<%BANNER%>

Impact of Thermal and Non-Thermal Processing Techniques on Apple and Grapefruit Juice Quality

Permanent Link: http://ufdc.ufl.edu/UFE0041105/00001

Material Information

Title: Impact of Thermal and Non-Thermal Processing Techniques on Apple and Grapefruit Juice Quality
Physical Description: 1 online resource (170 p.)
Language: english
Creator: Azhuvalappil, Zareena
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: apple, aroma, flavor, grapefruit, juice, non, pef, rfef, thermal, uv
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: IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNIQUES ON APPLE AND GRAPEFRUIT JUICE QUALITY Increased consumer demand for fresh-like products with minimum flavor and nutritional losses has paved the way for alternatives to heat processing. The impact of non-thermal techniques, including pulsed electric field (PEF), radio frequency electric field (RFEF) or ultraviolet irradiation (UV) and thermal pasteurization on quality of fruit juices after 4 weeks of storage at 4masculine ordinalC were studied in this project. All treatments were optimized to achieve equivalent five log reduction in E. coli K12 levels. The effect of PEF, UV and thermal treatments on apple cider quality was investigated based on microbial, color, physical properties, volatile and sensory analysis. PEF and thermally processed cider maintained good microbial quality during 4 weeks of storage, but UV treated cider fermented after 2 weeks. Thermal and UV pasteurized cider color faded significantly (p < 0.05) during storage. CIE L* (lightness) and b* (yellow) values increased compared to PEF cider. Significant differences (p < 0.05) in the level of key cider odorants were observed between treated apple ciders after 4 weeks of storage. Thermal samples lost 30% and PEF cider lost less than 2% of the total ester and aldehydes during storage. In UV cider, hexanal and 2-(E)-hexenal were completely lost after 4 weeks of storage. Microbial spoilage in UV cider was chemically confirmed by the detection of the microbial metabolite 1,3 -pentadiene. Triangle sensory analysis indicated a significant difference (p < 0.05) in aroma between treatments after 4 weeks storage. PEF treated cider was preferred over thermally treated cider by 91% of the sensory panel. PEF treated apple cider had a longer shelf life than UV treated cider and a better aroma and color than the thermally processed sample. The effect of PEF, RFEF, UV and thermal techniques on the quality of red grapefruit juice were investigated based on microbial, color, physical properties, vitamin C content, non-enzymatic browning, pectin esterase inactivation, aroma volatiles and sensory analysis. Thermal treatment was more effective compared to non-thermal treatment in terms of microbial stability after 4 weeks storage. However, RFEF treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with significantly (p < 0.05) higher Vitamin C content and equivalent PME inactivation compared to thermally treated juice. PEF and UV treatments maintained good microbial quality for 1 week with significantly (p < 0.05) higher Vitamin C content, as well as better color preservation compared to thermal. Thermal, as well as non-thermal, pasteurization affected the aroma profile of fresh grapefruit juice considerably. The effects of treatments on juice aroma were highlighted by loss in desirable fruity and citrus odorants, along with an increase in undesirable cooked/catty odorants. Aroma of treated juice differed significantly (p < 0.05) from fresh juice by sensory evaluation. Non-thermally treated juice had a shorter shelf life but higher vitamin C content and superior physical qualities compared to thermally treated juice.
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.
Statement of Responsibility: by Zareena Azhuvalappil.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Rouseff, Russell L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041105:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041105/00001

Material Information

Title: Impact of Thermal and Non-Thermal Processing Techniques on Apple and Grapefruit Juice Quality
Physical Description: 1 online resource (170 p.)
Language: english
Creator: Azhuvalappil, Zareena
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: apple, aroma, flavor, grapefruit, juice, non, pef, rfef, thermal, uv
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: IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNIQUES ON APPLE AND GRAPEFRUIT JUICE QUALITY Increased consumer demand for fresh-like products with minimum flavor and nutritional losses has paved the way for alternatives to heat processing. The impact of non-thermal techniques, including pulsed electric field (PEF), radio frequency electric field (RFEF) or ultraviolet irradiation (UV) and thermal pasteurization on quality of fruit juices after 4 weeks of storage at 4masculine ordinalC were studied in this project. All treatments were optimized to achieve equivalent five log reduction in E. coli K12 levels. The effect of PEF, UV and thermal treatments on apple cider quality was investigated based on microbial, color, physical properties, volatile and sensory analysis. PEF and thermally processed cider maintained good microbial quality during 4 weeks of storage, but UV treated cider fermented after 2 weeks. Thermal and UV pasteurized cider color faded significantly (p < 0.05) during storage. CIE L* (lightness) and b* (yellow) values increased compared to PEF cider. Significant differences (p < 0.05) in the level of key cider odorants were observed between treated apple ciders after 4 weeks of storage. Thermal samples lost 30% and PEF cider lost less than 2% of the total ester and aldehydes during storage. In UV cider, hexanal and 2-(E)-hexenal were completely lost after 4 weeks of storage. Microbial spoilage in UV cider was chemically confirmed by the detection of the microbial metabolite 1,3 -pentadiene. Triangle sensory analysis indicated a significant difference (p < 0.05) in aroma between treatments after 4 weeks storage. PEF treated cider was preferred over thermally treated cider by 91% of the sensory panel. PEF treated apple cider had a longer shelf life than UV treated cider and a better aroma and color than the thermally processed sample. The effect of PEF, RFEF, UV and thermal techniques on the quality of red grapefruit juice were investigated based on microbial, color, physical properties, vitamin C content, non-enzymatic browning, pectin esterase inactivation, aroma volatiles and sensory analysis. Thermal treatment was more effective compared to non-thermal treatment in terms of microbial stability after 4 weeks storage. However, RFEF treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with significantly (p < 0.05) higher Vitamin C content and equivalent PME inactivation compared to thermally treated juice. PEF and UV treatments maintained good microbial quality for 1 week with significantly (p < 0.05) higher Vitamin C content, as well as better color preservation compared to thermal. Thermal, as well as non-thermal, pasteurization affected the aroma profile of fresh grapefruit juice considerably. The effects of treatments on juice aroma were highlighted by loss in desirable fruity and citrus odorants, along with an increase in undesirable cooked/catty odorants. Aroma of treated juice differed significantly (p < 0.05) from fresh juice by sensory evaluation. Non-thermally treated juice had a shorter shelf life but higher vitamin C content and superior physical qualities compared to thermally treated juice.
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.
Statement of Responsibility: by Zareena Azhuvalappil.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Rouseff, Russell L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041105:00001


This item has the following downloads:


Full Text





IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNIQUES ON
APPLE AND GRAPEFRUIT JUICE QUALITY



















By

ZAREENA AZHU VALAPPIL


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

2010
































2010 Zareena Azhu Valappil
































To my daughter Suha and my husband, Niyas









ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Russell Rouseff, for his guidance and

unending support throughout the course of my research work. It would not have been

possible to conduct my research at the United States Department of Agriculture without

his help and encouragement. I would also like to thank my committee members: Dr. M.

Marshall, Dr. B. Ache, Dr. W. Yang and Dr. H. Zhang. I would also like to thank my

previous committee members, Dr. M. Balaban and Dr. S. Talcott, who have left

University of Florida. My specials thanks to Dr. Fan, Dr. Geveke and Dr. Zhang at

USDA for helping me successfully conduct my research at USDA.

I would also like to thank my lab mates Kim, Jen, Kym, Glen and Tim for their

constant help and support and for making my life easier and enjoyable at USDA. A

special thanks to Kim for all her help in the lab and participating in GCO and sensory

work. I also want to thank my Mom and Dad for their love and guidance throughout my

life. Finally, I would like to thank my husband Niyas and daughter Suha for supporting

and helping me achieve my goals. Lastly, I want to thank God for everything.









TABLE OF CONTENTS

page

A C K N O W LE D G M E N T S ......................................................................................... ... 4

LIST O F TA BLES ............... ................................................................................... 9

LIST O F FIG U R ES ........................................................................................................ 11

A BSTRA CT ................................................................................................. ............... 13

CHAPTERS

1 IN T R O D U C T IO N .................................................................................................... 1 5

2 LITERATU R E R EV IEW ........................................................................................ 18

F lavor P perception .................................................................................................. 18
F ru it F la v o rs ............................................................................................................ 2 2
A pple Juice/C ider A rom a................................................................................ 22
G rapefruit Juice A rom a .................................................................................. 25
V olatile Extraction ................................................................................................... 28
Gas Chromatography Olfactometry ...................................................................... 30
P ro c e s s in g .............................................................................................................. 3 1
Therm al Pasteurization .................................................................................. 32
Citrus juice therm al processing ................................................................ 33
Apple juice therm al processing ................................................................ 33
Effect of Thermal Pasteurization on Juice Flavor ........................................... 34
Non-Thermal Processing Methods ................................................................. 36
P ulsed electric field .................................................................................. 38
Radio frequency electric field................................................................... 45
U ultraviolet radiation .................................................................................. 46

3 COMPARISON OF THERMAL AND NON-THERMAL TECHNIQUES ON
APPLE CIDER STORAGE QUALITY UNDER EQUIVALENT PROCESS
C O N D IT IO N S ......................................................................................................... 55

Introduction ....................................................................................... ............... 55
M materials and M ethods ............................................................................................ 56
Determination of Processing Conditions That Achieve 6-log Reduction of E.
coli K 12 ........................................................................... ................. 56
U ultraviolet process ................................................................................... 56
Pulsed electric field process ..................................................................... 57
Therm al process ........................................................................................ 58
Shelf-Life of Processed Apple Cider ............................................................... 58
Processing and packaging ....................................................................... 58
S to rage ...................................................................................... 59









M icro b ia l tests ......................................................................................... 59
p H .............................................................................................. ...... 6 0
B rix .............................................................................. ........................ 6 0
C o lo r ........................................................................... ........................ 6 0
S statistical A analysis ......................................................................................... 6 1
Results and D discussion ........................................................................................... 61
Optimization of Equivalent Processing Conditions ....................... ................. 61
Effect of Thermal and Non-Thermal Processing on Microbial Stability During
Storage of A pple C ider ...................... .. .................................. ................. 63
Effect of Thermal and Non-Thermal Processing on Appearance of Apple
Cider During Storage ................. ......... ................... 65
Effect of Thermal and Non-thermal Processing on the pH and Brix of Apple
C id e r D u rin g S to ra g e ................................................................ ... ................. 6 5
Effect of Thermal and Non-Thermal Processing on Color of Apple Cider
D during S torage ........................................................................................... 66
C conclusion .............................................................................. ...................... 68

4 IMPACT OF THERMAL AND NON-THERMAL PROCESSING
TECHNOLOGIES ON APPLE CIDER AROMA.................................... ...............74

Introduction .............................................................................. ........................ 74
M materials and M methods ...................................................................................... 74
A pple C ider ........................................................................................ 75
A pple C ider Processing .............................................................................. 75
H eat treatm ent ........................................................................................ 76
U ultraviolet treatm ent ................................................................................ 76
Pulsed electric field treatm ent ................................................................. 76
Packaging and Storage ................................................................................. 77
M icrobia l S ability .......................................................................................... 77
V o latile E xtra ctio n ...................................... ............................ ............. 7 8
Gas Chromatography-Mass Spectrometry Analysis...................................... 78
Quantification of Apple Cider Volatiles ....................................................... 79
S ensory Evaluation ....................................................................................... 79
S statistical A analysis ......................................................................................... 80
R results and D discussion ........................................................................................ 81
M icrobial Stability D during Storage ............................................... ............... 81
V olatile C om position ...................................................................................... 81
O dor Activity Values ...................................................................................... 82
Effect of Treatment and Storage on Volatiles ............... ........ ............... 83
Effect of Treatment and Storage on Odor Activity Values of Volatiles .............. 85
A ro m a S enso ry S tud ies ............................................... ................... ............... 86
C o n c lu s io n ............................................................................................... 8 7

5 COMPARISON OF THERMAL AND NON-THERMAL TECHNIQUES ON
GRAPEFRUIT JUICE STORAGE QUALITY UNDER EQUIVALENT PROCESS
C O N D IT IO N S ....................................................................................... ... 9 2



6









In tro d u c tio n ............................................................................................................. 9 2
M materials and M ethods................................. .... ............ ............. 93
Determination of Equivalent Treatment Conditions ...................................... 93
H eat treatm ent ......................................................................................... 93
U ultraviolet treatm ent ................................................................................. 93
P ulsed electric field treatm ent ................................................. ............... 94
Radio frequency electric field treatm ent.................................. ............... 94
S h e lf L ife S tu d y ................................................................................................ 9 5
G rapefruit juice pasteurization ................................................ ............... 95
P ackaging and storage .......................................................... .............. 95
M icro b ia l assay ............................. ...................................................... 96
Pectin m ethyl esterase activity assay ..................................... ............... 96
Ascorbic acid assay ............................................................... .............. 97
N on enzym atic brow ning ......................................................... ............... 97
pH .......................................................................... ..................... 98
B rix ......................................................................................................... .. 9 8
C o lo r ..................................................................................................... 9 8
S tatistica l A na lysis .................................................................. .. .......... 99
Results and Discussion ................. ....... ..... ........ ........................ 99
Effect of Treatments on Microbial Inactivation.............................................. 99
Effect of Treatments on Microbial Stability During Storage .......................... 101
Effect of Treatments on Pectin Methyl Esterase Activity .............................. 102
Effect of Treatments on Non-enzymatic Browning ................ .................. 104
Effect of Treatments on Ascorbic Acid Degradation.................................. 105
Effect of Treatm ents on Color..................................................... ............... 106
Effect of Treatments on pH, Brix and Total Acidity................ .................. 107
C o n c lu s io n ........................................................................................................ .. 1 0 7

6 IMPACT OF THERMAL AND NON-THERMAL PROCESSING
TECHNOLOGIES ON AROMA OF RED GRAPEFRUIT JUICE: GC-
OLFATOMETRIC COMPARSION ....... ................ .................... 118

In tro d u c tio n ....................................................................................................... 1 1 8
M materials and M ethods..................................................................................... 119
G rapefru it Ju ice ....................................................................................... 119
G rapefruit Juice Processing ....... .......... ........... .................... 120
Heat treatment ................. ....... ................ ............... 120
Non-thermal juice treatments ...... .......................... 120
Packaging and storage ...... ........... ........... .................... 120
Extraction of G rapefruit Juice Volatiles...... .... .................................... 121
Volatile Analysis of Grapefruit Juice Volatiles............................................. 121
Sensory Evaluation ........... ... ...... ...................... 122
Screening and training of panelists ...... .... ..................................... 122
T est m ethod ................................................................. ........... 123
S tatistica l A na lysis................................................................................... 124
Results and Discussion.................................................. ............... 124
Effect of Treatment and Storage on Grapefruit Juice Volatiles..................... 124









Gas Chromatography Olfactometry Profile of Grapefruit Juice.................... 125
Gas Chromatography Olfactometry Profile Comparison of Fresh Untreated
and Treated Juice at W eek 0 ....... ....... ..................... 128
T herm a l treatm ent ............................................... ................ .......... 129
U ltravio let treatm ent............................................................ .......... 130
Pulsed electric field treatment .............. .......................... 131
Radio frequency electric field treatm ent........................... .................. 131
Gas Chromatography Olfactometry Comparison of Fresh Untreated and
Treated Juice at W eek 4 ....... ......... ........... ..................... 131
Sensory Evaluation ................................................................... .............. 132
C o n c lu s io n ....................................................................................................... .. 1 3 3

7 C O N C L U S IO N ...................................................................................................... 14 3

APPENDIX

A CHANGE IN APPLE CIDER APPEARANCE DURING STORAGE ................... 146

B CALIBRATION TABLE FOR APPLE CIDER VOLATILES.............................. 147

C GRAPEFRUIT JUICE VOLATILE CONCENTRATION .................................. 148

D GRAPEFRUIT JUICE SENSORY RESULTS ............................................. 152

E SAMPLE SENSORY BALLOT FOR DIFFERENCE FROM CONTROL TEST...... 154

LIST O F R EFER EN C ES ................................................................. .............. 155

B IO G RA PH ICA L SKETC H ..................................... .......................... ............... 170









LIST OF TABLES


Table page

2-1 Time temperature parameters for PE inactivation in citrus juices................. 52

3-1 Change in pH and Brix values for fresh*, thermal, UV and PEF treated apple
cider stored at 4 oC for 4 w eeks..................................................... ............... 72

3-2 Change in Hunter color values** for fresh*, thermal, UV and PEF treated
apple cider stored at 4 oC for 4 w eeks........................................... ............... 73

4-1 Effect of thermal and non-thermal treatments on apple cider volatiles
compared to fresh untreated cider after 4 weeks of storage at 4 C................ 89

4-2 Effect of thermal and non-thermal treatments on odor activity values (OAV) of
apple cider volatiles after 4 weeks of storage at 4 oC .................... ............... 91

5-1 Yeast & mold count in control, thermal, UV, PEF and RFEF treated grapefruit
juice stored at 4 oC for 4 weeks ....... ...... ..... ..................... 111

