 vs. 4.500 52.920 8.547 0.000 0.000 Yes
1.500 vs. 9.000 52.338 8.453 0.000 0.000 Yes
3.000 vs. 5.500 52.058 8.407 0.000 0.000 Yes
1.500 vs. 10.000 51.899 8.382 0.000 0.000 Yes
2.500 vs. 7.500 51.825 8.370 0.000 0.000 Yes
3.000 vs. 6.000 51.027 8.241 0.000 0.000 Yes
3.000 vs. 4.000 50.966 8.231 0.000 0.000 Yes
2.000 vs. 9.500 49.906 8.060 0.000 0.000 Yes
11.000 vs. 5.000 49.193 7.945 0.000 0.000 Yes
3.000 vs. 6.500 49.081 7.927 0.000 0.000 Yes
1.500 vs. 9.500 48.674 7.861 0.000 0.000 Yes
11.000 vs. 4.500 48.056 7.761 0.000 0.000 Yes
11.000 vs. 5.500 47.194 7.622 0.000 0.000 Yes
2.000 vs. 10.500 46.869 7.569 0.000 0.000 Yes
2.500 vs. 8.000 46.562 7.520 0.000 0.000 Yes
11.000 vs. 6.000 46.163 7.455 0.000 0.000 Yes
11.000 vs. 4.000 46.101 7.445 0.000 0.000 Yes
3.000 vs. 7.000 45.727 7.385 0.000 0.000 Yes
1.500 vs. 10.500 45.637 7.370 0.000 0.000 Yes
2.500 vs. 3.500 45.025 7.272 0.000 0.000 Yes
11.000 vs. 6.500 44.216 7.141 0.000 0.000 Yes
3.000 vs. 7.500 44.159 7.132 0.000 0.000 Yes
2.500 vs. 8.500 44.015 7.108 0.000 0.000 Yes
2.500 vs. 9.000 43.234 6.982 0.000 0.000 Yes
2.500 vs. 10.000 42.795 6.911 0.000 0.000 Yes
11.000 vs. 7.000 40.863 6.599 0.000 0.000 Yes
2.500 vs. 9.500 39.570 6.391 0.000 0.000 Yes
11.000 vs. 7.500 39.295 6.346 0.000 0.000 Yes
3.000 vs. 8.000 38.896 6.282 0.000 0.000 Yes
NC vs. 11.000 38.621 6.237 0.000 0.000 Yes
3.000 vs. 3.500 37.359 6.034 0.000 0.000 Yes
2.500 vs. 10.500 36.533 5.900 0.000 0.000 Yes
3.000 vs. 8.500 36.349 5.870 0.000 0.000 Yes
3.000 vs. 9.000 35.568 5.744 0.000 0.000 Yes
3.000 vs. 10.000 35.129 5.673 0.000 0.000 Yes
12.000 vs. 11.000 34.048 5.499 0.000 0.000 Yes
11.000 vs. 8.000 34.031 5.496 0.000 0.000 Yes
NC vs. 3.000 33.756 5.452 0.000 0.000 Yes
11.000 vs. 3.500 32.495 5.248 0.000 0.000 Yes










3.000 vs. 9.500
11.000 vs. 8.500
11.000 vs. 9.000
11.000 vs. 10.000
11.500 vs. 11.000
12.000 vs. 3.000
3.000 vs. 10.500
11.000 vs. 9.500
NC vs. 2.500
10.500 vs. 5.000
11.500 vs. 3.000
10.500 vs. 4.500
11.000 vs. 10.500
10.500 vs. 5.500
2.000 vs. 11.000
10.500 vs. 6.000
9.500 vs. 5.000
10.500 vs. 4.000


31.904
31.485
30.704
30.265
29.877
29.183
28.867
27.040
26.090
25.190
25.013
24.054
24.003
23.191
22.866
22.161
22.153
22.099


5.152
5.085
4.959
4.888
4.825
4.713
4.662
4.367
4.214
4.068
4.040
3.885
3.876
3.745
3.693
3.579
3.578
3.569


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Two Way ANOVA of Salt Solubility data

Two Way Analysis of Variance
Dependent Variable: Solubility


Source of Variation
Treatment pH
Salt Conc
Treatment pH x Salt (
Residual
Total


DF SS
6 5.995
4 2.018
24 2.021
244 0.559
278 10.612


MS
0.999
0.505
0.0842
0.00229
0.0382


F
436.432
220.381
36.777


P
<0.001
<0.001
<0.001


Conc


Main effects cannot be properly interpreted if significant interaction is determined. This is
because the size of a factor's effect depends upon the level of the other factor.

The effect of different levels of Treatment pH depends on what level of Salt Cone is present.
There is a statistically significant interaction between Treatment pH and Salt Conc. (P = <0.001)


Power of performed test with alpha
Power of performed test with alpha
Power of performed test with alpha


0.0500: for Treatment pH : 1.000
0.0500: for Salt Cone : 1.000
0.0500: for Treatment pH x Salt Cone : 1.000


All Pairwise Multiple Comparison Procedures (Holm-Sidak method):
Overall significance level = 0.05











Comparisons for factor: Salt Cone within 2
Comparison Diff of Means t
450M vs. OM 0.0953 3.982
600M vs. OM 0.0883 3.691

Comparisons for factor: Salt Cone within 2.5
Comparison Diff of Means t
450M vs. OM 0.0814 3.405
600M vs. OM 0.0676 2.824


Unadjusted P Critical Level
0.000 0.005
0.000 0.006


Significant?
Yes
Yes



Significant?
Yes
Yes


Unadjusted P
0.001
0.005


Critical Level
0.005
0.006


Comparisons for factor: Salt Cone within 3: No Significant Differences


Comparisons for factor: Salt Cone within 10.5


Comparison
600M vs. OM
600M vs. 150M
450M vs. OM
450M vs. 150M
300M vs. OM
300M vs. 150M
600M vs. 300M
450M vs. 300M
600M vs. 450M


Diff of Means
0.345
0.334
0.286
0.275
0.210
0.198
0.135
0.0768
0.0586


t Unadjusted P
14.420 0.000
13.954 0.000
11.971 0.000
11.505 0.000
8.759 0.000
8.294 0.000
5.660 0.000
3.211 0.001
2.449 0.015


Critical Level
0.005
0.006
0.006
0.007
0.009
0.010
0.013
0.017
0.025


Critical Level
0.005
0.006
0.006
0.007
0.009
0.010
0.013


Critical Level
0.005
0.006
0.006
0.007
0.009


Critical Level
0.005


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes



Significant?
Yes


Comparisons for factor: Salt Cone within 11
Comparison Diff of Means t
600M vs. 150M 0.153 6.406
600M vs. OM 0.151 6.308
450M vs. 150M 0.121 5.059
450M vs. OM 0.119 4.961
300M vs. 150M 0.0886 3.706
300M vs. OM 0.0863 3.608
600M vs. 300M 0.0646 2.700


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.007


Comparisons for factor: Salt Cone within 11.5
Comparison Diff of Means t Unadjusted P
600M vs. OM 0.105 4.223 0.000
450M vs. OM 0.0941 3.934 0.000
300M vs. OM 0.0831 3.474 0.001
600M vs. 150M 0.0818 3.302 0.001
450M vs. 150M 0.0713 2.981 0.003

Comparisons for factor: Salt Cone within control
Comparison Diff of Means t Unadjusted P
600M vs. OM 0.659 27.559 0.000










600M vs. 150M
300M vs. OM
450M vs. OM
300M vs. 150M
450M vs. 150M
600M vs. 450M
600M vs. 300M
150M vs. OM


0.590
0.547
0.522
0.478
0.453
0.137
0.112
0.0697


24.647
22.883
21.839
19.972
18.927
5.720
4.675
2.912


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.004


0.006
0.006
0.007
0.009
0.010
0.013
0.017
0.025


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Treatment pH within OM
Comparison Diff of Means t Unadjusted P
control vs. 2.500 0.131 5.463 0.000
control vs. 2.000 0.124 5.200 0.000
control vs. 11.500 0.120 5.025 0.000
control vs. 3.000 0.0758 3.168 0.002
control vs. 10.500 0.0729 3.048 0.003

Comparisons for factor: Treatment pH within 150M


Critical Level
0.002
0.003
0.003
0.003
0.003



Critical Level
0.002
0.003
0.003
0.003
0.003
0.003



Critical Level
0.002
0.003
0.003
0.003
0.003
0.003
0.003
0.004
0.004
0.004
0.005
0.005
0.006



Critical Level
0.002
0.003


Significant?
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes



Significant?
Yes
Yes


Comparison
control vs. 2.500
control vs. 11.500
control vs. 2.000
control vs. 11.000
control vs. 3.000
control vs. 10.500


Diff of Means
0.174
0.167
0.160
0.142
0.136
0.131


t
7.281
6.984
6.692
5.924
5.684
5.494


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000


Comparisons for factor: Treatment pH within 300M


Comparison
control vs. 2.500
control vs. 2.000
control vs. 11.500
control vs. 3.000
control vs. 11.000
control vs. 10.500
10.500 vs. 2.500
10.500 vs. 2.000
10.500 vs. 11.500
10.500 vs. 3.000
10.500 vs. 11.000
11.000 vs. 2.500
11.000 vs. 2.000


Diff of Means
0.619
0.615
0.585
0.567
0.531
0.411
0.208
0.205
0.174
0.156
0.120
0.0884
0.0846


t
25.884
25.726
24.435
23.688
22.190
17.172
8.712
8.553
7.262
6.515
5.018
3.694
3.536


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Comparisons for factor: Treatment pH within 450M
Comparison Diff of Means t Unadjusted P
control vs. 2.500 0.572 23.898 0.000
control vs. 2.000 0.552 23.056 0.000










control vs. 11.500
control vs. 3.000
control vs. 11.000
control vs. 10.500
10.500 vs. 2.500
10.500 vs. 2.000
10.500 vs. 11.500
10.500 vs. 3.000
10.500 vs. 11.000
11.000 vs. 2.500
11.000 vs. 2.000
11.000 vs. 11.500
11.000 vs. 3.000


0.549
0.542
0.473
0.309
0.263
0.243
0.240
0.233
0.164
0.0982
0.0781
0.0751
0.0683


22.930
22.648
19.792
12.916
10.981
10.140
10.014
9.732
6.876
4.105
3.264
3.138
2.856


0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.002
0.005


0.003
0.003
0.003
0.003
0.003
0.004
0.004
0.004
0.005
0.005
0.006
0.006
0.007


Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


Comparisons for factor: Treatment pH within 600M


Comparison
control vs. 2.500
control vs. 2.000
control vs. 3.000
control vs. 11.500
control vs. 11.000
control vs. 10.500
10.500 vs. 2.500
10.500 vs. 2.000
10.500 vs. 3.000
10.500 vs. 11.500
10.500 vs. 11.000
11.000 vs. 2.500
11.000 vs. 2.000
11.000 vs. 3.000
11.000 vs. 11.500


Diff of Means
0.722
0.695
0.685
0.675
0.578
0.387
0.335
0.308
0.298
0.288
0.191
0.144
0.117
0.107
0.0968


Unadjusted P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000


Critical Level
0.002
0.003
0.003
0.003
0.003
0.003
0.003
0.004
0.004
0.004
0.005
0.005
0.006
0.006
0.007


Significant?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes


t
30.198
29.067
28.633
27.257
24.165
16.187
14.011
12.880
12.445
11.618
7.978
6.033
4.902
4.467
3.911









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BIOGRAPHICAL SKETCH

Matthew Paul Davenport was born in Lexington, KY, and moved to Gainesville, FL two

years later where he was raised. Matthew attended Gainesville High School graduating in 1998.

During the last year and a half of high school, Matthew started working as a laboratory

technician for Dr. Carlotta Grooves at the University of Florida in the College of Veterinary

Medicine, Department of Basic Sciences; the focus of that work was on environmental

toxicology. After graduation from Gainesville High School, Matthew attended Greenville

College in Greenville, Illinois, graduating in 2002 with a Bachelor of Arts degree in biology.

While at Greenville College Matthew began work in the area of food science, investigating

carbon monoxide processing of yellow fin tuna and the effect of consumption on the consumer.

This proj ect was done in collaboration with the Food Science and Human Nutrition Department

of the University of Florida. After graduation from Greenville College, Matthew began work in

the Food Science and Human Nutrition Department at the University of Florida as a laboratory

technician for Dr. W. Steven Otwell during the summer of 2002. Matthew began his graduate

work in the fall of 2002 under the tutelage of Dr. Hordur G. Kristinsson. During his

matriculation at the University of Florida, Matthew j oined Alpha Zeta, agricultural honors

fraternity, and Phi Tau Sigma, food science honors fraternity, where he served as vice president

from 2005-2006 and president from 2006-2007. Upon graduation Matthew hopes to contribute

to the scientific community, both academic and industrial through his pursuit of knowledge in

the sciences, specifically food science.





PAGE 1

1 STRUCTURE/FUNCTION RELATIONS HIP OF CHANNEL CATFISH ( Ictalurus punctatus) MUSCLE PROTEINS SUBJECTED TO pH-SHIFT PROCESSING By MATTHEW PAUL DAVENPORT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Matthew Paul Davenport

PAGE 3

3 To my Lord and Savior Jesus Christ, my wife Alyson and the past three generations of my family, thank you for paving the way making this milestone possible and welcoming me to your ranks

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4 ACKNOWLEDGMENTS I thank m y supervising committee chair for hi s mentorship, his friendship, and for giving me the opportunity to pursue a life long dream. I thank my supervisory committee (Jess F. Gregory, W. Steve Otwell, and James F. Preston) for their valuable expertise, instruction, and contributions to the scientific community and general public through exceptional scholarship and genuine concern. I thank Southe rn Pride Catfish LLC for providi ng raw materials and extending my experiences during this process. I thank my Lord Jesus Christ for His stead fa st love. I thank my parents Dr. Paul and Cherith Davenport for a lifetime of support, enco uragement and tolerance; though your acts of love this accomplishment was not only possible but given true meaning. To my wife thank you for your years of love, patients and the embodiment of love. Your devotion over the years has allowed me to reach this point. To Jeremy Jacobson, Jeremy Hershey, Ross Dubose and my many friends for the encouragement and joyful ti mes allowing me to reach this goal with some sanity intact. I thank all of my lab peers for giving me their time, kno wledge, adventures and laughs.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................14 CHAP TER 1 INTRODUCTION..................................................................................................................16 Background.............................................................................................................................16 pH-Shift Processing............................................................................................................ ....18 Protein Gelation......................................................................................................................21 Rheology.................................................................................................................................27 Key Muscle Proteins of Intere st for the pH-shift Process ...................................................... 31 Objectives...............................................................................................................................33 Experimental Design............................................................................................................ ..35 2 METHODS.............................................................................................................................40 Raw Material..........................................................................................................................40 Preparation of Pr otein Isolates ................................................................................................40 Protein Concentration.......................................................................................................... ...41 Protein Surface Hydrophobicity............................................................................................. 41 Circular Dichroism (CD)........................................................................................................42 Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HCl) .......................... 42 Differential Scanning Calorimetry (DSC).............................................................................. 43 Reactive Sulfhydryl Groups...................................................................................................43 Total Sulfhydryl Groups.........................................................................................................44 Myosin ATPase Activity Assay.............................................................................................. 44 Susceptibility of Proteins to Tran sglutam inase-Induced Cross Linking................................ 45 Oscillatory Rheology........................................................................................................... ...46 Torsion Gel Testing............................................................................................................ ....46 Punch Test..............................................................................................................................47 Fold Test.................................................................................................................................47 Gel Water Holding Capacity.................................................................................................. 48 Cook Loss...............................................................................................................................48 Heating Rate Gelation Studies................................................................................................ 48 Protein Solubility as a Function of pH...................................................................................49 Protein Solubility as a Function of Salt Concentration .......................................................... 50 Statistical Analysis........................................................................................................... .......50

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6 3 CHANNEL CATFISH ( I ctalurus punctatus ) MUSCLE PROTEIN ISOLATE PERFORMANCE PROCESSED UNDER DI FFERING ACID-AIDED AND ALKALIAIDED pH VALUES............................................................................................................. 53 Introduction................................................................................................................... ..........53 Methods..................................................................................................................................55 Raw Material...................................................................................................................55 Preparation of Pr otein Isolates .........................................................................................55 Protein Concentration...................................................................................................... 56 Oscillatory Rheology....................................................................................................... 57 Torsion Gel Testing......................................................................................................... 57 Punch Test.......................................................................................................................58 Fold Test..........................................................................................................................58 Gel Water Holding Capacity........................................................................................... 58 Cook Loss........................................................................................................................59 Statistical Analysis.......................................................................................................... 59 Results.....................................................................................................................................60 Rheological Changes in Protein Is olates During T hermal Gelation............................... 60 Gel Quality of Isolates as Assessed by Torsion Testing................................................. 63 Gel Quality of Isolates as Assessed by Punch Testing....................................................63 Gel Quality of Isolates as Assessed by Expressible Moisture......................................... 64 Gel Quality of Isolates as Assessed Fold Test................................................................. 64 Gel Quality of Isolates as Assessed by Cook Loss.......................................................... 64 Discussion...............................................................................................................................65 Conclusions.............................................................................................................................74 4 CHEMICAL PROPERTIES OF ACID AIDED AND ALKALI AIDED PROTEIN ISOLATES FROM C ATFISH ( Ictalurus punctatus )...........................................................133 Introduction................................................................................................................... ........133 Methods................................................................................................................................136 Raw Material.................................................................................................................136 Preparation of Pr otein Isolates .......................................................................................136 Protein Concentration.................................................................................................... 137 Protein Surface Hydrophobicity.................................................................................... 137 Reactive Sulfhydryl Groups..........................................................................................138 Total Sulfhydryl Groups................................................................................................139 Myosin ATPase Activity Assay....................................................................................139 Protein Solubility as a Function of pH..........................................................................140 Protein Solubility as a Function of Salt Concentration ................................................. 141 Statistical Analysis........................................................................................................ 141 Results...................................................................................................................................142 Myosin ATPase.............................................................................................................142 Surface Hydrophobicity................................................................................................. 142 Total Sulfhydryl Groups................................................................................................142 Reactive Sulfhydryl Groups..........................................................................................143 Solubility of Proteins at Different Salt Levels .............................................................. 143

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7 Solubility of Proteins at Different pH Values ............................................................... 143 Discussion.............................................................................................................................143 Conclusions...........................................................................................................................147 5 THE EFFECT OF pH-SHIFT PROCESSING ON THE STRUCTURAL AND THERMAL PROPERTI ES OF CATFISH ( Ictalurus punctatus ) PROTEIN ISOLATES.. 155 Introduction................................................................................................................... ........155 Methods................................................................................................................................156 Raw Material.................................................................................................................156 Preparation of Pr otein Isolates .......................................................................................156 Protein Concentration.................................................................................................... 157 Circular Dichroism (CD)...............................................................................................157 Isolate Susceptibility to Unfolding in Guanidine Hydrochloride (Gu-HCl) ................. 158 Differential Scanning Calorimetry (DSC)..................................................................... 158 Susceptibility of Proteins to Tran sglutam inase-Induced Cross Linking....................... 159 Statistical Analysis........................................................................................................ 159 Results...................................................................................................................................160 Circular Dichroism (CD)...............................................................................................160 Guanidine Hydrochloride Denaturation (Gu-HCl)........................................................ 160 Micro-Differential Scanning Calorimetry (DSC).......................................................... 161 Susceptibility of Proteins to Cross-linking by Transglutam inase................................. 162 Discussion.............................................................................................................................163 Conclusions...........................................................................................................................169 6 THE EFFECT OF HEATING RATE ON pH-SHIFT PROCESSED CATFISH ( Icta lurus punctatus ) MUSCLE PROTEINS....................................................................... 181 Introduction................................................................................................................... ........181 Methods................................................................................................................................182 Production of Protein Isolates....................................................................................... 182 Protein Composition...................................................................................................... 182 Torsion...........................................................................................................................182 Rheology........................................................................................................................183 Statistical Analysis........................................................................................................ 183 Results...................................................................................................................................184 Torsion...........................................................................................................................184 Rheology........................................................................................................................185 Discussion.............................................................................................................................186 Conclusions...........................................................................................................................188 7 GENERAL DISCUSSION................................................................................................... 197 APPENDIX STATSTICAL TABLES......................................................................................................201

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8 REFERENCES............................................................................................................................232 BIOGRAPHICAL SKETCH.......................................................................................................238

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9 LIST OF TABLES Table page 5-1: Thermal events ( cal) of isolates.................................................................................... 180

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10 LIST OF FIGURES Figure page 1-1 Experimental design of the structure/function relationship of channel catfish. ................. 36 1-2 Experimental design of the physic al testing of channel catfish. ........................................37 1-3 Experimental design of the chem ical testing of channel catfish. ....................................... 38 1-4 Experimental design of the struct ural testing of channel catfish. ......................................39 2-1 The process used in acid and alkali pH shift processing. .................................................. 52 3-1 The storage modulus (G) of catfish protein isolates trea ted at pH 2.0 without NaCl. ..... 76 3-2 The loss modulus (G) of catfish protei n isolates treated at pH 2.0 without NaCl........... 77 3-3 The tan delta (G/G) of catfish protei n isolates treated at pH 2.0 without NaCl............. 78 3-4 The storage modulus (G) of catfish protein isolates trea ted at pH 2.5 without NaCl. ..... 79 3-5 The loss modulus (G) of catfish protei n isolates treated at pH 2.5 without NaCl........... 80 3-6 The tan delta (G/G) of catfish protein isolates treated at pH 2.5 without NaCl.............81 3-7 The storage modulus (G) of catfish protein isolates trea ted at pH 3.0 without NaCl. ..... 82 3-8 The loss modulus (G) of catfish protein isolates treated at pH 3.0 without NaCl...........83 3-9 The tan delta (G/G) of catfish protein isolates treated at pH 3.0 without NaCl.............84 3-10 The storage modulus (G) of catfish protein isolates trea ted at pH 10.5 without NaCl. ... 85 3-11 The loss modulus (G) of catfish protein isolates treated at pH 10.5 without NaCl......... 86 3-12 The tan delta (G/G) of catfish protein isolates treated at pH 10.5 without NaCl........... 87 3-13 The storage modulus (G) of catfish protein isolates trea ted at pH 11.0 without NaCl. ... 88 3-14 The loss modulus (G) of catfish protein isolates treated at pH 11.0 without NaCl......... 89 3-15 The tan delta (G/G) of catfish protein isolates treated at pH 11.0 without NaCl........... 90 3-16 The storage modulus (G) of catfish protein isolates trea ted at pH 11.5 without NaCl. ... 91 3-17 The loss modulus (G) of catfish protein isolates treated at pH 11.5 without NaCl......... 92 3-18 The tan delta (G/G) of catfish protein isolates treated at pH 11.5 without NaCl........... 93

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11 3-19 The storage modulus (G) of untreat ed catfish m uscle without NaCl............................... 94 3-20 The loss modulus (G) of untreated catfish m uscle without NaCl.................................... 95 3-21 The tan delta (G/G) of untreat ed catfish m uscle without NaCl...................................... 96 3-22 The storage modulus (G) of catfish protein isolates tr eated at pH 2.0 with NaCl. ........... 97 3-23 The loss modulus (G) of catfish protei n isolates treated at pH 2.0 with NaCl................ 98 3-24 The tan delta (G/G) of catfish protei n isolates treated at pH 2.0 with NaCl.................. 99 3-25 The storage modulus (G) of catfish protein isolates tr eated at pH 2.5 with NaCl. ......... 100 3-26 The loss modulus (G) of catfish protei n isolates treated at pH 2.5 with NaCl.............. 101 3-27 The tan delta (G/G) of catfish protei n isolates treated at pH 2.5 with NaCl................ 102 3-28 The storage modulus (G) of catfish protein isolates tr eated at pH 3.0 with NaCl. ......... 103 3-29 The loss modulus (G) of catfish protei n isolates treated at pH 3.0 with NaCl.............. 104 3-30 The tan delta (G/G) of catfish protei n isolates treated at pH 3.0 with NaCl................ 105 3-31 The storage modulus (G) of catfish protein isolates tr eated at pH 10.5 with NaCl. ....... 106 3-32 The loss modulus (G) of catfish protei n isolates treated at pH 10.5 with NaCl............ 107 3-33 The tan delta (G/G) of catfish protei n isolates treated at pH 10.5 with NaCl.............. 108 3-.34 The storage modulus (G) of catfish protein isolates tr eated at pH 11.0 with NaCl. ....... 109 3-35 The loss modulus (G) of catfish protei n isolates treated at pH 11.0 with NaCl............ 110 3-36 The tan delta (G/G) of catfish protei n isolates treated at pH 11.0 with NaCl.............. 111 3-37 The storage modulus (G) of catfish protein isolates tr eated at pH 11.5 with NaCl. ....... 112 3-38 The storage modulus (G) of catfish protein is olates treated at pH 11.5 with NaCl. ...... 113 3-39 The tan delta (G/G) of catfish protei n isolates treated at pH 11.5 with NaCl.............. 114 3-40 The storage modulus (G) of untre ated catfish m uscle with NaCl..................................115 3-41 The loss modulus (G) of untreated catfish m uscle with NaCl....................................... 116 3-42 The tan delta (G/G) of untreated catfish muscle with NaCl......................................... 117 3-43 The storage modulus (G) before he ating catfish protein without NaCl. ........................118

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12 3-44 The storage modulus (G) after heating catfish protei n to 80C without NaCl. .............. 119 3-45 The storage modulus (G) after cooling catfish protei n to 5C without NaCl. ................ 120 3-46 The storage modulus (G) before he ating catfish prot ein with NaCl. .............................. 121 3-47 The storage modulus (G) after heating catfish protein to 80C with NaCl. ................... 122 3-48 The storage modulus (G) after cooli ng catfish protein to 5C with NaCl. ..................... 123 3-49 The shear stress (torsion) cat fish m uscle with 2% NaCl.................................................124 3-50 The shear strain (torsion) cat fish m uscle with 2% NaCl.................................................125 3-51 The shear stress (punch test) catfish muscle with 2% NaCl. ........................................... 126 3-52 The shear stress (punch test) catfish muscle with 2% NaCl. ........................................... 127 3-53 The jelly strength (stress*stra in, punch test) with 2% NaCl. ...........................................128 3-54 The expressible moisture of catfish muscle with 2% NaCl............................................. 129 3-55 The quality score (fold test) of catfish m uscle with 2% NaCl......................................... 130 3-56 The cook loss of catfish muscle with 2% NaCl. .............................................................. 131 3-57 Torsion texture profile of pH-s hift processed catfish m uscle.......................................... 132 4-1 Myosin ATPase activity of catfish muscle......................................................................149 4-2 The surface hydrophobicity of catfish muscle................................................................. 150 4-3 Total sulfhydryl content of catfish muscle....................................................................... 151 4-4 Reactive sulfhydryl content of catfish muscle................................................................. 151 4-5 Solubility (NaCl) of catfish muscle................................................................................. 152 4-6 Solubility (pH) of acid-treated catfish m uscle.................................................................153 4-7 Solubility (pH) of alka li-treated catfish m uscle...............................................................154 5-1 Circular dichroism of catfish m uscle, acid-treated and control....................................... 170 5-2 Circular dichroism of catfish mu scle, alkali-treated and control. .................................... 170 5-3 -helix, -structureand random coil in catfish muscle..................................................... 171 5-4 Guanidine Hydrochloride (Gu-HCl, 0-2M) on catfish m uscle, acid-aided and control..172

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13 5-5 Guanidine Hydrochloride (Gu-HCl, 0-2M) on catfish m uscle, alkali aided and control..............................................................................................................................173 5-6 Thermograms (5-80oC) of aci d treated catfish m uscle.................................................... 174 5-7 Thermograms (5-80oC) of alkali treated catfish muscle. ................................................ 174 5-8 Enzyme susceptibility (TGase) of catfish muscle............................................................ 175 5-9 Rheogram of pH treatment 2.0 with and without TGase treatm ent. .............................. 176 5-10 Rheogram of pH treatment 2.5 with and without TGase treatm ent. .............................. 176 5-11 Rheogram of pH treatment 3.0 with and without TGase treatm ent ............................... 177 5-12 Rheogram of pH treatment 10.5 w ith and without TGase treatm ent............................... 177 5-13 Rheogram of pH treatment 11.0 w ith and without TGase treatm ent............................... 178 5-14 Rheogram of pH treatment 11.5 w ith and without TGase treatm ent............................... 178 5-15 Rheogram of the control with and without TGase treatment........................................... 179 6-1 Stress response of acida nd alkali-aided catfish muscle. ...............................................189 6-2 Strain response of acida nd alkali-aided catfish muscle. ................................................ 190 6-3 Rheological response of acidand al kali-aided catfish protein isolates. ......................... 191 6-4 Rheogram of alkali treated catfish m uscle heated at 20oC/minute, no hold.................... 192 6-5 Rheogram of alkali treated catfish m uscle heated at 20oC/minute, 20 minute hold........ 193 6-6 Rheogram of alkali treated catfish m uscle heated at 0.5oC/minute, no hold...................193 6-7 Rheogram of acid treated catfish muscle heated at 20oC/minute, no hold...................... 194 6-8 Rheogram of acid treated catfish muscle heated at 20oC/minute, 20 minute hold.......... 194 6-9 Rheogram of acid treated catfish muscle heated at 0.5oC/minute, no hold..................... 195 6-10 Rheograms of acid and alkali treated catfish m uscle heated at 20oC/minute.................. 196

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE STRUCTURE/FUNCTION RELATI ONSHIP OF CHANNEL CATFISH ( Ictalurus punctatus ) MUSCLE PROTEINS SUBJECTE D TO pH-SHIFT PROCESSING By Matthew Paul Davenport May, 2008 Chair: Hordur G. Kristinsson Major: Food Science and Human Nutrition Novel processing utilizing pH-s hift technology applied to muscle-based systems modifies or increases raw product utiliza tion. The use of pH-shift proc essing on muscle products and byproducts allows for the separation of muscle prot eins from lipids, collagen, skin, bones and other undesirable components. Undesirable materials ar e not easily separated from whole muscle or other comminuted muscle products such as surimi During pH-shift processing, animal and fish muscle including channel catfish muscle is modi fied by the reduction of lipid and heme content, altering protein composition and thermal gelatio n properties. Reduction of lipid and heme content increases whiteness and oxidative stability of protein isolates. Changes in protein composition are associated with the removal of sarcoplasmic prot eins including heme proteins, some endogenous enzymes, collagen and other protein components. Modification of thermal gel properties induced variable gel strengths. It was hypothesized that pH-shift processing will structurally modify channel catf ish muscle proteins resulting in functional properties leading to the expansion, utilization and pr oduction of muscle based products The model system in this study was developed to provide the basis for implementation of economical and environmental utilization of current seafood byproducts by pH-shift processing.

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15 The results show that pH-shift processing m odifies muscle proteins structurally and functionally. pH-shift processing reduced the relative content of al pha helix to beta structure of muscle proteins after isoelectri c precipitation indicating a molten globular state. Muscle proteins in a molten globular state showed reduced myos in ATPase activity, altered protein surfaces, modified thermal sensitivity, increased suscepti bility to enzymatic crosslinking and modified solubility. These changes lead to changes in the physical properties of pH-shift processed muscle proteins. Alkali proce ssed catfish showed increased ge l rigidity, gel strength and gel flexibility compared to acid processed catfi sh which exhibited inconsistent functional performance, increasing and decreasing in gel rigidity, gel strength and gel flexibility. These results show that pH-s hift processing of channel ca tfish muscle provides highly functional isolates with a broad range of applications by induc ing structural modification of muscle proteins. This innovative process yields muscle proteins with good color, high oxidative stability and modified functi onality, which can find many a pplications in formulated comminuted muscle food products. Furthermor e, this technology can be an important contribution to better utilization of seafood and land animal by-products.

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16 CHAPTER 1 INTRODUCTION Background Presently th e world demand for seafood products exceeds the sustainable limits of the available fisheries (Hultin and Kelleher 2000). The supply traditionally has been based on wild caught products; however, the demand for high qua lity seafood products has not been fulfilled. The investigation into economica lly viable alternative producti on methods and utilization of currently harvested products is of interest to fill th e pressing needs of the consuming public. The advent of commercially viable a quaculture in the past 50 years ha s helped to alleviate some of the stress on wild stocks (Har vey 2002). The supply of aquacultured seafood products has come under fire for reducing the price of high value wild species such as salmon, shrimp and grouper (Eaglea and others 2004). Aqu aculture also has been criticized for unmanaged waste production, increase in fish disease and damage to the aquifer (Barton 1997). Thus the need for value-added products is of great interest to commercial pr oducers as the profit margin s are getting smaller and the cost of production is rising (Harvey 2002). Th e utilization of value addition with modern processing and preservation has allowed for in creased commercial production of high value products from lower value fish species and byproducts, but many useabl e protein sources are still diverted into animal feed or waste (Kristinsson and Rasco 2002). One of the products commercially produced as a value addition produc t is surimi (Park 2005). Surimi is not a new product. Surimi orig inated centuries ago as a high value product in Japan (Park 2005). A variety of structured products can be form ed from surimi, including the traditional kamaboko (Park 2005). Kamaboko was traditionally made by first producing surimi by mincing freshly ground fish meat, washing it with pure water or water with low concentrations of salt, dewateri ng the washed tissue, forming a sausage, and cooking the sausage

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17 to form kamaboko. The value of quality surimi and kamaboko was high due to the requirement to use fresh fish and the very short shelf-life of either the raw or cooke d material (Park 2005). Commercialization of surimi production and subs equent products made it necessary to extend their shelf-life. Freezing surimi was one of the prerequisites to its success as a global commodity, but surimi is particularly sens itive to freeze thaw denaturation (Park 2005). Cryoprotection increased protein stability to freeze thaw treatments by utilizing small carbohydrates which stabilized the muscle proteins during freeze thaw processes. This has been attributed to paving the way for the commercializa tion of surimi (Park 2005). The current surimi production uses lower value white fish, primarily Alaskan Pollack and Pacific Whiting as the base material. The use of Alaskan Pollack and Pacific Whiting for the production of surimi allows for commercial processors to fill the commodities market with two products, surimi and frozen fillet blocks (Park 2005). Surimi processing does have drawbacks such as still mainly requiring fresh fish (Park 2005). This has led to the utiliz ation of large trawlers which catch, process and freeze surimi either on the water or on shore (Park 2005). Concurrently on th ese trawlers the alternative product, frozen fish fillet blocks and mince al so are produced (Park 2005). Even though surimi can allow for increased utilization and profit ma rgins from these species, there are many other commercially harvested species which are not used for surimi and go to animal feed or other lower value products (Hultin and Kelleher 2000). Traditional commercial surimi processing cannot easily handle complex materials, such as w hole fish, species rich in dark muscle, high in fat or processing byproducts (Hultin and Kelle her 2000). Not only is it difficult to recover proteins effectively from comp lex materials, but these materi als lead to major color and oxidation problems resulting in surimi with poor functionality (Hultin a nd Kelleher 2000). The

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18 by-products from fish processing which contain the most muscle tissue are the frame and the trimmings. The frame is the backbone and ribs le ft after the fillet is removed. The trimmings are the parts of the fillet which are removed to increase the quality and acceptability of a fillet; a useable trimming from catfish is the nugget or belly flap. Large amounts of unprocessed byproducts such as frames and trimmings are not us ed for human consumption but rather end up in animal feed, bone meal or fish meal if used at all (Hultin and others 2000). Unutilized byproducts end up as waste material and add dispos al costs and increase the biological effluent from the processing plant (Hultin and others 2000 ). Usable meat left on the frame and other processing by-products can be used for human consumption leaving enough remaining material to satisfy the animal feed market (Hultin and Kelleher 1999). Therefore a major need exists to develop and use alternative techniques to provi de economical advantag es and recover these proteins for high value products to minimize the impact on wild stoc ks and also satisfy the large demand for seafood based protein. pH-Shift Processing A recently d eveloped technology in fish and m eat processing allows for the potential use of discarded raw materials for human consump tion. The primary drawback to using these materials is the high level of pro-oxidants in particular heme proteins collagen, phospholipids and triacylglycerols all of which contribute to reduced consumer acceptability (Kristinsson and Hultin 2003b). Removal of these components resulti ng in a relatively pure protein product made possible by acid or alkali solubiliza tion of muscle proteins with r ecovery at or near the average isoelectric point of a muscle homogenate (Hul tin and Kelleher 1999; Hultin and others 2000; Kristinsson and others 2005b). Isoelectric recovery is done after unwanted components are removed by either filtration or centrifugation at low or high pH (Hultin and Kelleher 1999; Hultin and others 2000; Kristinsson and Hultin 2 004b; Kristinsson and others 2005b). This pH-

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19 shift process allows for the rec overy of proteins with high functi onality at a relatively low cost to the producer (Hultin and Ke lleher 1999; Hultin and others 2000; Kristinsson 2002; Kristinsson and Ingadottir 2006; Kristinsson and others 2005b). pH-shift processing relies on electrostatic repulsion between muscle proteins to impart solubility (Kristinsson and Hultin 2003b). This is possible after the muscle has b een thoroughly homogenized and d iluted with water. Dilution allows for the dispersion of protei ns and reduction of viscosity to a sufficient degree facilitating solubilization by allowing for the necessary mo lecular spacing to occur. HCl and NaOH are used to adjust the pH to extreme low or high pH however the effect of other acids and bases are under investigation (Raghavan and Kristinsson 2007b). After the appr opriate pH has been reached, i.e. the pH necessary to achieve solubil ity, the insoluble material is removed (Hultin and Kelleher 1999; Hultin and others 2000). If centr ifugation is used, the insoluble material will produce a three phase separation; a top layer and bottom sediment with the soluble proteins comprising the middle phase (Kristinsson and ot hers 2005b). The top fat layer is comprised primarily of triacylglycerides a nd some emulsified proteins (Kristinsson and others 2005b). The sediment layer consists of phospholipids, collag en, bones, skin, scales and some poorly soluble proteins, including a small percentage of the actomyosin complex, actin and myosin among other proteins also found in the soluble fraction (Kri stinsson and others 2005b). After separation, the middle soluble layer is collected and adjusted to the average pI (~5.3-5.5) of the muscle protein slurry (Hultin and Kelleher 1999; Hultin and others 2000). The proteins are recovered by dewatering using centrifugation, fi ltration or screening (Hultin and Kelleher 1999; Hultin and others 2000). This gives a partly dewatered pr otein isolate of 70-80% moisture. The proteins retained, for the most part, are the myofibrill ar proteins, although some of the sarcoplasmic proteins are retained as well, increasing the recovery over conventiona l washing processes to