5-2 pH, Brix and total acidity (TA) values for control, thermal, UV, PEF and RFEF
treated grapefruit juice stored at 4 oC for 4 weeks................. .................. 117

6-2 GCO analysis results of pasteurized and unpasteurized grapefruit juice....... 136

6-3 Calculated total aroma intensity values for pasteurized and unpasteurized
grapefruit juice belonging to five odor categories .................. .................. 138

6-4 GCO comparison of pasteurized grapefruit juice at week 4 to unpasteurized
grapefruit juice at w eek 0 ............................................. ................ .............. 140

6-5 Results of ANOVA for mean values from difference from control test ........... 142

6-6 Difference in means between pasteurized and unpasteurized grapefruit juice. 142

6-7 Results of ANOVA of mean values from difference control test at week 4 ....... 142

6-8 Difference in means between pasteurized and unpasteurized grapefruit juice
at w eek 4 ........................................................................ ........... ............... 142

B-1 Regression equation for standard compounds...................... .................. 147

C-1 Volatile concentration (pg/L) in untreated (control) and treated grapefruit juice
at w eekO ......................................................................... ........... ............... 149

C-2 Volatile concentration (pg/L) in untreated (control week 0) and treated
grapefruit juice at w eek 4 ............................................. ................ .............. 150









D-1 Difference from control data between hidden control (unpasteurized juice)
and pasteurized juices (thermal, PEF, RFEF and UV) at week 0.................. 152

D-2 Difference from control data between hidden control (unpasteurized fresh
juice ) and pasteurized juices (thermal, PEF, RFEF and UV) at.................... 153









LIST OF FIGURES


Figure page

2-1 Key odorants of grapefruit and apple............................................. ............... 52

2-2 Schematic diagram of pulsed electric field processing unit. (Jia and other,
1 9 9 9 ) ............................................................................................................... ... 5 3

2-3 Schematic diagram of radio frequency electric field processing unit (Geveke
and other, 2007) .. .................................................................. .............. 53

3-1 UV inactivation of inoculated E. coil K12 in apple cider at different treatment
times. Treatment conditions: wavelength 254 nm, outlet temperature < 15 C... 69

3-2 PEF inactivation of inoculated E. coli K12 in apple cider at different electric
field strengths. Treatment conditions: pulse duration of 2.5ps, total treatment
time ofl 50 ps, outlet temperature 49 51 C. ................................................. 69

3-3 Thermal inactivation of inoculated E. coli K12 in apple cider at different
temperatures. Treatment condition: 1.3 s hold time, outlet temperature <15
C .............................................. ....................................................................... 7 0

3-4 Total aerobic plate count for control, thermal, UV and PEF treated apple
cider samples stored at 4 oc for 4 weeks. Vertical bar indicates standard
deviation (n=4 ) .. ..................................................................... ........... 70

3-5 Yeast and mold count in control, thermal, UV and PEF treated apple cider
samples stored at 4 oc for 4 weeks. Vertical bar indicates standard deviation
(n = 4 ) ............................................................................................................ 7 1

3-6 Change in total color difference (de) in thermal, UV and PEF treated apple
cider during storage. Vertical bar indicates standard deviation (n=3).............. 71

4-1 Effect of treatment and storage (after weeks) on major volatiles of apple
cider 1 = butyl acetate, 2 = hexanal, 3 = 2-methyl butyl acetate, 4 = 2-(E)-
hexanal, 5 = hexyl acetate, 6 = benzaldehyde, 7 = 1-hexanol, a,b,c,d =
different letters for each volatile indicate significant difference(p<0.05),
control refers to fresh unpasteurized cider maintained at 0 C for 4 weeks........ 88

5-1 Thermal inactivation of inoculate E. coli K12 in grapefruit juice ........................ 109

5-2 UV Inactivation of inoculated E. coli K12 in grapefruit juice........................... 109

5-4 RFEF inactivation of inoculated E. coil K12 in grapefruit juice....................... 110

5-5 Total aerobic plate count for control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 C for 4 weeks....... ....................................... 111









5-6 Change in PME activity in control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 oC for 4 weeks....... ... .................................... 112

5-7 Change in browning index of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 oC for 4 weeks. ...... ... .................................... 112

5-8 Change in ascorbic acid content of control, thermal, UV, PEF and RFEF
treated grapefruit juice stored at 4 oC for 4 weeks................. .................. 113

5-9 Linear regression plot of ascorbic acid vs. browning index for thermal and
RFEF treated grapefruit juice...... ........ .. ....... ..................... 113

5-10 Change in color L* value of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 oC for 4 weeks....... ... .................................... 114

5-11 Change in color b* values of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 oC for 4 weeks....... ... .................................... 115

5-12 Linear regression plot of brown color vs. color b* values for thermal and
RFEF treated grapefruit juice...... ........ .. ....... ..................... 115

5-13 Change in total color difference (dE) in control, thermal, UV, PEF and RFEF
treated grapefruit juice stored at 4 oC for 4 weeks................. .................. 116

6-1 Comparative aroma profile for unpasteurized and pasteurized grapefruit juice
at w eek 0 ........................................................................ ........... ............... 134

A-1 Change in appearance in treated and untreated apple cider during 4 weeks
of storage at 40C ................................................................... .............. 146

C-1 TIC of grapefruit volatiles extracted by HS-SPME A. untreated juice (week 0)
B. UV treated juice (week 4). Separation on DB-Wax column. Highest
change in peak nos. 4 = hexanal, 7 = ethyl hexanoate, 10 = octanal, 14 =
d e c a n a l ......................................................................................................... .. 1 5 1









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

IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNIQUES ON
APPLE AND GRAPEFRUIT JUICE QUALITY

By

Zareena Azhu Valappil

August 2010

Chair: Russell Rouseff
Major: Food Science and Human Nutrition

Increased consumer demand for fresh-like products with minimum flavor and

nutritional losses has paved the way for alternatives to heat processing. The impact of

non-thermal techniques, including pulsed electric field (PEF), radio frequency electric

field (RFEF) or ultraviolet irradiation (UV) and thermal pasteurization on quality of fruit

juices after 4 weeks of storage at 40C were studied in this project. All treatments were

optimized to achieve equivalent five log reduction in E. coli K12 levels.

The effect of PEF, UV and thermal treatments on apple cider quality was

investigated based on microbial, color, physical properties, volatile and sensory

analysis. PEF and thermally processed cider maintained good microbial quality during 4

weeks of storage, but UV treated cider fermented after 2 weeks. Thermal and UV

pasteurized cider color faded significantly (p<0.05) during storage. CIE L* (lightness)

and b* (yellow) values increased compared to PEF cider. Significant differences

(p<0.05) in the level of key cider odorants were observed between treated apple ciders

after 4 weeks of storage. Thermal samples lost 30% and PEF cider lost less than 2% of

the total ester and aldehydes during storage. In UV cider, hexanal and 2-(E)-hexenal

were completely lost after 4 weeks of storage. Microbial spoilage in UV cider was









chemically confirmed by the detection of the microbial metabolite 1,3 -pentadiene.

Triangle sensory analysis indicated a significant difference (p<0.05) in aroma between

treatments after 4 weeks storage. PEF treated cider was preferred over thermally

treated cider by 91% of the sensory panel. PEF treated apple cider had a longer shelf

life than UV treated cider and a better aroma and color than the thermally processed

sample.

The effect of PEF, RFEF, UV and thermal techniques on the quality of red

grapefruit juice were investigated based on microbial, color, physical properties, vitamin

C content, non-enzymatic browning, pectin esterase inactivation, aroma volatiles and

sensory analysis. Thermal treatment was more effective compared to non-thermal

treatment in terms of microbial stability after 4 weeks storage. However, RFEF

treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with

significantly (p<0.05) higher Vitamin C content and equivalent PME inactivation

compared to thermally treated juice. PEF and UV treatments maintained good microbial

quality for 1 week with significantly (p<0.05) higher Vitamin C content, as well as better

color preservation compared to thermal. Thermal, as well as non-thermal, pasteurization

affected the aroma profile of fresh grapefruit juice considerably. The effects of

treatments on juice aroma were highlighted by loss in desirable fruity and citrus

odorants, along with an increase in undesirable cooked/catty odorants. Aroma of treated

juice differed significantly (p<0.05) from fresh juice by sensory evaluation. Non-thermally

treated juice had a shorter shelf life but higher vitamin C content and superior physical

qualities compared to thermally treated juice.









CHAPTER 1
INTRODUCTION

Public perception of juices as a healthy natural source of nutrients and heightened

public interest in health issues have led to increased fruit juice consumption worldwide.

However, in recent years many outbreaks of foodborne illness have been reported after

consumption of unpasteurized juice due to the presence of microorganisms like

Salmonella sp., Escherichia coil 0157:H7 and Cryptosporidium parvum (Besser and

others). The 1999 Salmonella outbreak in the United States was caused by

consumption of unpasteurized orange juice. Under the federal Juice HACCP rule

published in 2001, juice processors must implement treatments to reduce populations of

"pertinent" microorganism by 5 log cycles. The "pertinent" microorganism is defined as

the most resistant microorganism of public health significance that is likely to occur in

the juice in question. At present, E. coil 0157:H7 and Cryptosporidium parvum are

accepted as the pertinent organisms for apple juice and Salmonella are the accepted

pertinent organisms in citrus juice.

Thermal processing is the most commonly used technique to reduce spoilage and

pathogenic microorganisms in fruit juices (Choi and Nielsen 2005). Unfortunately,

thermal pasteurization can produce undesirable quality changes like loss of color and

flavor in addition to reducing the nutritional quality of juice (Rouseff and others 2001;

Vikram and others 2005). Recently, non-thermal processing alternatives like high

pressure, dense phase CO2, pulse electric fields, oscillating magnetic fields, pulsed high

intensity light and ultraviolet irradiation have been examined for their efficacy in

extending shelf life and enhancing juice, pulp or cider microbial safety while minimizing









quality and nutritional losses (Cserhalmi and others 2006; Tandon and others 2003;

Donahue and others 2004).

Even though there are many non-thermal processing techniques available for the

pasteurization of juices, a lot more research is required to make them commercially

successful. Particularly, studies about the effects of non-thermal techniques on quality

and consumer acceptability compared to the effects of thermal pasteurization are

required. Most often the conditions used for thermal processing are severe in terms of

temperature and time of exposure to heat. Thus, shelf life studies comparing the effects

of non-thermal to thermal techniques on acceptability of juice using conditions that do

not achieve the same reduction of a concerned microorganism would result in incorrect

conclusions (Rivas and others 2006; Aguillar-Rosas and others 2007; Yeom and others

2000b) Although a large amount of microbial information concerning thermal and non-

thermal processed fruit juice can be found in the literature, little information exists on the

effects of these processes on the flavor of juice. A major motivation for non-thermal

processing technologies is a minimal change to organoleptic properties. Therefore an

in-depth analysis of the effect of the above processes on the flavor profile of juice and

its relation to sensory quality is necessary.

A recent study by Sentandreu and others (2006) compared the sensory

acceptability of citrus juices (Valencia, Clementine and Ortanique) pasteurized by

pulsed electric field (PEF) and heat treatment. Each treatment was optimized to achieve

comparable 90% pectin methyl esterase (PME) inactivation. A simple ranking test

revealed no significant difference in sensory acceptability between PEF (25kV/cm for

330 ps) and heat (85 C for 10s) treated juices.









It is hypothesized that processing of juices by thermal and non-thermal techniques

at equivalent microbial inactivation conditions would have comparable effects on juice

quality. The main objectives of this study were to:

a) determine optimal conditions for apple cider and red grapefruit juice

pasteurization by thermal and non-thermal (PEF, RFEF & UV) techniques

b) compare the impact of thermal and non-thermal pasteurization techniques

on quality of apple cider and red grapefruit juice

c) study the change in quality of apple cider and red grapefruit juice during 4

weeks storage at 40C









CHAPTER 2
LITERATURE REVIEW

Flavor Perception

Flavor is one of the major influences on consumer purchases and consumption. It

is an amalgamation of aromatic character, gustatory stimuli and trigeminal sensations.

The aromatic character is imparted by a complex mixture of volatiles, whereas taste and

trigeminal components are mostly non-volatile, hydrophobic molecules soluble in saliva

(Laing and Jinks 1996). Aroma is sensed in the nasal cavity and encompasses most of

what is considered flavor. Odorants first interact with olfactory sensory neurons located

in the olfactory epithelium lining the nasal cavity. Conformational changes in the

receptor, due to binding of odorant, results in a cascade of biochemical changes leading

to transmission of signals to the olfactory bulb. These signals are further relayed to

other brain areas where the information is processed to given an integrated flavor profile

(Hatt 2000; Lancet and others 1993). A major breakthrough with regards to odorant and

receptor specificity came from the Nobel-Prize winning work of Buck and Axel (Buck

and Axel 1991). The three major points they presented were: a) each odor receptor can

recognize multiple odorants, b) each odorant can be detected by multiple receptors, and

c) the olfactory system uses a combinatorial receptor coding scheme to encode odor

identity. The receptor specificity for certain molecular groups is evident in the case of

the rat 17 odor receptor. This receptor binds to seven to nine carbon aliphatic aldehydes

with maximum affinity to octanal and lesser interaction with molecule-like heptanal

(Krautwurst and others 1998). However, with substitution of the aldehyde group with

any other functional group, this affinity is completely lost (Araneda and others 2000).

The functional group of compounds appears to be significant for recognition of odorants.









On the other hand, each odorant can be recognized by multiple receptors and thus

stimulate several receptors at same time. It is predicated that those odor receptors

which interact with the same odorant would have a similar protein sequence. For

example, highly related odor receptors S6, S50 and S79 interact with odorant

nonanedioic acid (Malnic and others 1999). Each odorant thus activates a unique

combination of olfactory cells and send a unique pattern of neuronal activity to the

olfactory bulb. The signal is then relayed to brain to encode the odor identity. Humans

have about 350 different odor receptors; hence, the olfactory system is well adapted to

recognize an endless list of odorants and to discriminate them (Zozulya and others

2001).

Gustatory stimuli (or taste) is the perception of sweet, sour, salty, bitter, and

umami sensations in the mouth. Umami is the characteristic taste imparted by

glutamate and 5'-nucleotides like inosine and guanine. Taste cells are clustered in taste

buds on the tongue, palate and pharynx. The tastants interact either with ion channels

(salty, sour) or specific G-protein coupled receptors (bitter, sweet, umami) on the taste

cells and transducts the signal to brain. Generally, chemicals that elicit sour and salty

tastes are ionic in nature and are associated with high concentrations of H+ and Na+,

respectively (Lindemann 1996). Ions enter the cell via selective membrane channels

resulting in depolarization of cell. This causes the release of neurotransmitters between

taste cells and taste neurons. Bitter, sweet and umami taste components bind to G-

protein coupled receptors similar to olfactory receptors, although the chemical

recognition is not as discriminating as it is in the case of the olfactory system (Zhang

and others 2003). Bitter receptors belong exclusively to a class known as the T2R









family (Mueller and others 2005; Chandrashekar and others 2000). Bitter tasting

compounds have an important role in survival, as they signal the presence of potentially

dangerous components. In nature, numerous and biologically diverse bitter compounds

exist, such as alkaloids, flavonoids, glycosides, steroids, phenols, N-heterocyclic

compounds, and urea and related compounds. In comparison to the wide range of bitter

compounds, there are far fewer T2Rs (~ 25) identified in mammals. Studies using

functional expression of T2R transcripts in taste receptor cells have shown that each

bitter sensing taste cell co-expresses the majority of T2R genes (Adler and others

2000). Meyerhof and others (2010) suggested that T2Rs are broadly tuned to perceive

a large variety of bitter molecules. When 25 hTAS2Rs (human bitter taste receptors)

were challenged with 104 bitter chemicals, they found that 3 of these hTAS2Rs were

able to detect ~50% of the bitter compounds. However, the ability of bitter receptors to

discriminate between the hTAS2Rs was limited (Adler et al. 2000). Thus, quite often

many bitter compounds taste the same. Both sweet and umami receptors belong to the

family of receptors called T1 R (Nelson and others 2001; Nelson and others 2002).

Heterodimers of these receptors, namely T1 R1 -T1 R3, T1 R2-T1 R3 or T1 R3, function as

receptors for sweet tastes. These receptors expressed differentially among the taste

cells enable binding of a wide range of structurally dissimilar molecules which are

perceived as sweet, like sweet sugars, sweet proteins and artificial sweeteners (Nelson

et al. 2001). Heterodimer T1 R1 -T1 R3 acts as umami taste receptors and binds to

monosodium L-glutamate and other L-amino acids (Nelson et al. 2002). Because this

type of heterodimer is common to both umami and sweet tastes, there may be a

relationship between these two taste modalities. Compared to the olfactory system, the









taste system is incapable of detailed chemical analysis due to the small number of taste

receptors and ion channels.

Another system found in olfactory epithelium is the trigeminal nerve receptors. The

trigeminal nerve extends to second set of nerve endings in areas around the mouth,

eyes and nasal cavity. They are responsible for tactile sensations, pressure, pain and

temperature. Some food components like capsaicin, menthol, flavonoids and alkaloids

are responsible for trigeminal sensations like pungency, cooling and astringency. The

trigeminal system influences the perception of taste and smell. A study by Prescott and

others (1993) showed that capsaicin inhibits the perceived sweetness of sucrose. In

another study, the burning sensation produced by capsaicin was influenced by the

presence of sodium chloride but not by the presence of sucrose (Prescott and

Stevenson 1995). More studies of the role of trigeminal system in flavor perception are

required.

The perception of aroma is also influenced by whether the odorant is perceived via

the orthonasal or retronasal path. Orthonasal refers to when the aroma is taken directly

through the nose (Linforth and others 2002). Retronasal refers to the aroma volatiles

produced after the food is swallowed and air from the lungs is exhaled through the nasal

cavity. In an orthonasal case, the release of aroma compounds from foods depends on

the partition coefficient between air phase and food matrix. In retronasal olfaction,

aroma release depends on the partition coefficient between the water phase (saliva)

and the food matrix. The eating process is a dynamic process, and the release of

volatiles from food depends on the different physical and chemical conditions in the

mouth like pH, temperature, mastication and enzymes. The rate of release of odorants









is also affected by the degree of interactions between aroma compounds and both

macro (carbohydrates, proteins and lipids) and micro (acids, salt) food constituents. The

retronasal impact is thus a combination of olfaction and gustatory perception.

Retronasal aroma would most likely leave a lasting impression on the consumer.

Fruit Flavors

Fruit and fruit juice consumption have increased recent past years due to their

perceived health benefits. Because flavor is one of the major influences of consumer

purchases and consumption, a significant amount of research in last 30 years has been

conducted in area of fruit flavors. The focus of research has been on characterizing the

key volatile components that deliver the characteristic flavor unique to particular fruits.

Apple Juice/Cider Aroma

Apple cider (sometimes called soft cider) is the name used in the United States for

an unfiltered, unsweetened, non-alcoholic beverage produced from apples. It is opaque

due to the fine apple particles in suspension and may be tarter than conventional filtered

apple juice depending on varietal characteristics of the apples used. In European

countries, cider refers to an alcoholic beverage made from the fermented juice of

apples.

Volatile compounds in fresh apples have been a subject of interest to many

investigators. Over 300 volatile compounds have been detected in apples comprising of

alcohols, aldehydes, carboxylic esters, ketones and ethers (Dimick and Hoskin 1983).

Esters are present in highest amounts (78-92%) followed by alcohols (6-16%). Ester

volatiles in apples give a fruity odor and are classified as ethyl esters, butyric esters,

propanoic esters and hexanoic esters. The alcohols mostly are ethyl, butyl and hexyl

alcohols.









The volatile composition and content in apple varies according to variety, maturity

and storage conditions. Most of the changes in flavor and aroma characteristics

develop after harvest. The aroma volatiles in whole, intact fruits are mostly formed via

beta-oxidation or the lipoxygenase pathway from fatty acids, which are the major

precursors of volatiles (Dimick and Hoskin 1983). The straight chain esters are

synthesized via beta-oxidation of fatty acids to give acetic, butanoic and hexanoic acids.

These acids are then reduced to corresponding alcohols and esters using Acyl CoA

enzyme (Paillard and Rouri 1984a). As the fruit ripens, the cell wall and membranes

becomes more permeable and the lipoxygenase enzyme comes in contact with fatty

acids like linoleic and linolenic acid, converting them into C6 and C9 aldehydes (Dever

and others 1992). These aldehydes are responsible for the green notes in apple aroma.

The C6 and C9 aldehydes formed in homogenized fruit reach their maximum

concentration within the first hour after homogenization.

Varietal differences affect volatile profile of apples. Granny Smith and Nico

varieties are characterized by a high concentration of ethyl butyrate and hexanol,

whereas Cox's Orange Pippin and Jonathan apples are characterized by high levels of

hexanal and 2-E-hexenal (Dixon and Hewett 2000; Schamp and others 1988). In

addition to the ripening and varietal effect, storage time and environment also impacts

apple flavor (Aaby and others 2002; Plotto and others 1999). Apples are mostly held in

controlled atmospheres during storage to delay ripening and reduce respiration. The

reduced oxygen environment induces acetaldehyde and ethanol accumulation and

decreases ester and aldehyde levels. Exposure of apples to low temperatures for more

than 3 months decreases volatile concentration (Streif and Bangerth 1988).









One of the earliest studies on sensory contribution of apple volatiles to aroma was

by Flath and others (1967b). They performed GC sniffing of Delicious apple essence. In

total, 56 compounds were identified by mass spectrometry. Compounds with apple

characteristics were listed as ethyl 2-methyl butyrate, hexanal and 2-E-hexenal. A study

by Durr and others (1981) on 48 samples of commercial apple essence showed that the

concentration of 2-E-hexenal and butyl acetate had a correlation of 0.78 and 0.72,

respectively, to the odor intensity of apple essence. Good apple essence was

characterized by high levels of C6 aldehydes and esters and low alcohol levels.

In Red Delicious apples, ethyl butyrate, ethyl 2-methyl butyrate, propyl 2-methyl

butyrate, hexyl acetate, ethyl hexanaote and 1,3,5 (E,Z)-undecatriene were identified as

key odorants by GCO analysis (Cunningham and others 1986). In Gala apples stored in

a regular atmosphere, the key contributors to aroma were butyl acetate, hexyl acetate,

butyl 2-methyl butyrate, hexyl 2-methyl butyrate and hexyl propanoate (Plotto and

others 2000). Its profile changed in a controlled atmosphere by a decrease in esters

hexyl acetate and butyl acetate.

Fuhrmann and others (2002) compared GCO data of three apple cultivars, Cox

Orange, Elstar and Royal Gala, to determine the key contributing odorants. They

studied the odor profile in the headspace of whole fruit as well as homogenized fruit.

The key ester volatiles in whole fruit for different apple varieties were ethyl 2-methyl

butyrate, ethyl butyrate (Elstar), ethyl 2-methyl propanoate, ethyl butyrate, 2-methyl

butanol (Cox) and methyl 2-methyl butyrate, ethyl 2-methyl butyrate, and propyl 2-

methyl butyrate (Royal Gala). In homogenized fruit, the FD profile changed compared to

whole fruit. High FD values for compounds hexanal, cis-3-hexenal and hexyl acetate in









Cox and Elstar varieties were noted. The increase in levels of aldehydes was due to

oxidation of linoleic acid and linolenic acid by liopxygenase released from crushed cells.

Other volalites noted with high FD values were P3-damascenone and p-allyl anisole.

Beta-damascenone has a rosy, fragrant odor, which adds intensity to the fruity odor of

an apple. Para-allyl anisole with musk herbaceous odor, along with its isomer anethole,

imparts spicy notes to an apple's aroma.

Mehanigic and others (2006) characterized odor volatiles of three apple cultivars

(Fuji, Golden Delicious and Braeburn) at different maturity stages. They found 15

odorants common to all varieties, the important ones being ethyl butyrate, ethy-2-methyl

butyrate, butyl acetate, hexyl acetate and hexanal. The overall quantity of odorants

increased during maturation in Fuji and Golden Delicious apples with more alcohols and

esters (butyric & hexanoic) and less aldehydes. The data from various literature reports

on different varieties of apples indicate esters like ethyl butyrate (4), ethyl-2-methyl

butyrate (5), butyl acetate (6) and hexyl acetate (7) (Figure 2-1) as key odorants of

apple aroma.

Grapefruit Juice Aroma

Grapefruit juice has a unique flavor due to its sweet-tart and bitter taste combined

with a characteristic aroma. Quantitative and qualitative studies on volatile constituents

of grapefruit juice, essence and oils have been carried out by many groups (Buettner

and Schieberle 1999; Lin and others 2002a; Coleman and others 1972). Moshonas and

others (1971) reported a total of 32 compounds in grapefruit essence extracted from

canned grapefruit juice Based on the relative amounts by GC-FID, they reported major

volatiles as ethanol, acetal, ethyl acetate, ethyl butyrate, isoamyl alcohol, cis and tran-

linalool oxide, linalool, octanol, alpha-terpineol, ethyl 3- hydroxyhexanoate and terpinen-









4-ol as major components. In contrast to essence, fresh juice had high ethanol content

followed by volatiles limonene, beta-caryophyllene, ocimene and myrcene. Other

compounds found in lower quantities were linalool, citral, a-terpeniol, P3-phelladrene,

copaene and nootkatone (Shaw and others 2000; Nunez and others 1985).

Pino and others (1986) studied the relationship between sensory response and 32

compounds present in grapefruit juice. Some of the important aroma compounds

identified were limonene, acetaldehyde, decanal, nootkatone, ethyl acetate, methyl

butyrate and ethyl butyrate. They determined the odor threshold value of the

compounds in water and quantified the amount of each compound in juice. The relative

flavor contribution of each compound was calculated based on the ratio of amount in

juice to odor threshold in water. Ethyl acetate and acetaldehyde had the highest

relative threshold values and nootkatone the lowest. The authors performed a sensory

analysis wherein panelists scored the odor of juice with four different dilutions of

compounds in increasing order. These sensory scores were then related to quantities of

compounds from GC data using linear regression analysis. Nootkatone, limonene,

decanal, ethyl butyrate and methyl butyrate showed positive correlation, which indicates

that they contribute significantly to good grapefruit juice flavor. However, an increase in

concentration of a-terpineol, cis and trans epoxydihydro linalool had a negative

correlation with sensory scores. In another study by Jella and others (1998), a positive

correlation in grapefruit juice acceptability with beta-caryophyllene levels and negative

correlation with myrcene and linalool levels were reported.

Two compounds, nootkatone (1) and p-1-menthene-8-thiol (2) (Figure 2-1), have

been identified and proposed as character impact components of grapefruit juice aroma









(Macleod and Buigues 1964; Demole and others 1982). The flavor threshold of

nootkatone is at 1ppm in water and 6ppm in reconstituted grapefruit juice (Berry and

others 1967). Nootkatone at levels more than 6-7ppm impart an unpleasant bitterness

to juice. The importance of nootkatone to grapefruit juice flavor is debatable, as Shaw

and others (1981) found that addition of nootkatone to grapefruit juice had very little

impact on juice flavor. They suggested the presence of other aroma components in

juice that are significant to grapefruit flavor. Sulfur compound 1-p-menthene-8-thiol is a

character impact odorant in grapefruit juice. Demole and others (1982) isolated 1-p-

menthene-8-thiol (1-PMT) and its bicyclic epimer 2, 8-epithio-cis-p-menthane, from

canned grapefruit juice. They reported 1 -PMT as the most potent odorant in nature with

a very low odor threshold at 0.1 x 10-9 g/L. It occurs at 200 fold higher concentration in

juice, implying its significance to grapefruit flavor. The threshold of thel-PMT isomer

was about 105 greater than the thiol, indicating lesser contribution to grapefruit flavor.

To investigate other important odorants in grapefruit juice, further studies by the same

authors led to the identification of 15 related sesquiterpenes ketones in grapefruit juice

(Demole and Enggist 1983). One of these ketones, 8,9-didehydronootkatone, was a

more intense (lower threshold) grapefruit aroma component than nootkatone.

Most of the odor active volatiles important to grapefruit aroma are present in very

low levels and often not detected by mass spectrometry or FID. Numerous GC-

olfactometry studies have been performed to identify the character impact volatiles in

grapefruit juice. A recent study by Buettner and others (1999) detected 37 odor-active

volatiles in fresh grapefruit juice. Although acetaldehyde and limonene were the most

abundant compounds present in grapefruit juice, they had a minor impact on juice









flavor. The authors reported high FD values for: ethyl butyrate, 1-p-menthene-8-thiol,

(Z)-3-hexenal, 4, 5-epoxy-(E)-2-decenal, 4-mercapto-4-methyl pentan-2-one (4-MMP)

(3), 1-hepten-3-one and wine lactone. The trace component 4-MMP had the highest

odor activity value. The same authors conducted further studies by quantitation,

reconstitution and omission methods to determine the contribution of above odorants

(Buettner and Schieberle 2001). Sensory evaluation of the reconstituted grapefruit

aroma model using 20 odor active components in water showed the sulfurous

grapefruit-like odor quality due to 4-MMP and 1-p-menthene -8-thiol to be important.

Single compound omission of 4-MMP from aroma models indicated that it had a higher

impact on grapefruit odor than 1-p-menthene-8-thiol. Other grapefruit juice sulfur

compounds reported in other studies included: 4-mercapto 4-methyl pentanol, 3-

mercapo hexanol and 3-mercapto hexyl acetate (Lin and others 2002b). Hydrogen

sulfide was also reported as a potential contributor to grapefruit juice aroma as it is

present at levels above its odor threshold in juice (Shaw and others 1980). Grapefruit

juice has 2-4X more sulfur components compared to orange juice (Rouseff and others

2003).

Volatile Extraction

The first step when characterizing odor active chemicals in a complex mixture is to

separate them from nonvolatile components. This is accomplished through a variety of

techniques such as solvent extraction, headspace concentration or distillation. The

results can vary significantly in terms of final composition and flavor-component mixture,

depending on the method used for volatile collection and concentration of aromas. For

example, low impact methods of flavor isolation like hydrodistillation and solvent

extraction are preferred for concentrating aromas from fruits and blossoms, as they are









less invasive and use mild temperatures. However, these methods might not be suitable

for reaction flavors that have already been subjected to heat treatment.

The extraction methods used in the past for apple volatiles include: vacuum hydro

distillation, dynamic and static headspace and liquid extraction (Komthong and others

2006a; Matich and others 1996; Mehinagic and others 2003). Young and others (2004)

found that butanol was detected at the highest levels in Royal Gala apples by vacuum

hydrodistillation, while butyl acetate was detected at the highest levels using headspace

methods. Matich and others (1996) compared solid phase micro extraction (SPME) and

solid phase extraction (SPE) method for Granny Smith apple volatiles. SPME provided

a greater adsorption of high molecular components but required long period of time for

equilibration and exposure times.

Various methods like solvent extraction, vacuum distillation, purge and trap, steam

distillation extraction (SDE) and SPME have been used for extraction of grapefruit juice

volatiles (Nunez and others 1984; Cadwallader and Xu 1994; Yoo and others 2004).

SDE method was good for extraction of medium- and low-volatile components, but the

high temperature used during the procedure combined with the presence of oxygen

could alter composition of volatiles (Nunez et al. 1985). Purge and trap extraction

eliminates some artifact formation. However, the method showed a few drawbacks

when used for grapefruit juice volatile analysis. Compounds having lower vapor

pressure, like oxygenated mono and sesquiterpenes, could not be effectively collected

and therefore could not be detected. Moreover, sulfur compounds significant for

grapefruit juice aroma were also not detected using the above extraction method

(Cadwallader and Xu 1994). Solvent extraction is reported to be a better method of









volatile extraction for grapefruit juice, but it is cumbersome and loss of volatiles may

occur during solvent evaporation (Buettner and Schieberle 1999; Jella and Rouseff

1997). Reports on SPME extraction of grapefruit volatiles are limited (Yoo et al. 2004).

Static headspace analysis by solid phase micro extraction (SPME) is a simple,

rapid and solventless technique for extraction of volatiles (Kataoka and others 2000).

Equilibrium is established among the analyte concentrations above the sample

headspace and the polymer coating on the fused silica fiber. The amount of analyte

adsorbed by the fiber depends on the thickness of polymer coating, the type of fiber

coating and the distribution constant for the analyte. In addition to fiber, the transfer of

analyte from food matrix to SPME fiber also depends on the matrix composition, time

and temperature of extraction. Some of the fiber coatings used are polydimethylsiloxane

(PDMS), divinylbenzene (DVB), carbowax (CW), carboxen and polyacrylate. Most often

a combination of these coatings is used for broad-range volatile extraction.

Gas Chromatography Olfactometry

Even though food aroma is a complex mixture of volatiles, only a fraction of these

volatiles are odor active. Most of the potent odorants are not often detected by

instrumental techniques due to very low odor thresholds. Human olfaction has superior

sensitivity and selectivity to odor compounds compared to many instrumental systems.

The hyphenation of olfactometry to gas chromatography technique is an effective tool

for the discrimination of relevant food flavor components, enabling the differentiation of

a multitude of volatiles into odor active and non-odor active categories. In GCO,

assessors judge the olfactory impressions elicited by the volatile compounds

immediately after elution from a GC column in order to associate odor activity with the









eluting compounds. GC/O methods are classified into three categories: dilution

methods, time- intensity methods and frequency of detection methods.