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20 recover muscle proteins (e.g. surimi processing) (Hultin and Kelleher 19 99; Hultin and others 2000). This is particularly the case for the acid -aided process where significant amounts of heme proteins (denatured and oxidized ) are retained whereas in the alkali-aided process very low levels of heme proteins are retained and they remain in their native and reduced state (Kristinsson and Hultin 2004a). The sarcoplasmic pr oteins include not only the heme proteins but also endogenous enzymes and other small pr oteins which can have negative effects on the quality of the final protein product (Kristinss on and others 2005a). The removal of the sarcoplasmic proteins, especially the heme prot eins increases the whiteness of the proteins isolate, leads to less oxidation and improves gel strength (Kristinsson and others 2005a). Research to date has shown that during pH-shi ft processing, protein st ructure is disrupted and may be partially responsible for the effect s on the functional prope rties of the pH-shift process isolate as compared to conventional methods (e.g. surimi processing) (Choi and Park 2002; Kristinsson and Hultin 2003b; Shann-Tzong a nd others 1998; Yongsawatdigul and Park 2004). Conformational changes occurring during the pH-shift isolation pr ocess are linked to differences observed between acid a nd alkali solubilization on the physical nature of the isolate (Kristinsson and Hultin 2003a). When utilizing the acid solubilization process higher puncture force and lower deformation values are observe d than with conventional surimi. However isolate gels from the alkali process show bot h higher puncture and deformation values than isolate gels from the acid process and gels from conventional surimi processing (Choi and Park 2002; Perez-Mateos and others 2004; Undeland and others 2002). Kristinsson and Demir demonstrated that among four species, catfish, mack erel, mullet and croaker, alkali solubilization produced higher gel storage modulus (i.e. gel rigid ity/firmness) after cooli ng than either surimi or acid processing (Kristinsson and Demir 2003). In all three species except croaker, acid

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21 processing produced a much lower storage modulus than the other pro cesses (Kristinsson and Demir 2003). The acid and alkali-aided technology is expe cted to provide the seafood industry a new powerful tool in the production of high quality muscle protein is olates from inexpensive sources (Kristinsson and others 2005b). The effect isolati on conditions have on muscle proteins is still under investigation and is relativ ely unknown. There are changes noted in the function, structure and conformation of single prot eins (e.g. myosin) subjected to the process as well as whole protein isolates from various species compared to proteins subjected to conventional processes (e.g. surimi processing) or no processing (Hul tin and Kelleher 2000; Kristinsson and Demir 2003; Kristinsson and Hultin 2003a; Undeland and ot hers 2002). The pH-shift process has been successful on many aquatic species as well as turkey and beef (Kristinsson and Hultin 2003b; Kristinsson and Ingadottir 2006; Kristinsson and others 2005b; Liang and Hultin 2003; Mireles Dewitt and others 2002; Undeland and others 2002; Yongsawatdigul and Park 2004). The proteins extracted and concentrated during pH-s hift processing, when compared to similar processes such as surimi, show altered physic al properties (Kristinss on and others 2005b). Further understanding on changes that occur to muscle proteins during the pH-shift processing and the mechanism responsible may lead to tailor -made protein isolates with the functionality desired for specific products, additives or utilizations. Protein Gelation The prim ary functionality of interest for surimi and protein isolates is gelation. Gelation is the interaction of molecules, primarily polymer s giving rise to a three dimensional network entrapping solvent within the network (Stone and Stanley 1992). Water is typically the solvent in food polymers (Stone and Stanley 1992). Protein gelation can and does occur via different mechanisms, leading to differences in textur e and quality (Stone and Stanley 1992). During

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22 protein gelation, especially fish protein gelation, there are three phases of the protein paste/gel formation. Phase one involves the uncooked ra w product or the sol which has little to no detectable tensile strength. The ne xt phase is variable depending on the treatment. Suwari is the process when the sol is set at medium to high temperatures and this is also the point at which protein cross-linking enzymes (e.g. transglutaminase) may be added to increase the setting effect at the elevated temperature. Suwari setting temperatures ar e highly species dependent, but usually do not exceed 50C. Then the final phase of fish protein gelation is kamaboko the final cooked and cooled gel product (Stone and Stanley 1992). The three main types of protein gels are revers ible gels, chemically/enzymatically set gels and thermally set gels (Lanier 2000). The latter two are irreversible (L anier 2000). Reversible gels are unique since they are initiated by hea ting during which the solution becomes highly fluid and upon cooling the gel is formed (Lanier 2000). Collagen forms these type of gels due to its unique structure imparted by the high pe rcentage of prolin e and hydroxyproline (Ko odziejska and others 2004). Collagen structure is comprised of a super structure forming a triple helix with three collagen strands interacting to make large long stra nds (Ockerman and Hansen 2000). The collagen complex loses most of its structure during the heating proce ss as its non-covalent interactions are broken leadi ng to an unfolded scrambled chai n, then upon cooling the regions rich in proline and hydroxyproline reform hydr ophobic patches and associate, next hydrogen bonds form and, thus a, cold set gel is formed (Oakenfull and others 1997). This gel is reversible, and melts on heating, due to hydrophobic interactions and hydrogen bonding providing the basis for the three dimensiona l structure (Oakenfull and others 1997). Enzymatically and chemically set gels are form ed with either enzymes or chelating agents respectively. The enzymes and chelating agents will not, for the most part, independently form

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23 a gel but are used in setting or curing prior to heati ng the protein paste or solution to complete gel formation. Transglutaminase (TGase) is one of the most common enzymes used to increase the strength of protein gels (Walsh and others 2003). TGase is a calcium dependent enzyme which mediates an acyl transfer between gl utamine and lysine bound in a peptide/protein forming an -( -glutamyl) lysine cross-link. The covalent cross-linking of these amino acids gives increased strength the network (Lee and others 1997; Walsh and others 2003). The final and most common method of protein gel formation is thermally setting the gel. Thermally setting a gel is done primarily by h eating a protein solution. The heating process unfolds the proteins which allows for in teraction of hydrophobic groups and disulfide interchanges. Upon cooling the protein solution, the hydrophobic interactions are further set and reformation of hydrogen bonds in a different order from the native system leading to the three dimensional network of the newly created the gel structure (Oakenfull and others 1997). Many different factors affect protein-protein interaction duri ng the gel forming process. Muscle proteins specifically include ionic strength, ionic type, pH, protein hydrophobicity, reactive groups on proteins (e.g. SH groups), protein source and t ype, post-mortem age, chemical additives and thermal treatment regiment (Oakenfull and others 1997). The need to solubilize muscle proteins with salt is a believed prerequisite of well dispersed gel network formation (Feng and Hultin 2001). Solubility was attributed to the increased binding and interaction of protein systems with water leading to incr eased protein-protein interaction, water binding and reduced cook loss (F eng and Hultin 2001). High ionic strength is not necessary and low ionic strength, depending on pH leads to favorab le conditions improving gel formation (Chang and others 2001). If the pH of a low ionic strength muscle protein system is slightly alkaline (e.g. pH 7.2-7.6), enough electrostatic repulsion will form between the

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24 proteins to produce a well disp ersed, ordered protein network (Chang and others 2001) This network will form a strong gel on heating and coo ling, capable of binding high levels of water due to the gel pressure formed because of stro ng repulsive forces between proteins making up the gel network (Kristinsson and Hultin 2003c). The pH of a sol paste is very important to form a good gel (Feng and Hultin 2001). Characteristics of a good gel include incorporatin g water into the gelled network and retaining the water after setting (Kristinsson and Hultin 2003c ). The effect of pH on proteins is very specific due to the ionic nature of proteins (Feng and Hultin 2001). The interaction of proteins with both acid and base can re sult in different structural m odifications and intermolecular interactions greatly affecting the taste, texture, tensile strength and wate r-holding capacity of raw and cooked gels (Kristinsson and Hultin 2003a, 2003b, 2003c). A change in pH leads to a change in surface amino acid ionization of th e protein (Feng and Hultin 2001). A good muscle protein paste and gel has a proper balance between electrostatic repulsion and attraction (Feng and Hultin 2001). If the pH of the system is re duced close to the isoelectric point (pI) of the proteins, they will aggregate in the raw system a nd associate so closely that they have no water holding capacity and will form a brittle, dry gel (Lanier and Lee 1992). As mentioned above, at a higher pH there is enough electros tatic repulsion that the proteins will still associate via other means and produce open areas which are filled by wate r, giving rise to the texture and mouth feel desired in a good muscle protein ge l (Feng and Hultin 2001). If th e pH is changed too far above or below the pI, the total surface charge increases repulsion betwee n proteins and results in the behavior of muscle proteins to the point that the interactions necessary to form a gel are not possible even on cooking (Kristinsson 2003; Vega-Warner and Smith 2001).

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25 Proteins have hydrophobic amino acid residues, normally associated in groups to produce hydrophobic patches, which gives rise to a cert ain hydrophobicity of a protein molecule which may vary depending on solution conditions (Lan ier 2000). Hydrophobic residues play a very important role in the development of all levels of protein stru cture. Interactions of hydrophobic patches (and their exposur e) during heating (and cooling) are one of the main driving forces of protein gelation as they provide much of the initia l interaction and associat ion of proteins (Lanier 2000). Hydrophobic interactions are attributed to the increase in gel strength during heating (Lanier 2000). Hydrophobicity an d hydrophobic interaction of protei ns are mediated by a) the structure of the protein and b) by the external factors of the matrix and physical treatment (Lanier 2000). These factors include temper ature, pH, salts, lipid content, lipid oxidation products, free fatty acids and many others (Lanier 2000). Protein source has a great influence on the gelation characteristics of the system by dictating the types of pr oteins which are present to participat e in gelation (Lanier 2000). Protein source is not limited to species differences, be it between fish species or between land animal and fish species (Lan and others 1995a; Lanier 2000; Luo and others 2001b). Protein source also pertains to the location within the animal, th e difference between dark and light muscle or between muscle types (Vega-Warner and Smith 2001). The presence of specific proteins and concentrations of specific proteins can vary gr eatly between muscle types (Nowsad and others 2000). Differences observed between proteins, isolated under the same conditions, have been seen from various physical and chemical analys es done on the proteins, including gel strength, solubility, temperature stabil ity, hydrophobicity, and other chemi cal indicators (Oakenfull and others 1997).

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26 The postmortem age of the raw material from which proteins are rec overed from is highly important (Lanier 2000). Some an imal species have a shelf life of 2-3 weeks, e.g. beef and pork, and thus will still form an acceptable gel based pr oduct after that time, whereas some fish species have a very short shelf life and their proteins can be negatively affected very quickly (Lan and others 1995b). This is because of endogenous enzymes and the rigor state of the muscle (Lanier 2000). Processing fish muscle prevs. post-rigor can have an eff ect on the recovery and state of the isolated proteins (Lanier 2000). Additives such as sugars, polyols and polysaccharides are commonly added to minimize muscle protein denaturation a nd retain functional ity during freezing and thawing. These additives allow for long term frozen storage and make large scale processing of muscle protein ingredients, such as surimi possible. The addition of these com pounds affects the gelation characteristics of the muscle pr oteins (Lanier and Lee 1992). Thermal input has a profound affect on the gela tion and gel strength of muscle proteins (Riemann and others 2004a). The effect of therma l input in a gelation system can be equated to the D value during canning, i.e. there is a relatio nship between time and temperature and the gel quality (Riemann and others 2004a). During heat induced gelation, it has been demonstrated that the longer the time taken to reach the end point temperature the greater the gel strength (Riemann and others 2004a). This is assumed to be due to the increased energy adde d to the system during slow heating in contrast to rapid heating. Studies where gels have been heated rapidly but held at the end point temperatures to give equivalent energy input as the slow he ating rate have also shown to give similar increases in gel strength as slow heat ing, supporting the energy input theory (Riemann and others 2004a).

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27 Rheology Gelation and gel form ation can be studied and described using rheology (Steefe 1992). Rheology is the study of the flow properties of a material (Owusu-Apenten 2005). Rheological tests apply a rotational or tangentia l force to a material and the re sponse of the material to this force and its mode of application provide information which relates to its characteristics of flow (Owusu-Apenten 2005). The three main parameters which are controlled in the determination of flow are force of application, distance of head movement or degree of oscillation and the frequency of application (Steefe 1992). Macro-rheology is the st udy of tensile properties prior, during and after breaking and/or reforming of the materials three dimensional structure (Steefe 1992). Micro-rheology studies the same tensile properties as macro-rheology, however the breakage/resolution of the three dimensional stru cture may not be involved (Steefe 1992). There is a linear viscoelastic range of most if not a ll gels (Owusu-Apenten 2005). This region is the area where there is deformation of the three di mensional network, however the bond interactions are not broken (Owusu-Apenten 2005). Macro-rheology is utilized as either an end-point determinat ion or stop-point determination of gel properties (Owusu-Apenten 2005). As macr o-rheology is destructive to the material, except in reformation or continuous flow studies, it renders the material unus able for future tests (Owusu-Apenten 2005). The other method of macr o-rheology, which is continuous testing, is flow properties which may or may not render the sample unusable for future studies (OwusuApenten 2005). Common rheology parameters follo wed during testing are break point or shear strength, initial viscosity, whic h is the force required to begin flow, and continuous viscosity which his the force required to maintain flow (Steefe 1992). Two character istics associated with continuous flow tests are shear thinning, where vi scosity is reduced, and shear thickening, where viscosity is increased (Owusu-Apenten 2005). These tests provide very valuable information

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28 and are the primary methods of quality control (QC) for many food products, especially in starches (Ta 2001). Among products evaluated using this method ar e pork, surimi, food starches and many others (Steefe 1992). Micro-rheology determines different aspects of structure and change in structure than macro-rheology (Owusu-Apenten 2005). The cha nges monitored utilizing micro-rheology occur mostly during the formation of a gel (Steefe 1992) Micro-rheology is less characterized on a broad scale for QC utilization. Micro-rheology is very sensitive in its measurements and values; thus a greater degree of variation may be s een between samples and instruments (Ta 2001). Micro-rheology, when dealing with food polymers, remains the predominant method of choice in basic research of materials (St eefe 1992). Micro-rheology is, however, also used in QC for other polymer research pertaining to fluids, adhesive s and rubbers (Ta 2001). The crossover for food polymer analysis on a basic level to QC requires not a greater understand ing of micro-rheology but a standardization and data bank development of micro rheological data for specific materials and the correlation of those materials to currently held standard values for the other tests used in QC labs (Ta 2001). The basis for all rheological testing is on the deformation of a material when under stress, ie., the stress strain relations hip. The differences between micro-rheology and macro-rheology are the use of shear stress and shear strain or the force and distance a material can withstand at failure or when the structure breaks. Macro-rh eology utilizes th e shear stress and shear strain where as micro-rheology utilizes stress and strain parameters which may not shear the material. To do this in micro-rheology, osci llatory stress is used in place of continuous stress. The parameters which are then calcula ted in oscillatory stress testing are different that those observed in continuous stress tests. The to tal response of the material to os cillatory stress is made up of

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29 the two components of the stress applied the in-phase stress, that is the stress applied moving away from the zero point and the out-of-phase stress which is the removal of the stress from the material or the probe moving back to the zero pos ition. The in-phase stress is referred to as the storage modulus (G) and can be viewed as the re sistance of the material to movement or the solid fraction of a material. The out-of-phase stress is referred to as the loss modulus (G) and is considered the liquid phase of the material. The G represents the material as it is resistant to motion, this resistance to an applied force, when a small enough stress and strain are used, which does not break, shear, the three dime nsional structure of the material is testing the intramolecular strength of the material to tangential compressi on. As the probe returns to the zero position the G is testing for the compliance of the material to a non-fracture stress. This is the ability of the material, though neither the stress nor strain is large enough to induce flow or breakage of the structure, to move into space created by the positive stress applied by the probe and thus the resistance of the material to the negative stress. The effect of the sinusoidal movement of the head, G and G, the resistance of the material to the movement of the head both forward and back alter sin wave during the motion of the probe. The alteration of the sin wave is represented at the tan delta. Tan delta ranged from 0-1 (G/G), or is the tange nt of the degrees of change of the sin wave during the motion of the probe, 0 be ing a pure solid and 1 being a pure liquid or 0 difference being there is no modification of the sin wave G and G are equal and 1 being there is no G pressure of the material or no space fill ing occurring. This relationship and the analysis of tan delta in rheological studies allows for the determination of the phase change of a material as it transitions from a more liquid like material to a more solid like material or vice versa. This transition period of a material from a more liqui d like to a more solid like material is also determined by the change of G and G or th e transition from greater out-of-phase resistance

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30 (G) to greater in-phase resistan ce (G). As the G increases and the G decreases or vice versa, if these two values cross during a treatment, this point is known as the gelation point, or where the material is equally solid and liquid. The transition of the materi al at this point is either going from a more solid to a more liquid material (me lting) or a more liquid to a more solid material (gelling) (Oakenfull and others 1997; Owusu-A penten 2005; Steefe 1992; Stone and Stanley 1992; Ta 2001). The micro-rheology as discussed above provides valuable information about what is occurring during a treatmen t study. After the tests have been done to determine what is going on in a system it is then necessary to test how strong the gel is. This is done using shear stress and shear strain, causing the failure of the material and thus determin ing how strong it is after or at some point during a treatment. This is done in one of two ways 1) torsional stress or 2) tangential stress. That is either twisting or puncturing a material The shear values of each of these types of testing, both shear stress and shea r strain, are dependent on the type of force applied to a material. The torsional resistance of a material may not be the same as the tangential resistance of a material as the structure of some materials are very directional sensitive. In food systems the tangential and torsi on resistance of a material ha s been shown to provide a good indication of the mouth feel and texture. Both of these tests are also used as quality determinants in muscle products, especially comminuted muscle products. The use of shear stress and shear strain have lead to th e development of texture profile an alysis of foods and is a primary component used to determine the grade and thus value of comminut ed muscle products, especially surimi (Oakenfull and others 1997; Owusu-Apenten 2005; Park 2005; Steefe 1992; Stone and Stanley 1992; Ta 2001).

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31 Key Muscle Proteins of Interest for the pH-shift Process Myosin is the prim ary muscle protein comprising ~50% of the myof ibrillar fraction of muscle (Kristinsson and Hultin 2003a). Myosin is composed of a long helical rod, where most of its secondary structure comes from, and a la rge bulky head group where most of its tertiary structure is found. This structur e of myosin allows it to be a good gel former (Kristinsson and Hultin 2003a). The head is globular in nature with hydrophobic sites on the exterior of the protein designed to interact with actin duri ng muscle contraction (R uppel and Spudich 1996). These hydrophobic sites are blocked by meromyosin s and troponin, primarily (Berne and others 2004). In a living system the rele ase of calcium moves troponin a nd in the presence of ATP the light chains are moved by a conf ormational shift in myosin, whic h looks like the myosin head sitting up and making the exposed hydrophobic site on the S-1 fr action of the myosin head available to actin (Berne and ot hers 2004). The structural and f unctional design of the proteins as they are in the living system still holds the pr edominant role in the mechanism of gelation. In a non-living system when muscle is processed and cooked to form a gel, the sequence of events is quite different. The role of myosin in muscle gel formation is a cascade of events, the sequence of which is vaguely proposed to be myosin-myosin head interaction at hydr ophobic sites causing close association between myosin molecules at lower te mperatures as heat dena turation is taking place (Hettiarachchy 1994). The heat denaturation moves and or removes both tropomyosin and meromyosin from the hydrophobic patches on the m yosin head allowing for this interaction (Hettiarachchy 1994). After this association, di sulfide interchanges, mediated by increasing temperatures, are proposed to take place to some extent in the lower S-2 fragment of the myosin head (Stone and Stanley 1992). These disulfid e interchanges hold the myosin-myosin linkage, created by the hydrophobic attraction, increasing the overall tensile st rength of the system (Stone

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32 and Stanley 1992). During the heating process, th e myosin tail is partia lly uncoiled exposing the buried hydrophobic patches in the tail portion as th e myosin tail has on its surface predominantly hydrophilic residues (Stone and Stanley 1992). The heat denaturation of the myosin tail causes a scrambling effect in the myosin system allowi ng for the now exposed hydrophobic patches of the myosin tail to interact and fo rm new associations with other uncoiled myosin tails (Stone and Stanley 1992). This rearrangement of the myos in tail along with the polymerization of the myosin head is the strengthening effect of m yosin gelation (Stone and Stanley 1992). This is because of the increased disorder in the gel syst em which is a consequence of the disruption of the natural organized tight packing of myosin, and other proteins, in the myofibrillar unit (Hettiarachchy 1994). Protein solubility based on pH and ionic strength is attributed to the effect of ionization of the surface of the protein. Myosin has more than half of its amino acids as hydrophilic residues and 80% of these are exposed to the solv ent (Lanier and Lee 1992). The increasing concentration of either positive or negative ions depending on the pH and/or ionic strength of the solvent, water in this case, proceed to interact on the surface of the protein, forming a charged shell around the protein (L anier 2000). This shell shields the ionic bridges between the proteins, allowing for separation of the proteins and eventually so lubilizing protei ns. The ion shell around the protein also increases the as sociation of water around the protein surface, resulting in soluble proteins (Lanier 2000). Once the salt bri dges are weakened/broken by ion shielding, increased water asso ciation is possible well beyond the hydration level of the ion alone. This increased amount of water associated with the protein increa ses its total level of hydration and, thus, that level of hydration is perceived as solubili ty of the protein (Stefansson and Hultin 1994). This mechanism of solubility is more evident in the myofibrillar fraction of

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33 muscle than in the sarcoplasmic fraction, as sarc oplasmic proteins are fairly soluble under most conditions (Kristinsson and others 2005b). It is this aspect of sarcoplasmic proteins, mainly hemoglobin and myoglobin, that allows for thei r separation (remaining soluble in the aqueous phase) from the protein isolate during isoelect ric precipitation where primarily myofibrillar proteins aggregate (Hultin and Kelleher 1999; Hul tin and others 2000). This removes off color associated with pelagic surimi and increases the stability of the protein isolate as both myoglobin and hemoglobin are active in lipid and prot ein oxidation (Hultin and Kelleher 2000). Objectives The overall objective of this project was to investigate how different acid (pH 2.0, 2.5 and 3.0) and alkali (pH 10.5, 11.0, 11.5) treatm ents followe d by isoelectric precipitation influenced the structure, conformation and functionality of catfish muscle proteins. The ultimate goal of this work was to attempt to build an unders tanding how specific pH unfolding and refolding regimes affect the proteins and connect structural/conformational ch anges to functional changes. We hypothesized that different pH treatments lead to differences in structure and conformation which are responsible for changes in functionality (gelation). Basic work in this area is expected to further our knowledge of muscle protein gelation and provide insight for production of muscle proteins with specialized functional characteristics. The specific objectives of this project were: 1) Investigate the effect of different acidand alkali-aided unfolding followed by isoelectric precipitation on the gel forming properties pr otein isolates during thermal treatment. 2) Investigate the effect of different acidand alkali-aided unfolding followed by isoelectric precipitation on the strength, elasticity and qua lity of thermally set protein isolate gels.

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34 3) Investigate the effect of different acidand alkali-aided unfolding followed by isoelectric precipitation on the chemical properties of protein isolates (surface hydrophobicity, ATPase activity, sulfhydryl groups and protein solubility). 4) Investigate the effect of different acidand alkali-aided unfolding followed by isoelectric precipitation on the conformation, structure and structural stability of protein isolates. 5) Investigate the effect of heating rate on the gel formation, strength, elasticity and quality of protein isolates made from acidand al kali-aided unfolding followed by isoelectric precipitation.

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35 Experimental Design The study of pH-shift processing in catfish m u scle was conducted on catfish fillets. The use of catfish fillets as the raw material was chos en to provide a stable, high quality raw material. Catfish was chosen as the raw material due to its importance in the southeastern United States and increasing consumption within this region and through out the entire United States. The experiments used to study pH-shift processing, outlined in figures 1-1 through 1-4 and described in the next chapter, were conducted due to their common use in meat and meat products. Each method used to investigate the properties of pH-s hift processed catfish muscle was replicated on independent batches of catfish fillets. This was determined acceptable by power analysis ( =0.05, =0.80). The chemical analyzes used were sp ecifically chosen for their previously established correlation to the physic al/textural properties of muscle products. The determination of the physical properties of pH-s hift processed catfish muscle was the initial focus, as previous research had indicated that pH-s hift processing will modify physic al properties. The reason for the modification of the physical properties (fig ure 1-2) would be furt her understood through the investigation of the chemical pr operties (figure 1-3) and structur al properties (1-4) of muscle proteins. The combination of these three properties will provide a deeper look into how different the physical/textural properties in meat are formed.

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36 Catfish Fillet Ground Control pH-Shift Isolation As described previously Physical Properties Chemical Properties Structural Properties Post-isolation Variable Heating Rate Structure Function Relationship Catfish Fillet Ground Control pH-Shift Isolation As described previously Physical Properties Chemical Properties Structural Properties Post-isolation Variable Heating Rate Structure Function Relationship Figure 1-1: Figure summarizing the overall experimental design of the structure/function relationship of channel catfish ( Ictalurus punctatus ) muscle proteins subjected to pHshift processing.

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37 Physical Properties Torsion Properties Punch Properties Cook Loss Fold Test Expressible Moisture Rheological Properties Physical Properties Torsion Properties Punch Properties Cook Loss Fold Test Expressible Moisture Rheological Properties Figure 1-2: Experimental design of the investigations into the physical properties of channel catfish (Ictalurus punctatus) muscle prot eins subjected to pH-shift processing.

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38 Chemical Properties Surface Hydrophobicity TotalSulfhydrylgroups Salt Solubility pH Solubility Reactive Suflhydryl Groups Myosin ATPase Activity Chemical Properties Surface Hydrophobicity TotalSulfhydrylgroups Salt Solubility pH Solubility Reactive Suflhydryl Groups Myosin ATPase Activity Figure 1-3: Experimental design of the investigation into the chemical properties of channel catfish ( Ictalurus punctatus) muscle proteins subjected to pH-shift processing.

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39 Structural Properties Guanidine HCl Denaturation Differential Scanning Calorimetry Susceptibility to Enzymatic Cross-linking Circular Dichroism Structural Properties Guanidine HCl Denaturation Differential Scanning Calorimetry Susceptibility to Enzymatic Cross-linking Circular Dichroism Figure 1-4: Experimental design of the investigation into the st ructural properties of channel catfish ( Ictalurus punctatus) muscle proteins subjected to pH-shift processing.

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40 CHAPTER 2 METHODS Raw Material The raw m aterial used in these studies was fresh catfish fillets obtained 1-3 days post harvest from a local supplier. Catfish fillets were only purchased which were determined to be within 3 days of packaging. The catfish fillet s were purchased and im mediately transported on ice to the laboratory and pr ocessed the same day. Preparation of Protein Isolates Protein isolates were p repared according to figure 2-1. Fresh fillets were initially ground in an Oster heavy duty food grinder (Niles, Il l., U.S.A.) for the prel iminary disruption and collection of the muscle tissue. Following gri nding, the comminuted meat was diluted 1:2 (w/v) with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds. Following homogenization, the resulting muscle tissu e slurry was further diluted to give a final dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manuall y stirred with a plastic spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the methods described below, using either 2N NaOH or 2N HCl as needed for the pH desired, with continuous manual mixing. Upon reaching the desi red pH, insoluble material was removed by centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products, Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at 5oC. Following centrifugation, the soluble middle layer was collected through a kitche n strainer with a mesh size of approximatly 0.25 mm to minimize contamination with other sepa rated materials. The soluble material was readjusted to pH 5.5 as described above. After readjustment the solu tion was centrifuged to remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The

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41 precipitated protein was collected by decanting the supernatant containing the unprecipitated proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing until the moisture content was below 80%. Mois ture content was determined using a Cenco infrared moisture analyzer (CSC Scientific, Fairfax, Va., U.S.A.). Upon completion of manual dewatering the protein isolation was complete. Preliminary unpublis hed investigation of protein isolates in this laborato ry found the shelf life of catfish protein isolates to be 5-7 days on ice. All protein isolates were stored on ice at the precipitation pH and used within 5 days. Protein Concentration Protein concentration in the is olates and the subsequent solu tions was determ ined using the Biuret method, as described by Torten and Whita ker (1964), with of 10% w/v deoxycholic acid in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any remaining lipids in the samples. Protein concentration was measured based on a standard curve based on BSA. Protein Surface Hydrophobicity Protein su rface hydrophobicity was conducted acco rding to Liang and Kristinsson (2005). To measure surface hydrophobicity th e isolate was diluted to give a stock solution of 10 mg/ml in a 20 mM tris-HCl buffer, 0.6 M NaCl, pH 7.2. The stock solution was serially diluted to obtain a concentration curve. Increasing volume (100 l, 200 l, 300 l, 400 l and 500 l) of the stock solution were added to Tris-HCl buffer to a final volume of 4.0 ml. Then, 10 l of 6propionyl-2-(dimethylamino) naphthalene (PR ODAN) (11.35 g/ml in methanol) was added and the samples mixed for ~15 seconds. After mixing the samples were then incubated for 15 min in the dark. All sample preparations and incubations were performed on i ce in disposable test tubes.

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42 After incubation, the sample wa s transferred to a fluorescence cuvette and the fluorescence emission intensity scanned between 380-560 nm with excitation at 365 nm in a Perkin Elmer LS 45 Luminescence Spectrophotometer (Norwalk, CT). As the isolate is a collection of proteins, the fluorescence peak of the samples is a rela tively flat and broad peak between 430-460 nm. The maximal fluorescence of the protein isolat e was taken and used as the wavelength for analysis. The surface hydrophobicity was calculated as the slope of the ne t fluorescence versus protein concentration (mg/ml) of the samples. Circular Dichroism (CD) CD was done accord ing to Kristinsson and Hultin (2003a). The isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris -HCl buffer, pH 7.2 with 600 mM NaCl to a concentration of 10 mg/ml. This protein stock solution of 10 mg/ml was pr epared and diluted to 2 mg/ml 1 hr before analysis and held on ice. Fo r analysis of secondary structure the sample was scanned from 260-200 nm in a 0.1 cm quartz cuvette 0.2 nm resolution scanned at 50 nm/min on a Jasco J-500C circular dich roism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The time duration of the scan was no more than 10 minutes. Differences in total alpha helix beta structure and random coil st ructures present were determined from DICHROWEB using the K2D analysis program (Lobley et. al., 2002). Isolate Susceptibility to Unfolding in Guanidine Hydrochloride ( Gu-HCl) Catfish protein isolate and the control, untr eated ground catfish muscle were treated with Gu-HCl over the range of 0-6M in 0.5 M increments. For assessment of denaturation, 222 nm was used as an indicator wave length of alpha helical content, scanning from 220-225 nm in a 0.10 cm quartz cuvette, 0.2 nm resolution sca nned at 50nm/min on a Jasco J-500C circular dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The protein

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43 concentration was 1 mg/ml. Samples were allowed to incubate for 5 minutes on ice prior to reading. Differential Scanning Calorimetry (DSC) DSC was conducted according to Fukushim a and others (2003) on a MicroCal DSC (MicroCal, LLC, North Hampton, MA). Isolate we re diluted in sample buffer (20 mM tris-HCl, pH 7.2 with 600 mM NaCl) to a protein concen tration of 10 mg/ml and homogenized for 1 minute on ice at speed 2 in a Bio-hom ogenizer (M133/1281-0, Bio Spec Products Inc., Bartlesville, OK). The sample was degassed under vacuum for 5 min at 5oC. After degassing, 0.6 ml was loaded into the sample cell, the refe rence cell contained sample buffer. The sample was then linearly heated at 1oC/min from 5oC-80oC. Analysis of the data was conducted on the software provided by the manufacturer, Origin Pr o 7.5, for determination of both exothermic and endothermic events. Reactive Sulfhydryl Groups Reactive sulfhydryl grou ps were determined according to Kim and others (2003). The isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCl buffer, pH 7.2 with 600 mM NaCl to 250 g/ml. After dilution, 80 l of 10 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) was added to the sample. This mixtur e was then incubated for 1 hr on ice. After incubation the sample was read at 420 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). Micromolar determination of SH groups per gram in the sample were done using the following equation: mlmgion concentrat sample cmmol factor dilution absorbance gSH 13600100

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44 Total Sulfhydryl Groups Total su lfhydryl groups were determined accordi ng to Kim and others (2003) The isolate was diluted and homogenized for 1 minute on i ce at speed 2 in a Bio-homogenizer (M133/12810, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCl buffer, pH 7.2 with 600 mM NaCl to 250 g/ml. After dilution, 0.5 ml of dilu ted isolate was mixed with 2.5 ml urea buffer. The urea buffer contained 8 M urea, 0.2 M Tris-H Cl, 2% SDS, 10 mM EDTA and was adjusted to pH 8.5. After mixing with urea, 50 l of 10 mM DTNB was added to the sample. The mixture was incubated in a water bath for 15 minut es at 40C. After incubation the sample was read at 420 nm using an Agilent 8453 diode ar ray UV-visible spectroscopy system (Agilent Technologies Deutschland Gm bH, Waldbronn, Germany). To tal sulfhydryl groups were calculated using the following equation. mlmgion concentrat sample cmmol factor dilution absorbance gSH 13600100 Myosin ATPase Activity Assay Myosin ATPase activity was determ ined by th e method of Perry (1955). The buffers to initiate the enzymatic hydrolysis of ATP by myosin were prepared prior to sample preparation. The reaction buffers used in this assay were 0.2 M glycine-NaOH buffer (pH 9.0), 0.1 M calcium chloride, 0.05 M ATP, sodium salt (pH 6.8) and 15% trichloroacetic acid (TCA). Liberation of inorganic phosphate was monitored in this reac tion as the determinant of enzymatic function. For the estimation of inorgani c phosphate the following buffers were used: 12% TCA, 10% (w/v) ammonium molybdate stock solution in 10 N sulfuric acid. This ammonium molybdate stock was used in the preparation of the ferr ous sulfate ammonium molybdate reagent which was made fresh the day of analysis. For analys is of myosin ATPase activity the isolate was diluted to 1 mg/ml in Tris-HCl buffer c ontaining 600 mM NaCl pH 7.2 and homogenized

PAGE 45

45 thoroughly. This was kept on ice until needed. In separate tubes for each reaction, prior to isolate addition, the reaction buffer was prepar ed by adding 1.3 ml glycine-NaOH buffer, 0.2 ml calcium chloride, 0.3 ml ATP. This mixture was then incubated at 25oC for 5 min to allow the temperature to equilibrate. After temperature equilibration, 0.2 ml of the individual isolate solution was added to each reaction tube with proper mixing and allowed to incubate at 25oC for 5 min. After 5 min, 1 ml of 15% TCA was added to each reaction tube to stop the reaction. This was centrifuged for 10 minutes at 25,000 x g to precip itate the proteins from solution and leaving liberated phosphate from ATP in solution. Next 0.5 ml of supernatant was added to 3.2 ml of 12 % TCA with good mixing and left to stand at 25oC for 10 minutes. This so lution (3.0 ml) of this solution was removed and mixed with 2 ml of the ferrous sulfate ammonium molybdate reagent and incubated at room temperature for 1 min. After 1 min of incubation the solution was transferred to a cuvette and read at 363 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The color of this reaction in tensifies with time so only one reactio n was done at a time to standardize the incubation time of all samples to 1 min. The results of myosin ATPase activity were assayed as relative results to the c ontrol. The control absorbance was set to 100% and the percent activity of the isolates was calculated from the ab sorbance of the samples relative to the control. sample control sample treatedpH activity Susceptibility of Proteins to Transg lutaminase-Induced Cross Linking The susceptibility of pH-shift processed cat fish m uscle to protein cross linking was assayed using the commercial transglutaminase (TGase) Activia TI from Ajinomoto LLC (Ajinomoto Food Ingredients LLC Chicago Il). 0.2% TGase (w/w) was added to isolate diluted to 10% solids in 20mM Tris-HCl buffer, 600mM NaCl. TGase activity was assayed by using an

PAGE 46

46 AR2000 advanced research rheometer (TA Instrument New Castle, DE) with a head with a flat cross-hatched polyacrylic surface and thermally controlled plate. The gap was 1000 microns and the head lowered onto the sample using the cont rolled speed function provided by the software. The samples were tested in oscillatory mode under controlled frequency at 0.1 Hz, and strain controlled at 0.01. TGase activity was monitore d by the increase in G over 1 hour at 30oC. Activity was compared against samples with no TG ase added. G was plotted against time and the slope of the line from linear regression was used to calculat e the TGase activity. The activity coefficient was calculated as the ratio of treated to untreated samples. Oscillatory Rheology For the gelation tests, the isolates were rehydr ated to 10% protein and pH adjusted to 7.2 with 20 m M Tris-HCl and HCl/NaOH. For the added NaCl samples, NaCl was added at 2% w/w. The sample was homogenized at speed 1 using an Ultra-Turrax T 18 homogenizer. The protein paste was then transfe rred to a peltier plate at 5C attached to an AR2000 advanced research rheometer (TA Instrument, New Castle, DE) and a head with a flat cross-hatched polyacrylic surface. The gap was 1000 microns and the head lowered onto the sample using the controlled speed function provided by the software. The isolates were subjected to heating (580C) and cooling (80-5C), with testing conducted in oscillation mode. The storage modulus (G), loss modulus (G) and tan delta ( ) was followed as a functi on of heat and time. All experiments were done under a controlled stra in of 0.01 and controlled frequency of 0.1 Hz. Torsion Gel Testing Protein paste was prepared as above, except at 20% protein concentra tion. Proteins were stuffed in cylindrical tubes 10 cm long by 1.8 cm in diameter and cooked at 80C for 20 min in a water-bath. The interior wall of the tube was coated with a lecithin based spray. The cooked samples were then removed from the casing afte r cooling for 30 min on ice, held overnight on

PAGE 47

47 ice, and subsequently cut into 2.54 cm long pieces. These pieces were glued to plastic disks (Gel Consultants Inc., Raleigh, N.C.) using an inst ant adhesive, Krazy glue (Toagosel Co., LTD. Nishi-shinnbashi Minato-Ku, Tokyo). Dumbbell shaped samples were then milled from each sample to 1 cm diameter in a machine mill (Electro Sales Co., Somerville, Mass., U.S.A.), wrapped in plastic wrap to prevent moisture lo ss, and brought to room temperature prior to torsion testing. For testing, 4 gel specimens were vertically mounted and sh eared to the point of fracture at 2.5 rpm in a modified Brookfield viscometer (Gel C onsultants Inc., Raleigh, N.C). Stress (kPa) and strain (dimensionless) at frac ture were calculated with the manufacturers software for each sample, corresponding to the strength and deformation of the gels, respectively (Hamann and others 1990). Punch Test Punch test sam ples were prepared by adjusting protein isolates to 20% solids according to the moisture content and stuffed into plastic tubi ng, 1.25 inches in diameter. These samples were cooked for 20 min at 80C, and then cooled rapidly in ice water. Samples were air freighted the same day they were cooked to the laborator y of Dr. Herbert O. Hultin in Gloucester MA (University of Massachusetts Ma rine Station) where punch tes ting was conducted The samples were cut to 1.0 inch length and 1.25 inch diamet er. The sample was placed on the machine table and the head adjusted to just above the sample surface. A 5 mm head was used at a speed of 1.0 cm/min with force and distance at failure repo rted. Punch testing was conducted on a Rheo Tex Model AP-83 (Sun Science Co., Seattle, Wa). Fold Test A gel fold test was conducted according to Nowsad and others (2000). The test was conducted by slicing 2-mm thick sample disc fro m the cylindrical gels, mentioned above, and folding them into halves and quarters. The scales are: 5= no crack when folded into a quarter,

PAGE 48

48 4=no crack when folded into half but crack when folded into quarter, 3=crack when folded into half, 2=broke and split into halves when folded into half, 1=broke and split prior to being folded into half. Gel Water Holding Capacity Gel water holding capacity was conducted according to Nowsad and others (2000). Expressible moisture of the gels was determ ined by com pressing a 1.0 g spherical gel slice between 4 double layers of filter paper at a pressure of 20 Kg/cm2 for 1 min and calculated from weight before and after the compression. Expressi ble moisture was determined as the percentage of original weight after compression. Cook Loss Cook loss of gels was determ ined as the weight of the protein paste before heat treatment compared to the weight of the gels after cooking. Gels were prepared and heated as described above (torsion gel testing). Af ter cooking the gel was removed from the tube, lightly dried and reweighed. The weight of the tube was subtracted from the total initial weight and then compared to the final weight of the cooked gel. Percent cook loss was determined by: 100 1 WeighTube Weight Initial WeightGel LossCook Cook loss is in percent, gel we ight is the weight of the c ooked protein paste after being removed from the tube, initial weight is the ca pped tube plus the raw ge l stuffed inside, tube weight is the weight of the tube after the gel has been cooked and removed from the tube. Heating Rate Gelation Studies Heating rate gelation studies were conducted at North Carolin a State University in the laboratory of Dr. Tyre Lanier, Raleigh, NC. To rsion tubes, 10 cm in length and 1.8 cm in diameter were filled with protein pa ste and subsequently heated from 10oC to a 70oC internal endpoint, either rapidly by a cylindrical mi crowave (Riemann and others 2004b), at 20 or

PAGE 49

49 98oC/min to test rapid heati ng effects, slowly at 1oC/min by immersion in a programmable water bath or placed directly in an 70C water bath for 15 minutes. Temperatures were measured by fiber optic probe (Riemann and others 2004b). Upon reaching 70C, rapidly heated samples were held for 0 or 20 min prior to rapid cooling by immersion in ice water for sufficient internal cooling to <10C for ~30 min. Samples were held static in a cylindrical microwave applicator (length 16 cm, radius 12.5 cm) and heated usi ng power settings calcula ted previously. After reaching a final temperature of 70C, this te mperature was maintained in gels during the subsequent holding period by utilizing feedb ack software (Riemann and others 2004b). Rheological changes (storage modulus, G) of pastes/gels were non-destructively and continuously measured as pastes were heated, held and cooled using a 40 mm, 4 degrees slope cone and plate attachment of a constant stress, small strain rheometer (Stresstech, Rheologica instruments AB, Lund, Sweden). Oscillation parame ters were those used in Riemann and others (2004). Heating conditions were at either 20C/mi n, the most rapid heating rate possible for this apparatus, 0.5C/min, 1C/min, 2C/min or 5C/min to an endpoint temperature of 70C followed by holding for 0 or 20 min prio r to cooling at 5C/min to 10C. Protein Solubility as a Function of pH The isolate was diluted to 10 m g/ml in DI water with thorough ho mogenization using a tissue homognizer (Ultra-Turrax T18, IKA Works Inc. Wilmington, NC). After dilution in water, 2 ml diluted protein was added to 2 ml buffer with good mixing. The pH range was from 1.5-12 in 0.5 increments. For pH 1.5-6.5, 2 mM citric acid buffer was used and for pH 7.0-12, 2 mM sodium phosphate buffer was used. The buffers were adjusted to the desired pH prior to protein addition. After protein addition, the mixture was allowed to incubate on ice for 10 minutes. After incubation the protein solu tion was centrifuged at 3,000 x g for 10 minutes. After centrifugation, the supernat ant was sampled for protein c oncentration using the biuret

PAGE 50

50 method (Torten and Whitaker 1964). Protein concen tration was calculated fr om a standard curve based on bovine serum albumin (BSA). Percent sol ubility was then calculated as a percentage of an uncentrifuged control by the following equation: totalmlmgprotein ernant mlmgprotein / /sup/ Protein Solubility as a Function of Salt Concentration The isolate was diluted to 5 m g/ml in 20 mM Tris-HCl at pH 7.2 without added NaCl with thorough homogenization using an U ltra-Turrax T18. After dilution in buffer, 4 ml was taken and the appropriate amount of NaCl added to each tube to achieve 0 mM, 150 mM, 300 mM 450 mM or 600 mM concentrations. After the appropriate NaCl concentration was achieved, the samples were centrifuged at 3000 x g for 10 minutes at 5C. After centrifugation, the supernant was collected in duplicate and soluble protein was determined according to the biuret method (Torten and Whitaker 1964). The soluble protein was compared to an uncentrifuged control. Percent soluble protein was calculated as totalmlmgprotein ernant mlmgprotein / /sup/ Statistical Analysis Experim ental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on replicate isolations as discussed above. A repl icated (N=2) was determined as acceptable due to achieving an acceptable power ( =0.80, =0.05). One-way independent measures analyses of va riance were used to examine the effects of all methods used except for the analysis of pH solubility and salt solubility which were tested with 2-way ANOVA analysis. The Kruskal-Wallis ANOVA testing by ranks was used on data which did not pass normality. Post hoc anal ysis was conducted only in the presence of significant population differences. ANOVA sta tistical comparisons were conducted with

PAGE 51

51 SigmaStat, (Systat Software Inc. San Jose Ca) with a significance level of p<0.05. After SigmaStat completed the ANOVA analysis, the post hoc analysis recommended by SigmaStat was used. In most cases this was Tukeys test. Dunns test was used fo r all punch test analysis; force, distance and jelly streng th. The Holm-Sikak method was used with the torsion stress, expressible moisture and ATPase activity. Th e two-way ANOVA analysis used the Holm-Sidak pair wise comparisons. The two-way ANOVA tables and the pos t hoc tests are reported in appendix A. The heating rate study was analyzed with Graph Pad QuickC alcs online calculator software (Graph Pad Software Inc. San Diego Ca). Analysis used an unpaired t-test with a manual bonferoni correction, overa ll significance of p<0.05 with th e individual significant pvalues of p<0.004.