Time intensity methods are dynamic and odor intensity is recorded with the time

during peak elution. "Osme" is a time-intensity approach for evaluating the significance

of odor compounds in the GC effluent. Trained subjects sniffing the GC effluent mixed

with humidified air directly record the odor intensity and duration time of each odor-

active compound while describing its odor quality. The plot of the retention time/index

versus odor intensity provides an easily interpretable representation of the compound's

odor significance in the flavor extract, called an "Osmegram" (Bazemore and others

1999). Higher peaks suggest greater sensory importance of the compounds. Time-

intensity olfactometry has been used to identify key odorants in various fruits like

apples, oranges, grapefruits, blueberries and cashew apples (Plotto et al. 2000; Lin and

Rouseff 2001; Rouseff et al. 2001; Su and Chien 2010; Garruti and others 2003).

Processing

Fresh fruit juices can undergo quality degradation due to microbiological and

enzymatic activities, as well as chemical reactions. In recent years, there has been

increased concern about the safety of unpasteurized fruit juices due to outbreaks of

Escherichia coli 0157:H7, Salmonella spp. and Cryptosporidium parvum (Besser and

others 1993a; Parish 1997). On January 19, 2001, the FDA published a final rule in the

Federal Register that requires processors of juice to develop and implement Hazard

Analysis and Critical Control Point (HACCP) systems for their processing operations (66

FR 6138). HACCP is a systemic preventive process applied in the food industry to

address food safety by control and analysis of physical, biological and chemical hazards









at various processing stages. Under this rule, juice processors must comply with two

requirements:

(1) subpart A of the rule requires use of HACCP principles and systems in

their operations, and

(2) subpart B of the rule requires that processors implement treatments) to

reduce a theoretical population of "pertinent" microorganisms in the juice by 99.999% or

5-log cycles.

The "pertinent" microorganism is defined as the most resistant microorganism of

public health significance that is likely to occur in the juice. At the present time,

Salmonella is generally recognized as the pertinent organism for citrus juices and E. coli

0157:H7 and Cryptosporidium parvum for apple juice (FDA 2001).

Thermal Pasteurization

Thermal treatment has been widely used to inactivate spoilage, pathogenic

microorganisms and enzymes to extend shelf life of juice products. Initially,

pasteurization of fruit juices was done at low temperatures for long periods of time (63

C for 30 min). But extended heat treatment lowered the sensory and nutritional

qualities of juices (Moshonas and Shaw 2000). Current pasteurization methods in

practice are high temperatures, short time (HTST) and ultra high temperature (UHT).

UHT process is performed at 135-150 C for a few seconds (2 45 s). There are 2

methods: direct heating by steam injection and indirect heat transfer by heat exchanger.

The product is aseptically packaged after UHT processing in order to obtain a product

with a shelf life of 1 to 2 years at ambient temperatures. UHT destroys all pathogenic

microorganisms, as well as spores. It is commonly used for milk pasteurization (David









and others 1996). However, UHT causes considerable change to the quality of milk, as

it renders a heated flavor.

HTST, or flash pasteurization, is the most common method used for fruit

pasteurization (David et al. 1996). Flash pasteurization is used for perishable beverages

like milk, fruit and vegetable juices, and beer. It is done prior to pouring the beverages

into containers in order to kill spoilage microorganisms in an effort to make the products

safer and to extend their shelf life. The liquid moves in a controlled, continuous flow

through a heat plate exchanger system subjected to temperatures of 72 74 C for

about 15 to 30 s. It increases the shelf life by 2-3 weeks under refrigerated conditions.

Flash pasteurization destroys all pathogenic microorganisms but does not inactivate

spores. The time/temperature requirement for pasteurization of juice depends on the

initial microbial load, pH of the product and if enzyme inactivation is required.

Citrus juice thermal processing

In addition to microbial inactivation, citrus juice processing requires pectin

esterase (PE) inactivation, as this enzyme leads to cloud loss in stored citrus juices.

Normally, temperatures above 71 C for few seconds are enough to kill pathogens and

spoilage bacteria in orange juice (Kale and Adsule 1995). However, treatment of juice at

90 C for 0.8min to 1min is required for 99% inactivation of PE activity (Wicker 2004).

The food industry has different time and temperature parameters for enzymatic

inactivation and destruction of microbial population in citrus juices by heat (Table 2-1)

(Crupi and Rispoli 2002).

Apple juice thermal processing

Flash pasteurization of apple juice involves heating the juice to 71 C for 6s or

heating cider to an equivalent time /temperature combination (AFDO 2001). However,









the cost of flash pasteurization for small cider producers is unaffordable, and therefore

options like the hot fill technique are advantageous when the whole packaged product is

pasteurized. This reduces the cost of sterilizing the container. Hot filling is conducted by

passing the final product through a heat exchanger and raising the temperature such

that the temperature of the juice filling container reaches a recommended temperature

of 88 to 95 C. The juice is usually held for at least 3 min before cooling. The hot fill

technique is adequate for acidic beverages like apple juice, cranberry juice and grape

juice. Usually a shelf life extension from 9 to 12 months in glass bottles can be achieved

by this method (McLellan and Padilla-Zakour 2004).

Effect of Thermal Pasteurization on Juice Flavor

Conventional juice pasteurization by heat causes significant loss of fresh flavor,

color and nutritional content (Braddock 1999). There are few studies regarding the

effect of thermal processing on the volatile composition of apple juice. A decrease in

ester concentrations due to thermal pasteurization has been found in all these reports

(Kato and others 2003; Su and Wiley 1998; Aguillar-Rosas et al. 2007). Su and others

(1998) reported a decrease in apple juice aroma components iso-butyl acetate, ethyl

butyrate, ethyl 2-methyl butyrate and hexanal after pasteurization at 85 C for 10 min.

Apple juice processed by HTST at 90 C for 30 s resulted in more than 50% loss in

hexanal, ethyl acetate, ethyl butyrate, methyl butyrate and acetic acid concentrations

(Aguillar-Rosas et al. 2007). Kato and others (2003) correlated the change in apple juice

volatiles pasteurized at various time -temperatures (2-320s; 80-120 C) to sensory data

by principal component analysis. They found that the change in volatile concentrations

were very complicated with respect to different thermal processing conditions, as the

odor attributes of processed apple juice were not predictable. Principal component









analysis indicated that treatment temperature affected the volatile concentration more

than treatment time.

Citrus juice flavors are also sensitive to thermal pasteurization. Shaw and others

(2000) found a decrease in volatile constituents in heat pasteurized orange juice during

a 9 week storage study. Tatum and others (1975) reported occurrence of alpha-

terpineol, 4-vinyl guaiacol and Furaneol in thermally treated canned orange juice

stored at elevated temperatures. PVG was found to be the most detrimental to orange

juice flavor, as it gave a rotten flavor to the juice. Furanones like Furaneol are formed

from Maillard reactions between amino acids and sugars present in juice and impart a

caramel, cooked odor quality to juice (Perez-Cacho and Rouseff 2008). The acid

catalyzed degradation of limonene under acidic conditions results in formation of

compounds like ca-terpineol, cineole, carvone and carveol (Marcotte and others 1998).

Alpha-terpienol is considered to be a marker for thermally abused citrus juices.

In contrast to orange juice, there are few reports on the thermally-induced changes

in grapefruit juice aroma (Lin et al. 2002b; Lin and others 2001; Lin et al. 2002a). Shaw

and others (1982) compared the headspace levels of sulfur compounds in fresh and

commercially heat processed grapefruit juice. They reported higher levels of hydrogen

sulfide (tentatively identified) in fresh juice compared to processed juice, whereas

dimethyl sulfide was detected only in processed juice. In a more recent study, GCO

analysis of thermally processed grapefruit juice showed a loss of 6 odor active odorants

and formation of meaty, cooked odorants 2-methyl 3-furnathiol and 2-acetyl 2-thiazoline

(Lin and Rouseff 2001). These compounds negatively affect the flavor of juice. A sulfur

volatile quantitation using sulfur chemiluminescence detector showed that pasteurized









grapefruit juice had higher levels of sulfur compounds compared to untreated juice (Lin

et al. 2002a). Sulfur containing the amino acids methionine, glutathione and cysteine

were proposed as the possible precursors for these sulfur volatile compounds.

A comparison of single strength and grapefruit juice reconstituted from

concentrate without volatile restoration showed more than 90% loss of volatile

components in the reconstituted juice (Lin et al. 2002b). Olfactometric analysis showed

a drastic loss in fresh/citrusy and sulfur/grapefruit characteristic notes. The loss in fresh/

citrusy notes was attributed to a loss of limonene, octanal, and nonanal, whereas the

loss in 1, 10-dihrydronootkatone, 3-mercaptohexyl acetate, 3-mercapto hexanol and 4-

mercapto 4-methyl pentanol contributed to a decrease in sulfur/grapefruit odor. Thermal

instability, as well evaporation during heating, contributed to flavor loss. In addition to

flavor loss, thermal processing also causes degradation of vitamin C. Saguy and others

(1978) found 7-12% loss of Vitamin C content in grapefruit juice pasteurized at 95 oC for

1min.

Non-Thermal Processing Methods

Consumer demand for safer and minimally processed food has resulted in

investigation of non-thermal processing alternatives. The main objective of non-thermal

preservation methods is to minimize degradation of food quality by limiting heat damage

to foods. In addition to preservation of food quality, non-thermal process should also

achieve equivalent microbial safety level as thermal processes.

Juices are graded in United States based on standards such as Brix, Brix/acid

ratio, color and flavor (AMS 1983). Therefore, it is important to study the impact of non-

thermal processing on these quality parameters to determine if they maintain the









Agricultural Marketing Standards (AMS). Citrus juices are consumed for their nutritional

benefits (Vitamin C) as well as for their flavor. However, ascorbic acid is heat and

oxygen sensitive and undergoes degradation during processing and storage. Thus, the

effect of non-thermal processing on ascorbic acid content is an important parameter to

study.

Enzymes present in juice cause deterioration in the quality of juice by modification

of pectin, degradation of ascorbic acid and browning. They change the flavor, color and

texture of juice. In citrus juices, it is equally important to maintain the cloud stability

equally as flavor and color. About 5% of the cloud is pectin, and it has a dominating

effect on the behavior of clouds. Pectin methyl esterase enzyme present in juice

demethylates the pectin molecule, causing aggregation of pectin molecules. These

aggregates settle out of the juice, resulting in juice clarification or cloud loss (Guiavarc'h

and others 2005). Thermal pasteurization is the most common method used for PME

inactivation. The efficiency of non-thermal processing to inactivate PME is a crucial

factor in terms of commercial viability.

Some of the non-thermal techniques extensively studied for juice pasteurization

are high pressure (HPP), dense phase CO2, pulse electric fields (PEF), oscillating

magnetic fields, pulsed high intensity light and ultraviolet irradiation (UV) (Elez-Martinez

and others 2006; Boff and others 2003; Sampedro and others 2009; Donahue et al.

2004; Koutchma 2008). Many reports have been published in the last decade studying

the efficacy of non-thermal techniques to extend shelf life and enhance the safety of

fresh juice while minimizing changes to organoleptic and nutritional qualities of the juice

(Grahl and MarkI 1996; Tandon et al. 2003).









Regulatory approval is important for the commercialization of non-thermal

technologies. Some of the FDA approved technologies for juice processing are PEF, UV

and HPP. However, it has to be kept in mind that no single technology can be practically

applied to all products and, within technologies, pathogen reduction varies among

products treated.

Pulsed electric field

PEF processing involves treating liquid foods with high voltage pulses in the range

of 20- 80 kV for microseconds. A high electric field is generated between two electrodes

(treatment chamber), which results in a large electrical flux flow through the product. A

schematic diagram of a typical bench top PEF unit is given in Figure 2-2 (Jia and others

1999). The apparatus consists of a high voltage power supply, a pulse generator,

treatment chambers and a water bath for temperature control. PEF process achieves

both microbial and enzymatic inactivation while minimizing heating of foods and

associated detrimental changes to sensory and physical properties of foods. Though

there are several theories on microbial inactivation, the most accepted is by

electroporation. The high voltage pulses break cell membranes of vegetative

microorganisms, thus altering the membrane permeability. This results in protein

inactivation and leakage of cellular contents and eventually disruption of microbial cells

(Sale and Hamilton 1967). The factors that affect the efficiency of microbial inactivation

by PEF are product composition, microbial characteristics and treatment parameters.

Product Parameters. Only pumpable food products can be treated by PEF. The

critical parameters pertaining to food products are electrical conductivity, density,

viscosity, pH and water activity (Cserhalmi et al. 2006). The food products should have

low electrical conductivity, as conductivity increases the difference in conductivity









between medium and microbial cytoplasm resulting in a weakening of membrane

structure (VegaMercado and others 1996). An enhanced efficiency in microbial

inactivation in acidic environment was also reported by the same authors.

Microbial Characteristics. Generally, gram positive bacteria are more resistant to

PEF than gram negative bacteria (Hulsheger and others 1983). Yeasts and mold show

higher sensitivity to PEF than bacteria due most likely to their larger cell size (Qin and

others 1996). Wouters and others (2001) showed that Lactobacillus species in different

sizes or shapes had different membrane permeability to PEF. In addition to the type of

microorganism, the growth stage also changes the sensitivity to PEF. Bacteria and

yeasts at their logarithmic stage are more sensitive than those at the stationary or lag

growth stage (Pothakamury and others 1996). PEF processing is more efficient at

vegetative cell inactivation compared to spores. Raso and others (1998) compared the

effect of PEF treatment (32 -36.5 kV/cm) on vegetative cells and Zygosaccharomyces

baili ascospores. PEF treatment decreased the vegetative cell population by 4.5-5 log

cycles and ascoproes by 3.5-4 log cycles.

Treatment Parameters. The critical process factors for PEF treatment are electric

field intensity, treatment time, pulse wave shape, pulse length, number of pulses and

temperature. Microbial inactivation efficiency increases with an increase in electric field

strength above the critical electric field (Qin and others 1998). The critical electric field

is the intensity below which no microbial inactivation occurs. Hulseger and others

(1983) found a linear relationship between electric field strength and E. coli inactivation.

Treatment time is another important parameter that affects the efficiency of PEF

treatment. It is the product of number of pulses and pulse duration. Therefore, an









increase in any of the above two parameters results in increased microbial inactivation.

However, longer pulse width also increases food temperature. Peleg and others (1995)

showed that the rate of microbial inactivation by PEF at a constant electric field strength

increased with an increase in treatment time.

The electric field pulse can be applied in the form of exponential decaying, square

wave and oscillatory pulses. Zhang and others (1994) compared the effect of square

wave, exponentially decaying and charge reverse pulses on the shelf life of orange

juice. Square wave pulses yielded juice with longest shelf life. PEF treatment of juice

can lead to the formation of a shielding layer on electrodes in treatment chambers due

to migration of charged molecules to surface of electrode. This layer reduces the

efficiency of PEF treatment. Bipolar pulses prevent the formation of shielding layer, as

the polarity reversal causes a corresponding change in direction of charged molecules

(Evrendilek and Zhang 2005). Therefore, bipolar pulses are used with square wave or

exponential pulses to increase efficiency of PEF processing.

Moderate temperatures (40-60 C) exhibit synergistic effects with PEF on the

inactivation of organism. At moderate temperatures the membrane fluidity of cell is

altered, increasing sensitivity to PEF treatment. Heinz and others (Heinz and others

2003) found that E. coli inactivation increased from 1 to 6.5 log cycles with a

temperature increase from 32 C to 55 C in inoculated apple juice. However, since

application of electric field strength also increases the temperature of product, a proper

post treatment cooling is necessary.

Microbial stability studies in PEF treated juices. Microbial stability in PEF

treated juices have been studied in various fruit juices like apple juice, apple cider,









orange juice, cranberry juice, tomato juice, and mango juice, as well as in orange milk

beverages (Ayhan and others 2001; Charles-Rodriguez and others 2007; Jin and Zhang

1999; Yeom and others 2004; Min and Zhang 2003). There are no reports to date on

microbial stability in PEF treated grapefruit juice.

The commercial-scale processing of orange juice by PEF treatment at 40 kV/cm

for 97 [s reduced the total aerobic count and yeast and mold count by 6 log cycles (Min

and others 2003a). Commercially processed PEF juice had a shelf life of 196 days at 4

oC. Aseptically packaged PEF treated orange juice (29.5 kV/cm, 60 ps) had a shelf life

of 7 months at 4 oC (Qiu and others 1998).

In apple juice, PEF treatment (34 kV/cm, 166 [ts) using a bench scale system

resulted in a 4.5 log reduction in inoculated E. coil 0157:H7 levels (Evrendilek and

others 2000b). The same juice treated using a pilot scale unit at 35 kV/cm for 94 [s and

combination of heat treatment at 60 oC for 30 s gave a shelf life of more than 67 days at

both 4 oC and 22 oC.

Lately, the synergistic effect of PEF with antimicrobials to achieve improved

microbial safety has been a subject of interest. Liang and others (2002) reported a 5.9

log reduction in inoculated Salmonella levels in fresh orange juice at 90 kV/cm, 50

pulses and temperature at 55 oC. Addition of antimicrobials nisin and lysozyme

increased the inactivation by 1.37 logs. Interestingly, they also reported that PEF

inactivation was significantly more extensive in pasteurized juices than fresh juice,

probably due to differences in their composition. This is an important parameter to look

into when comparing the effect of varying PEF parameters on microbial inactivation in

juices. Similar studies of apple juice also showed an increase in microbial inactivation









by 1-2 log cycles using electric field strength in range 27-33 kV/cm in combination with

nisin and lysozyme (Liang and others 2006).

Though numerous studies on microbial stability and shelf life of products are

present in the literature, there are fewer studies on the effect of PEF technology on

different microorganisms. McDonald and others (2000) achieved more than 5 log

reductions of Leuconostoc mesenteroides, E. coli and Listeria innocua in orange juice at

30 kV/cm and 50 kV/cm electric field strength using varying pulses. Inactivation of

spores was more difficult, as a maximum of 2.5 log reductions of S. cerevisiae

ascospores was achievable at 50 kV/cm, 50 oC.

Garcia and others (2005) found that E. coli populations exhibited greater sensitivity

to subsequent holding of apple juice at refrigerated conditions than immediately after

PEF treatment (40kV/cm, 80 pulses). An increase in inactivation from 0.5 log to 5 log

was noted after 3 days of storage under refrigeration. This phenomenon was attributed

to increased sensitivity of injured cells to the acidic conditions of apple juice.

In summary, due to the numerous critical process factors and different

experimental conditions used, definite conclusions about the critical process parameter

effect on specific pathogen reductions are difficult to establish. Research that provides

conclusive data on the effect of critical PEF factors on microbial inactivation is required

for commercialization of the PEF process.

Effect of PEF processing on juice quality. The effect of PEF treatment on

aroma, nutrition, color, sensory and other physiochemical parameters of orange, apple,

cranberry and tomato juices are present in literature (Jin and Zhang 1999; Biasioli and

others 2003; Aguillar-Rosas et al. 2007; Ayhan and others 2002).









In apple juice, PEF extended the shelf life of fresh juice for up to 56 days with no

change in sensory or physiochemical properties compared to freshly squeezed juice

(Qin and others 1995). Evrendilek and others (2000b) found no significant change in

sensory acceptance of PEF treated (35kV/cm for 94 ps) apple juice compared to fresh

juice by paired preference test. Aguillar-Rosas and others (2007) reported a significantly

higher loss in eight select volatiles (acetic acid, hexanal, butyl hexanoate, ethyl acetate,

ethyl butyrate, methyl butyrate, hexyl acetate, and 1-hexanal) in HTST treated apple

juice (90 C, 30 s) compared to PEF treated juice (35kV/cm, 4ps bipolar pulse).

The effect of PEF treatment on orange juice has been more extensively studied

compared to other beverages. Jia and others (1999) noted that the loss of volatile

compounds in PEF (30kV/cm for 240 ps or 480 ps) and heat processed (90 C for 1 min)

orange juice were greatly influenced by compound type and processing methods. Loss

in volatiles a-pinene, limonene and ethyl butyrate were common in both treatments.

However, decanal and octanal were lost only in heat treated samples. The flavor loss by

PEF process was mainly due to the vacuum degassing system rather than PEF

application.

Yeom and others (2000b) investigated the effects of PEF treatment on

microorganisms and pectin methyl esterase activity. They compared vitamin C, volatile

and other attributes of PEF treated juice (35kV/cm, 59ps) to heat pasteurized (94.60C

for 30s) orange juice. PEF processing prevented growth of microorganisms at 4, 22 and

370C for 112 days and inactivated 88% of PME activity. PEF treated juice also retained

greater amounts of vitamin C and aroma compounds (pinene, myrcene, octanal,

limonene and decanal) compared to heat-treated samples.









In contrast, Ayhan and others (2002) reported a significant increase in

hydrocarbons d-limonene, alpha-pinene, myrcene and valencene for PEF treated single

strength orange juice (35kV/cm for 50 ps) compared to untreated juice. They proposed

that the hydrophobic flavor compounds present inside pulp of orange juice become

more available during PEF application.

Because most of the previous PEF studies were performed in laboratories or pilot

plant scales, Min and others (2003a) studied the effect of commercial scale PEF

processing (40kV/cm for 97 ps ) on microbial stability, ascorbic acid content, flavor

compounds and other sensory attributes compared to thermally processed (90 C for 90

s) and freshly squeezed orange juice. PEF and thermally processed juice had similar

microbial shelf lives at 4 C for 196 days. PEF processed juice retained more ascorbic

acid, flavor and color compared to thermally treated juice. Preference sensory analysis

indicated significantly higher sensory attributes of texture, flavor and overall

acceptability for PEF treated juice compared to thermally processed juice.

Only one reference in the literature on PEF processed grapefruit juice could be

found (Cserhalmi et al. 2006). The authors investigated the effect of PEF technology on

physical properties, color, non-enzymatic browning and flavor components. They

reported no significant changes in any of the quality parameters post processing

compared to fresh, untreated juice (Cserhalmi et al. 2006). The aroma analysis in the

study was limited to eight select volatiles (terpinen-4-ol, ethyl octanoate, decanal,

carvone, geraniol, geranyl acetate, geranyl acetone, and nootkatone) in grapefruit juice.

Inactivation of enzymes by PEF. Enzyme conformational change is suggested

as the possible mechanism of enzyme inactivation by PEF (Castro AJ 2001). The









authors found that alkaline phospatase molecules tend to associate and aggregate due

to polarization created by electric charges.

Yeom and others (2002) reported an 83.2 % inactivation of PME in fresh orange

juice at 35 kV/cm for 180 ms at 30 C. However, a 90% PME inactivation was achieved

at 25 kV/cm when the temperature was increased to 50 C, indicating a synergistic

effect of heat and PEF treatment on PME inactivation. In apple juice, PEF reduced the

PPO activity by 50-70% at treatment condition 38.5kV/cm, 300 pps and 50 C

(Sanchez-Vega and others 2009). Min and others (2003b) reported 88.1% inactivation

in lipoxygenase in tomato juice treated by PEF at 30 kV/cm for 50 [ts at 50 oC.

Radio frequency electric field

Radio frequency electric fields (RFEF) processing is a relatively new

pasteurization technique and is not FDA approved yet. The key equipment components

of the process include a radio frequency power supply and a treatment chamber that is

capable of applying high electric fields to liquid foods (Figure 2-3) (Geveke and others

2007). The process is similar to the pulsed electric fields process, except that the power

supply is continuous using an AC generator rather than pulses, thus reducing the capital

cost.

Ukuku and others (2008) worked on E. coli inoculated apple juice to understand

the mechanism of RFEF on microbial inactivation. They demonstrated a 4 log reduction

in inoculated apple juice at 15kV/ cm entirely due to the non-thermal effect of RFEF and

not due to temperature increase during treatment. Membrane damage in bacterial cell

wall leading to leakage of intracellular material was cited as a mechanism of microbial

inactivation.









The process parameters affecting RFEF efficiency are electric field strength,

frequency, treatment time, output temperature and number of treatment cycles. Radio

frequencies of 15 and 20 kHz were more effective in inactivation of E. coil than 30-70

kHz (Geveke and Brunkhorst 2004). Increasing electric field strength (20 30 kV/cm),

treatment time (140 420 ps) and outlet temperature (50 60 C) increased

inactivation of E. coli in apple cider (Geveke and others 2008). An increase in treatment

cycles also increased inactivation of inoculated E. coil K12 at conditions 18kV/cm, 170

ps and 50 C outlet temperature. No studies on the effect of RFEF on physiochemical

properties, flavor or color of apple juice are reported in the literature.

In orange juice, an electric field strength of 20 kV/cm and outlet temperature of 65

C gave a 3.9 log reduction in E. coli K12 population with no significant non-enzymatic

browning and loss in ascorbic acid content (Geveke et al. 2007). Because RFEF is a

relatively new pasteurization technique, not much information is available on its effect of

the quality of juices.

Ultraviolet radiation

UV light in the wavelength 220 -300nm exhibits germicidal properties and is used

for inactivation of bacteria and viruses. UV light absorbed by the DNA of microorganism

causes cross-linking between neighboring pyrimidine nucleoside bases thymidinee and

cytosine) in the same DNA strand and prevents cell replication. UV light has low

penetration in juices and other beverages due to the presence of color compounds,

organic solutes and suspended particles. Therefore, UV irradiation is limited to microbial

inactivation of food surfaces, packaging material or processing plants. In recent years,

due to many developments in design of treatment chambers by utilization of turbulent

flow to form continuous renewed surface, microbial inactivation in juices and other









beverages is enabled. In 2000, the FDA approved UV irradiation as an alternative to

thermal treatment for fresh juices. UV light is now commercially used for water

disinfection and apple cider pasteurization. A picture of UV processing unit used by

Geveke (2008) for pasteurization of liquid egg whites is given in Figure 2-4. The unit

consists of a bi-pin base, a 30 W germicidal UV bulb, UV transparent tubing and silicon

rubber tape. The lamp generated 90% of its energy at 254nm. The product flows

through tubing, and the time of exposure to UV light is calculated based on the flow

rate. The effectiveness of microbial inactivation by UV process depends on the UV light

source, the product composition and microbial characteristics.

UV light sources. The different light sources used for UV are mercury lamps,

excimer lamps, microwave lamps and broadband pulsed lamps (Koutchma 2009). The

correct UV source enhances microbial inactivation by increasing UV penetration in

liquid, as well as by employing higher UV intensity from pulsed sources. Mercury lamps

are generally used due to their low cost and effective microbial inactivation. Based on

the vapor pressure of mercury when lamps are operating, they are categorized as: low

pressure lamps, low pressure high-output lamps and medium pressure lamps. Low

pressure mercury lamps are generally used in food processing and are approved by the

FDA. In pulsed lamps, alternating current is stored in a capacitor and energy is

discharged in the form of intense emission of light within microseconds. This technology

has been applied to food surface treatment in fresh produce and meats, but it has not

yet been established in the field of liquid foods.

Product composition. The physical, chemical and optical properties of juices

impact the effectiveness of microbial inactivation by UV. The variation in UV absorption









between different juices is due to the difference in their pH, viscosity, soluble solid and

suspended solid content. Clear juices are better candidates for UV treatment compared

to opaque juices, as the presence of dissolved solutes in juice causes strong UV

attenuation effects (Koutchma and others 2007). Carrot and pineapple juices (opaque

juices) had higher UV absorptivity than semi-transparent watermelon and apple juice.

Ascorbic acid present in juices also has strong absorbance between 220 -300 nm even

at very low concentrations. Tran and others (2004a) found a 17% loss in vitamin C

content in UV treated orange juice. It is important to study the ascorbic acid content and

its destruction during UV treatment in terms of UV dose delivery. Suspended solids

present in juice not only attenuate UV dose but also provide a site for aggregation of

bacteria to particle surfaces. Suspended solids also cause scattering in light such that

microbes far away from the UV light are not effectively inactivated (Murakami and

others 2006).

Microbial characteristics. The information on the effect of UV on different

pathogens is very limited. Factors like microbial species, strain, growth media, and initial

microbial load, as well as stage of growth, affect the efficiency of microbial inactivation

by UV. Generally, gram positive bacteria are more resistant to UV than gram negative

bacteria due to difference in cell wall thickness and composition. Bacterial spores, like

Bacillus subtilis, are very resistant to UV requiring 36mJ/cm 2 for 1-log reduction in

water (Chang and others 1985). Mold spores are considered to be more UV resistant

than yeast and lactic bacteria. They are the major spoilage microorganisms in juices.

Microbial inactivation studies in UV treated juices. UV light inactivates

microorganisms by causing cross-linking between neighboring pyrimidine nucleoside in









the same DNA strand, thereby preventing cell replication. The extent of cross-linking is

directly proportional to the UV light exposure. However, photo reactivation can occur in

UV injured cells when exposed to visible light in blue spectra (Hoyer 1988). Therefore, it

is important to store UV treated juice under refrigeration and in dark conditions.

UV dose is calculated as a product of exposure time and intensity. It is assumed

that change in either time or intensity should not affect microbial inactivation as long the

UV dose used is same. However, recent studies have shown that some E. coil strains

do not follow time-intensity reciprocity (Sommer and others 1998). Time -intensity

reciprocity is crucial for the scale up from pilot to commercial scale. Scale up is possible

only if time-intensity reciprocity exists. Murakami and others (2006) showed that in

apple juice inoculated with E. coil K12, the reciprocity occurs within a range of

intensities only and the range decreases as UV dose level increases.

UV processing of apple juice and apple cider has previously been conducted to

study the inactivation kinetics of microorganisms like E. coli 0157:H7 and Listeria

innocua (Duffy and others 2000; Geveke 2005). Duffy and others (2000) showed a 5 log

reduction in E. coli in apple cider treated by UV irradiation using a CiderSure pasteurizer

(OESCO, Inc., Conway, MA). This unit has 8 UV lamps, and the UV light is set to

penetrate a 0.03" layer of cider. Geveke (2005) exposed apple cider inoculated with E.

coli and Listeria innocua to a UV lamp for varying lengths of time. They achieved a 3.4

log reduction in E. coli population at 19 s of UV exposure at 25 oC. L. innocua required a

longer exposure time of 58 s for 2.5 log reduction. In a different study by the same

authors on liquid egg white (Geveke 2008), the effect of exposure time, temperature

and pH on E. coli inactivation was investigated. They found an increase in inactivation









with an increase in exposure time (0 160 s) and that an exposure time of 160 s at 50

C reduced the microbial population by 4.3 log. They mentioned a direct dependency

between inactivation and temperature but an indirect relationship between pH and

inactivation. UV inactivation was greater at neutral pH than higher pH values.

In grapefruit juice inoculated with Saccharomyces cerevisiae, a UV dose of 450

kJ/m2 at flow rate of 1.02L/min using a two coupled UV disinfection system resulted in a

maximum of 2.4 log reduction (Guerrero-Beltran and others 2009). Keyser and others

(2008) treated apple juice, guava-pineapple juice, mango nectar, strawberry nectar, two

different orange juices and tropical juice using a PureUV pilot scale system. Reduction

in inoculated E. coli levels in apple juice from 7.92 log cfu/mL to 7.42 lof cfu/mL at a UV

dose of 1377J/L was observed. However, in the remaining juices no inoculation studies

were done by the authors. It was not possible to interpret the effective UV dose required

to achieve pasteurization in juice as the initial microbial load varied between the juices.

The authors suggested that UV treatment optimization is required for each new juice

product.

Effect of UV treatment on juice quality. Donahue and others (2004) showed a 5

log reduction in E. coli when exposed to UV light for 8.12 s and energy of 35.1 mJ/cm2.

UV-treated cider had a shelf life 7 days longer than untreated apple cider with no

significant sensory difference. Choi and others (2005) found that thermal pasteurized

apple cider had better microbial quality compared to UV treated cider after 21 days of

storage. However, UV treated cider preserved better color. Consumer acceptability

studies showed no significant difference between UV and untreated cider, whereas

thermally treated cider had the lowest acceptability scores.









Tandon and others (2003) compared UV treated apple cider at dose 14 mJ/cm2 to

hot fill pasteurized cider at 63 C. UV treated cider had a shelf life of 2 weeks compared

to 4 weeks for hot fill cider. UV treated cider was less acceptable than hot fill cider by

sensory preference tests due to fermentation after 2 weeks of storage.

Studies on UV treated orange juice are limited due to the low transmittance of UV

light through the high pulp juice (Tran and Farid 2004b). UV exposure of 73.8m J/cm2

extended the shelf life of orange juice by 5 days with a 17% loss of ascorbic acid.

Vitamin C is a light-sensitive component and is degraded by UV light. No PME

inactivation was noted by the authors in UV processed juice.

