PAGE 52

52 Ground Fish Homogenization 1 part fish : 6 parts water pH reduction or increase Acid (2M HCl) / Alkali (2M NaOH) SedimentLayer Membrane lipids Centrifugation Solution Phase Soluble muscle proteins Upper Layer Neutral lipids Protein aggregation pH adjusted to 5.5 Sediment = Protein isolate Centrifugation Supernatant Mostly water, can bereused Ground Fish Homogenization 1 part fish : 6 parts water pH reduction or increase Acid (2M HCl) / Alkali (2M NaOH) SedimentLayer Membrane lipids Centrifugation Solution Phase Soluble muscle proteins Upper Layer Neutral lipids Protein aggregation pH adjusted to 5.5 Sediment = Protein isolate Centrifugation Supernatant Mostly water, can bereused Figure 2-1: The process used in acid and alkali pH shift processing.

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53 CHAPTER 3 CHANNEL CATFISH ( I ctalurus punctatus ) MUSCLE PROTEIN ISOLATE PERFORMANCE PROCESSED UNDER DIFFERING ACID-AID ED AND ALKALI-AIDED pH VALUES. Introduction The need for an increase in the production of seaf ood products has become a clear concern with international seafood shorta ges projected to be seen as early as 2015 (FAO Subcommittee on Aquaculture, Beijing China 2002). One of th e methods which may alleviate some of the stress currently on the seafood marked is the utilization of byproducts. Byproducts are considered what is left over fr om the whole fish after the primary products have been removed. In catfish these are the fillet and the belly flaps (nugget). These byproducts provide an inexpensive raw material which increases the tota l utilization, profitabili ty and management of the fisheries resources. The production of catfish in the United States has been steadily increasing for the last 35 years (Harvey 2002). In the fille ting process the utilization based on live weight is at best 45%, comprising fillets and nuggets. The byproducts are the frame and cutoffs, these portions of the fillet/nugget are not acceptable to the consumer. This is a substantial amount of usable material that, with the appropriate pro cessing, may be used for the pr oduction of value-added protein isolates. A relatively new process develope d by Hultin and coworkers (Hultin and Kelleher 1999) utilizes the solubilization properties of specific muscle tissues, primarily myofibrillar proteins, at extreme low or high pH. This property of myofibril proteins allows for the removal of membranes and neutral lipi ds, connective tissue, bones, sc ales and other non-solublized components. The effect of pH-shift processing on muscle prot eins has been studied by several workers. Currently it has been demonstrat ed that pH-shift pr ocessing can modify the physical properties of the resulting protein isolate. This has been demonstrated in species ranging from cold water

PAGE 54

54 fish to land animals. The modification of physi cal properties of pH-shift produced isolates has resulted in a wide range of effects. Acid (pH 2.5) treatment of myofibrillar proteins showed a decrease in the storage modulus of cod muscle proteins where as alkali (pH 11.0) treatment has shown an increase in the storage modulus when compared to a surimi control (Kristinsson and Hultin 2003b). An increase in storage modulus refers to a stiffer gel and might indicate that a treatment may lead to stronger and better gels. In Atlantic croaker, however, both acid (pH 2.5) and alkali (pH 11.0) processing showed an increase in the storage modulus as compared to a surimi control (Kristinsson and Liang 2006). Y ongsawatdigul and Park (2004) made rockfish protein isolates using both acid and alkali pH -shift processing and compared their gelation properties with washed muscle and whole musc le homogenates. Compared to muscle and washed muscle, isolates from alkali-aided proces sing had an increase in gel elasticity and force needed to penetrate the gel while isolates from acid-aided processing showed a decrease in both (Yongsawatdigul and Park 2004). It is unknown what the specific effects of solubilizing muscle proteins at different high or low pHs will have on the functional properties of recovered muscle proteins. Muscle gelation is a multi step process with the final result being a three dimensional network. This network may be studied utilizi ng different methods whic h provide information on the various mechanisms involved in its forma tion and its properties. The modifications, treatments and additives of a formulated muscle product are a directly infl uence of the final gel network formed. The changes to the three dime nsional gel structure by utilizing the acid or alkali solubilizing process have shown changes in gel strength which can be determined using the Hamann torsion test and the punch test. Small st rain oscillatory testing on isolates made with the pH shift process has shown increases in G both after heating (~80C) and upon cooling

PAGE 55

55 (~5C) (Park 2005). However, decreases have al so been shown with acid processed protein isolates. Inconsistent results from acid pH processing raise the question of a species/pH relationship. Acid and alkali solubilization may be done ove r a range of pH. The muscle proteins of interest, mainly actin and myosin, are solubilized under acidic conditions between 2.0-3.0 and 10.5-11.5 under basic conditions. This can yield protei ns of varying levels of unfolding. It is hypothesized that utilizi ng different solubilizing conditions within this pH range will produce differences in gel forming properties and also differences in the physical properties in the final gel. Methods Raw Material The raw m aterial used in these studies was fresh catfish fillets obtained 1-3 days post harvest from a local supplier. Catfish fillets were only purchased which were determined to be within 3 days of packaging. The catfish fillet s were purchased and im mediately transported on ice to the laboratory and pr ocessed the same day. Preparation of Protein Isolates Protein isolates were p repared according to figure 2-1. Fresh fillets were initially ground in an Oster heavy duty food grinder (Niles, Il l., U.S.A.) for the prel iminary disruption and collection of the muscle tissue. Following gri nding, the comminuted meat was diluted 1:2 (w/v) with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds. Following homogenization, the resulting muscle tissu e slurry was further diluted to give a final dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manuall y stirred with a plastic spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the methods described below, using either 2N NaOH or 2N HCl as needed for the pH desired, with

PAGE 56

56 continuous manual mixing. Upon reaching the desi red pH, insoluble material was removed by centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products, Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at 5oC. Following centrifugation, the soluble middle layer was collected through a kitchen strainer with a mesh size of approximately 0.25 mm to minimize contamination with other sepa rated materials. The soluble material was readjusted to pH 5.5 as described above. After readjustment the solu tion was centrifuged to remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The precipitated protein was collected by decanting the supernatant containing the unprecipitated proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing until the moisture content was below 80%. Mois ture content was determined using a Cenco infrared moisture analyzer (CSC Scientific, Fairfax, Va., U.S.A.). Upon completion of manual dewatering the protein isolation was complete. Preliminary unpublis hed investigation of protein isolates in this laborato ry found the shelf life of catfish protein isolates to be 5-7 days on ice. All protein isolates were stored on ice at the precipitation pH and used within 5 days. Protein Concentration Protein concentration in the is olates and the subsequent solu tions was determ ined using the Biuret method, as described by Torten and Whita ker (1964), with of 10% w/v deoxycholic acid in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any remaining lipids in the samples. Protein concen tration was measured based on a standard curve based on BSA.

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57 Oscillatory Rheology For the gelation tests, the isolates were rehydr ated to 10% protein and pH adjusted to 7.2 with 20 m M Tris-HCl and HCl/NaOH. For the added NaCl samples, NaCl was added at 2% w/w. The sample was homogenized at speed 1 using an Ultra-Turrax T 18 homogenizer. The protein paste was then transfe rred to a peltier plate at 5C attached to an AR2000 advanced research rheometer (TA Instrument, New Castle, DE) and a head with a flat cross-hatched polyacrylic surface. The gap was 1000 microns and the head lowered onto the sample using the controlled speed function provided by the software. The isolates were subjected to heating (580C) and cooling (80-5C), with testing conducted in oscillation mode. The storage modulus (G), loss modulus (G) and tan delta ( ) was followed as a functi on of heat and time. All experiments were done under a controlled stra in of 0.01 and controlled frequency of 0.1 Hz. Torsion Gel Testing Protein paste was prepared as above, except at 20% protein concentra tion. Proteins were stuffed in cylindrical tubes 10 cm long by 1.8 cm in diameter and cooked at 80C for 20 min in a water-bath. The interior wall of the tube was coated with a lecithin based spray. The cooked samples were then removed from the casing afte r cooling for 30 min on ice, held overnight on ice, and subsequently cut into 2.54 cm long pieces. These pieces were glued to plastic disks (Gel Consultants Inc., Raleigh, N.C.) using an inst ant adhesive, Krazy glue (Toagosel Co., LTD. Nishi-shinnbashi Minato-Ku, Tokyo). Dumbbell shaped samples were then milled from each sample to 1 cm diameter in a machine mill (Electro Sales Co., Somerville, Mass., U.S.A.), wrapped in plastic wrap to prevent moisture lo ss, and brought to room temperature prior to torsion testing. For testing, 4 gel specimens were vertically mounted and sh eared to the point of fracture at 2.5 rpm in a modified Brookfield viscometer (Gel C onsultants Inc., Raleigh, N.C). Stress (kPa) and strain (dimensionless) at frac ture were calculated with the manufacturers

PAGE 58

58 software for each sample, corresponding to the strength and deformation of the gels, respectively (Hamann and others 1990). Punch Test Punch test sam ples were prepared by adjusting protein isolates to 20% solids according to the moisture content and stuffed into plastic tubi ng, 1.25 inches in diameter. These samples were cooked for 20 min at 80C, and then cooled rapidly in ice water. Samples were air freighted the same day they were cooked to the laborator y of Dr. Herbert O. Hultin in Gloucester MA (University of Massachusetts Ma rine Station) where punch tes ting was conducted The samples were cut to 1.0 inch length and 1.25 inch diamet er. The sample was placed on the machine table and the head adjusted to just above the sample surface. A 5 mm head was used at a speed of 1.0 cm/min with force and distance at failure repo rted. Punch testing was conducted on a Rheo Tex Model AP-83 (Sun Science Co., Seattle, Wa). Fold Test A gel fold test was conducted according to Nowsad and others (2000). The test was conducted by slicing 2-mm thick sample disc fro m the cylindrical gels, mentioned above, and folding them into halves and quarters. The scales are: 5= no crack when folded into a quarter, 4=no crack when folded into half but crack when folded into quarter, 3=crack when folded into half, 2=broke and split into halves when folded into half, 1=broke and split prior to being folded into half. Gel Water Holding Capacity Gel water holding capacity was conducted according to Nowsad and others (2000). Expressible moisture of the gels was determ ined by com pressing a 1.0 g spherical gel slice between 4 double layers of filter paper at a pressure of 20 Kg/cm2 for 1 min and calculated from

PAGE 59

59 weight before and after the compression. Expressi ble moisture was determined as the percentage of original weight after compression. Cook Loss Cook loss of gels was determ ined as the weight of the protein paste before heat treatment compared to the weight of the gels after cooking. Gels were prepared and heated as described above (torsion gel testing). Af ter cooking the gel was removed from the tube, lightly dried and reweighed. The weight of the tube was subtracted from the total initial weight and then compared to the final weight of the cooked gel. Percent cook loss was determined by: 100 1 WeighTube Weight Initial WeightGel LossCook Cook loss is in percent, gel we ight is the weight of the c ooked protein paste after being removed from the tube, initial weight is the ca pped tube plus the raw ge l stuffed inside, tube weight is the weight of the tube after the ge l has been cooked and removed from the tube. Statistical Analysis Experim ental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on replicate isolations as discussed above. A repl icated (N=2) was determined as acceptable due to achieving an acceptable power ( =0.80, =0.05). One-way independent measures analyses of va riance were used to examine the effects of all methods used. The Kruskal-Wallis ANOVA tes ting by ranks was used on data which did not pass normality. Post hoc analysis was conducted only in the presence of significant population differences. ANOVA statistical comparisons were conducted with SigmaStat, (Systat Software Inc. San Jose Ca) with a significance level of p<0.05. After SigmaS tat completed the ANOVA analysis, the post hoc analysis recommended by Si gmaStat was used. In most cases this was

PAGE 60

60 Tukeys test. Dunns test was used for all punch te st analysis; force, dist ance and jelly strength. The Holm-Sikak method was used with the to rsion stress and expressible moisture. Results Rheological Changes in Protein Iso lates Durin g Thermal Gelation The storage modulus (G) measures the resistance of a material to positive movement of a probe. Positive movement is the duration of mo vement during the oscillation of the head which is moving away from the zero point. G is used to determine the solid fraction of a material during testing. In this study G was followed with temperature to assess the change in the solidlike characteristics of prot ein isolates. G showed a decrease for all isolates without added NaCl from 5C until 45C, and after 45C, G began to increase (figure 3-1, 3-4, 3-7, 3-10, 3-13, 316). For isolates made under acidic conditi ons, G increased until 60C for pH 2.0 samples without added NaCl and pH 2.5 samples with and without NaCl and then began to decline until the final temperature of 80oC was reached (figures 3-1, 3-4, 325). The other isolates produced under acidic conditions, pH 2.0 with added NaCl and pH 3.0, exhibited a decrease in G until ~72C. Acid isolates with added NaCl showed an initial decrease in G which ended at ~30C. Further decrease in G was then observed at about 70-72C. Isolates produced under basic conditions with and without added NaCl did not show a second decrease in G af ter the initial decrease ending at 45C. The G of gel pastes with no salt added prio r to heat treatment (figure 3-43) were not significantly different (p>0.05) within the acid treated samples. The alkali processed gel paste treatment pH 11.5 was significantly higher (p<0.05) in G than pH treatment 10.5, but there were no other significant differences between the indi vidual alkali treated pH treatments. Between individual acid and alkali pH treatments, pH 2.5 was significantly lower (p <0.05) in G than all alkali pH treatments and pH treatment 2.0 was significantly (p<0.05) lower in G than pH

PAGE 61

61 treatment 11.5. The control sample was significan tly lower (p<0.05) in G than all alkali pH treatments but was not significantly different (p>0.05) from any of the acid treatments. The G of the gel paste with 2% added salt (figure 3-46 ) was not significantly di fferent (p>0.05) within the acid treated samples. The alkali treatment pH 11.5 was significantly (p<0.05) higher in G than pH treatments 2.5, 3.0 and 10.5. The control pa ste was significantly (p <0.05) higher in G than pH treatments 2.5, 3.0 and 10.5. No other si gnificant differences (p>0.05) were present between gel pastes. Samples treated at low pH had G values si gnificantly lower (p<0.05) than samples treated at high pH after heating at 80 C and after cooling to 5C (Fig ure 3-45). This difference was found for treatment conditions c ontaining NaCl and also for those without added NaCl. The differences between low and high pH treatment were not the same in the presence or absence of added salt. Without the addition of salt pH treatments 2.0 and 2.5 were significantly different (p<0.05) from pH treatment 11.5 with pH treat ment 2.5 also being significantly different (p<0.05) from pH treatment 11.0 when heated to 80oC and when cooled to 5oC. With the addition of salt pH treatments 3.0 and 2.5 were significantly different (p<0.05) from pH treatment 11.5 after cooling to 5oC however after heating to 80oC and before cooling pH treatment 3.0 was significantly different (p< 0.05) from pH treatments 11.0 and 11.5. The rheology studies also provi ded information about the loss modulus (G) of the protein isolates as well as the storage modulus. The loss modulus is considered th e viscous or liquid like fraction of a viscoelastic material The G of catfish protein isolates in the absence of salt are shown in figures 3-2 (pH 2.0), 3-5 (pH 2.5), 3-8 (pH 3.0), 3-11 (pH 10.5), 3-14 (pH 11.0) 3-17 (11.5) and 3-20 (whole muscle). The G of catfish protein isolates treated in the presence of salt are shown in figures 3-23 (pH 2.0), 3-26 (p H 2.5), 3-29 (pH 3.0), 3-32 (pH 10.5), 3-35 (pH 11.0),

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62 3-38 (pH 11.5) and 3-41 (whole muscle). The changes in G during both the heating and cooling phase in acid treated samples without ad ded salt showed a decline in G during heating with the minimum at the end of the heating phase, 80oC. During the cooling phase the G increased, after decreasing during heating. The G after cooling was slig htly higher than the starting G but did not show the in creases seen in G. Alkali tr eated samples in the absence of added salt showed a decrease in G during heating but the decrease during heating in all alkali samples was characterized by a sharp dip beginning below 40oC and ending just below 60oC. The increase in G after the minimum point of th e dip did not continue to increase but began to decrease again. Untreated muscle showed a similar trend to alka li treated samples but showed a more gradual decline before increasing. After th e increase in G during h eating of the untreated muscle, G began to decline at 70oC whereas the alkali treated sa mples after a minor increase, began to decrease at 60oC for pH treatments 11.0 and 11.5. Samples from the pH 10.5 treatment however showed a similar trend to the untreated muscle at higher temp eratures, beginning to decrease in G at 70oC. In the presence of added salt, pH treated samples showed a G trend similar to that of G though the value was lower. Tan delta is the relationship of G/G which provides an analysis of the transition of a material from a more viscous material to a more elastic material. A perfectly elastic material is represented as tan delta equal to 0, or stress a nd strain are perfectly in phase and a perfectly viscous material is represented as tan delta equal to 90 or stress and strain are perfectly out of phase (Park 2005). Tan delta was analyzed in all samples by merging and smoothing replicate rheograms. The tan delta results are shown in figures 3-3 (pH 2.0), 36 (pH 2.5), 3-9 (pH 3.0), 312 (pH 10.5), 3-15 (pH 11.0), 3-18 (pH 11.5), 3-21 (w hole muscle) for samples treated without salt. In the absence of salt the tan delta transiti on showed more of a peak increase in tan delta

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63 prior to the start of the decline in tan de lta. The decline in tan delta began at 37oC in samples subjected to pH-shift processing. The whole muscle samples did not show a peak increase but a gradual increase in tan delta leading to a gradual decline in the absence of added salt. Samples treated with either acid or alkali processing in th e presence of added salt is shown in figures 3-24 (pH 2.0), 3-27 (pH 2.5), 3-30 (pH 3.0), 3-33 (p H 10.5), 3-36 (pH 11.0), 3-39 (pH 11.5) and 3-42 (whole muscle). Samples treated with either acid or alkali processing sh owed a gradual decline in slope beginning between 19-22oC during heating and did not incr ease after this decline began. The tan delta for the control however increased to between 28oC and 42oC with the peak ranging over 5oC from 42-47oC with no change in tan delta then began the decrease, forming a bell curve. After the decline began at 47oC no increase in tan delta was observed. Gel Quality of Isolates as Assessed by Torsion Testing The results of torsion testing are shown in figures 3-49, 3-50. Alkali aided isolate gels 11.0 and 11.5 had significantly higher (p< 0.05) stress and strain values than acid aided isolate gels or control. Acid and alkali treated isolate gels were significantly higher in stress (p< 0.05) but not significantly different (p> 0.05) in strain from the contro l. Alkali aided isolate gels showed significant differences (p< 0.05) within the high pH gr oup, with pH 11 samples showing significantly higher stress differences (p< 0.05) from pH 10.5 and 11.5, with no significant differences (p>0.05) between isolates made at pH 10.5 or 11.5. There were no significant differences (p>0.05) in strain within the alkali aided isolate. Significant differences (p<0.05) were observed between all pH treatments compared to the control for stress but no differences were observed in strain values. Gel Quality of Isolates as Assessed by Punch Testing Acid and alkali aided isolate gels 2.5, 10.5, 11.0 and 11.5 had a significantly higher (p< 0.05) break force than control sam ples shown in figur es 3-51. The distance required to break all

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64 of the gels did not show signi ficant differences (p>0.05) except pH 3.0 was significantly lower (p<0.05) than pH 10.5, and the control. The contro l had a significantly lower (p<0.05) force than pH treated samples 2.0, 10.5, 11.0 and 11.5. The cont rol was significantly higher (p< 0.05) in the distance needed to break the gel than gels ma de with isolate from pH 3.0 treatment. No other significant differences (p>0.05) were observed between the cont rol and pH treated samples. Gel Quality of Isolates as Assessed by Expressible Moisture The results from expressible moisture testing of the samples are shown in figure 3-54. The results of the study show gels made with acid-ai ded isolates have higher expressible moisture than gels made with alkali-aided isolates. Significant differences (p<0.05) were observed between gels made with alkali treatments 11.0 an d 11.5 when compared to the acid treatment at pH 2.0 and the control. Other individual pH treatme nts from acidor alkaliaided isolates did not show any significant differences (p>0.05). Gel Quality of Isolates as Assessed Fold Test The fold test conducted in this study is show n in figure 3-55. These results did not show any significant differences (p>0.05) between gels from the pH-shift process and the control gels. All gels made with isolates from the alkali-aided process treatment and the control gels scored a 5, which is the highest score. The gel made with the isolate from the acid-aided process ranged from 2.5-4, but were not significantly different from the control and the alkali-aided gels. Gel Quality of Isolates as Assessed by Cook Loss The cook loss results of this st udy are shown in figure 3-56. Th e results of this study did not show any significant differences (p>0.05) between gels made with isolates from the acidaided process as compared to gels made with isolates from the alkali-ai ded process. Individual pH treatments within samples treated at low pH or at high pH did not show any significant differences (p>0.05). Individual pH treatments or grouped pH treatments did not show any

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65 significant differences (p>0.05) as compared to the whole muscle contro l except pH treatment 11.0 was significantly (p<0.05) lower than the control. Discussion The G or rigidity m odulus is the resistance to gel structural move ment of the network which is being formed. G is derived in osci llatory testing as the fraction of the sigmoid movement of the head which is positive or nega tive displacement from 0 as compared to the fluid fraction of oscillatory measurement which is the return to 0 from positive or negative movement of the head. As the myofibrillar prot eins set into a gel structure the resistance to movement of the curing system is increased by molecular interac tions. The rheological testing of isolate measured non-fracture parameters using micro-strain osci llatory testing. The resistance to movement, or decreasing fluidity of the gel network is shown as increased G. The rheological findings of this study show that pH treatments used to produce isolate significantly influence the final rigidity of the thermally set gel. The protein paste prior to heat treatment, in both the presence and ab sence of salt, showed the same trend as final gel rigidity of the thermally set and cooled gels (figures 3-43 and 3-45 (no salt, respectively) and 3-46 and 3-48 (added salt, respectively)) except for pH 3.0 treatment. The pH 3.0 treatment was equal to pH 2.5 treatment prior to heating, but after heating decreased in G relative to the other samples. Th e trends of the gel paste prior to heat treatment may provide an indication of the relationship of the final gel rigidity of the thermally treated muscle paste with the pH shift processed catf ish muscle used in this study; however the relative incr ease [(final G/ initial G)*100] in gel rigid ity from the raw paste to the final cooked gel ranged from 2002000%. Due to the consistent trend of the raw past e with the final gel rigidity, standardizing the start point of rheological testing (initial Gfinal G) did not affect the final results of the testing even with the out of trend raw paste seen in pH treatment 3.0.

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66 The acid treated samples had a higher suscep tibility to movement whereas alkali treated samples had a decreased susceptibility or increased structural rigidity (G ) when compared to untreated muscle. These results are inconsistent with results shown for Atlantic croaker which showed an increase in gel rigidity over musc le without pH-shift pr ocessing, for both acid (pH 2.5) and alkali (pH 11.0) treatments (Kristinsson and Liang 2006). Isolates produced at pH 2.0 with added salt increased above th at of the untreated catfish musc le. It can be concluded from the differentiation of gel rigidity as indicated by G that pH pr ocessing at differe nt low or high pH values allows for the modula tion of the gel strength. This modulation of gel strength can now be tailored as the final product requires. The rheological results of acid aided isolate ma de from the lowest pH treatment (highest degree of ionization at the low pH values tested) show gel rigidity increased above that of the untreated control in the presence of added salt (f igure 3-48). When the treatment pH used was to achieve solubility and separati on was less extreme (pH 2.5 and 3), the rigidity of the gel was equal to or less than the control. These re sults are similar to t hose shown by Raghavan and Kristinsson (2007a), where ca tfish myosin increased in gel rigi dity as pH of acid solubilization decreased. Previous studies on ac id solubilization of muscle proteins from sources other than catfish have shown acid solubili zation increasing gel strength above that of untreated controls (Kristinsson and Liang 2006; Mireles Dewitt and others 2007). However, the solubilization pH used in these experiments was not as low as the mo st extreme pH used here. The increase in gel rigidity above the control in only the most extr eme pH treated sample indicates that a high net positive charge which influences the level of unfolding is necessary to produce increased gel rigidity above that of an untreat ed control is species specific. Studies have demonstrated that extreme pH may lead to a partially refolded stat e, often called a molten globule. This modified

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67 structure may be predisposed to promote more protein-protein interact ions which can promote the formation of gels and produced stronger gels via more extensive protei n-protein interactions during thermal processing. Rheologi cal studies on isolated muscle proteins, specifically catfish myosin, have shown that acid trea tment resulted in a gel which in creased in G above untreated control (Raghavan and Kristinsson 2007a). Increa sing gel rigidity of catfish myosin with less extreme ionization/unfolding than that used here, indi cates the contribution of other myofibril proteins co-precipitated with myosin in the w hole protein system (Kristinsson and others 2005b) after pH adjustment will greatly in fluence the final rigidity an isolated muscle system is able to achieve. Thus, when using a whole muscle system it is important to optimize the pH used. The higher low pH values used to make acid aided cat fish protein isolates (i n the presence of salt) resulted in lower G, These results indicate that treatment with less [H+] subjected to the muscle proteins produces isolates wh ich do not promote the stronger in teractions occurring with the isolates made with the treatments with the highest level of [H+]. No significant differences (p>0.05) were obs erved between alkali treated gels in the presence or absence of added NaCl. Previous studies investigating multiple pH treatments on tilapia white muscle reported an improvement of gel properties with alkali treatment but decreased gelling ability of acid treated proteins (Ingadottir 2004). The increase in gel rigidity with alkali treated samples is consistent with pr eviously published data on pH-shift processed muscle at alkali pH. The lack of differences in alkali treated catfish sa mples indicates that the molecular transitions induced by alkali pH treatment produces prot ein structures (in the presence of 2% NaCl) with similar interact ion potentials at all al kali pH values teste d,, thus resulting in unfolded structures which have similar gel formi ng abilities. As muscle gels are formed in the presence of salt, it may be concluded that alkali solubilized isolate produ ces a gel network with

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68 the highest resistance to compressi bility of the samples tested and all alkali treatments tested results in gel networks with equi valent non-fracture compressibility. During the heating phase, all isolates and the control in the absence of added salt demonstrated a similar decline in G until about 45oC, which indicates that the molecular interactions induced by the therma l input during this range were similar for all isolates under the conditions tested. In the absen ce of salt, and at the pH tested there is a significant repulsion between the muscle proteins at low temperatures, which may be modified as the system is heated, leading to a more fluid system, hence lower G as seen here in this study. Above approximately 45oC, all isolates and control samples with no added salt demonstrated an increase in G, indicating the developmen t of thermally induced protein-protein in teractions. While alkali-aided isolates made from pH 11 and 11.5 treatments (figures 3-13 and 3-16) showed a gradual increase in G until 80C (maximum point of heating), the acid-aided isolates (figures 31, 3-4 and 3-7) peaked at about 60C (pH 2.0 and 2.5) and about 70C (pH 3.0), demonstrating they had different and weaker interactions than the alkali-aided isolates. The isolate made with pH 10.5 treatment (figure 3-10) had a similar ge lation behavior as the acid-aided isolates, peaking at about 70C, suggesting it had different prot ein-protein interacti ons than the other alkali-aided treatments. Gelation behavior of isolates in the presence of salt was different than in the absence of salt. This is not unexpected as the presence of salt can cause partial solub ilization of the proteins and can also induce protein unfoldi ng. Prior to heating the G of the samples with salt was lower than the sampled without added salt. This is most likely due to the fact that salt would screen the electrostatic repulsive ch arges between then proteins, and thus lead to a more fluid system, hence lower G. Small changes were seen in G dur ing heating, until between 35-40C for the acid-

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69 aided gels (figures 3-22, 3-25 and 3-28) and about 40-45C for the alkali-aided gels (figure 3-31, 3-34 and 3-37). Unfolding is a prerequisite to thermal gelation of muscle proteins, thus suggesting that proteins in the ac id-aided isolates were more susceptible to heat and presumably thermally unfolded at a lower temperature than the proteins in the alkali-aided process. Both the acidand alkali-aided isolates had a distinctly different gela tion behavior on heating in the presence of salt compared to the control (figure 3-40), which demonstrated an expected dip in G between 35 and 50C. This clearly shows that th e pH-shift process leads to changes with the proteins in the isolate which in turn ha ve different gel forming properties. All isolates, and control, rega rdless of the presence or absence of salt demonstrated a significant increase in G during th e cooling phase. The final G was in all cases substantially higher than the initial G, but the difference betw een the two varied greatl y between treatments. The difference in initial and final G can be an indicator of the ability of a sample to produce a strong gel. The larger the final G, the more ri gid and strong the gel could be. The increase in initial and final G for acid-aided gels in th e absence of salt ranged from 1.8-3.3 fold, while it was 5.9-6.4 fold for the alkali-aided gels. Altho ugh the control increased its G 5.5 fold, the final G was lower than the G of alka li-aided gels. In th e presence of salt the in crease in G was 3.613 fold for the acid-aided gels but 14-25 fold for the alkali-aided gels. Th e control had an 8 fold increase in G. The much larger increase in G during the heating phase of gelation exhibited by alkali processed muscle proteins, clearly indicates that high pH processing results in protein conformations and supramolecular structures which promote stronge r protein-protein interactions during heating and particularly cooling than acid-a ided gels and untreated minced catfish muscle. The larger increase in G on cooling for the alka li-aided gels compared to the acid-aided gels may suggest they lead to increased hydrogen bonding on cooling (Lanier 2000).

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70 Muscle protein gels are characterized from a quality standpoint by color, gel strength and flexibility. Gel strength is evaluated as resistance to structural breakage or the stress coefficient. The gel flexibility is the structural elasticity or the strain coeffici ent. The stress and strain at fracture of muscle gel may be anal yzed in two ways; 1) the rotati onal stress and strain and 2) the tangential stress and strain. Tors ion testing analyzes the rotationa l stress and strain by measuring the force and distance required to twist a cyli ndrical gel segment until it breaks in half. Tangential fracture measurement is done using th e punch test. The punch test lowers a probe into a flat cross-section of th e gel and measures the force and distance required to puncture the gel. These two tests are frequently used to dete rmine physical qualities attributed to the quality of muscle protein products. Torsion testing is the study of the rotational fr acture properties of musc le proteins gels and was used in this study to compare pH treated and untreated samples using a method widely accepted for surimi testing. Four primary texture profile properties which have been attributed to surimi gels by torsion testing (Park 2005), t hose are mushy, brittle, r ubbery, and tough (Park 2005). Mushy is described by having low stress and low strain, with brittle having high stress and low strain. The other two are high strain descriptors with rubbery having low stress and tough having high stress. However, the results with catfish protein isolate, as seen in figure 3-57, do not fall within the parameters of a typical te xture profile graph, as th e gel stress is too high except for the control which is considered brittle. As seen in figure 3-57 all treatments resulted in product that had brittle properties (Park 2005). The differentiation between acid and alkali tr eated samples in the way they respond to rotational fracture indicates alkali treatment fo rms both a stronger and more elastic gel. The individual pH treatments did show differences with pH 2.5 having the lo west stress and pH 11.0

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71 the highest. The desired textur es of a restructured muscle product are important in the processing chosen for the production of that product. Kim and others (1996) showed conventional processing of catfish by-products re sulted in a surimi product with acceptable textural properties for the production of shellf ish analogs with the a ddition of cryoprotectants and starch. The torsion results of pH-shift processing of catfish muscle when compared to the results of Kim and others (1996) showed that protein gels (with no added cryoprotectants) made from alkali processing produced a stronger gel than either acid or no processing. The increased strength of pH-shift processed gels are however categorized as brittle according to the torsion texture profile (Park 2005). Acid processing of catfish, however, has led to mixed results. The acid results reported here for catfish are similar to Mireles Dewitt and ot hers (2007) who showed that acid treatment resulted in an improvement in gel strength as compared to a ground muscle control. The pH-shift process exhibited higher shea r stress and lower shear strain than the processing and formulation additives used in the products tested to deve lop the torsion texture profile (Park 2005). The pH-shift processing of cat fish muscle proteins compared to croaker, whiting, rockfish and cod since alkali treatment increased gel strength and elasticity of all species compared to acid treatment and no treatmen t. These differences show that the pH used to produce isolate do in fact provi de protein isolates w ith different rotationa l physical resistance. Punch testing, which is a study of tangential fr acture properties of muscle protein gels, was also used in this study to compare pH treatment s and untreated samples. This is the other fracture testing method widely us ed in the surimi industry. Th e parameters recorded are the resistance and distance traveled at breakage. Brea kage is when the structure of the gel gives way and can no longer hold its cohesive form. The resu lts of this study indica te that pH treatment

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72 directly affects the punch test scores as shown in figures 351, 3-52, and 3-53. Other studies have shown surimi from carp form a gel with lo wer break force and distance than acid or alkali processing (Luo and others 2001a). The effect of changing the punch test scores is that the surimi quality grade may be based on the jelly score (Park 2005) obtained from punch test values, the jelly score is the multiplication of forc e times distance. The jelly score for pH-shift processed catfish muscle is shown in figure 353. The problems with the jelly score are many (Park 2005), but as the jelly scor e is the product of gel force and gel deformation the quality of the protein isolate is not repor ted accurately. The results of this study show that pH-shift processing does not change the gel structure as assessed by the punch test as dramatically as seen in other testing methods, specifically torsion testing. The fold test is a semi-quantitative test used to determine the flexibility of cooked muscle protein gels rapidly. Based on th e 1-5 or grading scale the fold test allows for a five point scoring of the gels. The fold test allows for the determination of gels which are considered to be too brittle. However the fold test does not acco unt for gels which may be too elastic. The fold test is one of the primary methods used in gr ading surimi. The lack of significant difference (p>0.05) between all pH treated sa mples and the control indicates that catfish muscle proteins with or without pH shift pro cessing will form acceptable gels under these test conditions. The alkali results are consistent with pH-shift pr ocessing of tilapia (Ingadot tir 2004). However, acid processed tilapia also resulted in fold test scores of 5 which the catfish isolate gels from the acidaided process did not. The lack of significant di fference (p>0.05) between the acid treatment and the alkali treatment or the control, but with inconsistent results in acid processed catfish muscle as compared to the consistent results of acid processed tilapia muscle, indicate that the acid processing of catfish muscle may lead to a more complex material than the same process did

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73 with tilapia. The production of surimi from catfish, however, showed acceptable results with the addition of cryoprotectants and starch (Kim and others 1996). This indicates that based on the fold test, pH-shift processing of catfish musc le, though inconsistent, under the acidic conditions tested here, provides a gel structure acceptable fo r the production of a surimi product without the addition of cryoprotectants or other additives. The press test investigates wa ter holding capacity of the gel matrix formed after cooking. The water holding capacity of muscle gels is dire ctly related to the firm ness of the gel network (Kim and others 1996). The results of the study show that alkali treate d isolate, pH 11.0 and 11.5, had significantly higher water holding capacity than acid treated isolate, pH 2.0, and the control. Water holding capacity of pH-shift pr ocessed catfish muscle when compared to catfish surimi indicates that pH-shift processing resulted in decreased wa ter holding capacity compared to washed catfish surimi with added cryoprotectan ts which had an expressible moisture of less than 2 percent (Kim and others 1996). The results of Kim and others (19 96) indicate that catfish mince with or without cryoprotec tion produced a weak gel with puncture forces less than 500g. These results are inconsistent wi th the results found in this st udy showing that even though there is a reduction in water holding capacity, the preparation of thermally set gels without cryoprotection does result in strong gel formation. The stability of pH-shift processed catfish muscle on frozen storage and the need fo r cryoprotection has yet to be determined. Cook loss determines the amount of weight a product may lose during thermal processing. This weight loss is very important as increased cook loss reduces the final retail weight of the finished product and is a direct economic loss for the processor. Cook loss is a form of water holding capacity of muscle gels as the minced musc le product transitions fr om a protein paste to a thermally set network. The lack of signifi cant difference (p>0.05) between the pH treated

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74 samples and the control except for pH treatment 11.0 indicates that the water holding capacity of pH processed isolate during thermal transition is not affected by all pH treatments except pH 11.0. The low cook loss of all samples in the cu rrent study (less than 3%) indicated that the isolates have a better water holding capacity than that reported for acid pr ocessed catfish surimi with added cryoprotectants and phosphates, which are known to aid in water retention (Mireles Dewitt and others 2007). This means that pH-shi ft processing of catfish muscle may provide a protein isolate with comparable or improved properties of phosphates in muscle systems but without the addition of phosphates. Conclusions The results of the gel propertie s of pH-shift processed catfis h m uscle indicate that pH processing results in improved ge l strength over conventional proces sing according to the catfish results published by Kim and others (1996). These results also indica te that the range of textural possibilities has not been fully explored. The brit tle gels formed, based on the textural schematic of Park (2000), indicate that optimization of th e gel properties of pH-s hift processed catfish muscle is crucial before commerc ial processing is possible. Increasing the shear strain above 2.25 and decreasing the shear stress below 70 kPa is just the first step in developing a pH-shift processed catfish muscle into a useable prim ary product. The most desirable method for achieving the reduction in shear stress and the increas e in shear strain is the addition of water to the gel paste. The pH-shift processed isolates were tested at 80% moisture compared to 78% moisture used by Kim and others (1996). This suggests that pH-shift processed isolates may require a lower protein concentration than conve ntionally processed catfish muscle. Additional benefits to the processor and the consumer if pH-shift processing is used include taking advantage of the different solubi lization pH values. pH-shift processing allows the processor to have greater control over the desired texture for specific applications as seen within the textural

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75 ranges published here. This data also suggests that individual isolates may be combined to provide an even broader range of textures within a single commi nuted product if the appropriate processing conditions are used.

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76 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-1: The storage modulus (G) of catfish protein isol ates produced at pH 2.0 during heating and cooling as tested by small strain rheology. The gel was developed at 10% solids, 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a fr equency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acry lic head set to a gap of 1000 m. The development of thermal gelation during h eating and cooling shows th e physical transitions of proteins subjected to pH-shift processing.

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77 0 500 1000 1500 2000 2500 3000 3500 020406080100 Temperature (oC)G" (Pa) heating Cooling 0 500 1000 1500 2000 2500 3000 3500 020406080100 Temperature (oC)G" (Pa) heating Cooling Figure 3-2: The loss modulus (G) of catfish protein isolates produced at pH 2.0 during heating and cooling as tested by small strain rheo logy. The gel was developed at 2C/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

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78 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-3: The tan delta (G/G) of catfish pr otein isolates produced at pH 2.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

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79 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-4: The storage modulus (G) of catfish protein isol ates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

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80 0 200 400 600 800 1000 1200 1400 1600 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 200 400 600 800 1000 1200 1400 1600 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-5: The loss modulus (G) of catfish protein isolates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

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81 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-6: The tan delta (G/G) of catfish pr otein isolates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

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82 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-7: The storage modulus (G) of catfish protein isol ates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 83

83 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-8: The loss modulus (G) of catfish protein isolates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 84

84 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-9: The tan delta (G/G) of catfish pr otein isolates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 85

85 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-10: The storage modulus (G) of catfish protein isol ates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 86

86 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-11: The loss modulus (G) of catfish protein isolates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 87

87 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-12: The tan delta (G/G) of catfish protein isolates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 88

88 0 10000 20000 30000 40000 50000 60000 70000 80000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 10000 20000 30000 40000 50000 60000 70000 80000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-13: The storage modulus (G) of catfish protein isol ates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 89

89 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-14: The loss modulus (G) of catfish protein isolates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 90

90 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-15: The tan delta (G/G) of catfish protein isolates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 91

91 0 10000 20000 30000 40000 50000 60000 70000 80000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 10000 20000 30000 40000 50000 60000 70000 80000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-16: The storage modulus (G) of catfish protein isol ates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 92

92 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-17: The loss modulus (G) of catfish protein isolates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Te sting was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm crosshatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of proteins subjected to pHshift processing.

PAGE 93

93 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-18: The tan delta (G/G) of catfish protein isolates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 94

94 0 5000 10000 15000 20000 25000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 5000 10000 15000 20000 25000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-19: The storage modulus (G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with cont rolled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 95

95 0 500 1000 1500 2000 2500 3000 3500 4000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 500 1000 1500 2000 2500 3000 3500 4000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-20: The loss modulus (G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with cont rolled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 96

96 0 0.05 0.1 0.15 0.2 0.25 0.3 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-21: The tan delta (G/G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min in the absence of added NaCl. Testing was conducted with cont rolled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 97

97 0 2000 4000 6000 8000 10000 12000 14000 16000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 2000 4000 6000 8000 10000 12000 14000 16000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-22: The storage modulus (G) of catfish protein isol ates produced at pH 2.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 98

98 0 500 1000 1500 2000 2500 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 500 1000 1500 2000 2500 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-23: The loss modulus (G) of catfish pr otein isolates produced at pH 2.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 99

99 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-24: The tan delta (G/G) of catfish protein isolates produced at pH 2.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 100

100 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-25: The storage modulus (G) of catfish protein isol ates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 101

101 0 200 400 600 800 1000 1200 1400 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 200 400 600 800 1000 1200 1400 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-26: The loss modulus (G) of catfish pr otein isolates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 102

102 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-27: The tan delta (G/G) of catfish protein isolates produced at pH 2.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 103

103 0 500 1000 1500 2000 2500 3000 3500 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 500 1000 1500 2000 2500 3000 3500 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-28: The storage modulus (G) of catfish protein isol ates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 104

104 0 100 200 300 400 500 600 700 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 100 200 300 400 500 600 700 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-29: The loss modulus (G) of catfish pr otein isolates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 105

105 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-30: The tan delta (G/G) of catfish protein isolates produced at pH 3.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 106

106 0 10000 20000 30000 40000 50000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 10000 20000 30000 40000 50000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-31: The storage modulus (G) of catfish protein isol ates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 107

107 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-32: The loss modulus (G) of catfish protein isolates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 108

108 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-33: The tan delta (G/G) of catfish protein isolates produced at pH 10.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 109

109 0 5000 10000 15000 20000 25000 30000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 5000 10000 15000 20000 25000 30000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-.34: The storage modulus (G) of catfish protein isolates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 110

110 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-35: The loss modulus (G) of catfish protein isolates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 111

111 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-36: The tan delta (G/G) of catfish protein isolates produced at pH 11.0 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 112

112 0 5000 10000 15000 20000 25000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 5000 10000 15000 20000 25000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-37: The storage modulus (G) of catfish protein isol ates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 113

113 0 500 1000 1500 2000 2500 3000 3500 4000 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 500 1000 1500 2000 2500 3000 3500 4000 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-38: The storage modulus (G) of catfish protein isol ates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geom etry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 114

114 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-39: The tan delta (G/G) of catfish protein isolates produced at pH 11.5 during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was co nducted with controlled strain, 0.01 at a frequency of 0.1 Hz. Plate on plate geomet ry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical trans itions of proteins subjected to pH-shift processing.

PAGE 115

115 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G' (Pa) Heating Cooling 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 020406080100 Temperature (oC)G' (Pa) Heating Cooling Figure 3-40: The storage modulus (G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controll ed strain, 0.01 at a fr equency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 116

116 0 200 400 600 800 1000 1200 1400 020406080100 Temperature (oC)G" (Pa) Heating Cooling 0 200 400 600 800 1000 1200 1400 020406080100 Temperature (oC)G" (Pa) Heating Cooling Figure 3-41: The loss modulus (G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controll ed strain, 0.01 at a fr equency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 117

117 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 020406080100 Temperature (oC)Deg Heating Cooling 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 020406080100 Temperature (oC)Deg Heating Cooling Figure 3-42: The tan delta (G/G) of untreated catfish muscle during heating and cooling as tested by small strain rheology. The gel was developed at 2oC/min with 2% added NaCl. Testing was conducted with controll ed strain, 0.01 at a fr equency of 0.1 Hz. Plate on plate geometry was used with a 40 mm cross-hatched acrylic head set to a gap of 1000 m. The development of thermal gelation during heating and cooling shows the physical transitions of protei ns subjected to pH-shift processing.

PAGE 118

118 0 2000 4000 6000 8000 10000 12000 14000 16000 22.5310.51111.5control TreatmentG' AB B ABC A AC B C 0 2000 4000 6000 8000 10000 12000 14000 16000 22.5310.51111.5control TreatmentG' AB B ABC A AC B C Figure 3-43: The storage modulus (G) of catfi sh protein isolates compared to unprocessed muscle (control) prior to h eating, in the absence of added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant difference (p>0.05) between treatments.

PAGE 119

119 0 2000 4000 6000 8000 10000 12000 14000 16000 22.5310.51111.5control TreatmentG' (Pa)AB C A BC ABC ABC ABC 0 2000 4000 6000 8000 10000 12000 14000 16000 22.5310.51111.5control TreatmentG' (Pa)AB C A BC ABC ABC ABC Figure 3-44: The storage modulus (G) of catfi sh protein isolates compared to unprocessed muscle (control) after hea ting to 80C, in the absence of added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar lette rs represent no signi ficant difference (p>0.05) between treatments.

PAGE 120

120 0 10000 20000 30000 40000 50000 60000 70000 22.5310.51111.5control TreatmentG' (Pa)A A AB ABC ABC BC C ABC 0 10000 20000 30000 40000 50000 60000 70000 22.5310.51111.5control TreatmentG' (Pa)A A AB ABC ABC BC C ABCFigure 3-45: The storage modulus (G) of catfish protein isol ates compared to unprocessed muscle (control) after coo ling to 5C, in the absence of added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar lette rs represent no signi ficant difference (p>0.05) between treatments.

PAGE 121

121 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2.0 salt 2.5 salt 3.0 salt10.5 salt11.0salt11.5saltcontrol salt TreatmentG'AB A AA AB B B 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2.0 salt 2.5 salt 3.0 salt10.5 salt11.0salt11.5saltcontrol salt TreatmentG'AB A AA AB B B Figure 3-46: The storage modulus (G) of catfi sh protein isolates compared to unprocessed muscle (control) prior to heati ng, in the presence of 2% added Na Cl. The significant differences present between samples (p<0.05) are represente d by the letters above the column. Similar letters represent no significant diffe rence (p>0.05) between treatments.

PAGE 122

122 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 22.5310.51111.5control TreatmentG' (Pa)A B B AB AB AB AB 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 22.5310.51111.5control TreatmentG' (Pa)A B B AB AB AB AB Figure 3-47: The storage modulus (G) of catfish protein isol ates compared to unprocessed muscle (control) after hea ting to 80C, in the presence of 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant difference (p>0.05) between treatments.

PAGE 123

123 0 5000 10000 15000 20000 25000 30000 35000 40000 22.5310.51111.5control TreatmentG' (Pa)AB A A AB AB B AB 0 5000 10000 15000 20000 25000 30000 35000 40000 22.5310.51111.5control TreatmentG' (Pa)AB A A AB AB B AB Figure 3-48: The storage modulus (G) of catfish protein isol ates compared to unprocessed muscle (control) after coo ling to 5C, in the presence of 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant difference (p>0.05) between treatments.

PAGE 124

124 0 20 40 60 80 100 120 140 160 22.5310.51111.5control TreatmentStress (kPa)AB A AB D E C BE 0 20 40 60 80 100 120 140 160 22.5310.51111.5control TreatmentStress (kPa)AB A AB D E C BE Figure 3-49: The torsion shear stress of catfish protei n isolates and unprocessed muscle (control) heat set gels as tested by torsion shear. The gel was de veloped with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant difference (p>0.05) between treatments.

PAGE 125

125 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 22.5310.51111.5control TreatmentStrainAB AB A AB B B AB 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 22.5310.51111.5control TreatmentStrainAB AB A AB B B AB Figure 3-50: The torsion shear st rain of pH-shift processed catfish protein isolates and unprocessed muscle (control) heat set gels as tested by torsion shear. The gel was developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar lett ers represent no significant difference (p>0.05) between treatments.

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126 0 100 200 300 400 500 600 700 800 900 1000 22.5310.51111.5control pH treatmentBreak force (g)AB AAB A A A B 0 100 200 300 400 500 600 700 800 900 1000 22.5310.51111.5control pH treatmentBreak force (g)AB AAB A A A B Figure 3-51: The tangential shear stress of pH-shift processed catfish protein isolates and unprocessed muscle (control) heat set gels as tested by punch testing. The gel was developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar lett ers represent no significant difference (p>0.05) between treatments.

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127 0 2 4 6 8 10 12 14 16 22.5310.51111.5control pH treatmentdistance (mm)AB AB A B AB AB B 0 2 4 6 8 10 12 14 16 22.5310.51111.5control pH treatmentdistance (mm)AB AB A B AB AB B Figure 3-52: The tangential shear strain of pH-shift processed catfish protein isolates and unprocessed muscle (control) heat set gels as tested by punch testing. The gel was developed with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2% added NaCl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar lett ers represent no significant difference (p>0.05) between treatments.

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128 0 100 200 300 400 500 600 700 800 900 22.5310.51111.5control SampleJelly Strength (g x cm)A AB AB A A A B 0 100 200 300 400 500 600 700 800 900 22.5310.51111.5control SampleJelly Strength (g x cm)A AB AB A A A B Figure 3-53: The jelly strength of pH-shift processed catfish protein isolates and unprocessed muscle (control) heat set gels. Jelly streng th was calculated from the punch test values and is tangential shear stress*shear stra in.. The gel (with 2% added NaCl) was developed with static heating (80oC for 20 min) followed by 48 hrs cold setting time. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant difference (p>0.05) between treatments.

PAGE 129

129 0 2 4 6 8 10 12 22.5310.51111.5control Treatment% Weight ReductionA B B A AB AB AB 0 2 4 6 8 10 12 22.5310.51111.5control Treatment% Weight ReductionA B B A AB AB AB Figure 3-54: The expressible moisture of pH-s hift processed catfish protein isolates and unprocessed muscle (control) heat set gels as tested by placing a 3 Kg weight on a gel slice, 1.8 cm in diameter and 3 mm in thic kness. The weight loss is assumed to be water leakage from the gel matrix and is a determinant of water holding capacity of the gel matrix. The gel was deve loped with static heating (80oC for 20 min) and 48 hrs cold setting time, with 2% added Na Cl. The significant differences present between samples (p<0.05) are represented by the letters above the column. Similar letters represent no significant diffe rence (p>0.05) between treatments.

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130 0 1 2 3 4 5 6 22.5310.51111.5control TreatmentQuality Score Figure 3-55: The shear stress of pH-shift pro cessed catfish protein is olates and unprocessed muscle (control) heat set gels as test ed by folding a 3 mm gel slice, 1.8 cm in diameter, in half then quarte rs. The gel score is assessed as 5= no crack when folded into a quarter, 4=no crack when folded into half but crack when folded into quarter, 3= no crack when folded in half but split when folded into quarter, 2=crack when folded into half, 1=broke and split into ha lves. The gel was developed with static heating (80oC for 20 min) 48 hrs cold setting time, with 2% added NaCl. No significant differences were present between any of the samples tested.

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131 0 0.5 1 1.5 2 2.5 22.5310.51111.5control Treatment% Weight Reduction Figure 3-56: The weight loss of pH-shift processed catfish pr otein isolates and unprocessed muscle (control) heat set gels with 2% added NaCl developed with static heating (80oC for 20 min), 48 hrs cold setting (4C), as compared to the weight before cooking and setting. No si gnificant differences were present between samples (p>0.05).

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132 Figure 3-57: Torsion shear strain vs. shear stress of pH-shift processed catfish muscle and an untreated control. The texture profile associ ated with torsion testing is according to Park (2000). The X in the middle represen ts the crossing points of the different textures. The shaded box is the extent wh ich the texture profile was determined. The points from pH-shift proce ssing outside of the shaded box (all points except the untreated control) are above the maximum stress value listed in the texture profile. The pH-shift samples are pH 2.0( ), pH 2.5 ( ), pH3.0 ( ), pH 10.5 ( ), pH 11.0 ( ), pH 11.5 ( ), untreated control ( ).

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133 CHAPTER 4 CHEMICAL PROPERTIES OF ACID AIDED AND ALKALI AIDED PROTEIN ISOLATES FROM C ATFISH ( Ictalurus punctatus ) Introduction Channel catf ish ( Ictalurus punctatus ) is one of the fastest grow ing aquatic products in the United States. The production and utilization of aquacultured catfish in the United States has increased over the past 20 years and recen tly stabilized to a production level of 600,000 pounds from 1999-2001 (Harvey 2002). The primary product of catfish is the fillet, with the secondary product being the belly flaps, or the nugget. The quality and potential utilization of catfish for other purposes is under on-going investigation w ith promising results (Kim and others 1996; Kristinsson and others 2005b; S uvanich and others 2000). The over-harvesting of all aquatic resources was recently highlighted by Worm and others (2006) with the projected depletion of aquatic resources by 2048. This becomes more of an issue since approximately 40% of the live weight of many harvested species is discarded. Methods are curre ntly being investigated which will put more of the total seafood catch into the human consumable market and potentially diminish or greatly relieve some of the current strain on the fish eries. The present seafood catch totals approximately 100 million tons (Kristin sson and others 2005b) and can be far better utilized if new economic technol ogies are developed. To achieve optimally utilize material currently being discarded or divert ed into other products not intende d for human use, it is of the utmost importance to understand the chemistry of these material so novel processes can be developed. The chemical properties of muscle proteins in meat and meat products are known to affect quality, application and formulation ranging from whole meat to comminuted products (Lanier 2000). The chemistry of muscle proteins direct ed for human consumption is investigated to better understand the effect of inherent chemi cal properties on the f unctional properties and

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134 quality of proteins. The main functional properties of importance to protei ns are their gelation properties which dictate the difference textural properties you can achieve with muscle protein products, and also their water holding capacity. Myosin, actin and the complex actomyosin are known to be the primary contributors of textural properties of muscle gels (Park 2005). The chemical properties of proteins in muscle vary widely, but are known to affect the utilization of muscle proteins for food applications, includi ng protein gels. The ch emical properties of proteins include but are not limited to myosin ATPase activity, reactiv e and total sulfhydryl groups and surface hydrophobicity (Lanier 2000). Myosin ATPase activity is used as an indica tor of structural changes in myosin and/or actomyosin integrity during post-harvest handling and processing (Park 2005). These structural changes have been found to correlate to changes in the gelling properties of muscle proteins. Yongswawatdigul and Park (2002) st ate that the gelling differences are due to the oxidation of SH groups at the active site on the head portion of the myosin heavy chain. Modification of -SH groups ma y be monitored by measurem ent of reactive and total sulfhydryl groups (Liang and Kristinsson 2005). Prot ein sulfhydryl groups in gelled systems are believed to provide structural stability to the gel network by undergoing disulfide interchanges and -SH oxidation which contributes to disulfide bridges formed during ge lation (Visschers and De Jongh 2005). The presence and availability of -SH groups to participate in forming disulfide bridging increases protein-protei n interactions and thus the integrity of th e gel network (Visschers and De Jongh 2005). Surface hydrophobicity of proteins in gel networks is known to impart structural integrity to the gel (Lanier 2000). As the gel is forme d, the hydrophobic patches of proteins exposed to the solvent interact to bu ry these exposed hydrophobic pa tches through protein-protein

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135 interactions. Protein surface hydrophobicity and physical properties (i.e. with torsion testing) have been found to correlate (L anier 2000). Hydrophobic interactions are belie ved to occur after disulfide interchanges as menti oned above during the heat indu ced gel formation of muscle proteins (Smyth and others 1998). The hydrophobic interactions which are partly directed by disulfide links bring proteins into the preliminary three-dimensional network allowing for hydrogen bonding to occur during th e cooling phase of heat induced gels (Smyth and others 1998). Modification of sulfhydryl and/or hydrophobic properties of muscle proteins would be one way of directing protein ge lation as well as investigating the chemical mechanisms responsible for differences observed in the gel networks formed when implementing new technologies of muscle protei n recovery (Lanier 2000). Acid-aided and alkali-aide d processing is a new technology aimed at utilizing underutilized products and byproduc ts of muscle-based system s (Hultin and Kelleher 2000). Acid aided and alkali aided processing utilizes the solubility properties of muscle proteins, specifically the myofibrillar proteins. Solubility is imparted to myofibrillar proteins by the increase or decrease in pH to a point where th e proteins are highly ioni zed resulting in their solubility. Solubility of myofibril proteins is important because it allows for the separation and removal of unwanted materials such as collagen and fat. These unwanted materials are not easily separated or removed in other r ecovery processes, such as surimi. Acid-aided and alkali-aided processing of channel catfish has been shown to provide an improved pr otein isolate by reducing the lipid and heme content. The reduction of lipid and heme content increases whiteness and increases the oxidative stability of the isolate (Kristinsson and others 2005b). Acid aided and alkali aided processing has been shown to alter the structural states of muscle proteins in cod and catfish myosin (Davenport and Kristinsson. 2003; Kristinsson and Hultin 2003a; Raghavan and

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136 Kristinsson 2007a, 2007b) and whiting and rockfish protein isolate made from whole ground muscle (Choi and Park 2002; Y ongsawatdigul and Park 2004). However, the structural stability of catfish muscle proteins as a whole when subj ected to the acid aided or alkali aided processing has not yet been investigated. We hypothesize that acid-aided and alkali-aided processing will structurally modify channel catfis h muscle proteins. The goal of th is study is to characterize the effect of acid and alkali processing on some key structural properties of catfish muscle protein. Methods Raw Material The raw m aterial used in these studies was fresh catfish fillets obtained 1-3 days post harvest from a local supplier. Catfish fillets were only purchased which were determined to be within 3 days of packaging. The catfish fillet s were purchased and im mediately transported on ice to the laboratory and pr ocessed the same day. Preparation of Protein Isolates Protein isolates were p repared according to figure 2-1. Fresh fillets were initially ground in an Oster heavy duty food grinder (Niles, Il l., U.S.A.) for the prel iminary disruption and collection of the muscle tissue. Following gri nding, the comminuted meat was diluted 1:2 (w/v) with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds. Following homogenization, the resulting muscle tissu e slurry was further diluted to give a final dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manuall y stirred with a plastic spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the methods described below, using either 2N NaOH or 2N HCl as needed for the pH desired, with continuous manual mixing. Upon reaching the desi red pH, insoluble material was removed by centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products, Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at 5oC. Following centrifugation, the

PAGE 137

137 soluble middle layer was collected through a kitche n strainer with a mesh size of approximatly 0.25 mm to minimize contamination with other sepa rated materials. The soluble material was readjusted to pH 5.5 as described above. After readjustment the solu tion was centrifuged to remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The precipitated protein was collected by decanting the supernatant containing the unprecipitated proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing until the moisture content was below 80%. Mois ture content was determined using a Cenco infrared moisture analyzer (CSC Scientific, Fairfax, Va., U.S.A.). Upon completion of manual dewatering the protein isolation was complete. Preliminary unpublis hed investigation of protein isolates in this laborato ry found the shelf life of catfish protein isolates to be 5-7 days on ice. All protein isolates were stored on ice at the precipitation pH and used within 5 days. Protein Concentration Protein concentration in the is olates and the subsequent solu tions was determ ined using the Biuret method, as described by Torten and Whita ker (1964), with of 10% w/v deoxycholic acid in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any remaining lipids in the samples. Protein concen tration was measured based on a standard curve based on BSA. Protein Surface Hydrophobicity Protein su rface hydrophobicity was conducted acco rding to Liang and Kristinsson (2005). To measure surface hydrophobicity th e isolate was diluted to give a stock solution of 10 mg/ml in a 20 mM tris-HCl buffer, 0.6 M NaCl, pH 7.2. The stock solution was serially diluted to obtain a concentration curve. Increasing volume (100 l, 200 l, 300 l, 400 l and 500 l) of

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138 the stock solution were added to Tris-HCl buffer to a final volume of 4.0 ml. Then, 10 l of 6propionyl-2-(dimethylamino) naphthalene (PR ODAN) (11.35 g/ml in methanol) was added and the samples mixed for ~15 seconds. After mixing the samples were then incubated for 15 min in the dark. All sample preparations and incubations were performed on i ce in disposable test tubes. After incubation, the sample wa s transferred to a fluorescence cuvette and the fluorescence emission intensity scanned between 380-560 nm with excitation at 365 nm in a Perkin Elmer LS 45 Luminescence Spectrophotometer (Norwalk, CT). As the isolate is a collection of proteins, the fluorescence peak of the samples is a rela tively flat and broad peak between 430-460 nm. The maximal fluorescence of the protein isolat e was taken and used as the wavelength for analysis. The surface hydrophobicity was calculated as the slope of the ne t fluorescence versus protein concentration (mg/ml) of the samples. Reactive Sulfhydryl Groups Reactive sulfhydryl grou ps were determined according to Kim and others (2003). The isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCl buffer, pH 7.2 with 600 mM NaCl to 250 g/ml. After dilution, 80 l of 10 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) was added to the sample. This mixtur e was then incubated for 1 hr on ice. After incubation the sample was read at 420 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). Micromolar determination of SH groups per gram in the sample were done using the following equation: mlmgion concentrat sample cmmol factor dilution absorbance gSH 13600100

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139 Total Sulfhydryl Groups Total su lfhydryl groups were determined accordi ng to Kim and others (2003) The isolate was diluted and homogenized for 1 minute on i ce at speed 2 in a Bio-homogenizer (M133/12810, Bio Spec Products Inc., Bartlesville, OK) in 20 mM Tris-HCl buffer, pH 7.2 with 600 mM NaCl to 250 g/ml. After dilution, 0.5 ml of dilu ted isolate was mixed with 2.5 ml urea buffer. The urea buffer contained 8 M urea, 0.2 M Tris-H Cl, 2% SDS, 10 mM EDTA and was adjusted to pH 8.5. After mixing with urea, 50 l of 10 mM DTNB was added to the sample. The mixture was incubated in a water bath for 15 minut es at 40C. After incubation the sample was read at 420 nm using an Agilent 8453 diode ar ray UV-visible spectroscopy system (Agilent Technologies Deutschland Gm bH, Waldbronn, Germany). To tal sulfhydryl groups were calculated using the following equation. mlmgion concentrat sample cmmol factor dilution absorbance gSH 13600100 Myosin ATPase Activity Assay Myosin ATPase activity was determ ined by th e method of Perry (1955). The buffers to initiate the enzymatic hydrolysis of ATP by myosin were prepared prior to sample preparation. The reaction buffers used in this assay were 0.2 M glycine-NaOH buffer (pH 9.0), 0.1 M calcium chloride, 0.05 M ATP, sodium salt (pH 6.8) and 15% trichloroacetic acid (TCA). Liberation of inorganic phosphate was monitored in this reac tion as the determinant of enzymatic function. For the estimation of inorgani c phosphate the following buffers were used: 12% TCA, 10% (w/v) ammonium molybdate stock solution in 10 N sulfuric acid. This ammonium molybdate stock was used in the preparation of the ferr ous sulfate ammonium molybdate reagent which was made fresh the day of analysis. For analys is of myosin ATPase activity the isolate was diluted to 1 mg/ml in Tris-HCl buffer c ontaining 600 mM NaCl pH 7.2 and homogenized thoroughly. This was kept on ice until needed. In separate tubes for each reaction, prior to

PAGE 140

140 isolate addition, the reaction buffer was prepar ed by adding 1.3 ml glycine-NaOH buffer, 0.2 ml calcium chloride, 0.3 ml ATP. This mixture was then incubated at 25oC for 5 min to allow the temperature to equilibrate. After temperature equilibration, 0.2 ml of the individual isolate solution was added to each reaction tube with proper mixing and allowed to incubate at 25oC for 5 min. After 5 min, 1 ml of 15% TCA was added to each reaction tube to stop the reaction. This was centrifuged for 10 minutes at 25,000 x g to precip itate the proteins from solution and leaving liberated phosphate from ATP in solution. Next 0.5 ml of supernatant was added to 3.2 ml of 12 % TCA with good mixing and left to stand at 25oC for 10 minutes. This so lution (3.0 ml) of this solution was removed and mixed with 2 ml of the ferrous sulfate ammonium molybdate reagent and incubated at room temperature for 1 min. After 1 min of incubation the solution was transferred to a cuvette and read at 363 nm using an Agilent 8453 diode array UV-visible spectroscopy system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). The color of this reaction in tensifies with time so only one reactio n was done at a time to standardize the incubation time of all samples to 1 min. The re sults of myosin ATPase activity were assayed as relative results to the c ontrol. The control absorbance was set to 100% and the percent activity of the isolates was calculated from the ab sorbance of the samples relative to the control. sample control sample treatedpH activity Protein Solubility as a Function of pH The isolate was diluted to 10 m g/ml in DI water with thorough ho mogenization using a tissue homognizer (Ultra-Turrax T18, IKA Works Inc. Wilmington, NC). After dilution in water, 2 ml diluted protein was added to 2 ml buffer with good mixing. The pH range was from 1.5-12 in 0.5 increments. For pH 1.5-6.5, 2 mM citric acid buffer was used and for pH 7.0-12, 2 mM sodium phosphate buffer was used. The buffers were adjusted to the desired pH prior to

PAGE 141

141 protein addition. After protein addition, the mixture was allowed to incubate on ice for 10 minutes. After incubation the protein solu tion was centrifuged at 3,000 x g for 10 minutes. After centrifugation, the supernat ant was sampled for protein c oncentration using the biuret method (Torten and Whitaker 1964). Protein concen tration was calculated fr om a standard curve based on bovine serum albumin (BSA). Percent sol ubility was then calculated as a percentage of an uncentrifuged control by the following equation: totalmlmgprotein ernant mlmgprotein / /sup/ Protein Solubility as a Function of Salt Concentration The isolate was diluted to 5 m g/ml in 20 mM Tris-HCl at pH 7.2 without added NaCl with thorough homogenization using an U ltra-Turrax T18. After dilution in buffer, 4 ml was taken and the appropriate amount of NaCl added to each tube to achieve 0 mM, 150 mM, 300 mM 450 mM or 600 mM concentrations. After the appropriate NaCl concentration was achieved, the samples were centrifuged at 3000 x g for 10 minutes at 5C. After centrifugation, the supernant was collected in duplicate and soluble protein was determined according to the biuret method (Torten and Whitaker 1964). The soluble protein was compared to an uncentrifuged control. Percent soluble protein was calculated as totalmlmgprotein ernant mlmgprotein / /sup/ Statistical Analysis Experim ental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on replicate isolations as discussed above. A repl icated (N=2) was determined as acceptable due to achieving an acceptable power ( =0.80, =0.05). One-way independent measures analyses of va riance were used to examine the effects of all methods used except for the analysis of pH solubility and salt solubility which were tested

PAGE 142

142 with 2-way ANOVA analysis. The Kruskal-Wallis ANOVA testing by ranks was used on data which did not pass normality. Post hoc anal ysis was conducted only in the presence of significant population differences. ANOVA sta tistical comparisons were conducted with SigmaStat, (Systat Software Inc. San Jose Ca) with a significance level of p<0.05. After SigmaStat completed the ANOVA analysis, the post hoc analysis recommended by SigmaStat was used. In most cases this was Tukeys test The Holm-Sikak method was used with ATPase activity. The two-way ANOVA analysis used the Holm-Sidak pair wise comparisons. The twoway ANOVA tables and the post hoc test s are reported in appendix A. Results Myosin ATPase Myosin ATPase activity was reduced by pH-shi ft processing (Figure 4-1). This suggests that the pH treatm ent leads to changes in the myosin recovered with the pH-shift isolates. Myosin ATPase activity was significantly re duced (p<0.05) by pH treatments 2.0, 2.5, 3.0 and 10.5. No significant differences (p>0.05) were seen between any of the pH-shift isolates. The isolates retained from 35% to 61% myos in ATPase activity of the raw material. Surface Hydrophobicity Surface hydrophobicity of the proteins in th e isolates was not significan tly (p>0.05) reduced compared to the proteins in the raw mate rial (Figure 4-2). All isolate treatments had lower surface hydrophobicities but no main effect was determined. As with the myosin ATPase results, there were no significant differences (p>0.05) between any of the acid or alkali treatments. Total Sulfhydryl Groups When total sulfhydryls were analyzed no significant differences (p>0.05) w ere seen between any of the acid or alkali treatments within groups or betw een groups (figure 4-3). All

PAGE 143

143 the pH treatments were not significantly diffe rent (p>0.05) from the control as individual treatments or as a population. Reactive Sulfhydryl Groups There were no significant differences (p>0.05) in surface sulf hydryl grou ps between any of the acid or alkali treatments within groups or between groups (figure 4-4). No significant differences were present between any of the pH-shift treated is olates and the control. No significant differences were present between any individual treatments or within the population. Solubility of Proteins at Different Salt Levels The results of the salt solubility study are shown in figure 4-5. The isolates and untreated control were compared with the Holmes-Sidak multiple pair wise comparison with a p<0.05. The pair wise comparison of the salt solubility is shown in appendix A. Significant differences were present between all comparisons made, both within pH treatme nts and within salt concentrations. Solubility of Proteins at Different pH Values The results of pH solubility of isolates and untreated catfish muscle are shown in figure 46. The isolates and untreated control were comp ared with the Holmes-Sid ak multiple pair wise comparison with a p<0.05. The pair wise compar ison of the pH solubility is shown in appendix A. The isolates showed significant differences (p<0.05) at extreme pH values but no significant differences were present between pH 4 to pH 7.5. Discussion The globular head of m yosin has an enzyma tic function where ATP is hydrolyzed to ADP and phosphate. The purpose of the enzymatic functi on is to break the myosin/actin bridge which is important for the muscle c ontraction/relaxation cycle (Ber ne and others 2004). Myosin ATPase activity has been reported as a good indicator of protein quality a nd quality of processed

PAGE 144

144 fish muscle food products, such as surimi (Park, 2005). The results here for catfish show that there were no significant differe nces (p>0.05) in myosin ATPase activity between the isolate treatments. The isolate treatments had however significantly (p<0.05) lower ATPase activity than the control (Figure 4-1). Is olate treatments did not completely inactivate myosin ATPase activity but did reduce th e activity, suggesting high and low pH treatments the proteins were subjected to modified the struct ure of the myosin catalytic site The change in myosin ATPase activity has been attributed to a shifting of the sulfhydryl groups at the myosin active site (Yongsawatdigul and Park 2004). Th e reduction in myosin ATPase activity was highly variable as indicated from a large standard deviation (Figure 4-1). In the pH 10.5 treatment group, the myosin ATPase activity ranged from 0-62%. This range may be due to the inconsistent exposure of the myosin active site to the solvent at high pH. These inconsistencies were present in all samples with some varying more than others. Th e lack of significant difference (p>0.05) in the myosin ATPase activity (figure 4-1) may be due to the variability of the pH treated systems and differentiation between acid and alkali treatments of muscle proteins wa s not possible. Isolate production with either acid or al kali may or may not go through the same structural changes, however, the product is similar in that the acid or alkali trea tments produce a reduction of ATPase activity which is not significantly diffe rent (p>0.05) among the treatment groups. This means that though there is a redu ction in the ATPase activity when acid or alkali processing is used there is not a complete inactivat ion of myosin ATPase activity. Surface hydrophobicity measurement is an an alysis of the hydrophobic patches on the surface of a protein that bind a fluorescent probe. In this expe riment, PRODAN was used as the fluorescent probe. Figure 4-2 shows no significant difference (p>0.05) in surface hydrophobicity between treatment groups. However, the contro l was higher in surface hydrophobicity than all

PAGE 145

145 pH treatmented samples though no main effect was determined. Previous studies have shown that pH-shift processing increased the surface hyd rophobicity of catfish m yosin (Kristinsson and Hultin 2003a; Raghavan and Kristinsson 2007a, 2007b). The surface hydrophobicity of isolated catfish myosin shows pH treatment 2.5 having the greatest surface hydrophobicity followed by pH treatment 11.0 and then followed by untreated control (Davenport a nd Kristinsson 2003).The reduction of surface hydrophobic patches during acid a nd alkali treatment could be the result of structural modification during the pH readju stment to 5.5, burying the hydrophobic patches previously on the surface of the protein. Howe ver this reduction of surface hydrophobicity in the whole isolate system could also be due to micro-aggregation preventing the exposure of hydrophobic patches on the surface of the proteins. Total sulfhydryls are the total number of -SH groups exposed after treatment in 8 M urea. Reactive sulfhydryls are the -SH gr oups of the protein which are exposed to the solvent (Tris buffer with 0.6M NaCl) and are able to reac t with DTNB. The di fference between total sulfhydryls and reactive sulfhydryl s is the molar content of sulfhydryl groups which are buried within the protein structure of the system. There were no significant differences (p>0.05) between total sulfhydryls in all treatment groups and the control. Figure 4-4 shows no SH groups were altered during acid or alkali processing. The total su lfhydryl groups present in the samples, which may participate in gel formati on, remained the same. Figure 4-3 depicts the reactive sulfhydryls measured in the control and all treatments No significant differences (p>0.05) were observed between any of the trea tment groups or between any of the treatment groups and the control. Due to a lack of signifi cant differences (p>0.05) in total sulfhydryl and reactive sulfhydryl groups it may be concluded that sulfhydryl content was not significantly (p>0.05) linked to changes in gel strength (see gela tion data in Chapter 3). However, structural

PAGE 146

146 susceptibility during heating denaturation to ex pose sulfhydryl groups du ring thermal treatment was not assayed. Thus the molar content of disu lfide bonds in the thermally set gel is currently unknown. Salt solubility of muscle proteins is one way to assess whether functionality has been modified by pH-shift processing. Muscle proteins myofibrillar protein in particular, are highly sensitive to changes in ionic stre ngth and type of ions in their environment. The ability of the proteins in the isolate to enter into solution at varying concentra tions of salt was tested at pH 7.0 (Figure 4-5). Myofibrill ar proteins are the contractile protei ns of muscle, those proteins which are salt soluble, or more specifi cally go into solution above an i onic strength of ~0.3. Figure 4-5 shows a reduction in solubility of the protein isolates in the pres ence of high salt concentrations. This reduction may be due to structural change s and/or micro-aggregation which prevent the continued solubility of these proteins under conditions which normally would solublize myofibrillar proteins. The reduction in salt solubil ity of isolates follows the same trend as the reduction of surface hydrophobicity. These tests provided results when compared to previous results on cod muscle protein isolates a nd cod myosin where surface hydrophobicity was increased while solubility was decreased (Kri stinsson and Hultin 2003b) indicating a species effect of pH-shift processing. These results indicate that in catf ish isolates, the protein-protein interactions are changed in such a way to prevent ionization of the protein surface thus resulting in a reduction of solubility. As stated above the reduction in surface hydrophobicity may be due to microaggregation of misfolded proteins, t hus leaving little exposed hydrophobic surface. Increased salt will solubilize musc le proteins, but it is possible the interactions between these microaggregates were too strong for the salt to overcome, thus leading to only a small increase in solubility. Due to the similarity of results obse rved between isolated catfish myosin (Raghavan

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147 and Kristinsson 2007a) and whole muscle isolate, the same molecular mechanism is most likely responsible for these differences. The modification of catfish muscle proteins by pH -shift processing result ed in isolates with different pH solubility profiles. The increased solubility of the control in the pH range 3.5-10 is most likely due to a higher amount of sarcoplasmic proteins compared to isolates. Previous work on pH-shift processing of catfish muscle (Krist insson and others 2005b) has shown an increased retention of sarcoplasmic proteins when catfish is processed at pH 2.5 as compared to alkali processing at pH 11.0. This suggests that sarcopla smic proteins which are largely soluble in the control co-aggregate with the myofibrillar proteins and thus become largely insoluble, explaining the different pH profile seen in the pH 3.5-10 range. The low so lubility in this range would indicate that the protein isolates are more prone to aggregation th an untreated control. This is likely due to the fact that the isolates are compos ed of only partially refo lded protein structures which favor interactions with each other more than th e solvent. It is also interesting to note that all isolates expect the pH 10.5 treatment, became soluble at a lower pH than the control, suggesting more protonation was needed to brea k the interactions betw een them. The pH 10.5 treatment may have been milder than the othe r treatment, thus giving isolates with weaker protein-protein interactions. It is also interesting to note that contrary to the results seen at low pH the isolate solubilized at lowe r high pH values than the control, indicating that they were more sensitive to increases in negative charges than positive charges. These data therefore suggest the pH-shift process modi fied the protein structure and th eir interactions compared to control. Conclusions No significant differences (p>0.05) w ere seen between total or reac tive sulfhydryl groups of catfish protein isolates compared to contro l. No significant differences (p>0.05) were seen

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148 between the surface hydrophobicity or myosin ATPase activity of the isolates but all isolate samples showed reduction in both compared to c ontrol. Significant differences (p<0.05) were observed in saltand pH-dependent solubility of isolates compared to control. These results indicate that, although significant differences are not detected (p>0.05) in some of the commonly used tests to describe the chemical nature of pr otein products, isolates su bjected to different pHshift processing conditions do in fact have diffe rent properties and pH-s hift processed muscle proteins may have chemical modi fications which are different from traditional muscle systems. The chemical modification of catfish muscle prot eins by pH-shift processing may also lead to structural and functional modifica tion of catfish muscle proteins. Further studies are needed to determine whether the structural and functiona l effects when using this new technology are dictated by any chemical changes to specific am ino acids during processing. With further study and characterization the formulation of custom pr otein isolates to provi de application specific utilization may be possible.