SH


0



(4) -
(4)


0




(5)


SH 0



(3)


0




(6)


Figure 2-1. Key odorants of grapefruit and apple juice


Table 2-1. Time


Temperature
Time


- temperature parameters for PE inactivation
Orange, mandarin Grapefruit juice
and tangerine
juice
90-98 C 85-90 C
60 s 30-40 s


in citrus juices
Lemon juice


75-85 C
30 s


o

A



































Figure 2-2. Schematic diagram of pulsed electric field processing unit (Jia et al. 1999)


Treatment Chamber Enclosure


2 Cables Each ConsisTing of 3 #14 Ga.,
25 kV Silicone Insulation


Figure 2-3. Schematic diagram of radio frequency electric field processing unit (Geveke
et al. 2007)



























Figure 2-4. Picture of Ultraviolet processing unit (Geveke 2008)









CHAPTER 3
COMPARISON OF THERMAL AND NON-THERMAL TECHNIQUES ON APPLE
CIDER STORAGE QUALITY UNDER EQUIVALENT PROCESS CONDITIONS

Introduction

Non-thermal processing techniques like pulse electric field (PEF) and ultraviolet

(UV) treatment have been explored for their efficacy to extend shelf life and enhance

the safety of fresh juice while preserving organoleptic and nutritional qualities

(Evrendilek and others 2001; Min and Zhang 2003). Studies on the inactivation of

microorganisms by PEF processing of orange, apple and tomato juices have shown

better preservation of flavor, color and nutrients in comparison to heat pasteurized

juices (Min et al. 2003b; Evrendilek and others 2000a; Ayhan et al. 2001). UV

processing of apple juice and apple cider has previously been conducted to study the

inactivation kinetics of microorganisms like E. coil 0157:H7 and S. typhimurium (Duffy

et al. 2000; Uljas and others 2001). Even though there are many non-thermal

processing techniques available for the pasteurization of juices, a lot more research is

required to make them commercially successful. Particularly, studies about the effects

of non-thermal techniques on quality and consumer acceptability in comparison to

thermal pasteurization are required. Most often, the conditions used for thermal

processing are severe in terms of temperature and time of exposure to heat. Thus,

shelf-life studies comparing non-thermal to thermal techniques on acceptability of juice

using conditions that do not achieve the same reduction of a target microorganism

would result in incorrect conclusions (Rivas et al. 2006; Aguillar-Rosas et al. 2007;

Yeom et al. 2000b). For proper comparison of thermal and non-thermal treatment

effects on product quality, both processes must achieve similar levels of microbial

inactivation. The current work focuses on comparing the effect of thermal and non-









thermal processing techniques on microbial population and overall quality of apple cider

to that of untreated cider. Each treatment is optimized to achieve approximately 6-log

reduction of added E. coil K12, a surrogate of E. coil 0157:H7.

Materials and Methods

Determination of Processing Conditions That Achieve 6-log Reduction of E. coli
K12

Pasteurized apple cider (Ziegler Juice Co., Lansdale, PA) inoculated with E. coli

K12 (ATCC 23716) was used to standardize processing parameters to achieve a 6 log

reduction. E. coli K12 obtained from the American Type Culture Collection (ATCC)

(Manassas, VA) was maintained on Tryptic Soy Agar (Remel, Lenexa, KS, USA) at 4

oC. It was cultured in Tryptic Soy Broth (Remel) with shaking at 37 oC for 16-18 h. Apple

cider inoculated from the stationary phase culture gave an approximately 7 log CFU/mL

population.

Ultraviolet process

Pasteurization was performed using a simple apparatus consisting of four low-

pressure mercury lamps, each surrounded by a coil of UV transparent tubing. The UV

apparatus was scaled up from a smaller-sized apparatus developed by Geveke (2005).

The UV lamp assemblies contained a bi-pin base (model S130 120 LPF, Lithonia

Lighting, Conyers, GA) and a 30-W bulb (G30T8, Buylighting.com, Burnsville, MN) that

generated 90% of its energy at a wavelength of 254 nm. Norton Chemfluor 367 tubing

(Cole-Parmer, Vernon Hills, IL) with an ID of 3.2 mm and a wall thickness of 1.6 mm

was wrapped around the entire length of each UV lamp. The four UV lamps were

connected in a series with 14m of tubing wrapped around them. The experimental

system included a feed tank, a peristaltic pump and four UV lamps of the same









dimensions connected in a series. Cider was pumped through the tubing at a flow rate

of 25 L/hr, which translated to an exposure time of 17 s per bulb. Microbial assays were

conducted using 1, 2, 3 and 4 UV lamps corresponding to UV treatment times of 17, 34,

51 and 68 s.

Pulsed electric field process

A bench scale continuous PEF system (OSU-4F, Ohio State University,

Columbus, OH, USA) was used to treat the inoculated apple cider. The system

consisted of 6 co-field treatment chambers with a diameter of 0.23 cm and a gap

distance of 0.29 cm between electrodes connected in a series. Applied voltage and

current were monitored by a digital real time oscilloscope (Tektronix DS210, Beaverton,

OR). The inlet and outlet cider temperatures were continuously monitored via

thermocouples. Cider was pumped through the system using a digital gear pump (Cole

Parmer 75211-30, Vernon Hills, IL) at a flow rate of 7.2 L/hr. The electric field strengths

tested were 5, 10, 13, 17, 21 and 23 kV/cm at a square wave pulse duration of 2.5 ps.

Apple cider sequentially flowed through all the treatment chambers via steel coils

immersed in a water bath set at 48 oC. The mean total treatment time (t) was calculated

as 150 ps using the following equation 1:

t= n*T* f*d/v (3-1)

Where n is the number of treatment chambers (n = 6), T is pulse width (T = 2.5

ps), f is the repetition frequency (f = 1670 Hz), d is the distance between two electrodes

(d = 0.29 cm), v is the velocity of flow inside treatment chamber (v = cm/s), which is

determined by flow rate and the diameter of the treatment chamber (0.23 cm).









Thermal process

Inoculated apple cider was heat pasteurized using a miniature-scale Armfield

HTST processing system (model FT74-30-Mklll-33-34, Jackson, NJ, USA). The system

included a feed tank, a peristaltic pump, a plate heat exchanger (comprising a

regeneration section, a heating section and a cooling section that mimics industrial

scale systems), a holding tube, thermocouples, and an electric-powered hot water boiler

and pump. A flow rate of 15 L/hr was maintained through the system, which translated

to a hold time of 1.3 s. Microbial inactivation was studied at holding tube outlet

temperatures of 60, 63, 66, 69, 72, 74 and 76 oC.

Shelf-Life of Processed Apple Cider

Unpasteurized apple cider procured from Ziegler Juice Company (Lansdale,

Pennsylvania) was frozen at -17 C in gallon plastic containers. Samples were thawed

at 4 C overnight prior to use.

Processing and packaging

All processing equipment was sanitized by pumping 5% bleach solution through

the system, followed by a distilled water rinse. Heat pasteurization was performed at 76

C for 1.3 s. For UV treatment, apple cider was exposed for 51 s at an outlet

temperature of 15 C. PEF processing was conducted at an electric field strength of 23

kV/cm, 2.5 ps pulse duration, a total treatment time of 150 ps and a treatment

temperature of 48 C. The treatment conditions were selected based on 6 log reduction

of inoculated E. coli K12 as determined in the above experiments. As a control sample,

apple cider was passed through the HTST processing system without any heat at room

temperature.









Cider was packaged in clean 1 L media bottles inside a sanitary laminar hood after

processing. The laminar hood contained a UV lamp and HEPA air filter. The hood was

sanitized by a UV lamp at 254 nm for 30 min before use and then wiped with alcohol.

Storage

Bottled samples stored at 4 oC for four weeks were periodically analyzed for

quality, including microbiology, pH, Brix and color attributes. Microbial analyses were

performed every week, while all other analyses were done at 0, 2 and 4 weeks. Control

apple cider consisted of untreated cider stored at 4 oC, while fresh apple cider was

untreated cider stored at -17 oC.

Microbial tests

Microbial inactivation and growth were examined by preparing appropriate

dilutions of samples with Butterfield's Phosphate Buffer (Hardy Diagnostics, Santa

Maria, CA). Duplicate samples (1 mL) were then pour plated with Tryptic Soy Agar

(Remel, Lenexa, KS, USA) and incubated at 37 oC for 24 h. Plates with 30-300 colonies

were enumerated using a Bantex model 920 manual colony counter (Burlingame, CA,

USA). The E. coil K12 population was expressed as CFU/mL of apple cider. Data for

each replicate were normalized against the control and plotted as the log reduction

versus temperature, time or electric field strength.

For the shelf-life study, total aerobic plate count (TPC) and yeast and mold count

(YMC) were determined using plate count agar (PCA) and yeast and mold (YM)

pertrifilms. The PCA plates were incubated at 37 oC for 24 hrs while YM plates were

incubated at room temperature for 5 days before counting. All samples were analyzed in

duplicate, and two replicates of each dilution were prepared and plated.











The pH meter used for all analysis was an Orion 420A+ (Thermo Electron Corp.,

Beverly, MA). The pH meter was calibrated at pH 4.0 and pH 7.0 using standard

solutions on each day of analysis. The pH measurements for all samples were

performed in triplicate.

Brix

A Bausch & Lomb Abbe refractometer (B&L Corp., Rochester, NY) was used. To

measure oBrix of juice samples, a drop of the sample was placed on the refractometer

and the corresponding refractive index was recorded, which gave the measure of

soluble solids in juice. All measurements of oBrix for each cider sample were performed

in triplicate.

Color

Color was measured in CIE L* a* b* 3-dimesional color space using a ColorQuest

XE spectrophotometer (Hunter Associates Lab, Reston, VA) where L designated

lightness and measured the relative lightness and darkness of juice with L = 0

corresponding to black and L = 100 corresponding to white. The "a" value gave the

measure of green to red with positive values indicating more red and negative values

indicating more green. The "b" value measured blue to yellow with positive value

signaling more toward yellow and negative value signaling more toward blue. A dual

beam xenon lamp was used as light source. The lamp was allowed to warm up for 30

min prior to measurement. The system was calibrated using black and white standard

tiles on each day of analysis. Forty mL of cider sample in a 20 mm path length glass cell

were used for measuring color. All measurements of color for each sample were

performed in triplicate. The effect of processing methods on the color of apple cider was









represented using total color difference (dE) calculated for all the samples using the

following equation (3-2) (Lee and Coates 1999):

dE = (L- Lo)2 +(a-ao)2 + (b-bo)2 (3-2)

where L = lightness of treated sample at time t; LO = lightness of untreated sample

at day 0; a = redness of treated sample at time t; aO = redness of untreated sample at

day 0; b = yellowness of treated sample at time t; and bO = yellowness of untreated

sample at day 0.

Statistical Analysis

All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc.,

Raleigh, N.C., USA). Statistical significance of the differences among the treatments

was tested using the analysis of variance (ANOVA) at the significance level of a = 0.05.

The effect of storage and processing techniques was compared using the Duncan's

multiple comparison test (a = 0.05). The interactive effect of storage and treatments was

also determined statistically.

Results and Discussion

Optimization of Equivalent Processing Conditions

Pasteurized apple cider was inoculated with E. coli K12 to give an approximately 7

log CFU/ml population. The initial microbial count in pasteurized apple cider was below

20 CFU/mL, indicating very low microbial load before inoculation. Inactivation of

inoculated E. coil K12 in apple cider increased with increasing exposure time,

temperature and electric field strength for UV, PEF and thermal process, respectively

(Figures 3-1, 3-2, 3-3).









The population of E. coli K12 decreased with increasing UV treatment time in

apple cider (Figure 3-1). UV exposure for 34 s reduced the population by 3.7 logs, and

lengthening the exposure time to 51 s reduced the population by 6.3 logs. Using the

same UV apparatus, Geveke (2005) was able to achieve 4.7 log reduction of the same

bacterium in apple cider for a treatment time of 30 s and temperature at 25 C. The inlet

and outlet temperatures recorded in this study were below 15 C during processing

since refrigerated apple cider was inoculated and passed through the UV processing

unit. Thus, no detrimental effect of heat on the inactivation of E. coli K12 could be

expected in the UV processed cider. The energy applied was calculated to be 13 J/mL

for 51 s of exposure time based on the flow rate and UV lamp wattage. UV

pasteurization energy is less compared to conventional thermal pasteurization energy

of 34 J/mL (Kozempel and others 1998).

The effect of PEF processing on E. coil K12 inactivation at electric field strength

from 5 to 27 kV/cm with a pulsed duration of 2.5 ps and a mean total treatment time of

150 ps is shown in Figure 3-2. Many authors have reported significant synergistic effect

against microorganisms when PEF is used in conjunction with moderate heat (Heinz et

al. 2003). PEF processing was performed with minimal heat of 48 C. The outlet

temperatures recorded were between 49-51 C during the length of treatment. The

increase in temperature is a consequence of the electric energy supplied to the medium

during PEF treatment. The incubation of inoculated cider at 48 C in the absence of PEF

treatment did not achieve any reduction in microbial population. Inactivation of the E.

coli K12 population increased with an increase in electric field strength from 13 to 23

kV/cm. An electric field strength of 23 kV/cm reduced the population in apple cider by









6.1 log cycles. Evrendilek and others (2000b) achieved a 4.5 log reduction in E. coli

0157:H7 population in apple juice using a similar bench scale PEF system with

treatment conditions of 34 kV/cm electric field strength, 4 ps bipolar pulse duration, a

total treatment time of 166 ps and temperature below 38 C The increased reduction in

bacterial population at a lower electric field strength in the current study is evidence of

the synergistic effect of PEF with heat on microbial inactivation.

Thermal processing of inoculated cider performed at a flow rate of 15 L/hr gave a

hold time of 1.3 s. The inlet and outlet product temperatures were recorded continuously

and did not increase beyond 15 C due to built-in cooling section in the equipment. A

significant decrease in E. coli K12 population was noted with increase in temperature

beyond 63 C (P < 0.05). Heat treatment at 76 C for 1.3 s is recommended by the FDA

for pasteurization of apple juice of pH 4.0 or less (CFSAN 2004). Treatment of apple

cider by heat at the same conditions resulted in 6.0 log reduction of inoculated E. coil

K12 population in this study (Figure 3-3).

Effect of Thermal and Non-Thermal Processing on Microbial Stability During
Storage of Apple Cider

Fresh unpasteurized apple cider was treated by heat, PEF and UV at conditions

achieving approximately 6 log reduction in E. coil K12 population. The microbial stability

of treated apple cider was studied for a storage period of 4 weeks at 4 C. The TPC and

YMC of apple cider stored at 4 C for 4 weeks in both treated and untreated cider are

given in Figures 3-4 & 3-5, respectively.

The initial TPC for untreated control apple cider was 2.4 log CFU/mL and reached

3.1 log CFU/mL at the end of the storage period. The TPC for thermal, UV and PEF

treated cider was reduced to 1.8, 1.6 and 1.8 log CFU/mL, respectively, at week 0.









Thermal and UV processed cider did not show any significant increase (P < 0.05) in log

values during storage. However, TPC of PEF-processed cider increased to 2.4 log

CFU/mL by the end of the 4 week storage period. The increase in microbial population

during storage is possibly due to a lower inactivation of spores by PEF and their

subsequent growth during storage. Grahl and others (1996) have reported PEF to be

ineffective for inactivation of microbial spores.

Apple juice/cider is highly susceptible to yeast spoilage due to low pH and high

sugar content (Deak and Beuchat 1993). Examination on petrifilm showed yeast as the

major microorganism in apple cider. The initial YMC in untreated control cider was

around 2.5 log CFU/mL, which steadily increased to 4.7 log CFU/mL by week 2 of

storage (Figure 3-5). No significant increase (P < 0.05) in YMC was noticed by week 2

and 3, indicating a stationary phase in the growth cycle of microorganisms. However, a

decline in YMC to 3.7 log CFU/mL by week 4 in the control cider was observed,

suggesting that the microorganisms were in the death phase of their cycle. Analysis of

UV treated cider showed an increase in YMC to 4.0 log CFU/mL after 2 weeks of

storage at 4 C. The YMC decreased to 3.5 log by the end of 4 weeks of storage similar

to untreated cider. Donahue and others (2004) proposed that the decrease in efficiency

of microbial inactivation by UV is due to high turbidity and inadequate mixing of apple

cider as it flows through the tubes. However, TPC of UV cider shows effective bacterial

inactivation and stability during storage in this study. The inefficient inactivation of yeast

and mold is due to their apparent higher resistance to UV-radiation compared to

bacteria. Yeast and mold have different chemical composition and thicker cell walls than

bacteria, which is a major factor in determining the relative UV resistance of an









organism (Tran and Farid 2004b). The YMC for PEF and thermally processed cider

were <1 log CFU/mL throughout storage, indicating effective inactivation of spoilage

microorganisms. The PEF process is known to be more effective in yeast inactivation

compared to bacteria due to their larger cell size (Jeyamkondan and others 1999).

Larger cells are more permeable than smaller cells.

Effect of Thermal and Non-Thermal Processing on Appearance of Apple Cider
During Storage

No visual changes were noted in treated apple cider samples compared to

untreated cider at week 0. However, after 2 weeks of storage both untreated and UV

treated apple cider samples were accompanied by gas formation due to fermentation.

Also, visually both the samples clarified after 2 weeks of storage (Appendix A-1).

Previous studies also showed clarification of juice due to production of fungal enzymes,

as well as deterioration in quality of apple cider, after 2 weeks of storage due to mold

and yeast growth (Tandon et al. 2003; Donahue et al. 2004). No clarification was noted

in PEF and thermally treated cider during 4 weeks of storage.

Effect of Thermal and Non-thermal Processing on the pH and Brix of Apple Cider
During Storage

The changes in pH and Brix for untreated and treated apple cider during 4 weeks

of storage at 4 C are shown in Table 3-1. Untreated apple cider from the same juice lot

maintained at -17 C during 4 weeks of storage was used as the fresh sample.