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149 2 2.5 3 10.5 11 11.5 con 0 10 20 30 40 50 60 70 80 90 100Relative Activity (%)AA AA B ABAB 2 2.5 3 10.5 11 11.5 con 0 10 20 30 40 50 60 70 80 90 100Relative Activity (%)AA AA B 2 2.5 3 10.5 11 11.5 con 0 10 20 30 40 50 60 70 80 90 100Relative Activity (%)AA AA B ABAB Figure 4-1: Myosin ATPase activ ity of catfish muscle isolates compared to a control sample. The control was normalized to 100% activity and the percentage of activity was calculated relative to the control. Th e significant differences present between samples (p<0.05) are represented by the lett ers above the colum n. Similar letters represent no significant differen ce (p>0.05) between treatments.

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150 2 2.5 3 10.5 11 11.5 con 0 200 400 600 800 1000 1200 1400Surface Hydrophobicity Figure 4-2: The surface hydrophobicity of catfish muscle isolates and untreated control. Hydrophobicity was assayed by developing a protein concentr ation curve and measuring the maximal fluorescence response of the fluorescent probe PRODAN at ~400 nm. A main effect was not determin ed for the sample population (p>0.05).

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151 2 2.5 3 10.5 11 11.5 con 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07uM -SH/g protein Figure 4-3: Total sulfhydryl cont ent of catfish protein isolates compared to untreated control. Total sulfhydryl content was assayed by the reactivity of catfish proteins to DTNB after being treated for 10 min in 8M ur ea at 40C. No significant differences (p>0.05) were present between any of the samp les tested, thus it is concluded that pHshift processing did not affect total sulfhydryl content. 2 2.5 3 10.5 11 11.5 con 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014uM -SH/g protein Figure 4-4: Reactive sulfhydryl content of catfish protein isolates compared to untreated control. Reactive sulfhydryl content was assayed by th e reactivity of catfish muscle proteins to DTNB in T-HCl buffer. No significant differences (p>0.05) were present between any of the samples tested, thus it is concluded that pH-shift processing did not affect reactive sulfhydryl content.

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152 0 10 20 30 40 50 60 70 80 90 100 0mM150mM300mM450mM600mM mM NaClSolubility (%) pH 2.0 pH 2.5 pH 3.0 pH 10.5 pH 11.0 pH 11.5 control Figure 4-5: Solubility of catfish protein isolates and untreated c ontrol in varying concentrations of sodium chloride at pH 7.2 in 25 mM tris -HCl buffer. The percent solubility was determined by dividing protein concentrati on in the supernatant after centrifugation by the protein concentration in an uncentrifuged sample. Significant differences compared within pH treatments and salt co ncentrations are tabulated and reported in appendix A.

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153 0 20 40 60 80 1001.52.53.54.55.56.57.58.59.510.511.5NC pHSolubility (%) pH 2.0 pH 2.5 pH3.0 Control Figure 4-6: The pH solubility pr ofile of catfish protein acid-aided isolates and untreated control from pH 1.5-12 with the non-centrifuged samp les reported as the right most point on the graph. Solubility was calculated as expl ained in Fig. 4-5. Si gnificant differences present between samples are shown in appendix A.

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154 0 20 40 60 80 1001.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 NCpHSolubility (%) pH10.5 pH 11.0 pH 11.5 Control Figure 4-7: The pH solubility pr ofile of catfish protein acid-aided isolates and untreated control from pH 1.5-12 with the non-centrifuged samp les reported as the right most point on the graph. Solubility was calculated as expl ained in Fig. 4-5. Si gnificant differences present between samples are shown in appendix A.

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155 CHAPTER 5 THE EFFECT OF pH-SHIFT PROCESSING ON THE ST RUCTURAL AND THERMAL PROPERTIES OF CATFISH ( Ictalurus punctatus ) PROTEIN ISOLATES Introduction The structure function relationship of muscle pr oteins is important to the application and performance of muscle based food products. Th e modification of protei n structure from the native state is critical for meat functionality. The native struct ures of muscle proteins, though crucial for the function of the muscle proteins in the living system, do not exhibit functional characteristics associated with processed meat. Th e change in secondary st ructures are related to the functionality of muscle proteins, however the intermediary transition of secondary structures from native to denatured are considered to bear a more direct relations hip to muscle protein functionality (Xiong 1997). Muscle based products are first influe nced by the growth conditions, slaughter method and muscle type of the animal The production of functional comminuted meat products is further dictated by the processing conditions and chemical additives used. The production of comminuted meat products frequently includes th e addition of salts, primarily sodium chloride. Addition of salt induces structural changes of muscle proteins due to the electrostatic interaction between proteins and the sodium and ch loride ions which results in muscle fiber swelling (Xiong 1997). The swelling of muscle fibers allows for increased water retention of the meat system, and thus modifi es the functional properties and ultimately the perception of the product. These changes are direct ed by the state of the in termediate structural characteristics of the muscle proteins. The change in intermediate structures can thus be dictated by processing conditions and chemi cal additives resulting in the functional properties of muscle proteins being determined by ex ternal factors (Xiong 1997).

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156 The use of acid or alkali processing has been shown to change the structural conformations of isolated muscle proteins, specifically myosin (Kristin sson and Hultin 2003a; Raghavan and Kristinsson 2007a, 2007b), resulting in molten globular or stable interm ediate protein structures. In this study it is hypothesized that the utilization of acid-ai ded and alkali-aided extraction method on a warm water species (catfish) will lead to unique protein structural properties that are pH dependent. This hypothesis was investigated by determining the effect of pH shift processing at different pH on the structure and stability of catfish protein isolates produced at low and high pH treatments. Methods Raw Material The raw m aterial used in these studies was fresh catfish fillets obtained 1-3 days post harvest from a local supplier. Catfish fillets were only purchased which were determined to be within 3 days of packaging. The catfish fillet s were purchased and im mediately transported on ice to the laboratory and pr ocessed the same day. Preparation of Protein Isolates Protein isolates were p repared according to figure 2-1. Fresh fillets were initially ground in an Oster heavy duty food grinder (Niles, Il l., U.S.A.) for the prel iminary disruption and collection of the muscle tissue. Following gri nding, the comminuted meat was diluted 1:2 (w/v) with deionized (DI) water and homogenized in a Waring blender for two bursts of 30 seconds. Following homogenization, the resulting muscle tissu e slurry was further diluted to give a final dilution ratio of 1:6 (w/v) muscle to DI water. This slurry was manuall y stirred with a plastic spatula to achieve good homogeneity. The pH of the slurry was adjusted according to the methods described below, using either 2N NaOH or 2N HCl as needed for the pH desired, with continuous manual mixing. Upon reaching the desi red pH, insoluble material was removed by

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157 centrifugation (Sorvall RC-5B centrifuge with a GS-3 rotor, Kendro Laboratory Products, Newtown, Conn., U.S.A.) at 10,000 x g for 20 minutes at 5oC. Following centrifugation, the soluble middle layer was collected through a kitche n strainer with a mesh size of approximatly 0.25 mm to minimize contamination with other sepa rated materials. The soluble material was readjusted to pH 5.5 as described above. After readjustment the solu tion was centrifuged to remove excess water and remaining soluble proteins at 10,000 x g for 20 minutes. The precipitated protein was collected by decanting the supernatant containing the unprecipitated proteins and removing it with a steel spatula. All of the precipitate from each solubilization pH was combined from the centrifuge bottles into one protein isolate. This dewatered protein isolate was further dewatered by placing the combined precipitate into cheesecloth and hand squeezing until the moisture content was below 80%. Mois ture content was determined using a Cenco infrared moisture analyzer (CSC Scientific, Fairfax, Va., U.S.A.). Upon completion of manual dewatering the protein isolation was complete. Preliminary unpublis hed investigation of protein isolates in this laborato ry found the shelf life of catfish protein isolates to be 5-7 days on ice. All protein isolates were stored on ice at the precipitation pH and used within 5 days. Protein Concentration Protein concentration in the is olates and the subsequent solu tions was determ ined using the Biuret method, as described by Torten and Whita ker (1964), with of 10% w/v deoxycholic acid in water added at 10% v/v of the protein-Biuret reagent to minimize turbidity from any remaining lipids in the samples. Protein concen tration was measured based on a standard curve based on BSA. Circular Dichroism (CD) CD was done accord ing to Kristinsson and Hultin (2003a). The isolate was diluted and homogenized for 1 minute on ice at speed 2 in a Bio-homogenizer (M133/1281-0, Bio Spec

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158 Products Inc., Bartlesville, OK) in 20 mM Tris -HCl buffer, pH 7.2 with 600 mM NaCl to a concentration of 10 mg/ml. This protein stock solution of 10 mg/ml was pr epared and diluted to 2 mg/ml 1 hr before analysis and held on ice. Fo r analysis of secondary structure the sample was scanned from 260-200 nm in a 0.1 cm quartz cuvette 0.2 nm resolution scanned at 50 nm/min on a Jasco J-500C circular dich roism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The time duration of the scan was no more than 10 minutes. Differences in total alpha helix beta structure and random coil st ructures present were determined from DICHROWEB using the K2D analysis program (Lobley et. al., 2002). Isolate Susceptibility to Unfolding in Guanidine Hydrochloride ( Gu-HCl) Catfish protein isolate and the control, untr eated ground catfish muscle, were treated with Gu-HCl over the range of 0-6M in 0.5 M increments. For assessment of denaturation, 222 nm was used as an indicator wave length of alpha helical content, scanning from 220-225 nm in a 0.10 cm quartz cuvette, 0.2 nm resolution sca nned at 50nm/min on a Jasco J-500C circular dichroism spectropolarimeter (Jasco Inc, Easton, MD) at room temperature. The protein concentration was 1 mg/ml. Samples were allowed to incubate for 5 minutes on ice prior to reading. Differential Scanning Calorimetry (DSC) DSC was conducted according to Fukushim a and others (2003) on a MicroCal DSC (MicroCal, LLC, North Hampton, MA). Isolate we re diluted in sample buffer (20 mM tris-HCl, pH 7.2 with 600 mM NaCl) to a protein concen tration of 10 mg/ml and homogenized for 1 minute on ice at speed 2 in a Bio-hom ogenizer (M133/1281-0, Bio Spec Products Inc., Bartlesville, OK). The sample was degassed under vacuum for 5 min at 5oC. After degassing, 0.6 ml was loaded into the sample cell, the refe rence cell contained sample buffer. The sample was then linearly heated at 1oC/min from 5oC-80oC. Analysis of the data was conducted on the

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159 software provided by the manufacturer, Origin Pr o 7.5, for determination of both exothermic and endothermic events. Susceptibility of Proteins to Transg lutaminase-Induced Cross Linking The susceptibility of pH-shift processed cat fish m uscle to protein cross linking was assayed using the commercial transglutaminase (TGase) Activia TI from Ajinomoto LLC (Ajinomoto Food Ingredients LLC Chicago Il). 0.2% TGase (w/w) was added to isolate diluted to 10% solids in 20mM Tris-HCl buffer, 600mM NaCl. TGase activity was assayed by using an AR2000 advanced research rheometer (TA Instrument New Castle, DE) with a head with a flat cross-hatched polyacrylic surface and thermally controlled plate. The gap was 1000 microns and the head lowered onto the sample using the cont rolled speed function provided by the software. The samples were tested in oscillatory mode under controlled frequency at 0.1 Hz, and strain controlled at 0.01. TGase activity was monitore d by the increase in G over 1 hour at 30oC. Activity was compared against samples with no TG ase added. G was plotted against time and the slope of the line from linear regression was used to calculat e the TGase activity. The activity coefficient was calculated as the ratio of treated to untreated samples. Statistical Analysis Experim ental design, as shown in figures 1-1 1-4, was conducted, in duplicate, on replicate isolations as discussed above. A repl icated (N=2) was determined as acceptable due to achieving an acceptable power ( =0.80, =0.05). One-way independent measures analyses of va riance were used to examine the effects of all methods. The Kruskal-Wallis ANOVA testing by ranks was used on data which did not pass normality. Post hoc analysis was conducted only in the presence of significant population differences. ANOVA statistical comparisons were conducted with SigmaStat, (Systat Software

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160 Inc. San Jose Ca) with a significance level of p<0.05. After SigmaS tat completed the ANOVA analysis, the post hoc analysis recomme nded by SigmaStat was Tukeys test. Results Circular Dichroism (CD) pH-shift processing led to changes in secondary structure of protein isolates as seen by the CD results (figures 5-1, 5-2, 5-3). The order of secondary structure was found to be control>alkali>acid. Acid treated pr otein isolates ha d lower relative -h elix content than the control and higher relative beta structure content than the co ntrol though no main effect was observed. Alkali treated protein isol ates also showed relatively lower -helical content and higher beta structure content th an the control though no main e ffect was observed. There were no significant differences (p>0.05) in secondary structure between acid and alkali treated protein isolates. No significant diffe rence (p>0.05) was observed in random coil content among any treatment groups as compared with each other or with the control. Guanidine Hydrochloride Denaturation (Gu-HCl) Subjecting protein sam ples to increasing concen tration of Gu-HCl gave unexpected results (figure 5-4 and 5-5). Increasing Gu-HCl to 0.5 M led to substantial protein denaturation of the control samples (70%), some add itional denaturation of alkali-aide d isolate samples, while acidaided isolate was less affected. Isolates made with the pH 11.5 treatment were most affected and had fully unfolded in 0.5 M Gu-HCl. All sample s demonstrated a refolding behavior in 1 M GuHCl, with all isolates (except the one made with the pH 3 trea tment) and control having higher level of structure than untrea ted control at 0 M Gu-HCl. At 1.5 M Gu-HCl, control samples showed some unfolding, while acid -aided isolate samples continue d to refold (figure 5-4). Alkali-aided samples were little affected going from 1 to 1.5 M Gu-HCl (f igure 5-5). Going from 1.5 M to 2 M Gu-HCl led to almost complete unfolding of all samples.

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161 Micro-Differential Scanning Calorimetry (DSC) The therm al events observed for all pH-shift processed samples are shown in figures 5-6, 5-7 and table 5-1. The event boxes in blue (tab le 5-1) are endothermic events and the event boxes in red are exothermic events. The numbers listed are first the temperature and then the specific heat, normalized from an internally deri ved baseline using Origin software. The table boxes are grouped on a per-degree basis with samples grouped together in one row if the thermal events happen within one degree of each other. The first event occurred at 11.8 +/0.09C. The event was endothermic in all samples except pH treatment 11.5 was exothermic. The next event occurred at 20.8-21.74C in pH treatment 10.5 and the control. Following this ev ent a single exothermic event occurred in pH treatment 10.5 at 23.73C. Exothermic events occurred in pH treatments 2.0, 2.5, 10.5, 11.0 and 11.5 between 28.27-29.35C. The next event was an endothermic event in all the acid treated samples over the range of 34.79-35.26C. The al kali treated samples had an event at 36.8337.31C. The control, however, had an exothermic event at 36.56C. The pH treatments 2.5 and 3.0, had an endothermic event at 39.65C and 39.82C, respectively. The pH-treatments, 2.0, 10.5, 11.0, 11.5, had an exothermic event between 41.21-42.10C. The control sample had an endothermic event at 43.47C. An endothermic event was recorded among all the pH-treated samples occurring between 44.52-45.67C The pH-treatments 2.0, 3.0, 10.5, 11.0, 11.5 had an endothermic event ranging from 47.69-48.34C. The control had an exothermic event at 47.20oC. In pH treatment samples 2.0, 3.0, 10,5, 11.0, 11.5 and the control, an endothermic event was recorded, while pH-treatment 2.5 had an exothermic event between 50.04-51.75C. An exothermic event in pH-treatment 2.5 occurred at 55.71C. The pH-treatments 11.0 and 2.0 had exothermic events at 56.83C and 57.17C resp ectively. An exothermic event occurred in pH treated samples 2.5, 10.5, 11.5 and the contro l between 57.82-58.81C. The pH-treatments

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162 3.0 and 11.5 had an endothermic event at 60.39C and 59.61C respectively. In pH treatment samples 2.0 and 2.5 an endothermic event occurred at 62.60C and 63.16C, respectively. The pH-treatments 3.0 and 10.5 had exothermic even ts occur at 63.63C and 63.81C respectively. An endothermic event occurred in the control at 64.97C. Exothermic events occurred between 66.49-67.38C in pH-treatments 2.0, 3.0, 10.5 and 11.5. Exothermic events occurred in pHtreatment 2.5 and the control at 68.42C and 69.36C respectively. Endothermic events at 71.18C occurred in pH-treatments 2.0 and 10.5. The pH-treatment 2.0 had an exothermic event occurring at 72.88C. The last events ranged from 74.18-76.39C. Endothermic events occurred in pH-treatments 2.0, 2.5 and the control at 74.18C, 75.95C and 75.12C, respectively. The pH-treatments 10.5 and 11.0 had exothermic events occurring at 74.83C and 76.39C, respectively. Susceptibility of Proteins to Cro ss-linking by Transglutaminase Susceptibility of protein isolat es and contro l samples to transglutaminase (TGase) induced cross-linking was measured by following increases in G of the system as a function of time and calculating the difference between final G of the unt reated and treated sample (i.e. susceptibility ratio). Figures 5-9 to 5-15 s how that G for all TGase contai ning samples, except control and isolates made with the pH 2.0 treatment, increased over untreated samples as time progressed however no main effect was determined. All sa mples however had a higher final G for TGase samples after 1 h compared to untreated samples, re sulting in a positive susc eptibility ratio for all samples (figure 5-8). No significant differences (p>0.05) were seen in the susceptibility ratio (figure 5-8) between the acid tr eated samples or the alkali treated samples. No significant differences in the susceptibility ratios (p>0.05) were found between acid treatment and alkali treatments compared to the control.

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163 Discussion Circular dichroism was used as a comparativ e method to study the average change in the structural properties of all pr oteins collected during pH-shift processing. The differences observed between the average structural content of pH-shift treated samples and control samples indicate a structural transition fr om alpha helix to beta structure as denaturation increases. The native secondary structural content of muscle pr oteins followed the order of control>alkali>acid. The increase in beta structure content indicates that extreme pH used during pH shift processing induced protein unfolding at the high or low pH used to extract the proteins. After adjustment of the protein system from high or low pH to pH 5.5, refolding occurs resulting in an increase in the beta structure content of prot ein isolates. Previous studies have shown that during the denaturation process of proteins th e beta structure secondary structur e is more structurally stable than the alpha helix secondary structure and al pha helix structures ma y transition to beta structures before completely unfolding into a random coil (Drummy a nd others 2005). The structural changes induced by pH-shift processing may be species specific because, in contrast to the present study, results shown for pH adjusted cod myosin with pH-shi ft processing showed a minimal effect on the secondary structure of re folded myosin (Kristinsson and Hultin 2003a). However, similar to the catfish used in the pr esent study, it was reporte d that acid treatment resulted in the reduction of secondary structur e with isolated catfish myosin (Raghavan and Kristinsson 2007a). This indicates that the stru ctural trends observed when testing the whole protein isolates system are specific to fish speci es and similar to the results obtained for isolated catfish myosin. However the differences obser ved between catfish myosin and cod myosin further indicate that the unfoldi ng and refolding parameters need to determined across species and different fish may possibly impart different gel properties to the isol ated protein. Though the refolding properties of catfish myosin (Ragha van and Kristinsson 2007a) showed similarities

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164 with the results attained from the present protein isolates thos e results applied best to the pH 2.5 treatment. The results show th at catfish myosin treated at pH 2.0 showed a larger decrease in secondary structure than treatm ent at pH 2.5 (Raghavan and Kristinsson, 2007a). These results are different from the effect of pH 2.0 treatment on protein isolates (figures 5-1 and 5-2) which showed that this pH did not significantly de crease (p>0.05) in seconda ry structure and in particular the alpha helix content. This means that similarities exist between the isolated individual proteins and the whol e muscle system; however, the stru cture of protein isolates has unique properties that are not dictated exclusively by myosin. Gu-HCl treatment is used for investigating th e susceptibility of proteins to chemical denaturation. Gu-HCl as denaturant can be used to establish the chemi cal denaturation kinetics of a protein system. Normally one would expect a somewhat sigmoidal denaturation curve at increasing levels of Gu-HCl, but this was not seen here for the catfish proteins. Gu-HCl induced a small change in protein struct ure at 0.5 M for acid-aided isolates while alkali-aided isolates were further denatured. Control became highly denatured at this low level of Gu-HCl. A further increase in Gu-HCl led to refolding (more negative response at 222 nm), followed by more unfolding (less negative response at 222 nm) as Gu-HCl increased. This data therefore does not allow for a two-state kinetic model to be estab lished. Structural even ts occurring in protein folding after pH-shift processing and apparent in creases in folding of protein isolates with increased Gu-HCl indicate molten globular st ructures are formed. However these molten globules are not unique to the is olates as the control also de monstrated apparent refolding behavior. Fan and others (1996) showed an incr ease in chicken liver dihydrofolate reductase activity in low concentrations of Gu-HCl with no significant ch ange in secondary structure according to circular dichroism results of second ary structural analysis (Fan and others 1996).

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165 The results of Fan and others (1996) and those shown here suppor t the presence of a range of conformational isomers with similar low energy states (Kumar and others 2000) resulting in structures which have modified functional properties. The DSC results from this study show that th e thermal transitions of pH-shift processed catfish muscle differ not only fr om the control but are also dependent on the pH used during processing. The first temperature range which an event was recorded at was 11C. This event occurred in all samples however pH treatment 11.5 showed an e xothermic event at this low temperature compared to all other sa mples showing endothermic events. The next thermal event was recorded between 20.8-21.74oC in pH treatment 10.5 and the control with pH treatment 10.5 having anot her small event closely following at 23.73oC. Major distinguishing events occurred from 34.79-35.26oC in the acid treated samples and from 36.8337.31oC in alkali treated samples. In these temper ature ranges endothermic events were recorded for pH-treated samples. At 36.56oC, within the alkali treated range, the control had an exothermic event. Catfish muscle subjected to pH-shift processing resulted in an endothermic event occurring rather than an exothermic event. Acid processing of catfish muscle further changed the thermal sensitivity of the isolated proteins reducing the temperature, 34.79-35.26oC, at which this reaction occurred. The change in the thermal response of acid treated catfish muscle may be due to the proteins co-precipitating with myosin as acid treated isolated myosin shows the first thermal event at 37oC (Raghavan and Kristinsson 2007a, 2007b). The next series of even ts occurred between 39.65-48.34oC. The event leading this off was catfish muscle processed at pH 2.5 and 3.0 wh ich had exothermic events occurring at 39.65oC and 39.82oC respectively whereas the other pH shift processed samples had endothermic events occurring between 41.22-42.1oC, with the control event occurring at 43.47oC. Catfish muscle

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166 subjected to pH-shift processi ng exhibited a lower temperature range of thermal events, (39.6542.1oC), than the control sample, (43.47oC). The control peak can be associated with the second transition of whole myosin peaks as seen for catfish myosin at pH 7.3 which exhibited a second transition peak at 44.9oC (Raghavan and Kristinss on 2007b). The control at 43.47oC and 47.2oC showed very large exothermic peaks as seen in figure 5-6, the 4. These higher temperature peaks however did not correlate with the two myos in peaks shown by Raghavan and Kristinsson (2007b) for isolated catfish myosin which occurred at 37oC and 44.9oC. The first large peak at 43.47oC was shifted from the pH treated samples as seen in table 5-1. The second large peak was consistent with the pH-treated samples ex cept for pH treatment 2.5. However within these two large peaks seen in the cont rol all the pH shift processed samples had an endothermic event between 44.52-45.66oC. The range observed here would fall within an expected range with the second myosin peak shown by Raghavan and Kristinsson (2007b) with catfish myosin. Over the 39.65-42.1oC temperature range Xiong (1997) reported the first transition peak of chicken breast myosin at 40oC. The results of this study and those of Raghavan a nd Kristinsson (2007b) do not indicate that catfish myosin has the first transition at 40oC but that an additi onal peak is observed either by a co-precipitated protein or due to the structural modification of catfish myosin during the precipitation process in the presence of other muscle proteins. Following these series of events, between 50.04-51.02oC an endothermic event occurred in all samples. This indicates that though pH-shift processing alters th e structure and thermal response of muscle proteins, there is this one temperature range wh ich is not affected by pH-shift processing and some part of myosin or anothe r co-precipitated protein. The thermal events occurring within this temperature region have been associated with the transitions of myosin S-2 subfragment (51oC) in rabbit and chicken br east, the light meromyosin chains in chicken breast

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167 (52oC) and the myosin light chains in rabbit (51oC) (Xiong 1997). The thermal transitions above the 50-51oC range show different patterns of thermal reactivity. These differences in the thermal events, of individual pH treatmen ts and the control, may provide insight into the differences observed in other properties of pH-s hift processed muscle proteins. Transglutaminase (TGase) is an enzyme whic h promotes the formation of covalent bonds in muscle proteins. This bond is based on the cross linking of lysine and glutamine to form the dipeptide -( -glutamyl)lysine (Perez-Mateos and Lanier 2007). The formation of covalent cross links in muscle proteins in the absence of TGase are primarily by the formation of disulfide bonds (Stone and Stanley 1992). The formation of covalent cross links in mu scle proteins promotes the formation of stronger heat set gels at temperatures below the point where muscle proteins form irreversible gels, normally between 25-50C. This increase in gel strength at low temperature is a setting effect referred to as suwari setting. The use of TGase in muscle systems has been employed to formulate muscle products from smaller pieces of meat such as scallops, steaks, shrimp and ham (Ajinomoto 2007). The use of TGase in surimi processing has been employed for the improvement of lower grade surimi or the mixt ure of lower grade surimi with higher grade surimi to form the gel strengths needed for appropriate product formulation. The susceptibility of muscle products to TGase activity is based on the availability of both lysine and glutamine on the surface of the proteins to interact with the ac tive site of TGase to form the covalent cross link. The protein motifs which facilitate TGase activity have not been characterized; however, TGase activity in muscle systems has been directly correlated with the decrease in myosin heavy chain (P erez-Mateos and Lanier 2007).

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168 The results of this study indica te that pH treatment of catfish muscle proteins may promote interactions of the isolated prot eins with TGase for all alkalineaided isolates but not all acidaided isolates. Thus these resu lts indicate that structural modi fications which occur with pH treatment result in the interactions of isolated proteins with TGase appear greater than those without pH treatment under most treatment conditi ons. Perez-Mateos and Lanier (2007) have indicated that myosin heavy chain is reduced wh en muscle is treated with TGase, suggesting cross-linking between myosin. The structural modifications during acid processing were more pH specific with respect to TGas e treatment as treatment at pH 2.0 was not significantly different from the control. These results, when compared with the structur al results indicate that the specific changes of muscle proteins isolated at pH 2.0 are not identifiable from other acid treatments by CD but result in surface modification of the proteins as shown by TGase treatment. The high variability of pH-treatment 10.5 indicates that the refolded surf ace structure at this pH is not as consistent as the refolding of proteins trea ted under more extreme alkali conditions. The use of pH-shift processing is also species specific as the result s of this study are in c ontrast to the results reported (Perez-Mateos and Lani er 2007) which found no increase in gel strength by TGase treatment to acid solubilization of protein isolates made from A tlantic Menhaden. The reactivity of muscle proteins with TGase indicates the accessibility of sp ecific amino acids to TGase. Changes in the accessibility of specific amino acids indicate structural changes in the proteins directly affect the surface struct ure of proteins. The characteri zation of the specific location and proteins involved in the inter action with TGase may lead to fu rther understanding of the protein motifs responsible for enzyme induced cross-linki ng and increase the understanding of pH-shift processing at specific sites within a protein or protein mixtures.

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169 Conclusions This study dem onstrated that pH shift proces sing decreases the alpha helical content of protein isolates, increases the structural flexibil ity and modified the temperatures and types of thermal events catfish muscle proteins underwent. These results support our hypothesis that pHshift processing will affect the st ructure and thermal reactivity of catfish muscle proteins and those effects were unique to the pH used during processing. It may be concluded that pH-shift processing affects catfish muscle protein secondary structure. This modification of secondary structure may lead to the modification of the ther mal reactivity and the susc eptibility of protein isolates to enzymatic modification and ultimat ely the modification of the thermal gelation properties of catfish muscle proteins. Additi onally the use of enzyme treatment, specifically TGase, may provide an effective method of anal ysis for determining the modification of the surface structure of muscle proteins and provide insight to the mechanisms involved in the interaction and gelation of a highly complex material.

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170 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 200 210 220 230 240 250 260 nmDelta Epsilon 2 2.5 3 control Figure 5-1: Circular di chroism of catfish protein isolate ma de with acid processing and untreated catfish muscle recorded from 200-260nm. Delta epsilon units reported were obtained from molar ellipiticity. Samples were scanned in 25 mM Tris-HCl and 600 mM NaCl, pH 7.2. The treatment pH is shown at the top. -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 200 210 220 230 240 250 260 nmDelta Epsilon 10.5 11 11.5 control Figure 5-2: Circular dichrois m of catfish protein isolate made with alkali processing and untreated catfish muscle recorded from 200-260nm. Delta epsilon units reported were obtained from molar ellipiticity. Samp les were scanned in 25 mM Tris-HCl and 600 mM NaCl, pH 7.2. The treatment pH is shown at the top.

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171 0.00 10.00 20.00 30.00 40.00 50.00 60.00 2.02.53.010.511.011.5control pH TreatmentStructure (%) %alpha helix % beta sheet % random coil Figure 5-3: Level of apparent -helix, -structureand random coil in cat fish protein isolates and untreated control. Percent structure was calculated from Dichroweb using the K2D model. The K2D model did not require a protein reference set and provided a secondary structural model without re quiring data points below 200 nm. No significant differences (p>0.05) were present between any of the samples for all structures.

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172 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 00 511 52 [GuHCl]Coefficent of Denaturation 2.0 2.5 3.0 control Figure 5-4: Effect of Gu-HCl (0-2 M) on appare nt structure of low pH processed catfish protein isolates and untreated control. GuHCl treatment used 222 nm as the indicator wavelength of alpha helix content. The cha nge in structural content was determined by whole muscle control in 0 M GuHCl having 100% native or 0% denatured structure. 100% denatured structure was calculated from the response at 222 nm of the control in 6 M GuHCl. The two stat e denaturation model was then used to calculate the fraction of denaturation of each sample.

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173 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 00 511 52 [GuHCl]Coefficent of Denaturation 10.5 11.0 11.5 control Figure 5-5: Effect of Gu-HCl (0-2M) on apparent structure of high pH processed catfish protein isolates and untreated control. GuHCl treatment used 222 nm as the indicator wavelength of structural content. The cha nge in structural content was determined by whole muscle control in 0 M GuHCl having 100% native or 0% de natured structure. 100% denatured structure was calculated from the response at 222 nm of the control in 6 M GuHCl. The two state denaturati on model was then used to calculate the fraction of denaturation of each sample.

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174 -0.2 -0.1 0 0.1 0.2 0.3 0.4 51525354555657585 Temperature oC cal 2 2.5 3 control Figure 5-6: DSC thermograms of low pH treated catfish protein is olates and untreated control. The thermograms represented here are th e average of 4 replicate scans from two separate isolations. The curves were smoothed together by 3 point averaging. Average smoothing was repeated 4 times to obtain the smoothed graph. The black line is the untreated control. -0.2 -0.1 0 0.1 0.2 0.3 0.4 51525354555657585 Temperature oC cal 10.5 11 11.5 control Figure 5-7: DSC thermograms of high pH treated catfish protein isolates and untreated control. The thermograms represented here are th e average of 4 replicate scans from two separate isolations. The curves were smoothed together by 3 point averaging. Average smoothing was repeated 4 times to obtain the smoothed graph. The black line is the untreated control.

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175 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 2.02.53.010.511.011.5control isolation pHaveraged treated:untreated ratio Figure 5-8:The susceptibility of catfish protei n isolates and untreated control to enzymatic treatment with transglutaminase (TGase). Measurement of TGase activity was the relative increase in the storage modulus between 0.02% TGase treated and untreated incubated at 30C for 1 hr. A main eff ect was not determined for the sample population (p>0.05).

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176 0 5 10 15 20 25 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-9: Compiled and smoot hed rheogram of pH treatment 2.0 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software. 0 20 40 60 80 100 120 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-10: Compiled and smoothed rheogram of pH treatment 2.5 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software.

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177 0 20 40 60 80 100 120 140 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-11: Compiled and smoothed rheogram of pH treatment 3.0 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software. 0 20 40 60 80 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-12: Compiled and smoothed rheogram of pH treatment 10.5 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software.

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178 0 10 20 30 40 50 60 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-13: Compiled and smoothed rheogram of pH treatment 11.0 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software. 0 10 20 30 40 50 60 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-14: Compiled and smoothed rheogram of pH treatment 11.5 with (black) and without (gray) TGase treatment. Protein pastes we re prepared at 10% solids in 20 mM trisHCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed us ing TA rheology advantage data analysis software.

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179 0 2 4 6 8 10 12 01000200030004000 Time (s)Storage modulus (G') TGase Untreated Figure 5-15: Compiled and smoothed rheogram of the control with (black) and without (gray) TGase treatment. Protein pastes were pr epared at 10% solids in 20 mM tris-HCl buffer with 0.6 M NaCl, pH 7.2. Samples were tested at 30oC for 1 hour. Rheograms were combined and smoothed using TA rheology advantage data analysis software.

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180Table 5-1: Thermal events of catfish protein isolates and untreated control as record ed by DSC. The smoothed transposed data was used to generate this peak table. The columns are separated into samples and the rows are thermal events. Thermal events are paired to ~1oC. Blue represents an endothermic event, orange represents an exothermic and yellow denotes no event was present. pH 2.0 pH 2.5 pH 3.0 pH 10.5 pH 11.0 pH 11.5 Control T (C) tCP T (C) tCP T ( C) tCP T (C) tCP T (C) tCP T (C) tCP T (C) tCP 11.79 0.1044 11.82 0.0476 11.80 0.2234 11.84 0.0386 11.76 0.2021 11.84 -0.1160 11.79 0.0622 20.80 -0.0103 21.74 -0.0600 23.73 -0.0007 28.55 -0.0285 28.27 -0.0215 29.35 -0.0297 29.14 -0.0214 28.54 -0.0342 34.80 0.0852 35.26 0.0722 34.79 0.0845 37.31 0.0803 37.25 0.1111 36.83 0.0908 36.56 -0.0758 39.65 0.0076 39.82 0.0010 41.22 -0.0091 41.85 -0.0188 41.94 -0.0133 42.10 -0.0484 43.47 0.3714 45.19 0.0563 44.52 0.1175 45.64 0.0838 45.42 0.0985 45.34 0.0719 45.67 0.0398 47.70 0.0007 47.84 0.0018 47.77 0.0007 47.69 0.0019 48.34 0.0155 47.20 -0.0118 51.75 0.0645 51.09 -0.0907 51.08 0.0238 51.66 0.0960 50.93 0.0817 51.02 0.0776 50.04 0.3260 55.71 -0.0028 56.83 -0.0108 57.82 -0.0101 57.17 -0.0047 58.62 -0.0355 58.15 -0.0623 58.81 -0.0844 60.39 -0.0284 59.61 0.0392 62.60 0.0400 63.16 0.0003 63.63 -0.0024 63.82 -0.0013 64.97 0.0109 66.49 -0.0522 67.11 -0.0354 67.38 -0.0143 67.22 -0.0140 68.42 -0.0710 69.36 -0.0339 71.18 0.0020 71.18 0.0001 72.88 -0.0502 74.18 0.0396 75.95 0.0502 74.83 -0.0385 76.39 -0.0214 75.12 0.0424

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181 CHAPTER 6 THE EFFECT OF HEATING RATE ON pH-SHIFT PROCESSED CATFISH ( Icta lurus punctatus ) MUSCLE PROTEINS Introduction The functional characteristics of m uscle pr oteins are determined by the structural conformation of the proteins in raw muscle and th e intermediate structures of proteins during the transition from the native to denatured state (Xiong 1997). One of the ways to dictate the structural transition of muscle proteins from the native state to the non-native state is by the application of heat (Phillips a nd others 1994). Temperatures a bove the denaturation temperature are usually required to achieve the structural intermediates necessary which result in the formation of a gel network of prot eins (Phillips and others 1994). Muscle proteins denature over a wide temperature range. Thermal transitions poin ts of isolated myosin are in the range of 40oC67oC (Xiong 1997). Muscle proteins show increases in gel strengt h when subjected to thermal treatment., However, at medium temperatures, or about 35-55oC, muscle proteins have exhibited weakening in gel strength which is thought to be due to the disassociation of the helix-coil of myosin. Above this temperature range strengtheni ng of protein-protein in teractions occur, and gel strength increases again, This dissociation is thought to disrupt the gel network as it is forming but further increases in te mperature result in the reforma tion of the gel network resulting in the final set form (Xiong 1997). Thus certain structural and conformational changes of muscle proteins during thermal processing are require d to achieve desirabl e functionalities (Xiong 1997). The modification of thermal processing by altering the heating rate has been shown to affect the functional properties of comminuted muscle protein products (Riemann and others 2004a). For example, slower heating rates allo ws progressive, sequential unfolding of proteins leading to a finer texture and increased water holding capacity of musc le proteins (Xiong 1997).