The initial pH values were in the range of 3.76 to 3.77 for both treated and fresh

apple cider with no significant difference (P < 0.05) in mean values. Storage at 4 C for

4 weeks resulted in a slight, but not significant (P < 0.05), decrease in pH for UV treated

cider. The decrease in pH in UV treated cider could be due to fermentation in cider as a

result of increased yeast and mold growth during storage. PEF and thermal processing









did not significantly alter the pH of apple cider during storage (P < 0.05). An increase in

pH in heat pasteurized apple juice (85 C for 27s) due to evaporation of organic acids

was reported by Charles-Rodriguez and others (2007) compared to PEF treated juice.

However, in the present study at equivalent process conditions, the heat treatment

conditions used were mild (76 C for 1.3s) and therefore, no increase in pH compared to

PEF was noted. The result thus confirms the importance of comparison of quality

parameters under equivalent process conditions.

Brix value, which indicates soluble solids content in cider, is an important

measure of juice quality. UV treated cider during storage showed a significant (P < 0.05)

decrease in Brix after 2 weeks of storage corresponding to yeast and mold growth.

Microorganisms utilize sugars for growth and therefore could alter the Brix value of

juice. Tandon and others (2003) also noted a significant decrease in pH and Brix values

in UV treated cider after 2 weeks of refrigerated storage due to microbial spoilage. Brix

values were neither significantly (P < 0.05) affected by processing nor storage for

thermal and PEF treated apple cider, which corresponds to their good microbial quality.

Effect of Thermal and Non-Thermal Processing on Color of Apple Cider During
Storage

The effect of treatment and storage on color of apple cider was determined using

Hunter L* a* b* values (Table 3-2). A significant (P < 0.05) increase in L* values were

noted in treated cider samples compared to fresh cider at week 0. Increase in L* values

for pasteurized juice indicates that the juice color became lighter or whiter. Genovese

and others (1997) attributed the initial increase in L* values in pasteurized cloudy apple

juice to partial precipitation of suspended particles in juice. After 4 weeks of storage, L*

and b* values increased significantly (P < 0.05) for thermal and UV pasteurized ciders.









Noticeable decrease in a* values for all samples indicates a loss in redness, probably

due to breakdown of anthocyanins during treatment (Shewfelt 1986).

To understand the impact of the L* a* b* value changes on color of apple cider, the

total color difference (dE) values were calculated, which gives the magnitude of color

difference between treated and fresh cider (Lee and Coates 1999). Thermally treated

cider had significantly higher dE (P < 0.05) compared to both PEF and UV samples at

week 0 (Figure 3-6). Thermal treatments are known to have pronounced effects on color

of juice due to degradation of color pigments and Maillard reactions between sugars

and amino acids, resulting in browning of juice (Min et al. 2003b; Clegg 1964; Lee and

Coates 1999). Browning is commonly observed in heat treated citrus juice and is

affected by storage temperature and time (Nagy and others 1990). The dE value

increased for all treated ciders during 4 weeks of storage, indicating loss in overall color

compared to fresh cider. The increase in dE from 1.17 to 1.91 in UV cider during

storage is possibly due to microbial growth. Donahue and others (2004) attributed the

color change in UV treated cider to by products formed during fermentation by yeast

and mold growth. The dE value for PEF treated cider did not show any significant (P <

0.05) increase during storage. In comparison to UV and thermally treated samples, PEF

processed cider showed maximum color stability during 4 weeks of storage. Color was

better preserved in PEF treated juice compared to heat pasteurized juice in this study,

and this has also been documented by other authors (Yeom et al. 2000b; Min and

Zhang 2003). The conditions used for heat treatment were severe in terms of

temperature and time (94.6 C for 30 s and 92 C for 90 s, respectively). The current









study shows improved color stability in PEF cider compared to thermal cider during

storage even under equivalent process conditions.

Conclusion

Non-thermal processing has been extensively researched over the past few years

as an alternative to heat pasteurization. However, the non-thermal process was often

compared to thermal processes using conditions that did not achieve the same

reduction in concerned microorganismss, which made fair comparison of their effects

on juice quality impossible. This is the first report where conditions were carefully

selected so that all three processes achieved a similar reduction (6-log) of E. coli. The

present study demonstrated that PEF was as efficient in microbial inactivation as heat

pasteurization while maintaining the color of the apple cider. Heat pasteurization

negatively affected the color of apple cider. UV pasteurization as a non-thermal

technique was successful only for a shelf life of 2 weeks after the processing conditions

used in this study. Comparing the three techniques, PEF shows promise as a

pasteurization technique for the preservation of apple cider.
























0 17 34 51 68

Time (sec)


Figure. 3-1. UV inactivation of inoculated E. coli K12 in apple cider at different treatment
times. Treatment conditions: wavelength 254 nm, outlet temperature < 15 C


0 5 9 13 17 21 23


Electric field strength (kV/cm)


Figure. 3-2. PEF inactivation of inoculated E. co/i K12 in apple cider at different electric
field strengths. Treatment conditions: pulse duration of 2.5ps, total treatment
time ofl 50 ps, outlet temperature 49 51 oC.






69

























0 60 63 66 69 72 74 76


Temperature (C)

Figure. 3-3. Thermal inactivation of inoculated E. coli K12 in apple cider at different
temperatures. Treatment condition: 1.3 s hold time, outlet temperature <15
oC.


-*-control ----thermal -A-UV -0--PEF




II


storage time (weeks)


Figure. 3-4. Total aerobic plate count for control, thermal, UV and PEF treated apple
cider samples stored at 4 oc for 4 weeks. Vertical bar indicates standard
deviation (n=4)










-*- control --- UV -A--thermal -0-- PEF


storage time (weeks)


Figure. 3-5. Yeast and mold count in control, thermal, UV and PEF treated apple cider
samples stored at 4 oc for 4 weeks. Vertical bar indicates standard deviation
(n=4)


-4- thermal -- UV -o- PEF


2


storage time (weeks)


Figure. 3-6. Change in total color difference (de) in thermal, UV and PEF treated apple
cider during storage. Vertical bar indicates standard deviation (n=3)









Table 3-1. Change in pH and Brix values for fresh*, thermal, UV and PEF treated apple
cider stored at 4 C for 4 weeks

Weeks of pH Brix
storage Fresh Thermal UV PEF Fresh Thermal UV PEF
0 3.76a" 3.770 a 3.76 a 3.76 a 13.13a 13.005a 13.00 a 13.10a
2 3.76a 3.750 a 3.75a 3.75a 13.13a 13.00a 12.90a 13.00 a
4 3.76a 3.750 a 3.72a 3.75a 13.13a 12.88a 12.10b 12.90a
* Fresh apple cider refers to untreated cider stored at -17 C through 4 weeks of
storage. **Values with different initials within same row are significantly different from
each other at p<0.05.









Table 3-2. Change in Hunter color values** for fresh*, thermal, UV and PEF treated apple cider stored at 4 C for 4 weeks


Weeks L A b
of
storage Fresh Thermal UV PEF Fresh Thermal UV PEF Fresh Thermal UV PEF

0 32.58a 32.94b 32.88b 32.80b 3.32a 2.01b 2.18b 2.21b 5.55a 5.93a 5.31a 5.74a
2 32.60a 33.33c 32.63b 32.80b 3.31a 2.00b 2.00b 2.13b 5.57a 6.63b 6.50b 6.19b
4 32.59a 33.38c 33.44C 32.55b 3.32a 1.960 1.980 2.14b 5.58a 6.780 7.40d 6.62b


* Fresh apple cider refers to untreated cider stored at -17 C through 4 weeks of storage. **Values with different initials
within same row for each hunter value are significantly different from each other at p<0.05.









CHAPTER 4
IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNOLOGIES ON
APPLE CIDER AROMA

Introduction

A large amount of microbial information concerning thermally and non-thermally

processed apple juice can be found in the literature, but little information exists on the

effects of these processes on the flavor of apple cider. A decrease in ester

concentrations due to thermal pasteurization of apple juice has been reported (Kato et

al. 2003; Su and Wiley 1998), and a single study compared 8 apple juice volatiles in

PEF and thermally processed juice (Aguillar-Rosas et al. 2007). Because a major

motivation for non-thermal processing technologies is a minimal change in organoleptic

and nutritional properties, an in-depth analysis of the effect of the above processes on

the flavor profile of apple cider and its relation to sensory quality is necessary. The

present work focused on: 1) comparing the aroma volatiles from thermally processed

apple ciders with non-thermal treatments where each treatment was optimized to

achieve an equivalent of 6 log reduction in microorganisms, and 2) examining changes

in aroma active and major volatiles in the treated apple ciders stored at 40C at week 0

and week 4.

Materials and Methods

The standard compounds ethyl acetate, ethyl propanoate, 2-methyl propyl

acetate, methyl butyrate, ethyl butyrate, ethyl 2-methyl butyrate, hexanal, butyl 2-methyl

acetate, butyl propanoate, 2-methyl-l-butanol, (E)-2-hexenal, ethyl hexanoate, hexyl

acetate, (E)-2- hexen-1-ol acetate, 6-methyl -5-hepten-2-one, hexyl propanoate, (Z)-2-

hexen-1-ol, hexyl butyrate, hexyl 2-methyl butyrate, hexyl hexanoate, a-farnesene,

methional, phenylacetaldehyde, octanal, P3-damascenone, hexane, methanol, and 1-









butanol were purchased from Aldrich (St. Louis, MO). Butyl acetate, benzaldehyde, 1-

octanol, butyl butyrate, propyl butyrate, 1-hexanol, p-allylanisole, butyl 2-methylbutyrate,

pentyl acetate, propyl hexanoate, dimethyl sulfide and an alkane standard solution (C8-

25) were purchased from Fluka (Steinheim, Switzerland).

Apple Cider

Two hundred and twenty seven liters of unpasteurized apple cider with no

additives was procured from Ziegler Juice Company (Lansdale, Pennsylvania). A

varietal blend of apples, including Ginger Gold, Golden Delicious, Red Delicious,

Empire, Macintosh, Gala and Cortland, were used for the juice in this study. Apples

were inspected for quality (no visible mold or decay) and were washed and graded to

remove loose stems, leaves and other foreign materials. After further inspection and

grading, apples were passed through a Westphalia decanter press to express juice and

separate solids. No additives were added to the cider. The cider was packaged in high

density polyethylene (HDPE) 3.78 L (1 US Gallon) containers. The containers were

transported to the USDA facility within 2 hours of juicing and stored in a -17 C deep

freezer. The juice remained for in frozen condition for 6 days before being thawed at 4

C overnight for processing.

Apple Cider Processing

Preliminary experiments were performed to determine equivalent processing

conditions using heat, pulse electric field and ultraviolet radiation for apple cider as

described in chapter 3. Pasteurized apple cider (Ziegler Juice Co., Lansdale, PA) was

inoculated with E. coil K12 (ATCC 23716) from a stationary phase culture to give

approximately 7 log CFU/mL population. Microbial assays were conducted for ciders

heated from 60-76 C, with UV exposure times of 17-68 s and 5-23 kV/cm electric field









strengths for PEF treatment. Final optimized processing conditions resulted in

approximately 5 log reductions of E. coli K12 and were used for the remainder of the

study.

Heat treatment

Apple cider was heat pasteurized using a miniature scale HTST processing

system (Armfield, Jackson, NJ, FT74-30-Mklll-33-34) as described in chapter 3. Apple

cider was introduced into the system via the feed tank at a flow rate of 15 L/hr with hot

water circulation set at 76 oC such that the juice was held at 76 oC for 1.3s and cooled

rapidly. The inlet and outlet temperatures were continuously monitored and were in the

range of 13-17 oC and 24-30 oC, respectively, during processing.

Unpasteurized apple cider passed through the thermal processing system without

heat at room temperature and was used as the control sample for microbial studies.

Ultraviolet treatment

A low-pressure mercury lamp surrounded by a coil of UV transparent Chemflour

tubing was used for UV processing of apple cider (Geveke 2005) as described in

chapter 3. Cider was pumped through the tubing at flow rates of 25 L/hr, which

translates to a treatment time of 17 s per bulb. The energy used was 34 J/ml. Apple

cider was exposed to a total treatment time of 51 s. The inlet and outlet temperatures

recorded were between 10 -15 oC during processing.

Pulsed electric field treatment

A bench scale continuous PEF system, described in chapter 3 (OSU-4F, Ohio

State University, Columbus, OH), was used to treat the inoculated apple cider. The

cider was pumped through the system at flow rate of 7.2 L/hr. The square wave pulse

duration was 2.5 ps and the electric field strength was 23 kV/cm. The mean total









treatment time was calculated as 150 ps. Apple cider sequentially flowed through all the

chambers via steel coils immersed in a water bath set at 48 oC. The inlet and outlet

cider temperatures were continuously monitored using thermocouples and were in a

range of 30-34 oC and 49-51 oC, respectively.

Packaging and Storage

Processed apple cider was collected directly from the processing unit outlet into

sterile 1L media glass bottles (Corning Inc, Corning, NY) inside a sanitary laminar hood

equipped with a HEPA air filter (Forma Scientific Inc., Marietta, OH). The hood was

sanitized by UV lighting at 254nm for 30min before use and then wiped with 100%

alcohol. The packaged juice was stored at 4 oC for storage studies. Storage studies

were conducted for 4 weeks on the various processed apple ciders stored at 4 oC.

Microbial analyses were performed every week, whereas volatile and sensory analyses

were done in weeks 0 and 4. Fresh unpasteurized cider samples maintained at 0 C

were used as controls for volatile and sensory analysis.

Microbial Stability

The microbial analysis was performed according to method by Fan and Geveke

(2007). Microbial tests were conducted every week during the four weeks of storage.

Total aerobic plate count and yeast and mold count were determined using plate count

agar (PCA) and yeast and mold (YM) pertrifilms. The PCA plates were incubated at 37

oC for 24 hours while YM plates were incubated at room temperature for 5 days before

counting. All samples were analyzed in duplicate and two replicates of each dilution

were prepared and plated.









Volatile Extraction

Aliquots (27 ml) of apple cider were placed in 40 ml glass vials with screw cap

containing Teflon coated septa similar to the procedure used by Dreher and others

(2003). The cider was equilibrated for 10 min at 36 oC with stirring. A 2 cm-50/30pm,

DVB/ CarboxenTM/ PDMS StableflexTM (Supelco,Bellefonte, PA) SPME fiber was

exposed in the equilibrated headspace for 45 min at 36 oC. The fiber was desorbed for 5

min in the GC injection port at 250 oC. All samples were analyzed in quadruplicate.

Gas Chromatography-Mass Spectrometry Analysis

A Gas chromatography/Mass spectrometry (6890N GC, 5973N MS, Agilent

Technologies, Santa Clara, CA) was used for separation and analysis of volatiles. The

instrument was also equipped with a Pulsed Flame Photometric Detector, PFPD,

(model 5380, 01 Analytical, College station, Texas, USA).

Samples were run separately on a polar DB-Wax and non-polar DB-5 column with

identical dimensions (30 m x 0.32 mm x 0.5 pm from J&W Scientific, Folsom, CA). The

column oven temperature was programmed from 40 oC to 110 oC at 7 oC/min, then

raised 15 oC/min to 250 oC with 3 min hold. Injector and detector temperature were 250

oC. Mass spectrometry conditions were as follows: transfer line temperature at 275 oC,

mass range 30 to 300 amu, scan rate 5.10 scan/s and ionization energy 70 eV. Helium

was used as the carrier gas at a flow rate of 2 mL/min. Mass spectral matches were

made by comparison with NIST 2002 standard spectra. Authentic standards were used

for confirmation. Alkane linear index values were determined on both columns (Girard

and Tv 1996).









Quantification of Apple Cider Volatiles

Volatile free apple cider was prepared according to Fan and others (2001) by

concentrating 500 mL of apple cider using a vacuum rotary evaporator (Brinkmann

Instruments Inc., Westbury, N.Y., U.S.A.) from 11.0 to 28.0 Brix. Any residual volatiles

were extracted with hexane and discarded. Any trace hexane residue in the

concentrated juice was removed using a vacuum rotary evaporator. The concentrated

juice was diluted back to initial Brix of 11.0 using distilled water and checked for

residual volatiles. A mixture of 29 standards in methanol was serially diluted with

deodorized apple cider and added in concentration of 0.5 to 3 times their estimated

concentrations in cider. Volatiles were analyzed by SPME-GC/MS using the same

conditions described in GC/MS analysis section. A quantitation database for standards

was created using MSD Chemstation software. Response factor curves were created by

plotting target ion count (base peak) against standard concentrations in volatile-less

apple cider. Parameters used for compound identification were retention time, target ion

and secondary ions. Compounds were quantified using target ion values and response

factors generated from standard curves.