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182 The objective of this study was to investigate the effect of various heating rates on the gel forming ability of protein isolates made from acid and alkali processed ca tfish protein isolates. Methods Heating rate gelation studies were conducted at North Carolin a State University (Raleigh, NC) in the laboratory of Dr. Tyre Lanier. Production of Protein Isolates Fresh channel catfish was obtained for the st udy from a local supplier. Protein isolates were produced as outlined in Figure 2-1: pH 2.5 for the acid-aided process and pH 11.0 for the alkali-aided process. Precipitation following filtration was preformed at pH 5.5. Removal of insoluble materials was accomplished by screeni ng the material 3 times through progressively tighter mesh. After precipitation of the clarif ied muscle protein homogenates chiffon cloth was used for dewatering protein past e to less than 78% moisture. Protein Composition Protein isolates were assayed for m oisture in a drying oven fo r 24hrs. Protein content was assumed to be 97% dry weight (Kristinsson and others 2005b). Torsion Torsion tubes, 10 cm in length and 1.8 cm in diameter were filled with protein paste at 78% moisture and subsequently heated from 10C to a 70C internal endpoint, either rapidly by a cylindrical microwave (Riemann and others 20 04b), at 20 or 98C/min to test rapid heating effects, slowly at 1C/min by immersion in a prog rammable water bath or placed directly in an 70C water bath for 15 minutes. Temperatures were measured by a fiber optic probe (Riemann and others 2004b). Upon reaching 70C, rapidly heat ed samples were held for 0 or 20 min prior to rapid cooling by immersion in ice water for sufficient internal cooling to <10C for ~30 min. Samples were held static in a cylindrical mi crowave applicator (length 16 cm, radius 12.5 cm)

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183 and heated using power settings calculated previously. After reaching a final temperature of 70C, this temperature was maintained in gels during the subsequent holding period by utilizing feedback software (Riemann and others 2004b). Rheology Rheological changes (storage m odulus, G) of pastes/gels at 78% moisture were nondestructively and continuously meas ured as pastes were heated, held and cooled using a 40 mm, 4 degrees slope cone and plate atta chment of a constant stress, small strain rheometer (Stresstech, Rheologica instruments AB, Lund, Sweden). Os cillation parameters were those used in Riemann et al 2004. Heating conditions were at either 20C/min, the most rapid heating rate possible for this apparatus, 0.5C/min, 1C/min, 2 C/min or 5C/min to an endpoint temperature of 70C followed by holding for 0 or 20 min prior to cooling at 5C/min to 10C. Statistical Analysis Prelim inary investigation of the affect of heating rate on pH-s hift processed catfish protein isolates was conducted on a single isolation of catfi sh fillets. The fillets used in this study were obtained from two lots of fillets shipped to a local supplier. Torsion studies were conducted on four to six cooked gels. Each gel was sampled 2 to 4 times. Rheological studies were conducted in duplicate. The heating rate study was analyzed with Gra ph Pad QuickCalcs online calculator software (Graph Pad Software Inc. San Diego Ca). Anal ysis used an unpaired t-test with a manual bonferoni correction, overall significance of p<0.05 with the in dividual significant p-values of p<0.004.

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184 Results Torsion The torsion stress and strain results were obtained by therm ally tr eating catfish muscle isolated at both acid (2.5) and alkali (11.0) pH by two heating me thods: microwave heating at a heating rate of 20C per minute with (20/20) a nd without (20/0) a 20 mi nute hold, a heating rate of 98C per minute with a 20 minute hold (98/20 ) and water bath heating rates of 0.5C (0.5 water), 1.0C (1 water), and 10C (10 water) per minute. Rapid heating provided total process times of 3 23 and 21 min, respectively, for 20C with and without 20 minute holding and 98C per minute with a 20 minute hold. The stress an d strain are shown in figures 6-1 and 6-2, respectively. The stress results showed that alkali trea tments were significantly higher (p<0.05) than acid treatment. Alkali processing showed an order of 20/20>1 water>0.5 water>98/20>10 water>20/0. Significant differen ces were not present (p>0.05) between alkali treated samples 20/0 and 10 water. Significant differences were not present (p>0.05) 0.5 water and1 water when compared to 20/20 and 98/20 however significant differences were present between 98/20 and 20/20. Acid processing showed an order of 0.5 water>1 water>98/20>20/20>10 water>20/0. Significant differences were not present (p>0.05) between acid treated samples 20/0, 20/20 and 10 water. Significant differences were not present (p>0.05) between acid treated samples 98/20 and 1 water. Acid treatment 20/20 was not si gnificantly different (p >0.05) from 1 water however it wsa significantly different (p<0.05)fr om 98/20. Significant diffe rences were present (p<0.05) between the acid treated sample 0.5 wa ter and all other acid treated samples. The strain results showed that alkali trea ted catfish protein was significantly higher (p<0.05) than acid treatment 10 water. The strain results showed alkali treated catfish protein 20/0 had significantly higher (p<0.05) strain valu es than acid treated ca tfish protein 20/20, 98/20,

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185 0.5 water and 10 water. The alkali treated is olate showed an order of 20/20>0.5 water>20/0>1 water>98/20>10 water. Acid treatment showed an order of 1 water= 20/20>98/20>0.5 water>10 water>20/0. Rheology The rheogram s shown in figures 6-4 6-10 show the gel formation process of varied heat treatments. During the heating ph ase all rapidly heated samples, both acid and alkali, followed almost exactly the same heating pattern (figur e 6-10) with differentiation between the gels occurring at either the onset of holding or cool ing, depending on the treatment. The acid sample, though not significant (p>0.05), did not reach as high a G during high temperature holding as the alkali treatment. Acid treated catfish muscle not held at high temperature exhibited a reduction in G on cooling at 32oC. No decrease on cooling was observed for the acid sample held at high temperature or the alkali treated samples. The ac id and alkali samples heated at 0.5oC per minute did not follow the same gelation pattern on heating or cooling after heating ~30oC (figure 6-6, 6-9). During the heati ng phase of the samples heated at 0.5oC the G decreased until ~30oC. After this decrease the G began to increase in both the acid and alkali treated samples. The alkali treated sample increased at a greater rate than the acid treated sample and did not exhibit a major decline in G after it began to increase at 30oC. The acid treated sample increased in G until reaching the maximu m G of the heating a nd cooling regime at ~42oC. Above ~42oC the acid treated sample decreased in G until the start of the cooling phase at 70oC. The rheology results shown figure 6-3 of this study show that prior to heating there were no significant differences (p>0.05) between any of the samples treated under acid or alkali conditions. After heating at 70C prior to either holding or cooling, th e G of alkali isolate heated at 20C per minute with no hold was si gnificantly higher (p<0.05) than acid treated

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186 isolate heated under the same conditions. Afte r holding at 70C no significant differences (p>0.05) were shown between any of the samp les, before holding or after holding. No significant differences (p>0.05) were observed based on heating re gimen within acid or alkali treated samples. When comparing acid treatment to alkali treatment h eating at 20C per minute with or without holding alkali treated protein isolates had a significantly higher (p<0.05) G, however, acid treated isolate held for 20 minutes at 70C did not show significant differences (p>0.05) between any of the acid treated or alkali treated samples. Discussion Structu ral shifting from the native state to inte rmediate states results in modifications of muscle protein functionality. The transition of musc le proteins from native to intermediate states may be induced by the heating method applied to a muscle protein paste Heating rate has shown to affect the functional properties of muscle proteins. Isolates heated at a high rate with holding at high temperature, had a significantly higher (p<0.05) stress than rapidly heated samples with no holding at high temperature. The increase gel strength of isolates treated held at high temperature may allo w the requisite time for muscle proteins to unfold into a conformation of hi gher functionality. Previous studies on high temperature holding with rapid he ating rates showed muscle prot eins improving in gel strength during short high temperature holding times. Th ese results follow previously published studies showing that rapid heating rates with no holding produce gels with lower strength than those cooked at slower rates (Riemann and others 2004). The application of high temperature holding for alkali treated catfish muscle proteins fo llowed the results reporte d by Riemann and others (2004) showing an increase in gel strength with increased high temper ature holding on rapidly heated comminuted muscle pastes (Riemann an d others 2004). The acid processed catfish isolate, however, did not follow this same trend with high temperature holding of rapidly heated

PAGE 187

187 gels increasing gel stre ngth. Heating at 20oC/min with or without holding did not significantly affect (p>0.05) the gel strength of acid treated catfish muscle. However the very high heating rate of 98oC/min with 20 min holding resulted in signifi cant increases (p<0.05) in gel strength. The increased gel strength of rapi dly heated gels were significantly lower than gels cooked at the slowest heating rate of 0.5oC/min (p<0.05) but were not significantly lower than gels cooked at 1oC/min heating rate (p>0.05). The rheological results from this study indica te that during rapid heating, the high heating rate has the predominating effect on the mechanis m of gelation of pH-shift processed muscle as seen by the similar patterns of gel formation in figure 6-10. The modification of catfish muscle proteins by pH-shift processing ch anging the gel rigidity on cooling, indicate that the modified protein structures alter the hydrogen bond formation associated with the cooling phase of gelation (Lanier 2000). The effect of high temper ature holding on the gel st ructure after cooling may be due to increased covalent linkages fo rmed at high temperatur e (Lanier 2000) or the application of enough energy into the muscle protein matrix to allow for a more complete denaturation of muscle proteins resulting in a more favorable structural rearrangement (Xiong 1997) which promotes the forma tion of hydrogen bonds (Lanier 2000). The proposed equivalence point calculat ion (Riemann and others 2004a) for the determination of functional perf ormance of heated muscle pastes does not apply to acid processed catfish muscle as the equivalent po ints in the heated products did not result in equivalent improvements in gel strength. The lack of improvement with the equivalent point method as proposed may be due to the modifi cation of the protein structure during acid processing, which resulted in larger decreases in alpha helical structur e and thus reduced the

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188 overall reactive potential of acid produced catfish isolate as shown in chapter 3 with the reduction in gel strength of catfish muscle processed at pH 2.5. Conclusions These resu lts show that acid and alkali processing form gels with different gel strengths at fracture regardless of the rate h eat is applied. However during rapid heating the effect on gel formation, as shown by the rheology results, in dicates that the predominant effect on gel formation is the rate heat is applied. Using the equivalent point method, rapid heating with extended high temperature holding for equal therma l input to the system, for the production and analysis of comminuted muscle products produ ced with alkali processing was shown to be effective whereas the structural modification of acid processing may not allow for the use of rapid heating to produce thermally set gels. The further modification of gel properties and the functional performance of muscle proteins subj ected to pH-shift processing by modulating the rate of heat application as show n here may increase the future utilization and appl ication of pHshift processed protein isolates by expanding the range of textural prop erties achievable and reducing the required cook time for high qual ity comminuted muscle products.

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189 0 10 20 30 40 50 60 70 80 90 100 20/min no hold micro 20/min hold 20 min micro 98/min 20 min hold water .5/min water 1/min water 10/minStress (kPa)A B C BC BC A D DG E FEG D 0 10 20 30 40 50 60 70 80 90 100 20/min no hold micro 20/min hold 20 min micro 98/min 20 min hold water .5/min water 1/min water 10/minStress (kPa)A B C BC BC A D DG E FEG D Figure 6-1: Stress response of acid-aided (pH 2.5) in dark grey and alkali-aided (11.0) in light grey catfish protein isolates (pH 11.0) r ecorded on the Torsion Gelometer. The microwaved samples were heated at 20oC/min with no high temperature holding (20/min no hold micro) or with a 20 minut e high temperature hold time (20/min hold 20 min micro) and at 98oC/min with a 20 minute hold (98/min 20 min hold) for both acidand alkali-aided isolates The lower heating rates were achieved in a water bath. The 0.5oC/min (water .5/min) and 1oC/min (water 1/min) heating rates were done using a computer controlled water bath. The 10oC/min (water 10/min) heating rate was achieved by preheating a water bath to 70oC and placing the torsion tubes directly into the hot water bath for 15 minutes. Gel diameter was 1 cm and the machine rotated the gel at 2.5 RPM. The significant differences present between samples (p<0.05) are represented by the lett ers above the colum n. Similar letters represent no significant differen ce (p>0.05) between treatments.

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190 0 0.5 1 1.5 2 2.5 20/min no hold micro 20/min hold 20 min micro 98/min 20 min hold water .5/min water 1/min water 10/minStrainA BC BC BC ABAB AB AB ABC AB C ABC 0 0.5 1 1.5 2 2.5 20/min no hold micro 20/min hold 20 min micro 98/min 20 min hold water .5/min water 1/min water 10/minStrainA BC BC BC ABAB AB AB ABC AB C ABC Figure 6-2: Strain response of acid-aided (pH 2.5) in dark grey and alkali-aided (11.0) in light grey catfish protein isolates (pH 11.0) r ecorded on the Torsion Gelometer. The microwaved samples were heated at 20oC/min with no high temperature holding (20/min no hold micro) or with a 20 minut e high temperature hold time (20/min hold 20 min micro) and at 98oC/min with a 20 minute hold (98/min 20 min hold) for both acidand alkali-aided isolates The lower heating rates were achieved in a water bath. The 0.5oC/min (water .5/min) and 1oC/min (water 1/min) heating rates were done using a computer controlled water bath. The 10oC/min (water 10/min) heating rate was achieved by preheating a water bath to 70oC and placing the torsion tubes directly into the hot water bath for 15 minutes. Gel diameter was 1 cm and the machine rotated the gel at 2.5 RPM. The significant differences present between samples (p<0.05) are represented by the lett ers above the colum n. Similar letters represent no significant differen ce (p>0.05) between treatments.

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191 0 20000 40000 60000 80000 100000 120000acid 20/20 aci d 2 0/no acid 0. 5 al k ali 2 0 /20 a l ka l i 2 0/ n o al k ali 0 .5Storage modulus (G') start G' 70C G' 70C after G' final G'A B A C C D D 0 20000 40000 60000 80000 100000 120000acid 20/20 aci d 2 0/no acid 0. 5 al k ali 2 0 /20 a l ka l i 2 0/ n o al k ali 0 .5Storage modulus (G') start G' 70C G' 70C after G' final G'A B A C C D D Figure 6-3: Rheological response of acid aided (pH 2.5) and alkali aided catfish pr otein isolates (pH 11.0) recorded on a ATS controlled stress rheometer. The first dashed column is starting G at loading prior to any therma l treatment. The second dashed column is G after heating to 70C at the heating rate stated. The third dotted column is G after holding for 20 min at 70C, shown only in 20 minute hold samples. The fourth dotted column is G after cooling to 1C at 10C/min. The thermal treatments for both acidand alkali-aided is olates were heating at 20oC/min with (acid 20/20, alkali 20/20) or without (acid 20/0, alka li 20/0) 20 minutes holding at 70oC and heating at 0.5oC/min (acid 0.5, alkali 0.5). The significan t differences (p<0.05) are denoted by letters above the column. The letters a a nd b represent the significant differences (p<0.05) between samples after heating to 70oC. The letters c and d represent the significant differences (p<0.05) between sa mples after heating and cooling to 5oC but before holding or cooling. No other significant differences were present between the samples.

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192 0 10000 20000 30000 40000 50000 60000 70000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-4: Rheogram of alkali tr eated catfish muscle heated at 20oC/minute with no high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz. CoolingHeating

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193 0 10000 20000 30000 40000 50000 60000 70000 80000 01020304050607080 Temperature oCStorage mosulus (G') Figure 6-5: Rheogram of alkali tr eated catfish muscle heated at 20oC/minute with 20 minutes high temperature holding. Duplicate sample s at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 a nd averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz. 0 10000 20000 30000 40000 50000 60000 70000 80000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-6: Rheogram of alkali tr eated catfish muscle heated at 0.5oC/minute with no high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz. CoolingHeating Holding HeatingCooling

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194 0 5000 10000 15000 20000 25000 30000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-7: Rheogram of acid trea ted catfish muscle heated at 20oC/minute with no high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz. 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-8: Rheogram of acid trea ted catfish muscle heated at 20oC/minute with 20 minutes high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz.

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195 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-9: Rheogram of acid trea ted catfish muscle heated at 0.5oC/minute with no high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz.

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196 0 10000 20000 30000 40000 50000 60000 70000 80000 01020304050607080 Temperature oCStorage modulus (G') Figure 6-10: Rheograms of acid and alkali treated catfish muscle heated at 20oC/minute with and without 20 minutes high temperature holding. Duplicate samples at 78% moisture were heated in the presence of 2% added NaCl at pH 7.2 and averaged. Acid treated samples heated at 20oC with no hold ( ), acid treated samples heated at 20oC with a 20 minute high temperature hold ( ). Alkali treated samples heated at 20oC with no hold ( ), alkali treated samples heated at 20oC with a 20 minute high temperature hold ( ). The sample was tested under a controlled stress of 100 pa at a frequency of 0.1 Hz.

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197 CHAPTER 7 GENERAL DISCUSSION The results of these studies show that pH-shi ft processing leads to structural m odification of catfish muscle proteins. The structural modification of catfish muscle proteins appears to be responsible for the modification in solubility of catfish muscle proteins but does not lead to significant differences in the chemical properties tested. The structural modification of catfish muscle proteins by pH-shift processing was foun d to lead to the change in the thermal and enzymatic sensitivity of catfish muscle proteins ultimately leading to the modifications observed in the physical properties of thermally treated catfish muscle proteins. By using pH-treatment on catfish muscle proteins, different functional properties are exhi bited based on the type of pHtreatment used. The modification of these functiona l properties and the use of one or more of the pH-treatments studied provide a protein product with a wide variety of physical properties possibly leading to the custom fo rmulation of muscle proteins pr oducts. The custom formulation of muscle products may be accomplished by the utilization of one or more pH-processing techniques studied here. For example using one pH treatment a soft gel could be formed, but a hard elastic gel with another pH treatment. Th e results of this study indicate that further investigation is needed to fu lly understand the mechanisms responsible for the changes in the physical properties observed, for example studying ot her chemical properties of muscle proteins not investigated here and the effect of multiple proteins on the refolding of mechanisms of muscle proteins. Chemical analysis of pH-shift processed ca tfish muscle proteins showed no differences between reactive or total sulfhydryl groups. Th ese results therefore show that pH-shift processing does not affect the sulfhydryl content of catfish muscle prior to thermal treatment. The change in surface hydrophobic groups did not show any significant difference (p>0.05) in

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198 the type of pH-shift processing used, however unt reated catfish muscle pr oteins indicated higher surface hydrophobicity than pH-shift proce ssed catfish muscle. The lower surface hydrophobicity for isolates was unexpected, as a higher hydrophobicity would have been expected since proteins were onl y partially refolded. The most plausible explanation is that microaggregates of proteins formed when th e bulk of hydrophobic groups were buried from the solvent. The decrease in myosin ATPase activit y by pH-shift processing i ndicates that the active site on the myosin head was modified. Reducti on in myosin ATPase has been attributed to modification in the SH group in the active site of the myosin head as previously concluded (Yongswawatdigul and Park 2002). Since no signifi cant difference was seen in the reactive and total sulfhydryl groups the reduction in myosin ATPase activity may be due to a structural shift at the active site of the myosin head as no conc lusive data was shown on the modification of SH groups. The change in solubility of catfish musc le proteins by pH-shift processing shows that pH-shift processing does reduce th e solubility in both sodium chloride and modify the pH solubility profile of muscle proteins. The reduction in solubility, especially for low pH treatments, indicates that though sarcoplasmic prot eins are retained duri ng low pH treatment (Kristinsson and others 2005b) the solubility of sarcoplasmic proteins is reduced by low pH treatment. High pH treatment also retains sarcop lasmic proteins, but to a lesser degree than low pH treatment (Kristinsson and others 2005b) however the solubility result s shown here indicate that high pH treatment does not reduce the solubility of sarcoplasmic proteins as drastically as low pH treatment. The reduction in solubility of the isolates is supported by the structural data which show only partial refolding after pH-treatment and thus pres umably structures more prone to protein-protein interactions.

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199 The rheological properties of catfish muscle proteins show that pH-shift processing reduces the temperature at which the phase chan ge from a more viscous state to a gel like material occurs. The reduction in temperature as shown by the phase angle to the onset of the thermal transition of pH-shift processed catfish muscle proteins does not correlate with an increase in gel rigidity but the results as shown in the storage modulus indicate that with the correct pH salt combination increases in gel rigid ity are possible. The range of differences seen within all the pH treated samples provides a text ural schematic of what pH shift processing can do for muscle proteins. The structural changes induced by pH-shift pr ocessing suggest that a molten globular form is induced after pH-shift processing. The modi fication of the protein structure into a molten globular form is presumed to be responsible fo r the chemical and physical changes seen in pHshift processed catfish muscle. The changes in st ructure also cause the muscle proteins to be more flexible as treatment with low concentr ations of guanidine hydrochloride induced an increased response at 222 nm which may indicate th e reformation of alpha helix structure in pHshift processed muscle proteins. The structural changes due to pH-shift processing caused a change in thermal sensitivity as seen by the DS C data. The shifting of thermal events in both type and temperature show the important effect of pH-shift processing on muscle proteins as thermal treatment is the most common processing treatment to muscle proteins. This molten globular form of the muscle proteins after pH-shift processing is at tributed to the changes seen in the chemical and functional prope rties of the protein isolates. The modifications of thermal events show that the reactivity of catfish mu scle proteins to ther mal treatment undergoes different transitions and reactions at different te mperatures. The change in structure of catfish muscle proteins also increased the susceptibility to enzymatic cross-linking. The increase in

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200 enzymatic sensitivity indicates that pH-shift processing changes the st ructure of the muscle proteins to promote interaction with transglutaminase. The change in the thermal treatment regime of pH-shift processed catfish muscle showed that pH-shift processed catfish muscle can be modified functionally by the thermal treatment regime. Further investigation in to the cooking methods used in pH-shift processed proteins may lead to an increased range of textural properties and reduced processing times. The reduced processing times may lower energy requireme nts and reduce the cost of processing. This new understanding of pH-shift proces sing allows the production of meat based products designed with specific phys ical properties. This is po ssible due to the range of gel textures generated by the different pH processing parameters. In addition the combination of gels produced with multiple isolates may provide a variety of textures in a single product which is more closely related to meat and fillets. While the studies pr esented here show that pH-shift processed catfish muscle protei ns undergo structural changes wh ich lead to changes in the physical properties, future research is needed to determine the mechanisms for transforming pHshift produced isolates in to manufactured fillets.

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201 APPENDIX STATSTICAL TABLES One Way Analysis of V ariance Kruskal-Wallis One Way Analysis of Variance on Ranks Cook Loss Group N Missing Median 25% 75% CLpH2 4 0 1.611 1.331 1.890 CLpH2.5 4 0 2.251 2.072 2.430 CLpH3 4 0 0.942 0.189 1.695 CLpH10.5 4 0 1.242 0.971 1.512 CLpH11 4 0 1.036 0.952 1.119 CLpH11.5 4 0 1.765 1.158 2.372 CL control 4 0 1.766 1.301 2.030 H = 7.621 with 6 degrees of freedom. (P = 0.267) One Way Analysis of Variance Press Test Group Name N Missing Mean Std Dev SEM PLpH2 4 0 9.802 1.032 0.516 PLpH2.5 4 0 9.101 2.149 1.075 PLpH3 4 0 6.949 1.242 0.621 PLpH10.5 4 0 8.347 1.798 0.899 PLpH11 4 0 6.170 1.183 0.592 PLpH11.5 4 0 6.271 1.481 0.741 PLcontrol 4 0 10.187 0.745 0.373 Source of Variation DF SS MS F P Between Groups 6 66.830 11.138 5.337 0.002 Residual 21 43.830 2.087 Total 27 110.660 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Fold Test Group N Missing Median 25% 75% FpH2 4 0 3.750 3.000 4.750 FpH2.5 4 0 2.500 2.000 3.000 fpH3 4 0 4.000 3.000 5.000 fpH10.5 4 0 5.000 5.000 5.000 FpH11 4 0 5.000 5.000 5.000 FpH11.5 4 0 5.000 5.000 5.000 Fcontrol 4 0 5.000 5.000 5.000 H = 20.016 with 6 degrees of freedom. (P = 0.003)

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202 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Transglutaminase treatment Group N Missing Median 25% 75% TGASE2 4 0 1.707 0.716 2.635 TGASE2.5 4 0 2.306 2.191 2.608 TGASE'3 4 0 2.598 1.410 3.369 TGASE10.5 4 0 3.783 1.444 10.555 TGASE11 4 0 4.031 2.899 4.290 TGASE11.5 4 0 2.863 2.526 3.346 TGASECONTROL 4 0 1.049 1.016 1.112 H = 11.121 with 6 degrees of freedom. (P = 0.085) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Rheology with salt, 80oC Group N Missing Median 25% 75% REHWS80CG'2 4 0 3103.000 2179.500 4052.000 REHWS80CG'2.5 4 0 2151.500 2042.000 2323.000 REHWS80CG'3 4 0 1232.000 1071.000 1393.000 REHWS80CG'10.5 4 0 4516.500 4194.500 4838.000 REHWS80CG'11 4 0 7151.500 4971.500 10221.000 REHWS80CG'11.5 4 0 4815.500 4468.500 5874.000 REHWS80CG' CONTROL 4 0 2224.500 2122.500 2320.000 H = 23.488 with 6 degrees of freedom. (P = <0.001) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Rheology without salt, 80oC Group N Missing Median 25% 75% REHNS80CG'2 4 0 3900.500 2296.000 5560.500 REHNS80CG'2.5 4 0 1522.500 1365.500 1750.000 REHNS80CG'3 4 0 8551.000 7724.500 10739.000 REHNS80CG'10.5 4 0 8353.000 8053.500 9306.500 REHNS80CG'11 4 0 13270.000 9532.000 16600.000 REHNS80CG'11.5 4 0 14050.000 13055.000 15400.000 REHNS80CG' CONTROL 4 0 6379.500 5375.500 7099.000 H = 23.675 with 6 degrees of freedom. (P = <0.001) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Rheology with salt af ter cooling to 5oC Group N Missing Median 25% 75% REHSALTG'2 4 0 10487.500 6824.500 15360.000 REHSALTG'2.5 4 0 6843.500 6458.500 7463.000

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203 REHSALTG'3 4 0 3018.500 2532.000 3505.000 REHSALTG'109.5 4 0 14825.000 13920.000 15545.000 REHSALTG'11 4 0 14190.000 11350.000 17260.000 REHSALTG'11.5 4 0 19810.000 18385.000 24010.000 REHSALTG' CONTROL 4 0 7688.000 7521.500 8113.000 H = 22.379 with 6 degrees of freedom. (P = 0.001) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Rheology without salt after cooling to 5oC Group N Missing Median 25% 75% REHG'2 4 0 12315.000 8375.500 19625.000 REHG'2.5 4 0 5190.500 3929.000 6506.500 REHG'3 4 0 35500.000 29775.000 44675.000 REHG'109.5 4 0 31550.000 30275.000 35610.000 REHG'11 4 0 51540.000 37565.000 63940.000 REHG'11.5 4 0 57295.000 55460.000 63645.000 REHG' CONTROL 4 0 21855.000 18650.000 24095.000 H = 24.214 with 6 degrees of freedom. (P = <0.001) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Torsion strain Group N Missing Median 25% 75% TORSTRAIN2 8 0 1.325 1.255 1.410 TORSTRAIN2.5 8 0 1.175 1.140 1.295 TORSTRAIN3 8 0 1.085 0.970 1.150 TORSTRAIN10.5 8 0 1.395 1.020 1.605 TORSTRAIN11 8 0 1.560 1.380 1.715 TORSTRAIN11.5 8 0 1.575 1.525 1.660 TORSTRAIN CONTROL 8 0 1.250 1.025 1.435 H = 23.804 with 6 degrees of freedom. (P = <0.001) One Way Analysis of Variance Torsion stress Group Name N Missing Mean Std Dev SEM TORSTRESS2 8 0 84.790 11.315 4.001 TORSTRESS2.5 8 0 73.833 5.836 2.063 TORSTRESS3 8 0 89.440 18.116 6.405 TORSTRESS10.5 8 0 99.359 14.489 5.123 TORSTRESS11 8 0 127.396 18.656 6.596 TORSTRESS11.5 8 0 108.529 13.181 4.660 TORSTRESS CONTROL 8 0 41.960 4.667 1.650 Source of Variation DF SS MS F P Between Groups 6 35383.363 5897.227 33.189 <0.001 Residual 49 8706.538 177.684

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204 Total 55 44089.901 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Punch Test Jelly Strength Group N Missing Median 25% 75% PTJS2 9 0 6292.500 3196.050 8430.250 PTJS2.5 9 0 4636.400 4177.200 5239.200 PTJS3 9 0 3872.600 3259.250 4764.600 PTJS10.5 7 0 6374.400 4860.575 7720.000 PTJS11 6 0 6153.500 5349.600 7173.200 PTJS11.5 7 0 7174.400 6230.325 7675.150 PTJS CONTROL 9 0 1728.000 1546.425 2127.375 H = 32.504 with 6 degrees of freedom. (P = <0.001) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Punch Test Distance Group N Missing Median 25% 75% PTD2 9 0 7.600 7.150 12.925 PTD2.5 9 0 6.700 6.500 8.600 PTD3 9 0 6.700 6.350 7.125 PTD10.5 7 0 8.300 7.975 9.325 PTD11 6 0 8.000 7.700 8.500 PTD11.5 7 0 7.900 7.600 8.950 PTD CONTROL 9 0 9.100 7.175 10.250 H = 19.340 with 6 degrees of freedom. (P = 0.004) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Punch Test Force Group N Missing Median 25% 75% PTF2 9 0 491.000 455.000 819.500 PTF2.5 9 0 618.000 605.000 688.250 PTF3 9 0 578.000 513.750 661.750 PTF10.5 7 0 768.000 576.000 877.500 PTF11 6 0 791.500 701.000 853.000 PTF11.5 7 0 876.000 608.250 938.500 PTF CONTROL 9 0 192.000 186.750 222.750 H = 30.333 with 6 degrees of freedom. (P = <0.001)

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205 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Reactive Sulfhydryl Groups Group N Missing Median 25% 75% RSG2 4 0 0.00973 0.00584 0.0139 RSG2.5 4 0 0.0104 0.00784 0.0126 RSG3 4 0 0.00927 0.00555 0.0128 RSG10.5 4 0 0.00858 0.00857 0.00872 RSG11 4 0 0.0109 0.00784 0.0143 RSG11.5 4 0 0.0101 0.00830 0.0119 RSG CONTROL 4 0 0.00706 0.00677 0.00738 H = 5.409 with 6 degrees of freedom. (P = 0.493) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Total Sulfhydryl Groups Group N Missing Median 25% 75% TSGpH2 4 0 0.0313 0.0303 0.0335 TSGpH2.5 4 0 0.0363 0.0294 0.0388 TSGpH3 4 0 0.0322 0.0318 0.0333 TSGpH10.5 4 0 0.0293 0.0224 0.0356 TSGpH11 4 0 0.0312 0.0301 0.0345 TSGpH11.5 4 0 0.0344 0.0234 0.0456 TSGcontrol 4 0 1.766 1.301 2.030 H = 11.209 with 6 degrees of freedom. (P = 0.082) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Surface Hydrophobicity Group N Missing Median 25% 75% SHpH2 4 0 534.735 413.460 632.420 SHpH2.5 4 0 452.370 387.580 529.155 SHpH3 4 0 495.025 435.430 531.250 SHpH10.5 4 0 472.125 410.955 509.775 SHpH11 4 0 398.455 361.400 456.695 SHpH11.5 4 0 431.795 388.550 474.115 SHcontrol 2 0 1131.840 989.180 1274.500 H = 8.701 with 6 degrees of freedom. (P = 0.191)

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206 One Way Analysis of Variance ATPase activity Group Name N Missing Mean Std Dev SEM ATPpH2 4 0 51.014 22.191 11.095 ATPpH2.5 4 0 51.858 17.213 8.606 ATPpH3 4 0 35.211 16.573 8.286 ATPpH10.5 4 0 36.729 28.726 14.363 ATPpH11 4 0 61.346 19.634 9.817 ATPpH11.5 4 0 59.045 22.770 11.385 ATPcontrol 4 0 100.000 0.000 0.000 Source of Variation DF SS MS F P Between Groups 6 11271.662 1878.610 4.709 0.003 Residual 21 8377.625 398.935 Total 27 19649.287 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Group N Missing Median 25% 75% CDRAN2 5 0 0.480 0.480 0.495 CDRAN2.5 5 0 0.480 0.480 0.515 CDRAN3 5 0 0.480 0.480 0.502 CDRAN10.5 5 0 0.490 0.480 0.560 CDRAN11 5 0 0.500 0.487 0.528 CDRAN11.5 5 0 0.490 0.480 0.520 CDRAN CONTROL 5 0 0.510 0.338 0.538 H = 2.266 with 6 degrees of freedom. (P = 0.894) One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Group N Missing Median 25% 75% CDA2 5 0 0.0800 0.0725 0.220 CDA2.5 5 0 0.0800 0.0650 0.255 CDA3 5 0 0.0800 0.0800 0.250 CDA10.5 5 0 0.150 0.108 0.340 CDA11 5 0 0.210 0.150 0.320 CDA11.5 5 0 0.110 0.0975 0.302 CDA CONTROL 5 0 0.310 0.292 0.563 H = 12.623 with 6 degrees of freedom. (P = 0.049)

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207 One Way Analysis of Variance Kruskal-Wallis One Way Analysis of Variance on Ranks Group N Missing Median 25% 75% CDB2 5 0 0.430 0.285 0.448 CDB2.5 5 0 0.440 0.232 0.455 CDB3 5 0 0.430 0.248 0.440 CDB10.5 5 0 0.370 0.1000 0.412 CDB11 5 0 0.290 0.153 0.368 CDB11.5 5 0 0.400 0.178 0.412 CDB CONTROL 5 0 0.130 0.0950 0.183 H = 12.285 with 6 degrees of freedom. (P = 0.056) Two Way ANOVA of pH Solubility data Two Way Analysis of Variance General Linear Model Dependent Variable: Solubility Source of Variation DF SS MS F P Sample pH 22 796410.028 36200.456 472.098 <0.001 Treatment pH 6 15843.955 2640.659 34.437 <0.001 Sample pH x Treatment pH132 32574.525 246.777 3.218 <0.001 Residual 479 36729.665 76.680 Total 639 883137.566 1382.062 Main effects cannot be properly in terpreted if significant inter action is determined. This is because the size of a factor 's effect depends upon the level of the other factor. The effect of different levels of Sample pH depe nds on what level of Treatment pH is present. There is a statistically significant interaction between Sample pH and Treatment pH. (P = <0.001) Power of performed test with alpha = 0.0500: for Sample pH : 1.000 Power of performed test with alpha = 0.0500: for Treatment pH : 1.000 Power of performed test with alpha = 0.0500: for Sample pH x Treatment pH : 1.000 All Pairwise Multiple Comparison Procedures (Holm-Sidak method): Overall significance level = 0.05 Comparisons for factor: Treatment pH within 1.5: No Significant Differences Comparisons for factor: Treatment pH within 2 Comparison Diff of Means t Unadjusted P Critical Level Significant? 10.500 vs. 11.500 31.839 5.142 0.000 0.002 Yes 3.000 vs. 11.500 31.523 5.091 0.000 0.003 Yes 11.000 vs. 11.500 24.463 3.951 0.000 0.003 Yes 2.500 vs. 11.500 23.433 3.784 0.000 0.003 Yes

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208 Comparisons for factor: Treatment pH within 2.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 57.028 9.210 0.000 0.002 Yes con vs. 11.500 53.758 8.682 0.000 0.003 Yes con vs. 2.500 53.306 8.609 0.000 0.003 Yes con vs. 11.000 49.815 8.045 0.000 0.003 Yes con vs. 3.000 44.938 7.258 0.000 0.003 Yes con vs. 10.500 35.612 5.751 0.000 0.003 Yes 10.500 vs. 2.000 21.416 3.459 0.001 0.003 Yes 10.500 vs. 11.500 18.146 2.931 0.004 0.004 Yes Comparisons for factor: Treatment pH within 3 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 11.500 57.549 9.294 0.000 0.002 Yes con vs. 3.000 57.127 9.226 0.000 0.003 Yes con vs. 2.500 56.663 9.151 0.000 0.003 Yes con vs. 2.000 56.613 9.143 0.000 0.003 Yes con vs. 11.000 54.283 8.767 0.000 0.003 Yes con vs. 10.500 49.738 8.033 0.000 0.003 Yes Comparisons for factor: Treatment pH within 3.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 11.500 24.689 3.987 0.000 0.002 Yes con vs. 3.000 23.942 3.867 0.000 0.003 Yes con vs. 2.000 23.548 3.803 0.000 0.003 Yes con vs. 11.000 20.575 3.323 0.001 0.003 Yes con vs. 2.500 20.255 3.271 0.001 0.003 Yes Comparisons for factor: Treatment pH within 4: No Significant Differences Comparisons for factor: Treatment pH within 4.5: No Significant Differences Comparisons for factor: Treatment pH within 5: No Significant Differences Comparisons for factor: Treatment pH within 5.5: No Significant Differences Comparisons for factor: Treatment pH within 6: No Significant Differences Comparisons for factor: Treatment pH within 6.5: No Significant Differences Comparisons for factor: Treatment pH within 7: No Significant Differences Comparisons for factor: Treatment pH within 7.5: No Significant Differences

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209 Comparisons for factor: Treatment pH within 8 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 20.053 3.239 0.001 0.002 Yes con vs. 2.500 18.845 3.043 0.002 0.003 Yes Comparisons for factor: Treatment pH within 8.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 19.850 3.206 0.001 0.002 Yes con vs. 2.500 19.338 3.123 0.002 0.003 Yes Comparisons for factor: Treatment pH within 9 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 19.903 3.214 0.001 0.002 Yes con vs. 2.500 19.273 3.113 0.002 0.003 Yes Comparisons for factor: Treatment pH within 9.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 22.572 3.645 0.000 0.002 Yes con vs. 2.500 19.634 3.171 0.002 0.003 Yes Comparisons for factor: Treatment pH within 10 Comparison Diff of Means t Unadjusted P Critical Level Significant? con vs. 2.000 20.492 3.309 0.001 0.002 Yes Comparisons for factor: Treatment pH within 10.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 10.500 vs. 2.000 26.817 4.331 0.000 0.002 Yes 10.500 vs. 2.500 24.607 3.974 0.000 0.003 Yes 11.000 vs. 2.000 24.195 3.907 0.000 0.003 Yes 11.000 vs. 2.500 21.985 3.551 0.000 0.003 Yes 10.500 vs. 3.000 21.658 3.498 0.001 0.003 Yes con vs. 2.000 21.241 3.430 0.001 0.003 Yes 10.500 vs. 11.500 19.350 3.125 0.002 0.003 Yes 11.000 vs. 3.000 19.036 3.074 0.002 0.004 Yes con vs. 2.500 19.031 3.073 0.002 0.004 Yes Comparisons for factor: Treatment pH within 11 Comparison Diff of Means t Unadjusted P Critical Level Significant? 3.000 vs. con 44.686 7.217 0.000 0.002 Yes 10.500 vs. con 38.914 6.285 0.000 0.003 Yes 11.500 vs. con 32.067 5.179 0.000 0.003 Yes 3.000 vs. 2.000 27.165 4.387 0.000 0.003 Yes 11.000 vs. con 27.067 4.371 0.000 0.003 Yes 2.500 vs. con 26.711 4.314 0.000 0.003 Yes 10.500 vs. 2.000 21.393 3.455 0.001 0.003 Yes

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210 Comparisons for factor: Treatment pH within 11.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 3.000 vs. 11.000 23.241 3.753 0.000 0.002 Yes Comparisons for factor: Treatment pH within 12 Comparison Diff of Means t Unadjusted P Critical Level Significant? 10.500 vs. 11.000 26.405 4.264 0.000 0.002 Yes 2.000 vs. 11.000 23.213 3.749 0.000 0.003 Yes 3.000 vs. 11.000 21.632 3.494 0.001 0.003 Yes con vs. 11.000 21.485 3.470 0.001 0.003 Yes Comparisons for factor: Treatment pH within NC: No Significant Differences Comparisons for factor: Sample pH within 2 Comparison Diff of Means t Unadjusted P Critical Level Significant? NC vs. 5.500 101.286 16.358 0.000 0.000 Yes NC vs. 5.000 100.893 16.294 0.000 0.000 Yes NC vs. 6.500 100.387 16.213 0.000 0.000 Yes NC vs. 6.000 99.892 16.133 0.000 0.000 Yes NC vs. 4.500 99.343 16.044 0.000 0.000 Yes 12.000 vs. 5.500 98.441 15.898 0.000 0.000 Yes 12.000 vs. 5.000 98.048 15.835 0.000 0.000 Yes 12.000 vs. 6.500 97.543 15.753 0.000 0.000 Yes NC vs. 4.000 97.352 15.722 0.000 0.000 Yes 12.000 vs. 6.000 97.047 15.673 0.000 0.000 Yes 12.000 vs. 4.500 96.499 15.585 0.000 0.000 Yes NC vs. 7.000 94.696 15.294 0.000 0.000 Yes NC vs. 3.500 94.664 15.288 0.000 0.000 Yes 12.000 vs. 4.000 94.507 15.263 0.000 0.000 Yes 11.500 vs. 5.500 93.528 15.105 0.000 0.000 Yes NC vs. 7.500 93.296 15.067 0.000 0.000 Yes 11.500 vs. 5.000 93.134 15.041 0.000 0.000 Yes NC vs. 8.000 92.704 14.972 0.000 0.000 Yes 11.500 vs. 6.500 92.629 14.960 0.000 0.000 Yes 11.500 vs. 6.000 92.133 14.880 0.000 0.000 Yes 12.000 vs. 7.000 91.852 14.834 0.000 0.000 Yes 12.000 vs. 3.500 91.819 14.829 0.000 0.000 Yes 11.500 vs. 4.500 91.585 14.791 0.000 0.000 Yes 12.000 vs. 7.500 90.451 14.608 0.000 0.000 Yes NC vs. 3.000 90.370 14.595 0.000 0.000 Yes NC vs. 8.500 89.956 14.528 0.000 0.000 Yes 12.000 vs. 8.000 89.860 14.512 0.000 0.000 Yes 11.500 vs. 4.000 89.593 14.469 0.000 0.000 Yes NC vs. 10.000 89.377 14.434 0.000 0.000 Yes NC vs. 9.000 89.227 14.410 0.000 0.000 Yes NC vs. 9.500 88.232 14.250 0.000 0.000 Yes

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211 12.000 vs. 3.000 87.525 14.135 0.000 0.000 Yes 12.000 vs. 8.500 87.111 14.069 0.000 0.000 Yes 11.500 vs. 7.000 86.938 14.041 0.000 0.000 Yes 11.500 vs. 3.500 86.905 14.035 0.000 0.000 Yes 12.000 vs. 10.000 86.532 13.975 0.000 0.000 Yes 12.000 vs. 9.000 86.383 13.951 0.000 0.000 Yes 11.500 vs. 7.500 85.537 13.814 0.000 0.000 Yes 12.000 vs. 9.500 85.387 13.790 0.000 0.000 Yes 1.500 vs. 5.500 85.196 13.759 0.000 0.000 Yes 2.000 vs. 5.500 85.133 13.749 0.000 0.000 Yes 11.500 vs. 8.000 84.946 13.719 0.000 0.000 Yes 1.500 vs. 5.000 84.803 13.696 0.000 0.000 Yes 2.000 vs. 5.000 84.740 13.686 0.000 0.000 Yes 1.500 vs. 6.500 84.297 13.614 0.000 0.000 Yes 2.000 vs. 6.500 84.235 13.604 0.000 0.000 Yes NC vs. 10.500 83.864 13.544 0.000 0.000 Yes 1.500 vs. 6.000 83.802 13.534 0.000 0.000 Yes 2.000 vs. 6.000 83.739 13.524 0.000 0.000 Yes 1.500 vs. 4.500 83.253 13.445 0.000 0.000 Yes 2.000 vs. 4.500 83.191 13.435 0.000 0.000 Yes NC vs. 2.500 83.119 13.424 0.000 0.000 Yes 11.500 vs. 3.000 82.611 13.342 0.000 0.000 Yes 11.500 vs. 8.500 82.197 13.275 0.000 0.000 Yes 11.500 vs. 10.000 81.619 13.181 0.000 0.000 Yes 11.500 vs. 9.000 81.469 13.157 0.000 0.000 Yes 1.500 vs. 4.000 81.262 13.124 0.000 0.000 Yes 2.000 vs. 4.000 81.199 13.114 0.000 0.000 Yes 12.000 vs. 10.500 81.019 13.085 0.000 0.000 Yes 11.500 vs. 9.500 80.474 12.997 0.000 0.000 Yes 12.000 vs. 2.500 80.274 12.964 0.000 0.000 Yes 11.000 vs. 5.500 80.186 12.950 0.000 0.000 Yes 11.000 vs. 5.000 79.793 12.887 0.000 0.000 Yes 11.000 vs. 6.500 79.287 12.805 0.000 0.000 Yes 11.000 vs. 6.000 78.792 12.725 0.000 0.000 Yes 1.500 vs. 7.000 78.606 12.695 0.000 0.000 Yes 1.500 vs. 3.500 78.574 12.690 0.000 0.000 Yes 2.000 vs. 7.000 78.544 12.685 0.000 0.000 Yes 2.000 vs. 3.500 78.511 12.680 0.000 0.000 Yes 11.000 vs. 4.500 78.243 12.636 0.000 0.000 Yes 1.500 vs. 7.500 77.206 12.469 0.000 0.000 Yes 2.000 vs. 7.500 77.143 12.459 0.000 0.000 Yes 1.500 vs. 8.000 76.615 12.373 0.000 0.000 Yes 2.000 vs. 8.000 76.552 12.363 0.000 0.000 Yes 11.000 vs. 4.000 76.252 12.315 0.000 0.000 Yes 11.500 vs. 10.500 76.105 12.291 0.000 0.000 Yes 11.500 vs. 2.500 75.360 12.171 0.000 0.000 Yes

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212 1.500 vs. 3.000 74.280 11.996 0.000 0.000 Yes 2.000 vs. 3.000 74.217 11.986 0.000 0.000 Yes 1.500 vs. 8.500 73.866 11.929 0.000 0.000 Yes 2.000 vs. 8.500 73.803 11.919 0.000 0.000 Yes 11.000 vs. 7.000 73.596 11.886 0.000 0.000 Yes 11.000 vs. 3.500 73.564 11.881 0.000 0.000 Yes 1.500 vs. 10.000 73.287 11.836 0.000 0.000 Yes 2.000 vs. 10.000 73.224 11.826 0.000 0.000 Yes 1.500 vs. 9.000 73.138 11.812 0.000 0.000 Yes 2.000 vs. 9.000 73.075 11.802 0.000 0.000 Yes 11.000 vs. 7.500 72.196 11.660 0.000 0.000 Yes 1.500 vs. 9.500 72.142 11.651 0.000 0.000 Yes 2.000 vs. 9.500 72.079 11.641 0.000 0.000 Yes 11.000 vs. 8.000 71.605 11.564 0.000 0.000 Yes 11.000 vs. 3.000 69.270 11.187 0.000 0.000 Yes 11.000 vs. 8.500 68.856 11.120 0.000 0.000 Yes 11.000 vs. 10.000 68.277 11.027 0.000 0.000 Yes 11.000 vs. 9.000 68.128 11.003 0.000 0.000 Yes 1.500 vs. 10.500 67.774 10.945 0.000 0.000 Yes 2.000 vs. 10.500 67.711 10.935 0.000 0.000 Yes 11.000 vs. 9.500 67.132 10.842 0.000 0.000 Yes 1.500 vs. 2.500 67.029 10.825 0.000 0.000 Yes 2.000 vs. 2.500 66.966 10.815 0.000 0.000 Yes 11.000 vs. 10.500 62.764 10.136 0.000 0.000 Yes 11.000 vs. 2.500 62.019 10.016 0.000 0.000 Yes Comparisons for factor: Sample pH within 2.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? NC vs. 4.500 100.668 16.258 0.000 0.000 Yes NC vs. 5.000 100.529 16.235 0.000 0.000 Yes NC vs. 6.500 100.132 16.171 0.000 0.000 Yes NC vs. 6.000 99.828 16.122 0.000 0.000 Yes NC vs. 5.500 97.908 15.812 0.000 0.000 Yes NC vs. 4.000 96.841 15.640 0.000 0.000 Yes NC vs. 7.000 95.503 15.424 0.000 0.000 Yes NC vs. 7.500 93.288 15.066 0.000 0.000 Yes 2.000 vs. 4.500 92.150 14.882 0.000 0.000 Yes 2.000 vs. 5.000 92.010 14.860 0.000 0.000 Yes 2.000 vs. 6.500 91.614 14.796 0.000 0.000 Yes NC vs. 8.000 91.497 14.777 0.000 0.000 Yes NC vs. 3.500 91.370 14.756 0.000 0.000 Yes 2.000 vs. 6.000 91.310 14.747 0.000 0.000 Yes NC vs. 3.000 90.419 14.603 0.000 0.000 Yes NC vs. 8.500 89.444 14.445 0.000 0.000 Yes 2.000 vs. 5.500 89.390 14.437 0.000 0.000 Yes 11.000 vs. 4.500 88.759 14.335 0.000 0.000 Yes

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213 11.500 vs. 4.500 88.737 14.331 0.000 0.000 Yes 1.500 vs. 4.500 88.725 14.329 0.000 0.000 Yes 11.000 vs. 5.000 88.619 14.312 0.000 0.000 Yes 11.500 vs. 5.000 88.597 14.309 0.000 0.000 Yes NC vs. 9.000 88.597 14.309 0.000 0.000 Yes 1.500 vs. 5.000 88.586 14.307 0.000 0.000 Yes 2.000 vs. 4.000 88.323 14.264 0.000 0.000 Yes 11.000 vs. 6.500 88.223 14.248 0.000 0.000 Yes 11.500 vs. 6.500 88.201 14.245 0.000 0.000 Yes 1.500 vs. 6.500 88.189 14.243 0.000 0.000 Yes 11.000 vs. 6.000 87.919 14.199 0.000 0.000 Yes 11.500 vs. 6.000 87.897 14.195 0.000 0.000 Yes 1.500 vs. 6.000 87.886 14.194 0.000 0.000 Yes 2.000 vs. 7.000 86.985 14.048 0.000 0.000 Yes 12.000 vs. 4.500 86.669 13.997 0.000 0.000 Yes 12.000 vs. 5.000 86.529 13.975 0.000 0.000 Yes NC vs. 10.000 86.409 13.955 0.000 0.000 Yes 12.000 vs. 6.500 86.133 13.911 0.000 0.000 Yes 11.000 vs. 5.500 85.999 13.889 0.000 0.000 Yes 11.500 vs. 5.500 85.977 13.885 0.000 0.000 Yes 1.500 vs. 5.500 85.965 13.883 0.000 0.000 Yes 12.000 vs. 6.000 85.829 13.861 0.000 0.000 Yes NC vs. 9.500 85.294 13.775 0.000 0.000 Yes 11.000 vs. 4.000 84.932 13.717 0.000 0.000 Yes 11.500 vs. 4.000 84.910 13.713 0.000 0.000 Yes 1.500 vs. 4.000 84.899 13.711 0.000 0.000 Yes 2.000 vs. 7.500 84.770 13.690 0.000 0.000 Yes 12.000 vs. 5.500 83.909 13.551 0.000 0.000 Yes 11.000 vs. 7.000 83.594 13.500 0.000 0.000 Yes 11.500 vs. 7.000 83.572 13.497 0.000 0.000 Yes 1.500 vs. 7.000 83.560 13.495 0.000 0.000 Yes 2.000 vs. 8.000 82.979 13.401 0.000 0.000 Yes 2.000 vs. 3.500 82.852 13.381 0.000 0.000 Yes 12.000 vs. 4.000 82.842 13.379 0.000 0.000 Yes 2.000 vs. 3.000 81.901 13.227 0.000 0.000 Yes NC vs. 10.500 81.654 13.187 0.000 0.000 Yes 12.000 vs. 7.000 81.504 13.163 0.000 0.000 Yes 11.000 vs. 7.500 81.379 13.143 0.000 0.000 Yes 11.500 vs. 7.500 81.357 13.139 0.000 0.000 Yes 1.500 vs. 7.500 81.345 13.137 0.000 0.000 Yes 2.000 vs. 8.500 80.925 13.069 0.000 0.000 Yes 2.000 vs. 9.000 80.079 12.933 0.000 0.000 Yes 11.000 vs. 8.000 79.588 12.853 0.000 0.000 Yes 11.500 vs. 8.000 79.566 12.850 0.000 0.000 Yes 1.500 vs. 8.000 79.554 12.848 0.000 0.000 Yes 11.000 vs. 3.500 79.461 12.833 0.000 0.000 Yes

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214 11.500 vs. 3.500 79.439 12.829 0.000 0.000 Yes 1.500 vs. 3.500 79.427 12.828 0.000 0.000 Yes NC vs. 2.500 79.397 12.823 0.000 0.000 Yes 12.000 vs. 7.500 79.289 12.805 0.000 0.000 Yes 11.000 vs. 3.000 78.510 12.679 0.000 0.000 Yes 11.500 vs. 3.000 78.488 12.676 0.000 0.000 Yes 1.500 vs. 3.000 78.476 12.674 0.000 0.000 Yes 2.000 vs. 10.000 77.890 12.579 0.000 0.000 Yes 11.000 vs. 8.500 77.534 12.522 0.000 0.000 Yes 11.500 vs. 8.500 77.512 12.518 0.000 0.000 Yes 1.500 vs. 8.500 77.501 12.516 0.000 0.000 Yes 12.000 vs. 8.000 77.498 12.516 0.000 0.000 Yes 12.000 vs. 3.500 77.371 12.496 0.000 0.000 Yes 2.000 vs. 9.500 76.776 12.399 0.000 0.000 Yes 11.000 vs. 9.000 76.688 12.385 0.000 0.000 Yes 11.500 vs. 9.000 76.666 12.382 0.000 0.000 Yes 1.500 vs. 9.000 76.654 12.380 0.000 0.000 Yes 12.000 vs. 3.000 76.420 12.342 0.000 0.000 Yes 12.000 vs. 8.500 75.444 12.184 0.000 0.000 Yes 12.000 vs. 9.000 74.598 12.048 0.000 0.000 Yes 11.000 vs. 10.000 74.499 12.032 0.000 0.000 Yes 11.500 vs. 10.000 74.477 12.028 0.000 0.000 Yes 1.500 vs. 10.000 74.466 12.026 0.000 0.000 Yes 11.000 vs. 9.500 73.385 11.852 0.000 0.000 Yes 11.500 vs. 9.500 73.363 11.848 0.000 0.000 Yes 1.500 vs. 9.500 73.351 11.846 0.000 0.000 Yes 2.000 vs. 10.500 73.136 11.811 0.000 0.000 Yes 12.000 vs. 10.000 72.409 11.694 0.000 0.000 Yes 12.000 vs. 9.500 71.295 11.514 0.000 0.000 Yes 2.000 vs. 2.500 70.878 11.447 0.000 0.000 Yes 11.000 vs. 10.500 69.745 11.264 0.000 0.000 Yes 11.500 vs. 10.500 69.723 11.260 0.000 0.000 Yes 1.500 vs. 10.500 69.711 11.258 0.000 0.000 Yes 12.000 vs. 10.500 67.655 10.926 0.000 0.000 Yes 11.000 vs. 2.500 67.488 10.899 0.000 0.000 Yes 11.500 vs. 2.500 67.465 10.896 0.000 0.000 Yes 1.500 vs. 2.500 67.454 10.894 0.000 0.000 Yes 12.000 vs. 2.500 65.398 10.562 0.000 0.000 Yes Comparisons for factor: Sample pH within 3 Comparison Diff of Means t Unadjusted P Critical Level Significant? 11.000 vs. 6.500 105.648 17.062 0.000 0.000 Yes 11.000 vs. 4.000 105.532 17.044 0.000 0.000 Yes 11.000 vs. 4.500 105.332 17.011 0.000 0.000 Yes 11.000 vs. 5.500 105.046 16.965 0.000 0.000 Yes 11.000 vs. 5.000 104.667 16.904 0.000 0.000 Yes

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215 11.000 vs. 6.000 104.523 16.881 0.000 0.000 Yes 11.500 vs. 6.500 101.939 16.463 0.000 0.000 Yes 11.500 vs. 4.000 101.823 16.444 0.000 0.000 Yes 11.500 vs. 4.500 101.622 16.412 0.000 0.000 Yes 11.500 vs. 5.500 101.336 16.366 0.000 0.000 Yes 11.000 vs. 3.500 101.122 16.331 0.000 0.000 Yes 11.000 vs. 7.000 100.984 16.309 0.000 0.000 Yes 11.500 vs. 5.000 100.958 16.305 0.000 0.000 Yes 11.500 vs. 6.000 100.814 16.282 0.000 0.000 Yes NC vs. 6.500 99.583 16.083 0.000 0.000 Yes NC vs. 4.000 99.467 16.064 0.000 0.000 Yes NC vs. 4.500 99.267 16.032 0.000 0.000 Yes 2.000 vs. 6.500 99.155 16.014 0.000 0.000 Yes 11.000 vs. 7.500 99.148 16.012 0.000 0.000 Yes 2.000 vs. 4.000 99.039 15.995 0.000 0.000 Yes NC vs. 5.500 98.981 15.985 0.000 0.000 Yes 2.000 vs. 4.500 98.838 15.962 0.000 0.000 Yes NC vs. 5.000 98.602 15.924 0.000 0.000 Yes 2.000 vs. 5.500 98.552 15.916 0.000 0.000 Yes NC vs. 6.000 98.458 15.901 0.000 0.000 Yes 2.000 vs. 5.000 98.174 15.855 0.000 0.000 Yes 2.000 vs. 6.000 98.030 15.832 0.000 0.000 Yes 11.500 vs. 3.500 97.413 15.732 0.000 0.000 Yes 11.500 vs. 7.000 97.275 15.710 0.000 0.000 Yes 11.000 vs. 3.000 96.948 15.657 0.000 0.000 Yes 11.000 vs. 8.000 95.706 15.457 0.000 0.000 Yes 11.500 vs. 7.500 95.438 15.413 0.000 0.000 Yes 12.000 vs. 6.500 95.157 15.368 0.000 0.000 Yes NC vs. 3.500 95.057 15.352 0.000 0.000 Yes 12.000 vs. 4.000 95.041 15.349 0.000 0.000 Yes NC vs. 7.000 94.919 15.329 0.000 0.000 Yes 12.000 vs. 4.500 94.841 15.317 0.000 0.000 Yes 2.000 vs. 3.500 94.629 15.283 0.000 0.000 Yes 12.000 vs. 5.500 94.555 15.271 0.000 0.000 Yes 2.000 vs. 7.000 94.490 15.260 0.000 0.000 Yes 12.000 vs. 5.000 94.176 15.210 0.000 0.000 Yes 12.000 vs. 6.000 94.032 15.186 0.000 0.000 Yes 11.500 vs. 3.000 93.239 15.058 0.000 0.000 Yes 11.000 vs. 8.500 93.210 15.053 0.000 0.000 Yes NC vs. 7.500 93.083 15.033 0.000 0.000 Yes 1.500 vs. 6.500 92.943 15.010 0.000 0.000 Yes 1.500 vs. 4.000 92.827 14.992 0.000 0.000 Yes 2.000 vs. 7.500 92.654 14.964 0.000 0.000 Yes 1.500 vs. 4.500 92.627 14.959 0.000 0.000 Yes 1.500 vs. 5.500 92.341 14.913 0.000 0.000 Yes 11.500 vs. 8.000 91.997 14.858 0.000 0.000 Yes

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216 1.500 vs. 5.000 91.963 14.852 0.000 0.000 Yes 1.500 vs. 6.000 91.818 14.829 0.000 0.000 Yes 11.000 vs. 9.000 91.790 14.824 0.000 0.000 Yes 11.000 vs. 10.000 90.920 14.684 0.000 0.000 Yes NC vs. 3.000 90.883 14.678 0.000 0.000 Yes 12.000 vs. 3.500 90.631 14.637 0.000 0.000 Yes 12.000 vs. 7.000 90.493 14.615 0.000 0.000 Yes 2.000 vs. 3.000 90.455 14.609 0.000 0.000 Yes 11.000 vs. 9.500 90.202 14.568 0.000 0.000 Yes NC vs. 8.000 89.641 14.477 0.000 0.000 Yes 11.500 vs. 8.500 89.500 14.454 0.000 0.000 Yes 2.000 vs. 8.000 89.212 14.408 0.000 0.000 Yes 12.000 vs. 7.500 88.657 14.318 0.000 0.000 Yes 1.500 vs. 3.500 88.418 14.280 0.000 0.000 Yes 1.500 vs. 7.000 88.279 14.257 0.000 0.000 Yes 11.500 vs. 9.000 88.080 14.225 0.000 0.000 Yes 11.500 vs. 10.000 87.211 14.085 0.000 0.000 Yes NC vs. 8.500 87.145 14.074 0.000 0.000 Yes 2.000 vs. 8.500 86.716 14.005 0.000 0.000 Yes 11.500 vs. 9.500 86.493 13.969 0.000 0.000 Yes 12.000 vs. 3.000 86.457 13.963 0.000 0.000 Yes 1.500 vs. 7.500 86.443 13.961 0.000 0.000 Yes NC vs. 9.000 85.725 13.845 0.000 0.000 Yes 2.000 vs. 9.000 85.296 13.775 0.000 0.000 Yes 12.000 vs. 8.000 85.215 13.762 0.000 0.000 Yes NC vs. 10.000 84.855 13.704 0.000 0.000 Yes 11.000 vs. 10.500 84.770 13.690 0.000 0.000 Yes 2.000 vs. 10.000 84.427 13.635 0.000 0.000 Yes 1.500 vs. 3.000 84.244 13.605 0.000 0.000 Yes NC vs. 9.500 84.137 13.588 0.000 0.000 Yes 2.000 vs. 9.500 83.709 13.519 0.000 0.000 Yes 1.500 vs. 8.000 83.001 13.405 0.000 0.000 Yes 12.000 vs. 8.500 82.719 13.359 0.000 0.000 Yes 12.000 vs. 9.000 81.299 13.130 0.000 0.000 Yes 11.500 vs. 10.500 81.061 13.091 0.000 0.000 Yes 1.500 vs. 8.500 80.505 13.002 0.000 0.000 Yes 12.000 vs. 10.000 80.429 12.989 0.000 0.000 Yes 12.000 vs. 9.500 79.711 12.873 0.000 0.000 Yes 1.500 vs. 9.000 79.085 12.772 0.000 0.000 Yes NC vs. 10.500 78.705 12.711 0.000 0.000 Yes 2.000 vs. 10.500 78.276 12.642 0.000 0.000 Yes 1.500 vs. 10.000 78.216 12.632 0.000 0.000 Yes 1.500 vs. 9.500 77.498 12.516 0.000 0.000 Yes 11.000 vs. 2.500 77.093 12.451 0.000 0.000 Yes 12.000 vs. 10.500 74.279 11.996 0.000 0.000 Yes 11.500 vs. 2.500 73.384 11.852 0.000 0.000 Yes

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217 1.500 vs. 10.500 72.065 11.639 0.000 0.000 Yes NC vs. 2.500 71.028 11.471 0.000 0.000 Yes 2.000 vs. 2.500 70.600 11.402 0.000 0.000 Yes 12.000 vs. 2.500 66.602 10.756 0.000 0.000 Yes 1.500 vs. 2.500 64.389 10.399 0.000 0.000 Yes 2.500 vs. 6.500 28.555 4.612 0.000 0.000 Yes 2.500 vs. 4.000 28.439 4.593 0.000 0.000 Yes 2.500 vs. 4.500 28.238 4.560 0.000 0.000 Yes 2.500 vs. 5.500 27.952 4.514 0.000 0.000 Yes 2.500 vs. 5.000 27.574 4.453 0.000 0.000 Yes 2.500 vs. 6.000 27.430 4.430 0.000 0.000 Yes 2.500 vs. 3.500 24.029 3.881 0.000 0.000 Yes 2.500 vs. 7.000 23.890 3.858 0.000 0.000 Yes Comparisons for factor: Sample pH within 10.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 12.000 vs. 6.000 99.546 16.077 0.000 0.000 Yes 11.000 vs. 6.000 99.493 16.068 0.000 0.000 Yes 12.000 vs. 5.500 99.449 16.061 0.000 0.000 Yes 11.000 vs. 5.500 99.396 16.053 0.000 0.000 Yes NC vs. 6.000 99.200 16.021 0.000 0.000 Yes NC vs. 5.500 99.103 16.005 0.000 0.000 Yes 2.000 vs. 6.000 99.087 16.003 0.000 0.000 Yes 2.000 vs. 5.500 98.990 15.987 0.000 0.000 Yes 12.000 vs. 5.000 97.840 15.801 0.000 0.000 Yes 11.000 vs. 5.000 97.787 15.793 0.000 0.000 Yes NC vs. 5.000 97.493 15.745 0.000 0.000 Yes 2.000 vs. 5.000 97.381 15.727 0.000 0.000 Yes 12.000 vs. 4.500 96.262 15.546 0.000 0.000 Yes 11.000 vs. 4.500 96.209 15.538 0.000 0.000 Yes NC vs. 4.500 95.915 15.490 0.000 0.000 Yes 2.000 vs. 4.500 95.802 15.472 0.000 0.000 Yes 12.000 vs. 6.500 95.542 15.430 0.000 0.000 Yes 11.000 vs. 6.500 95.489 15.421 0.000 0.000 Yes NC vs. 6.500 95.195 15.374 0.000 0.000 Yes 2.000 vs. 6.500 95.083 15.356 0.000 0.000 Yes 12.000 vs. 4.000 93.501 15.100 0.000 0.000 Yes 11.000 vs. 4.000 93.448 15.092 0.000 0.000 Yes NC vs. 4.000 93.154 15.044 0.000 0.000 Yes 2.000 vs. 4.000 93.041 15.026 0.000 0.000 Yes 12.000 vs. 7.000 91.919 14.845 0.000 0.000 Yes 11.000 vs. 7.000 91.866 14.836 0.000 0.000 Yes NC vs. 7.000 91.572 14.789 0.000 0.000 Yes 2.000 vs. 7.000 91.459 14.771 0.000 0.000 Yes 11.500 vs. 6.000 91.095 14.712 0.000 0.000 Yes 11.500 vs. 5.500 90.998 14.696 0.000 0.000 Yes

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218 12.000 vs. 7.500 90.445 14.607 0.000 0.000 Yes 11.000 vs. 7.500 90.391 14.598 0.000 0.000 Yes NC vs. 7.500 90.098 14.551 0.000 0.000 Yes 2.000 vs. 7.500 89.985 14.533 0.000 0.000 Yes 11.500 vs. 5.000 89.389 14.436 0.000 0.000 Yes 11.500 vs. 4.500 87.810 14.181 0.000 0.000 Yes 12.000 vs. 3.500 87.760 14.173 0.000 0.000 Yes 11.000 vs. 3.500 87.707 14.165 0.000 0.000 Yes NC vs. 3.500 87.413 14.117 0.000 0.000 Yes 2.000 vs. 3.500 87.300 14.099 0.000 0.000 Yes 11.500 vs. 6.500 87.091 14.065 0.000 0.000 Yes 1.500 vs. 6.000 86.172 13.917 0.000 0.000 Yes 1.500 vs. 5.500 86.075 13.901 0.000 0.000 Yes 11.500 vs. 4.000 85.049 13.736 0.000 0.000 Yes 1.500 vs. 5.000 84.465 13.641 0.000 0.000 Yes 12.000 vs. 3.000 83.841 13.540 0.000 0.000 Yes 11.000 vs. 3.000 83.788 13.532 0.000 0.000 Yes NC vs. 3.000 83.494 13.484 0.000 0.000 Yes 11.500 vs. 7.000 83.468 13.480 0.000 0.000 Yes 2.000 vs. 3.000 83.381 13.466 0.000 0.000 Yes 1.500 vs. 4.500 82.887 13.386 0.000 0.000 Yes 1.500 vs. 6.500 82.167 13.270 0.000 0.000 Yes 11.500 vs. 7.500 81.993 13.242 0.000 0.000 Yes 1.500 vs. 4.000 80.126 12.940 0.000 0.000 Yes 11.500 vs. 3.500 79.308 12.808 0.000 0.000 Yes 1.500 vs. 7.000 78.544 12.685 0.000 0.000 Yes 12.000 vs. 9.000 77.209 12.469 0.000 0.000 Yes 11.000 vs. 9.000 77.156 12.461 0.000 0.000 Yes 1.500 vs. 7.500 77.070 12.447 0.000 0.000 Yes NC vs. 9.000 76.862 12.413 0.000 0.000 Yes 2.000 vs. 9.000 76.749 12.395 0.000 0.000 Yes 12.000 vs. 8.500 76.447 12.346 0.000 0.000 Yes 11.000 vs. 8.500 76.394 12.338 0.000 0.000 Yes NC vs. 8.500 76.100 12.290 0.000 0.000 Yes 2.000 vs. 8.500 75.988 12.272 0.000 0.000 Yes 12.000 vs. 10.000 75.645 12.217 0.000 0.000 Yes 11.000 vs. 10.000 75.592 12.208 0.000 0.000 Yes 11.500 vs. 3.000 75.389 12.175 0.000 0.000 Yes NC vs. 10.000 75.298 12.161 0.000 0.000 Yes 2.000 vs. 10.000 75.186 12.143 0.000 0.000 Yes 1.500 vs. 3.500 74.385 12.013 0.000 0.000 Yes 12.000 vs. 8.000 88.031 11.608 0.000 0.000 Yes 11.000 vs. 8.000 87.977 11.601 0.000 0.000 Yes NC vs. 8.000 87.684 11.562 0.000 0.000 Yes 2.000 vs. 8.000 87.571 11.548 0.000 0.000 Yes 12.000 vs. 9.500 71.218 11.502 0.000 0.000 Yes

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219 11.000 vs. 9.500 71.165 11.493 0.000 0.000 Yes NC vs. 9.500 70.871 11.446 0.000 0.000 Yes 2.000 vs. 9.500 70.758 11.428 0.000 0.000 Yes 1.500 vs. 3.000 70.466 11.380 0.000 0.000 Yes 11.500 vs. 9.000 68.757 11.104 0.000 0.000 Yes 11.500 vs. 8.500 67.996 10.981 0.000 0.000 Yes 11.500 vs. 10.000 67.194 10.852 0.000 0.000 Yes 11.500 vs. 8.000 79.579 10.494 0.000 0.000 Yes 1.500 vs. 9.000 63.834 10.309 0.000 0.000 Yes 1.500 vs. 8.500 63.072 10.186 0.000 0.000 Yes 11.500 vs. 9.500 62.766 10.137 0.000 0.000 Yes 1.500 vs. 10.000 62.270 10.057 0.000 0.000 Yes 12.000 vs. 2.500 62.049 10.021 0.000 0.000 Yes 11.000 vs. 2.500 61.996 10.012 0.000 0.000 Yes NC vs. 2.500 61.702 9.965 0.000 0.000 Yes 2.000 vs. 2.500 61.590 9.947 0.000 0.000 Yes 1.500 vs. 8.000 74.656 9.844 0.000 0.000 Yes 1.500 vs. 9.500 57.843 9.342 0.000 0.000 Yes 12.000 vs. 10.500 57.394 9.269 0.000 0.000 Yes 11.000 vs. 10.500 57.341 9.261 0.000 0.000 Yes NC vs. 10.500 57.047 9.213 0.000 0.000 Yes 2.000 vs. 10.500 56.934 9.195 0.000 0.000 Yes 11.500 vs. 2.500 53.598 8.656 0.000 0.000 Yes 11.500 vs. 10.500 48.942 7.904 0.000 0.000 Yes 1.500 vs. 2.500 48.674 7.861 0.000 0.000 Yes 1.500 vs. 10.500 44.019 7.109 0.000 0.000 Yes 10.500 vs. 6.000 42.153 6.808 0.000 0.000 Yes 10.500 vs. 5.500 42.056 6.792 0.000 0.000 Yes 10.500 vs. 5.000 40.446 6.532 0.000 0.000 Yes 10.500 vs. 4.500 38.868 6.277 0.000 0.000 Yes 10.500 vs. 6.500 38.148 6.161 0.000 0.000 Yes 2.500 vs. 6.000 37.497 6.056 0.000 0.000 Yes 2.500 vs. 5.500 37.400 6.040 0.000 0.000 Yes 10.500 vs. 4.000 36.107 5.831 0.000 0.000 Yes 2.500 vs. 5.000 35.791 5.780 0.000 0.000 Yes 10.500 vs. 7.000 34.525 5.576 0.000 0.000 Yes 2.500 vs. 4.500 34.213 5.525 0.000 0.000 Yes 2.500 vs. 6.500 33.493 5.409 0.000 0.000 Yes 10.500 vs. 7.500 33.051 5.338 0.000 0.000 Yes 2.500 vs. 4.000 31.452 5.079 0.000 0.000 Yes 10.500 vs. 3.500 30.366 4.904 0.000 0.000 Yes 2.500 vs. 7.000 29.870 4.824 0.000 0.000 Yes 2.500 vs. 7.500 28.395 4.586 0.000 0.000 Yes 9.500 vs. 6.000 28.329 4.575 0.000 0.000 Yes 9.500 vs. 5.500 28.232 4.559 0.000 0.000 Yes 9.500 vs. 5.000 26.622 4.300 0.000 0.000 Yes

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220 10.500 vs. 3.000 26.447 4.271 0.000 0.000 Yes 2.500 vs. 3.500 25.711 4.152 0.000 0.000 Yes 9.500 vs. 4.500 25.044 4.045 0.000 0.000 Yes 10.500 vs. 8.000 30.637 4.040 0.000 0.000 Yes 9.500 vs. 6.500 24.324 3.928 0.000 0.000 Yes 10.000 vs. 6.000 23.901 3.860 0.000 0.000 Yes 10.000 vs. 5.500 23.804 3.844 0.000 0.000 Yes 8.500 vs. 6.000 23.099 3.731 0.000 0.000 Yes 8.500 vs. 5.500 23.002 3.715 0.000 0.000 Yes 9.000 vs. 6.000 22.338 3.608 0.000 0.000 Yes 9.500 vs. 4.000 22.283 3.599 0.000 0.000 Yes 9.000 vs. 5.500 22.241 3.592 0.000 0.000 Yes 10.000 vs. 5.000 22.195 3.585 0.000 0.000 Yes Comparisons for factor: Sample pH within 11 Comparison Diff of Means t Unadjusted P Critical Level Significant? NC vs. 5.000 97.821 15.798 0.000 0.000 Yes NC vs. 6.000 97.578 15.759 0.000 0.000 Yes NC vs. 6.500 96.964 15.660 0.000 0.000 Yes NC vs. 5.500 96.675 15.613 0.000 0.000 Yes NC vs. 4.000 95.928 15.493 0.000 0.000 Yes NC vs. 4.500 94.476 15.258 0.000 0.000 Yes NC vs. 7.000 93.276 15.064 0.000 0.000 Yes NC vs. 3.500 91.691 14.808 0.000 0.000 Yes 1.500 vs. 5.000 90.686 14.646 0.000 0.000 Yes 1.500 vs. 6.000 90.443 14.607 0.000 0.000 Yes 2.000 vs. 5.000 90.333 14.589 0.000 0.000 Yes 2.000 vs. 6.000 90.090 14.550 0.000 0.000 Yes 1.500 vs. 6.500 89.829 14.507 0.000 0.000 Yes NC vs. 7.500 89.585 14.468 0.000 0.000 Yes 1.500 vs. 5.500 89.540 14.461 0.000 0.000 Yes 2.000 vs. 6.500 89.476 14.451 0.000 0.000 Yes 2.000 vs. 5.500 89.187 14.404 0.000 0.000 Yes 1.500 vs. 4.000 88.793 14.340 0.000 0.000 Yes 2.000 vs. 4.000 88.441 14.283 0.000 0.000 Yes NC vs. 3.000 88.039 14.218 0.000 0.000 Yes 1.500 vs. 4.500 87.341 14.106 0.000 0.000 Yes 2.000 vs. 4.500 86.988 14.049 0.000 0.000 Yes NC vs. 8.000 86.317 13.940 0.000 0.000 Yes 11.000 vs. 5.000 86.268 13.932 0.000 0.000 Yes 1.500 vs. 7.000 86.141 13.912 0.000 0.000 Yes 11.000 vs. 6.000 86.025 13.893 0.000 0.000 Yes 2.000 vs. 7.000 85.788 13.855 0.000 0.000 Yes 11.000 vs. 6.500 85.411 13.794 0.000 0.000 Yes 11.000 vs. 5.500 85.122 13.747 0.000 0.000 Yes 1.500 vs. 3.500 84.555 13.656 0.000 0.000 Yes

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221 11.000 vs. 4.000 84.375 13.627 0.000 0.000 Yes 2.000 vs. 3.500 84.203 13.599 0.000 0.000 Yes 11.000 vs. 4.500 82.923 13.392 0.000 0.000 Yes 1.500 vs. 7.500 82.450 13.316 0.000 0.000 Yes NC vs. 9.000 82.287 13.289 0.000 0.000 Yes 2.000 vs. 7.500 82.097 13.259 0.000 0.000 Yes 11.000 vs. 7.000 81.723 13.198 0.000 0.000 Yes NC vs. 8.500 81.163 13.108 0.000 0.000 Yes 1.500 vs. 3.000 80.904 13.066 0.000 0.000 Yes NC vs. 10.000 80.674 13.029 0.000 0.000 Yes 2.000 vs. 3.000 80.551 13.009 0.000 0.000 Yes 11.000 vs. 3.500 80.137 12.942 0.000 0.000 Yes 1.500 vs. 8.000 79.182 12.788 0.000 0.000 Yes 2.000 vs. 8.000 78.829 12.731 0.000 0.000 Yes 11.000 vs. 7.500 78.031 12.602 0.000 0.000 Yes 11.500 vs. 5.000 76.935 12.425 0.000 0.000 Yes 11.500 vs. 6.000 76.693 12.386 0.000 0.000 Yes 11.000 vs. 3.000 76.486 12.352 0.000 0.000 Yes 11.500 vs. 6.500 76.079 12.287 0.000 0.000 Yes NC vs. 2.500 75.905 12.259 0.000 0.000 Yes 11.500 vs. 5.500 75.790 12.240 0.000 0.000 Yes NC vs. 9.500 75.254 12.154 0.000 0.000 Yes 1.500 vs. 9.000 75.151 12.137 0.000 0.000 Yes 11.500 vs. 4.000 75.043 12.119 0.000 0.000 Yes 2.000 vs. 9.000 74.799 12.080 0.000 0.000 Yes 11.000 vs. 8.000 74.764 12.074 0.000 0.000 Yes 1.500 vs. 8.500 74.028 11.956 0.000 0.000 Yes 2.000 vs. 8.500 73.676 11.899 0.000 0.000 Yes 11.500 vs. 4.500 73.591 11.885 0.000 0.000 Yes 1.500 vs. 10.000 73.539 11.877 0.000 0.000 Yes 2.000 vs. 10.000 73.186 11.820 0.000 0.000 Yes 11.500 vs. 7.000 72.391 11.691 0.000 0.000 Yes 12.000 vs. 5.000 71.763 11.590 0.000 0.000 Yes 12.000 vs. 6.000 71.520 11.551 0.000 0.000 Yes 12.000 vs. 6.500 70.907 11.451 0.000 0.000 Yes 11.500 vs. 3.500 70.805 11.435 0.000 0.000 Yes 11.000 vs. 9.000 70.733 11.423 0.000 0.000 Yes 12.000 vs. 5.500 70.618 11.405 0.000 0.000 Yes 12.000 vs. 4.000 69.871 11.284 0.000 0.000 Yes 11.000 vs. 8.500 69.610 11.242 0.000 0.000 Yes 11.000 vs. 10.000 69.120 11.163 0.000 0.000 Yes 1.500 vs. 2.500 68.770 11.106 0.000 0.000 Yes 11.500 vs. 7.500 68.699 11.095 0.000 0.000 Yes 12.000 vs. 4.500 68.418 11.050 0.000 0.000 Yes 2.000 vs. 2.500 68.417 11.049 0.000 0.000 Yes 1.500 vs. 9.500 68.119 11.001 0.000 0.000 Yes

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222 2.000 vs. 9.500 67.766 10.944 0.000 0.000 Yes 12.000 vs. 7.000 67.218 10.856 0.000 0.000 Yes 11.500 vs. 3.000 67.154 10.845 0.000 0.000 Yes 12.000 vs. 3.500 65.633 10.600 0.000 0.000 Yes 11.500 vs. 8.000 65.432 10.567 0.000 0.000 Yes 11.000 vs. 2.500 64.351 10.393 0.000 0.000 Yes 11.000 vs. 9.500 63.701 10.288 0.000 0.000 Yes 12.000 vs. 7.500 63.527 10.260 0.000 0.000 Yes 12.000 vs. 3.000 61.981 10.010 0.000 0.000 Yes 11.500 vs. 9.000 61.401 9.916 0.000 0.000 Yes 11.500 vs. 8.500 60.278 9.735 0.000 0.000 Yes 12.000 vs. 8.000 60.259 9.732 0.000 0.000 Yes 11.500 vs. 10.000 59.788 9.656 0.000 0.000 Yes NC vs. 10.500 59.669 9.637 0.000 0.000 Yes 12.000 vs. 9.000 56.229 9.081 0.000 0.000 Yes 12.000 vs. 8.500 55.106 8.900 0.000 0.000 Yes 11.500 vs. 2.500 55.019 8.886 0.000 0.000 Yes 12.000 vs. 10.000 54.616 8.821 0.000 0.000 Yes 11.500 vs. 9.500 54.369 8.781 0.000 0.000 Yes 1.500 vs. 10.500 52.534 8.484 0.000 0.000 Yes 2.000 vs. 10.500 52.181 8.427 0.000 0.000 Yes 12.000 vs. 2.500 49.847 8.050 0.000 0.000 Yes 12.000 vs. 9.500 49.196 7.945 0.000 0.000 Yes 11.000 vs. 10.500 48.115 7.771 0.000 0.000 Yes 11.500 vs. 10.500 38.783 6.264 0.000 0.000 Yes 10.500 vs. 5.000 38.152 6.162 0.000 0.000 Yes 10.500 vs. 6.000 37.909 6.122 0.000 0.000 Yes 10.500 vs. 6.500 37.295 6.023 0.000 0.000 Yes 10.500 vs. 5.500 37.006 5.977 0.000 0.000 Yes 10.500 vs. 4.000 36.260 5.856 0.000 0.000 Yes 10.500 vs. 4.500 34.807 5.621 0.000 0.000 Yes 12.000 vs. 10.500 33.611 5.428 0.000 0.000 Yes 10.500 vs. 7.000 33.607 5.428 0.000 0.000 Yes 10.500 vs. 3.500 32.022 5.172 0.000 0.000 Yes 10.500 vs. 7.500 29.916 4.831 0.000 0.000 Yes 10.500 vs. 3.000 28.370 4.582 0.000 0.000 Yes 10.500 vs. 8.000 26.648 4.304 0.000 0.000 Yes NC vs. 12.000 26.058 4.208 0.000 0.000 Yes 10.500 vs. 9.000 22.618 3.653 0.000 0.000 Yes 9.500 vs. 5.000 22.567 3.645 0.000 0.000 Yes 9.500 vs. 6.000 22.324 3.605 0.000 0.000 Yes Comparisons for factor: Sample pH within 11.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? NC vs. 5.500 100.248 16.190 0.000 0.000 Yes NC vs. 5.000 99.849 16.126 0.000 0.000 Yes

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223 NC vs. 6.000 99.816 16.120 0.000 0.000 Yes NC vs. 4.500 99.122 16.008 0.000 0.000 Yes NC vs. 6.500 97.396 15.730 0.000 0.000 Yes 11.500 vs. 5.500 96.012 15.506 0.000 0.000 Yes NC vs. 3.500 95.804 15.472 0.000 0.000 Yes 11.500 vs. 5.000 95.614 15.442 0.000 0.000 Yes 11.500 vs. 6.000 95.580 15.436 0.000 0.000 Yes 11.500 vs. 4.500 94.887 15.324 0.000 0.000 Yes NC vs. 4.000 94.510 15.263 0.000 0.000 Yes 11.000 vs. 5.500 93.694 15.132 0.000 0.000 Yes 11.000 vs. 5.000 93.296 15.067 0.000 0.000 Yes 11.000 vs. 6.000 93.262 15.062 0.000 0.000 Yes 11.500 vs. 6.500 93.160 15.045 0.000 0.000 Yes 11.000 vs. 4.500 92.569 14.950 0.000 0.000 Yes 11.500 vs. 3.500 91.568 14.788 0.000 0.000 Yes NC vs. 3.000 91.306 14.746 0.000 0.000 Yes NC vs. 7.500 91.246 14.736 0.000 0.000 Yes 11.000 vs. 6.500 90.842 14.671 0.000 0.000 Yes 11.500 vs. 4.000 90.274 14.579 0.000 0.000 Yes 11.000 vs. 3.500 89.250 14.414 0.000 0.000 Yes 12.000 vs. 5.500 88.361 14.270 0.000 0.000 Yes 12.000 vs. 5.000 87.963 14.206 0.000 0.000 Yes 11.000 vs. 4.000 87.956 14.205 0.000 0.000 Yes 12.000 vs. 6.000 87.929 14.201 0.000 0.000 Yes NC vs. 8.000 87.329 14.104 0.000 0.000 Yes 12.000 vs. 4.500 87.236 14.089 0.000 0.000 Yes 11.500 vs. 3.000 87.070 14.062 0.000 0.000 Yes 11.500 vs. 7.500 87.010 14.052 0.000 0.000 Yes 12.000 vs. 6.500 85.510 13.810 0.000 0.000 Yes 11.000 vs. 3.000 84.752 13.688 0.000 0.000 Yes 11.000 vs. 7.500 84.692 13.678 0.000 0.000 Yes NC vs. 8.500 84.336 13.620 0.000 0.000 Yes 12.000 vs. 3.500 83.918 13.553 0.000 0.000 Yes NC vs. 9.000 83.518 13.488 0.000 0.000 Yes 11.500 vs. 8.000 83.093 13.420 0.000 0.000 Yes NC vs. 10.000 82.628 13.344 0.000 0.000 Yes 12.000 vs. 4.000 82.624 13.344 0.000 0.000 Yes 1.500 vs. 5.500 80.914 13.068 0.000 0.000 Yes 11.000 vs. 8.000 80.775 13.045 0.000 0.000 Yes 1.500 vs. 5.000 80.516 13.003 0.000 0.000 Yes 1.500 vs. 6.000 80.482 12.998 0.000 0.000 Yes 11.500 vs. 8.500 80.100 12.936 0.000 0.000 Yes NC vs. 2.500 79.848 12.896 0.000 0.000 Yes 1.500 vs. 4.500 79.789 12.886 0.000 0.000 Yes 12.000 vs. 3.000 79.419 12.826 0.000 0.000 Yes 12.000 vs. 7.500 79.359 12.817 0.000 0.000 Yes

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224 11.500 vs. 9.000 79.282 12.804 0.000 0.000 Yes 11.500 vs. 10.000 78.392 12.660 0.000 0.000 Yes 1.500 vs. 6.500 78.063 12.607 0.000 0.000 Yes 11.000 vs. 8.500 77.783 12.562 0.000 0.000 Yes 11.000 vs. 9.000 76.964 12.430 0.000 0.000 Yes NC vs. 9.500 76.623 12.375 0.000 0.000 Yes 1.500 vs. 3.500 76.471 12.350 0.000 0.000 Yes NC vs. 10.500 76.397 12.338 0.000 0.000 Yes 11.000 vs. 10.000 76.074 12.286 0.000 0.000 Yes 11.500 vs. 2.500 75.612 12.211 0.000 0.000 Yes 12.000 vs. 8.000 75.442 12.184 0.000 0.000 Yes 1.500 vs. 4.000 75.177 12.141 0.000 0.000 Yes NC vs. 7.000 92.065 12.140 0.000 0.000 Yes 11.000 vs. 2.500 73.295 11.837 0.000 0.000 Yes 12.000 vs. 8.500 72.450 11.701 0.000 0.000 Yes 11.500 vs. 9.500 72.388 11.691 0.000 0.000 Yes 11.500 vs. 10.500 72.161 11.654 0.000 0.000 Yes 1.500 vs. 3.000 71.972 11.624 0.000 0.000 Yes 1.500 vs. 7.500 71.912 11.614 0.000 0.000 Yes 11.500 vs. 7.000 87.829 11.582 0.000 0.000 Yes 12.000 vs. 9.000 71.631 11.569 0.000 0.000 Yes 12.000 vs. 10.000 70.742 11.425 0.000 0.000 Yes 11.000 vs. 9.500 70.070 11.316 0.000 0.000 Yes 11.000 vs. 10.500 69.843 11.280 0.000 0.000 Yes 11.000 vs. 7.000 85.511 11.276 0.000 0.000 Yes 2.000 vs. 5.500 68.296 11.030 0.000 0.000 Yes 1.500 vs. 8.000 67.995 10.981 0.000 0.000 Yes 12.000 vs. 2.500 67.962 10.976 0.000 0.000 Yes 2.000 vs. 5.000 67.898 10.966 0.000 0.000 Yes 2.000 vs. 6.000 67.865 10.960 0.000 0.000 Yes 2.000 vs. 4.500 67.171 10.848 0.000 0.000 Yes 12.000 vs. 7.000 80.178 10.573 0.000 0.000 Yes 2.000 vs. 6.500 65.445 10.569 0.000 0.000 Yes 1.500 vs. 8.500 65.003 10.498 0.000 0.000 Yes 12.000 vs. 9.500 64.737 10.455 0.000 0.000 Yes 12.000 vs. 10.500 64.510 10.418 0.000 0.000 Yes 1.500 vs. 9.000 64.184 10.366 0.000 0.000 Yes 2.000 vs. 3.500 63.853 10.312 0.000 0.000 Yes 1.500 vs. 10.000 63.295 10.222 0.000 0.000 Yes 2.000 vs. 4.000 62.559 10.103 0.000 0.000 Yes 1.500 vs. 2.500 60.515 9.773 0.000 0.000 Yes 1.500 vs. 7.000 72.731 9.591 0.000 0.000 Yes 2.000 vs. 3.000 59.354 9.586 0.000 0.000 Yes 2.000 vs. 7.500 59.294 9.576 0.000 0.000 Yes 1.500 vs. 9.500 57.290 9.252 0.000 0.000 Yes 1.500 vs. 10.500 57.063 9.216 0.000 0.000 Yes

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225 2.000 vs. 8.000 55.377 8.943 0.000 0.000 Yes 2.000 vs. 8.500 52.385 8.460 0.000 0.000 Yes 2.000 vs. 9.000 51.567 8.328 0.000 0.000 Yes 2.000 vs. 10.000 50.677 8.184 0.000 0.000 Yes 2.000 vs. 7.000 60.113 7.927 0.000 0.000 Yes 2.000 vs. 2.500 47.897 7.735 0.000 0.000 Yes 2.000 vs. 9.500 44.672 7.215 0.000 0.000 Yes 2.000 vs. 10.500 44.445 7.178 0.000 0.000 Yes NC vs. 2.000 31.951 5.160 0.000 0.000 Yes 11.500 vs. 2.000 27.716 4.476 0.000 0.000 Yes 11.000 vs. 2.000 25.398 4.102 0.000 0.000 Yes 10.500 vs. 5.500 23.851 3.852 0.000 0.000 Yes 9.500 vs. 5.500 23.624 3.815 0.000 0.000 Yes 10.500 vs. 5.000 23.453 3.788 0.000 0.000 Yes 10.500 vs. 6.000 23.419 3.782 0.000 0.000 Yes 9.500 vs. 5.000 23.226 3.751 0.000 0.000 Yes 9.500 vs. 6.000 23.192 3.746 0.000 0.000 Yes 10.500 vs. 4.500 22.726 3.670 0.000 0.000 Yes 9.500 vs. 4.500 22.499 3.634 0.000 0.000 Yes Comparisons for factor: Sample pH within con Comparison Diff of Means t Unadjusted P Critical Level Significant? NC vs. 5.000 87.813 14.182 0.000 0.000 Yes NC vs. 4.500 86.677 13.998 0.000 0.000 Yes NC vs. 5.500 85.814 13.859 0.000 0.000 Yes NC vs. 6.000 84.784 13.693 0.000 0.000 Yes NC vs. 4.000 84.722 13.683 0.000 0.000 Yes 12.000 vs. 5.000 83.240 13.443 0.000 0.000 Yes NC vs. 6.500 82.837 13.378 0.000 0.000 Yes 12.000 vs. 4.500 82.104 13.260 0.000 0.000 Yes 12.000 vs. 5.500 81.241 13.121 0.000 0.000 Yes 12.000 vs. 6.000 80.211 12.954 0.000 0.000 Yes 12.000 vs. 4.000 80.149 12.944 0.000 0.000 Yes NC vs. 7.000 79.483 12.837 0.000 0.000 Yes 11.500 vs. 5.000 79.070 12.770 0.000 0.000 Yes 12.000 vs. 6.500 78.264 12.640 0.000 0.000 Yes 11.500 vs. 4.500 77.933 12.586 0.000 0.000 Yes NC vs. 7.500 77.915 12.583 0.000 0.000 Yes 11.500 vs. 5.500 77.071 12.447 0.000 0.000 Yes 11.500 vs. 6.000 76.040 12.281 0.000 0.000 Yes 11.500 vs. 4.000 75.978 12.271 0.000 0.000 Yes 12.000 vs. 7.000 74.911 12.098 0.000 0.000 Yes 11.500 vs. 6.500 74.093 11.966 0.000 0.000 Yes 12.000 vs. 7.500 73.342 11.845 0.000 0.000 Yes NC vs. 8.000 72.652 11.733 0.000 0.000 Yes 2.000 vs. 5.000 72.059 11.638 0.000 0.000 Yes

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226 NC vs. 3.500 71.116 11.485 0.000 0.000 Yes 2.000 vs. 4.500 70.922 11.454 0.000 0.000 Yes 1.500 vs. 5.000 70.827 11.439 0.000 0.000 Yes 11.500 vs. 7.000 70.740 11.425 0.000 0.000 Yes NC vs. 8.500 70.105 11.322 0.000 0.000 Yes 2.000 vs. 5.500 70.060 11.315 0.000 0.000 Yes 1.500 vs. 4.500 69.691 11.255 0.000 0.000 Yes NC vs. 9.000 69.324 11.196 0.000 0.000 Yes 11.500 vs. 7.500 69.172 11.171 0.000 0.000 Yes 2.000 vs. 6.000 69.029 11.148 0.000 0.000 Yes 2.000 vs. 4.000 68.968 11.138 0.000 0.000 Yes NC vs. 10.000 68.885 11.125 0.000 0.000 Yes 1.500 vs. 5.500 68.828 11.116 0.000 0.000 Yes 12.000 vs. 8.000 68.079 10.995 0.000 0.000 Yes 1.500 vs. 6.000 67.798 10.949 0.000 0.000 Yes 1.500 vs. 4.000 67.736 10.939 0.000 0.000 Yes 2.000 vs. 6.500 67.082 10.834 0.000 0.000 Yes 12.000 vs. 3.500 66.543 10.747 0.000 0.000 Yes 1.500 vs. 6.500 65.851 10.635 0.000 0.000 Yes NC vs. 9.500 65.660 10.604 0.000 0.000 Yes 12.000 vs. 8.500 65.533 10.584 0.000 0.000 Yes 12.000 vs. 9.000 64.751 10.457 0.000 0.000 Yes 12.000 vs. 10.000 64.312 10.387 0.000 0.000 Yes 11.500 vs. 8.000 63.908 10.321 0.000 0.000 Yes 2.000 vs. 7.000 63.729 10.292 0.000 0.000 Yes NC vs. 10.500 62.623 10.114 0.000 0.000 Yes 1.500 vs. 7.000 62.498 10.093 0.000 0.000 Yes 11.500 vs. 3.500 62.372 10.073 0.000 0.000 Yes 2.000 vs. 7.500 62.161 10.039 0.000 0.000 Yes 2.500 vs. 5.000 61.723 9.968 0.000 0.000 Yes 11.500 vs. 8.500 61.362 9.910 0.000 0.000 Yes 12.000 vs. 9.500 61.087 9.866 0.000 0.000 Yes 1.500 vs. 7.500 60.929 9.840 0.000 0.000 Yes 2.500 vs. 4.500 60.586 9.785 0.000 0.000 Yes 11.500 vs. 9.000 60.581 9.784 0.000 0.000 Yes 11.500 vs. 10.000 60.142 9.713 0.000 0.000 Yes 2.500 vs. 5.500 59.724 9.645 0.000 0.000 Yes 2.500 vs. 6.000 58.693 9.479 0.000 0.000 Yes 2.500 vs. 4.000 58.632 9.469 0.000 0.000 Yes 12.000 vs. 10.500 58.050 9.375 0.000 0.000 Yes 11.500 vs. 9.500 56.917 9.192 0.000 0.000 Yes 2.000 vs. 8.000 56.897 9.189 0.000 0.000 Yes 2.500 vs. 6.500 56.746 9.165 0.000 0.000 Yes 1.500 vs. 8.000 55.666 8.990 0.000 0.000 Yes 2.000 vs. 3.500 55.361 8.941 0.000 0.000 Yes 2.000 vs. 8.500 54.351 8.778 0.000 0.000 Yes

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227 1.500 vs. 3.500 54.130 8.742 0.000 0.000 Yes 3.000 vs. 5.000 54.057 8.730 0.000 0.000 Yes 11.500 vs. 10.500 53.880 8.702 0.000 0.000 Yes 2.000 vs. 9.000 53.570 8.652 0.000 0.000 Yes 2.500 vs. 7.000 53.393 8.623 0.000 0.000 Yes 2.000 vs. 10.000 53.131 8.581 0.000 0.000 Yes 1.500 vs. 8.500 53.120 8.579 0.000 0.000 Yes 3.000 vs. 4.500 52.920 8.547 0.000 0.000 Yes 1.500 vs. 9.000 52.338 8.453 0.000 0.000 Yes 3.000 vs. 5.500 52.058 8.407 0.000 0.000 Yes 1.500 vs. 10.000 51.899 8.382 0.000 0.000 Yes 2.500 vs. 7.500 51.825 8.370 0.000 0.000 Yes 3.000 vs. 6.000 51.027 8.241 0.000 0.000 Yes 3.000 vs. 4.000 50.966 8.231 0.000 0.000 Yes 2.000 vs. 9.500 49.906 8.060 0.000 0.000 Yes 11.000 vs. 5.000 49.193 7.945 0.000 0.000 Yes 3.000 vs. 6.500 49.081 7.927 0.000 0.000 Yes 1.500 vs. 9.500 48.674 7.861 0.000 0.000 Yes 11.000 vs. 4.500 48.056 7.761 0.000 0.000 Yes 11.000 vs. 5.500 47.194 7.622 0.000 0.000 Yes 2.000 vs. 10.500 46.869 7.569 0.000 0.000 Yes 2.500 vs. 8.000 46.562 7.520 0.000 0.000 Yes 11.000 vs. 6.000 46.163 7.455 0.000 0.000 Yes 11.000 vs. 4.000 46.101 7.445 0.000 0.000 Yes 3.000 vs. 7.000 45.727 7.385 0.000 0.000 Yes 1.500 vs. 10.500 45.637 7.370 0.000 0.000 Yes 2.500 vs. 3.500 45.025 7.272 0.000 0.000 Yes 11.000 vs. 6.500 44.216 7.141 0.000 0.000 Yes 3.000 vs. 7.500 44.159 7.132 0.000 0.000 Yes 2.500 vs. 8.500 44.015 7.108 0.000 0.000 Yes 2.500 vs. 9.000 43.234 6.982 0.000 0.000 Yes 2.500 vs. 10.000 42.795 6.911 0.000 0.000 Yes 11.000 vs. 7.000 40.863 6.599 0.000 0.000 Yes 2.500 vs. 9.500 39.570 6.391 0.000 0.000 Yes 11.000 vs. 7.500 39.295 6.346 0.000 0.000 Yes 3.000 vs. 8.000 38.896 6.282 0.000 0.000 Yes NC vs. 11.000 38.621 6.237 0.000 0.000 Yes 3.000 vs. 3.500 37.359 6.034 0.000 0.000 Yes 2.500 vs. 10.500 36.533 5.900 0.000 0.000 Yes 3.000 vs. 8.500 36.349 5.870 0.000 0.000 Yes 3.000 vs. 9.000 35.568 5.744 0.000 0.000 Yes 3.000 vs. 10.000 35.129 5.673 0.000 0.000 Yes 12.000 vs. 11.000 34.048 5.499 0.000 0.000 Yes 11.000 vs. 8.000 34.031 5.496 0.000 0.000 Yes NC vs. 3.000 33.756 5.452 0.000 0.000 Yes 11.000 vs. 3.500 32.495 5.248 0.000 0.000 Yes

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228 3.000 vs. 9.500 31.904 5.152 0.000 0.000 Yes 11.000 vs. 8.500 31.485 5.085 0.000 0.000 Yes 11.000 vs. 9.000 30.704 4.959 0.000 0.000 Yes 11.000 vs. 10.000 30.265 4.888 0.000 0.000 Yes 11.500 vs. 11.000 29.877 4.825 0.000 0.000 Yes 12.000 vs. 3.000 29.183 4.713 0.000 0.000 Yes 3.000 vs. 10.500 28.867 4.662 0.000 0.000 Yes 11.000 vs. 9.500 27.040 4.367 0.000 0.000 Yes NC vs. 2.500 26.090 4.214 0.000 0.000 Yes 10.500 vs. 5.000 25.190 4.068 0.000 0.000 Yes 11.500 vs. 3.000 25.013 4.040 0.000 0.000 Yes 10.500 vs. 4.500 24.054 3.885 0.000 0.000 Yes 11.000 vs. 10.500 24.003 3.876 0.000 0.000 Yes 10.500 vs. 5.500 23.191 3.745 0.000 0.000 Yes 2.000 vs. 11.000 22.866 3.693 0.000 0.000 Yes 10.500 vs. 6.000 22.161 3.579 0.000 0.000 Yes 9.500 vs. 5.000 22.153 3.578 0.000 0.000 Yes 10.500 vs. 4.000 22.099 3.569 0.000 0.000 Yes Two Way ANOVA of Salt Solubility data Two Way Analysis of Variance Dependent Variable: Solubility Source of Variation DF SS MS F P Treatment pH 6 5.995 0.999 436.432 <0.001 Salt Conc 4 2.018 0.505 220.381 <0.001 Treatment pH x Salt Conc 24 2.021 0.0842 36.777 <0.001 Residual 244 0.559 0.00229 Total 278 10.612 0.0382 Main effects cannot be properly in terpreted if significant inter action is determined. This is because the size of a factor 's effect depends upon the level of the other factor. The effect of different levels of Treatment pH depends on what level of Salt Conc is present. There is a statistically significant interaction between Treatment pH and Salt Conc. (P = <0.001) Power of performed test with alpha = 0.0500: for Treatment pH : 1.000 Power of performed test with alpha = 0.0500: for Salt Conc : 1.000 Power of performed test with alpha = 0.0500: for Treatment pH x Salt Conc : 1.000 All Pairwise Multiple Comparison Procedures (Holm-Sidak method): Overall significance level = 0.05

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229 Comparisons for factor: Salt Conc within 2 Comparison Diff of Means t Unadjusted P Critical Level Significant? 450M vs. 0M 0.0953 3.982 0.000 0.005 Yes 600M vs. 0M 0.0883 3.691 0.000 0.006 Yes Comparisons for factor: Salt Conc within 2.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 450M vs. 0M 0.0814 3.405 0.001 0.005 Yes 600M vs. 0M 0.0676 2.824 0.005 0.006 Yes Comparisons for factor: Salt Conc within 3: No Significant Differences Comparisons for factor: Salt Conc within 10.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 600M vs. 0M 0.345 14.420 0.000 0.005 Yes 600M vs. 150M 0.334 13.954 0.000 0.006 Yes 450M vs. 0M 0.286 11.971 0.000 0.006 Yes 450M vs. 150M 0.275 11.505 0.000 0.007 Yes 300M vs. 0M 0.210 8.759 0.000 0.009 Yes 300M vs. 150M 0.198 8.294 0.000 0.010 Yes 600M vs. 300M 0.135 5.660 0.000 0.013 Yes 450M vs. 300M 0.0768 3.211 0.001 0.017 Yes 600M vs. 450M 0.0586 2.449 0.015 0.025 Yes Comparisons for factor: Salt Conc within 11 Comparison Diff of Means t Unadjusted P Critical Level Significant? 600M vs. 150M 0.153 6.406 0.000 0.005 Yes 600M vs. 0M 0.151 6.308 0.000 0.006 Yes 450M vs. 150M 0.121 5.059 0.000 0.006 Yes 450M vs. 0M 0.119 4.961 0.000 0.007 Yes 300M vs. 150M 0.0886 3.706 0.000 0.009 Yes 300M vs. 0M 0.0863 3.608 0.000 0.010 Yes 600M vs. 300M 0.0646 2.700 0.007 0.013 Yes Comparisons for factor: Salt Conc within 11.5 Comparison Diff of Means t Unadjusted P Critical Level Significant? 600M vs. 0M 0.105 4.223 0.000 0.005 Yes 450M vs. 0M 0.0941 3.934 0.000 0.006 Yes 300M vs. 0M 0.0831 3.474 0.001 0.006 Yes 600M vs. 150M 0.0818 3.302 0.001 0.007 Yes 450M vs. 150M 0.0713 2.981 0.003 0.009 Yes Comparisons for factor: Salt Conc within control Comparison Diff of Means t Unadjusted P Critical Level Significant? 600M vs. 0M 0.659 27.559 0.000 0.005 Yes

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230 600M vs. 150M 0.590 24.647 0.000 0.006 Yes 300M vs. 0M 0.547 22.883 0.000 0.006 Yes 450M vs. 0M 0.522 21.839 0.000 0.007 Yes 300M vs. 150M 0.478 19.972 0.000 0.009 Yes 450M vs. 150M 0.453 18.927 0.000 0.010 Yes 600M vs. 450M 0.137 5.720 0.000 0.013 Yes 600M vs. 300M 0.112 4.675 0.000 0.017 Yes 150M vs. 0M 0.0697 2.912 0.004 0.025 Yes Comparisons for factor: Treatment pH within 0M Comparison Diff of Means t Unadjusted P Critical Level Significant? control vs. 2.500 0.131 5.463 0.000 0.002 Yes control vs. 2.000 0.124 5.200 0.000 0.003 Yes control vs. 11.500 0.120 5.025 0.000 0.003 Yes control vs. 3.000 0.0758 3.168 0.002 0.003 Yes control vs. 10.500 0.0729 3.048 0.003 0.003 Yes Comparisons for factor: Treatment pH within 150M Comparison Diff of Means t Unadjusted P Critical Level Significant? control vs. 2.500 0.174 7.281 0.000 0.002 Yes control vs. 11.500 0.167 6.984 0.000 0.003 Yes control vs. 2.000 0.160 6.692 0.000 0.003 Yes control vs. 11.000 0.142 5.924 0.000 0.003 Yes control vs. 3.000 0.136 5.684 0.000 0.003 Yes control vs. 10.500 0.131 5.494 0.000 0.003 Yes Comparisons for factor: Treatment pH within 300M Comparison Diff of Means t Unadjusted P Critical Level Significant? control vs. 2.500 0.619 25.884 0.000 0.002 Yes control vs. 2.000 0.615 25.726 0.000 0.003 Yes control vs. 11.500 0.585 24.435 0.000 0.003 Yes control vs. 3.000 0.567 23.688 0.000 0.003 Yes control vs. 11.000 0.531 22.190 0.000 0.003 Yes control vs. 10.500 0.411 17.172 0.000 0.003 Yes 10.500 vs. 2.500 0.208 8.712 0.000 0.003 Yes 10.500 vs. 2.000 0.205 8.553 0.000 0.004 Yes 10.500 vs. 11.500 0.174 7.262 0.000 0.004 Yes 10.500 vs. 3.000 0.156 6.515 0.000 0.004 Yes 10.500 vs. 11.000 0.120 5.018 0.000 0.005 Yes 11.000 vs. 2.500 0.0884 3.694 0.000 0.005 Yes 11.000 vs. 2.000 0.0846 3.536 0.000 0.006 Yes Comparisons for factor: Treatment pH within 450M Comparison Diff of Means t Unadjusted P Critical Level Significant? control vs. 2.500 0.572 23.898 0.000 0.002 Yes control vs. 2.000 0.552 23.056 0.000 0.003 Yes

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231 control vs. 11.500 0.549 22.930 0.000 0.003 Yes control vs. 3.000 0.542 22.648 0.000 0.003 Yes control vs. 11.000 0.473 19.792 0.000 0.003 Yes control vs. 10.500 0.309 12.916 0.000 0.003 Yes 10.500 vs. 2.500 0.263 10.981 0.000 0.003 Yes 10.500 vs. 2.000 0.243 10.140 0.000 0.004 Yes 10.500 vs. 11.500 0.240 10.014 0.000 0.004 Yes 10.500 vs. 3.000 0.233 9.732 0.000 0.004 Yes 10.500 vs. 11.000 0.164 6.876 0.000 0.005 Yes 11.000 vs. 2.500 0.0982 4.105 0.000 0.005 Yes 11.000 vs. 2.000 0.0781 3.264 0.001 0.006 Yes 11.000 vs. 11.500 0.0751 3.138 0.002 0.006 Yes 11.000 vs. 3.000 0.0683 2.856 0.005 0.007 Yes Comparisons for factor: Treatment pH within 600M Comparison Diff of Means t Unadjusted P Critical Level Significant? control vs. 2.500 0.722 30.198 0.000 0.002 Yes control vs. 2.000 0.695 29.067 0.000 0.003 Yes control vs. 3.000 0.685 28.633 0.000 0.003 Yes control vs. 11.500 0.675 27.257 0.000 0.003 Yes control vs. 11.000 0.578 24.165 0.000 0.003 Yes control vs. 10.500 0.387 16.187 0.000 0.003 Yes 10.500 vs. 2.500 0.335 14.011 0.000 0.003 Yes 10.500 vs. 2.000 0.308 12.880 0.000 0.004 Yes 10.500 vs. 3.000 0.298 12.445 0.000 0.004 Yes 10.500 vs. 11.500 0.288 11.618 0.000 0.004 Yes 10.500 vs. 11.000 0.191 7.978 0.000 0.005 Yes 11.000 vs. 2.500 0.144 6.033 0.000 0.005 Yes 11.000 vs. 2.000 0.117 4.902 0.000 0.006 Yes 11.000 vs. 3.000 0.107 4.467 0.000 0.006 Yes 11.000 vs. 11.500 0.0968 3.911 0.000 0.007 Yes

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232 REFERENCES Barton JR. 1997. Enviro nment, sustainability and regulation in commercial aquaculture: The case of chilean salmonid production. Geoforum 28(3-4):313-28. Berne RM, Levy MN, Koeppen BM, Stanton BA 2004. Physiology Vol 5. St Louis, MO: Mosby. Chang HS, Feng Y, Hultin HO. 2001. Role of pH in gel formation of washed chicken muscle at low ionic strength. Journal of Food Biochemistry 25(439-57). Choi YJ, Park JW. 2002. Acid-aided protein reco very from enzyme-rich pacific whiting. Journal of Food Science 67(8):2962-7. Davenport MP, Theodore AE, Kristinsson HG. 2003, 12-16 July. Low and high ph treatments induce a molten globular structure in myos in which improves its gelation properties Paper presented at the IFT Annual Meeting, Chicago Il. Drummy L, Phillips D, Stone M, Farmer B, Naik R. 2005. Thermally induced alpha-helix to beta-sheet transition in regenerated s ilk fibers and films. Biomacromolecules 6(6):332833. Eaglea J, Naylorb R, Smith W. 2004. Why fa rm salmon outcompete fishery salmon. Marine Policy 28(259-70). Fan YX, Ju M, Zhou JM, Tsou CL. 1996. Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conforma tional change at the active site. Biochemistry 315(97-102). Feng Y, Hultin HO. 2001. Effect of pH on the rheol ogical and structural properties of gels of water-washed chicken-breast muscle at physiological ionic stre ngth. Journal of Agriculture and Food Chemistry 49(8):3927-35. Harvey DJ. 2002. Aquaculture outlook. United States Department of Agriculture No LDP-AQS15. Hettiarachchy NS.1994. Protein f unctionality in food systems New York: Marcel Dekker, Inc.

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237 Stone AP, Stanley DW. 1992. Mechanisms of fish muscle gelation. Food Research International 25(381-8). Suvanich V, Jahncke ML, Marshall DL. 2000. Changes in selected chemical quality characteristics of channel catfish frame mince during chill and frozen storage. Journal of Food Science 65(1):24-9. Torten J, Whitaker JR. 1964. Evaluation of th e biuret and dye-binding methods for protein determination in meats. Journal of Food Science 29(168-74. Undeland I, Kelleher SD, Hultin HO. 2002. Recovery of functional proteins from herring (clupea harengus) light muscle by an acid or alkali ne solubilization pr ocess. Journal of Agricultural and Food Chemistry 50(25):7371-9. Vega-Warner V, Smith DM. 2001. Denaturation and aggregation of myosin from two bovine muscle types. Journal of Agriculture and Food Chemistry 49(2):906-12. Visschers RW, De Jongh HHJ. 2005 Disulphide bond formation in food protein aggregation and gelation. Biotechnology Advances 23(1):75-80. Walsh DJ, Cleary D, McCarthy E, Murphy S, Fi tzGerald RJ. 2003. Modifi cation of the nitrogen solubility properties of soy protein isolate following proteolysis and transglutaminase cross-linking. Food Res earch International 36(677-83. Xiong YL.1997. Structure-function re lationships of muscle proteins. In: S Damodaran Editor., Food proteins and their applications. New York: Narcel Dekker, Inc. p 157-81. Yongsawatdigul J, Park JW. 2004. Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins. Journal of Food Science 69(7):499-505. Yongswawatdigul J, Park JW. 2002. Biochemical and conformation changes of actomyosin from threadfin bream stored in ice. Journal of Food Science 67(3):985-90.

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238 BIOGRAPHICAL SKETCH Matthew Paul Davenport was born in Lexington, KY, and moved to Gainesville, FL two years later where he was raised. Matthew a ttended Gainesville High School graduating in 1998. During the last year and a half of high sc hool, Matthew started working as a laboratory technician f or Dr. Carlotta Grooves at the Univer sity of Florida in the College of Veterinary Medicine, Department of Basic Sciences; th e focus of that work was on environmental toxicology. After graduation from Gainesville High School, Matthew attended Greenville College in Greenville, Illinois, graduating in 200 2 with a Bachelor of Arts degree in biology. While at Greenville College Matthew began work in the area of food science, investigating carbon monoxide processing of yello w fin tuna and the effect of consumption on the consumer. This project was done in collaboration with th e Food Science and Human Nutrition Department of the University of Florida. After graduati on from Greenville College, Matthew began work in the Food Science and Human Nutrition Department at the University of Florida as a laboratory technician for Dr. W. Steven Otwell during the summer of 2002. Matthew began his graduate work in the fall of 2002 under the tutelage of Dr. Hordur G. Kristinsson. During his matriculation at the University of Florida, Matthew joined Alpha Ze ta, agricultural honors fraternity, and Phi Tau Sigma, f ood science honors fraternity, wher e he served as vice president from 2005-2006 and president from 2006-2007. U pon graduation Matthew hopes to contribute to the scientific community, both academic a nd industrial through his pursuit of knowledge in the sciences, specifically food science.


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