Sensory Evaluation

A discriminative triangle test (Meilgaard and others 1999) was employed to

orthonasally detect differences in aroma between unpasteurized and pasteurized apple

cider for "0" and "4" weeks storage at 4 oC. The statistical power for the triangle test was

recorded as 0.9. The analysis was conducted in a sensory panel facility at Eastern

Regional Research Center (Wyndmoor, PA), which has 6 booths with computers. The

sensory analysis was designed and conducted using Compusense five (Compusense

Inc., Ontario, CA). Samples were prepared by pouring 40 mL apple cider into 100 mL









glass bottles and closed with airtight caps. The bottles were stored in boxes at 4 C

overnight. On the day of testing, the bottles were taken out 1 hr before testing. Cider

temperature was 10-12 C during testing. Testing was performed under red light so that

color and other visible differences were masked from panelists. Each panelist was given

five sets of apple cider samples (3 samples per set) one at a time. All samples were

randomly assigned three-digit codes. The order of presentation of sample sets among

panelist was also randomized. In total, 50 untrained panelists from ERRC evaluated

the samples and each panelist performed five triangle tests, which included control

versus PEF, control versus UV, control versus thermal, thermal versus UV and thermal

versus PEF treated apple cider samples. After each test, the panelists were also asked

which sample was preferred among the three test samples and to give a reason for their

preference.

Statistical Analysis

All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc.,

Raleigh, N.C., USA). Data were from a single sample for each treatment. Each sample

was analyzed in quadruplicate. The tests for statistical significance of difference

between treatments for storage data on volatiles was performed by analysis of variance

(ANOVA) at a significance level of a = 0.05. The effect of treatments on means of

samples between treatments was compared using Duncan's multiple comparison test (a

= 0.05). Microbial data was analyzed using MS Excel 2003 and standard deviations

were presented.









Results and Discussion

Microbial Stability During Storage

The total aerobic count for processed apple cider was maintained below 20.24

log CFU/ mL through 4 weeks of storage at 4 C. The yeast and mold count in initial

cider at 0 day was 20.12 log CFU/ mL. PEF and thermal processed apple cider did not

show any yeast and mold growth throughout storage. After 2 weeks storage, the yeast

and mold count in UV treated samples increased to 3.8 0.52 log CFU/ mL. A visible

mold growth was observed after 4 weeks storage. Deterioration in quality of UV treated

apple juice due to yeast and mold growth after 2 weeks of storage has also been

observed by others (Tandon et al. 2003; Donahue et al. 2004). Donahue and

coworkers confirmed the presence of injured microbial cells in UV treated cider using

selective enrichment media. They suggested that the decrease in efficiency of cell

inactivation by UV could possibly be due to high turbidity and inadequate mixing of

apple cider as it flows through the tubes (Donahue et al. 2004).

Volatile Composition

A total of 34 volatile compounds were identified using MS in the initial untreated

apple cider after separation on a DB-Wax column (Table 4-1). Apple juice volatiles have

been previously quantified using both external and internal standard methods

(Komthong and others 2006b; Mehinagic et al. 2006). The current study is the first

report where apple cider volatiles have been quantified using external calibration from

an odorless apple cider to compensate for matrix effects. Response factor plots were

determined for 29 compounds in Appendix B-1. No response factors were determined

for peaks 2, 3, 6, 14 and 30 because their signal/noise ratio was less than 3. The









standard addition plots for 29 compounds are given in Appendix B-1. The plots for most

compounds had a linear correlation coefficient (R2) above 0.95.

Esters, aldehydes and alcohols comprised the major volatiles in apple cider,

accounting for 40%, 43% and 16% of the total volatiles identified, respectively.

However, it should be kept in mind that the volatile composition of apple juice depends

on various factors like variety, maturity and storage conditions of fruit used for pressing

(Dixon and Hewett 2000; Girard and Lau 1995). Certain apple cultivars like Jonathan

and Cox Orange Pippin are reported to have 5-100 fold higher aldehyde content

compared to Golden Delicious cultivars (Dixon and Hewett 2000). Apple juice also has

higher C6-aldehyde concentration compared to fruit due to oxidation of the fatty acids

linoleic and linolenic acid produced by lipoxygenases soon after crushing (Paillard and

Rouri 1984b). Hexanal and 2-(E)-hexenal were the most abundant aldehydes identified

in apple cider in this study (see Table 4-1).

Apple cider esters can be classified into acetic, butyric, propanoic and hexanoic

groups. Acetate esters are reported to be the major volatiles in apple juice, and high

concentrations of hexyl acetate and butyl acetate are considered to be normal

characteristics of many apple cultivars (Dixon and Hewett 2000; Young and others

1996; Girard and Lau 1995). The most abundant acetate esters in this study were hexyl

acetate, 2-methyl butyl acetate and butyl acetate.

Odor Activity Values

To assess the contribution of each volatile to apple cider aroma, OAV were

calculated as the ratio of concentrations found in apple cider to its odor threshold value

in water (Table 4-2) (Takeoka and others 1990; Flath and others 1967a; Buttery and

others 1988). Apple cider odor threshold values were not available. Aqueous threshold









values should be similar to actual apple cider values since apple cider is approximately

90% water. The highest OAV values found in initial apple cider were for hexyl acetate

(69), hexanal (41), 2-methyl butyl acetate (25), 2-methyl ethyl butyrate (23) and 2-(E)-

hexenal (14). These results are consistent with the work of Fuhrmann and others

(2002), where 2-methyl ethyl butyrate, hexyl acetate and 2-methyl butyl acetate were

reported to be major contributors to fruitiness of apple aroma in "Elstar" and "Cox

orange" apple cultivars. The C6 aldehydes, hexanal and 2-(E)-hexenal are responsible

for the green or fresh aroma of apple cider and are essential to apple juice aroma due to

their high correlation with apple aroma intensity (Durr and Schobinger 1981). Durr and

others (1981) have shown that even though esters give the fruity aroma to cider, the

concentration of aldehydes is essential for sensory impression of juice odor. Alcohols

like 1-hexanol are low aroma impact components and have been identified as a

negative contributor to apple aroma (Bult and others 2002). In this study, only UV

treated cider possessed a hexanol OAV value, which might suggest that it was aroma

active.

Effect of Treatment and Storage on Volatiles

The concentration of volatiles was not significantly (p<0.05) affected by treatments

immediately after processing (week 0 data not shown). However, pronounced volatile

differences between treatments were observed after 4 weeks at 4 C (Table 4-1). Figure

4-1 compares the levels of volatiles with significant differences between treated and

control apple ciders after 4 weeks of storage at 4 C. Hexyl acetate concentrations

decreased during storage in all processed apple cider samples with a concomitant

increase in 1-hexanol by the action of residual esterase present in apple cider pulp

(Schreier and others 1978). Thermally treated ciders lost 30% of their original ester and









aldehyde content during storage with significant decreases (p<0.05) in butyl acetate, 2-

methyl butyl acetate, hexanal and 2-(E)-hexanal concentrations (Table 4-1 and Figure

4-1). Although thermal treatment is known to inactivate most enzymes, the cider in this

study was exposed to a temperature of 76 C for only 1.3 s. This may not have

completely inactivated flavor altering enzymes even though it reduced microbial

concentrations to the desired level. UV treated cider was characterized by a complete

absence of hexanal and 2-(E)-hexenal, a decrease in hexyl acetate and an increase in

1-hexanol compared to control after 4 weeks of storage. The increase in 1-hexanol

concentration is associated with decreases in precursors such as hexanal, 2-(E)-

hexenal and hexyl acetate (Schreier et al. 1978). PEF cider lost less than 2% of total

ester and aldehyde volatiles during storage, suggesting that it more effectively

inactivated indigenous enzymes than thermal or UV treatments. A significant decrease

(p<0.05) in volatile concentrations were observed only in hexyl acetate and 2-(E)-

hexenal.

1, 3 -Pentadiene was detected only in UV samples after 4 weeks of storage. It is

an unsaturated hydrocarbon produced by molds such as Zygosaccharomyces and

Penicillium in beverages. It possesses a petroleum odor that is often associated with

microbial spoilage (Casas and others 1999).

An interesting change observed in both thermal and PEF cider was an increase in

benzaldehyde concentrations after 4 weeks storage. Sumitani and coworkers found a

similar increase in benzaldehyde in high pressure treated peaches during storage due

to release from its bound glycoside form amygdalinn) by action of P3-glucosidases

present in the peach (Sumitani and others 1994). Similar enzyme action is possible in









thermal and PEF ciders since apple seeds are known to have amygadalin as a major

constituent (Lu and Foo 1998).

Effect of Treatment and Storage on Odor Activity Values of Volatiles

Odor activity values were calculated for quantified volatiles in control and all

treated samples to assess the impact of storage and treatment on apple cider aroma

(Table4-2). Significant odor value losses (p<0.05) were observed for thermal and UV

cider samples after 4 weeks storage. Hexanal and 2-(E)-hexenal OAV decreased 35%

and 43%, respectively, in thermally treated cider during storage. Decreases in aldehyde

odor values in thermally treated cider would likely reduce aroma strength as well as

perceived freshness. Significant decreases (p<0.05) in essential esters such as hexyl

acetate and 2-methyl butyl acetate could further deteriorate the aroma quality of thermal

cider.

In UV treated cider, 100% of the original hexanal and 2-(E)-hexanal was lost

during 4 weeks of storage. During the same time, the OAV for 1-hexanol increased from

0 to 2 in UV cider only. This volatile has a green, musty aroma and is characterized as a

negative contributor to apple aroma (Bult et al. 2002). They demonstrated that an

increase in 1-hexanol concentration lead to a higher "nuts-musty" rating and a lower

"apple" rating in an apple model solution. In addition to odorant losses, UV treated cider

also developed a perceivable fermented odor after 4 weeks storage due to microbial

spoilage. Due to its high odor threshold, benzaldehyde OAV was less than one for all

treatments. Therefore, increases in benzaldehyde concentrations observed in PEF and

thermal ciders probably did not impact the aroma of these apple ciders.

PEF volatile losses during storage were minor (less than 2% loss of total ester and

aldehydes) except for hexyl acetate. This key odorant was greatly diminished during









storage in all treated ciders. In the case of PEF treated cider, only 35% was retained

after 4 weeks storage. However, losses of hexyl acetate were even greater in thermally

and UV treated ciders.

Aroma Sensory Studies

An aroma triangle test comparison between the control (unpasteurized cider

samples maintained at 0 C for 4 weeks) and all treated apple ciders at day 0 found no

significant difference at p<0.05. However, after 4 weeks of storage at 4 C, 22 of the 50

panelists detected a difference between the aroma of the thermally treated sample and

the untreated cider (control). The aroma of thermally processed cider was less preferred

compared to fresh untreated (control) samples. Aroma of UV treated cider differed

significantly (p<0.05) from the control. Thirty-six of the 50 panelists correctly detected

the difference. Of those 36, 35 panelists (97%) preferred control apple cider compared

to UV cider. No significant difference (p<0.05) was detected by panelists between PEF

treated and control apple cider. Triangle test results indicated that UV and PEF treated

samples differed significantly from heat treated cider (p<0.05). Ninety one percent of the

triangle test panelists who correctly differentiated between PEF and thermal cider

preferred the odor of PEF treated cider over that of thermally treated cider. The

preference for PEF cider coincides with the greater retention of apple aroma volatiles in

PEF cider compared to thermal cider. Preference results from this study do not

necessarily reflect true consumer preference, and further sensory studies would be

necessary to evaluate the processing effects on preference in the general consumer

population.









Conclusion

The key volatile components identified in apple cider based on OAV values were

hexyl acetate, hexnanal, 2-methyl butyl acetate, 2-methyl ethyl butyrate and 2-(E)-

hexenal. The pasteurization of apple cider by thermal and non-thermal techniques at

equivalent processing conditions resulted in a decrease in volatile components after 4

weeks of storage. PEF treated cider showed better volatile retention compared to both

thermal and UV cider. UV treated cider had a shelf life of 2 weeks. PEF cider aroma

was undistinguishable from fresh untreated cider by triangle sensory analysis after 4

weeks of storage. PEF shows good promise as a viable pasteurization technique for

apple cider.














1000


800
.-J
C control
a= 600 -
6 0 thermal
o
]0 PEF
400 -
U UV
u a a a t
8 200 a b a d -



1 2 3 4 5 6 7

major volatiles


Figure 4-1. Effect of treatment and storage (after weeks) on major volatiles of apple
cider 1 = butyl acetate, 2 = hexanal, 3 = 2-methyl butyl acetate, 4 = 2-(E)-
hexanal, 5 = hexyl acetate, 6 = benzaldehyde, 7 = 1-hexanol, a,b,c,d =
different letters for each volatile indicate significant difference(p<0.05), control
refers to fresh unpasteurized cider maintained at 0 C for 4 weeks









Table 4-1. Effect of thermal and non-thermal treatments on apple cider volatiles
compared to fresh untreated cider after 4 weeks of storage at 4 C
Pk Compound name LRI Mean concentration (pg/L)
no. Wax (week 4)
Control Thermal PEF UV


1 1,3-pentadiene
2 dimethyl sulfide*
3 ethyl acetate*
4 ethyl propanoate
5 methyl butyrate
6 2-methyl propyl
acetate*
7 ethyl butyrate
8 ethyl 2-methyl
butyrate
9 butyl acetate
10 hexanal
11 butyl -2-methyl
acetate
12 propyl butyrate
13 butyl propionate
14 1-butanol*
15 pentyl acetate"
16 2-methyl-1-
butanol
17 butyl butyrate"
18 2-(E)-hexenal
19 butyl-2-methyl
butyrate
20 ethyl hexanoate
21 hexyl acetate"
22 propyl
hexanaoate
23 (E)-2-hexen-1-ol
acetate
24 6-methyl-5-
hepten-2-one
25 hexyl propionate"
26 1-hexanol
27 (Z)-2-hexen-1-ol
28 hexyl butyrate
29 hexyl -2-methyl
butyrate
30 2-methyl-6-
hepten-1 -olt
31 benzaldehyde*


714
753
810
961
994
1024

1050
1067

1088
1098
1135

1137
1153
1158
1183
1214

1225
1227
1236

1238
1278
1326


nda
nq
nq
0.730.09b
2.530.17b
nq

3.000.52a
2.360.27a

1206.83a
2038.73a
1276.64 a

6.500.34b
4.340.36a
nq
1.300.31a
6.833.25d

7.660.41b
2312.62a
1.500.095b

nda
13710.74a
1.800.26a


1342 0.570.11b

1348 0.100.03a


1349
1366
1423
1432
1443

1480


0.070. 00ab
1645.91 c
0.170.23a
5.340.23b2
2.900.22a


nda
nq
nq
0.730.04b
2.460.90b
nq

2.86+0.20b
2.300.43a

98.82.09b
13313.22b
96.71.48b

5.700.12b
4.230.37a
nq
0.760.07b
15.061.17c

6.800.07b
14413.39c
1.430.12b

nd a
43.22.07b
nd b

0.600.17b

0.100.26a

0.00+0.00b
2536.24b
0.530.48a
4.260.29b
3.060.20a


nda
nq
nq
1.000.07a
2.730.20a
nq

3.650.55a
2.600.13a

131 9.38 a
21913.1a
1285.02a

10.00.25a
5.130.10a
nq
1.460.23a
32.13.73b

8.710.83a
18617.12b
1.530.10a

nd a
48.68.92b
nd b

0.730.1 0b

0.200.05a

0.130.01a
241 5.76b
0.1610.25a
6.930.22a
3.730.1 8a


nqb"
nq
nq
1.550.15
2.930.35a,b
nq

3.751.35"
2.000.12a

89.41.95b
ndc
121 9.53a

5.550.76b
4.560.26a
nq
0.360.1 0b
56.607.85a

4.560.20b
ndd
1.500.08b

nda
11.718.54c
nd b

1.700.12a

0.100.03a

ndc
80312.83a
ndc
1.550.12c
3.400.13a


1546 3.730.26c


62.212.92a


0.8612.20c









Table 4-1 Continued
Pk Compound LRI
no. name Wax


Mean concentration (pg/L)
(week 4)
Controls Thermal PEF UV


32 1-octanol** 1568 nda nda nda 10.45+0.13b"
33 hexyl 1620 0.430.00a 0.400.03a 0.460.00a ndb
hexanaote
34 p-allyl 1683 0.270.07a 0.200.10a 0.730.15b 0.130.05a
anisole
35 a- 1747 bq bq bq bq
farnesene
Mean concentrations given as pg/L standard deviations of quadruplicate analyses on
single samples aMean concentration ug/L, (n = 4) ;nq = not quantitated; bq = below
quantitation; nd = not detected d; different letters in the same row indicate significant
differences (p<0.05). b* 1,3-pentadiene was detected in stored UV cider but it was not
quantitated; ** represent 29 standards used for quantitation of volatiles; compounds
not quantitated as present in trace levels; t no standard available control = fresh
unpasteurized cider maintained at 0 C for 4 weeks









Table 4-2. Effect of thermal and non-thermal treatments on odor activity values (OAV) of
apple cider volatiles after 4 weeks of storage at 4 C
Compound b RI (Wax) OTa OAV
(pg/L) Control Therm PEF UV
al
Esters
ethyl propanoate 961 10 <1a <1 a <1 a <1 a
methyl butyrate 994 60 <1 a <1 a <1 a <1 a
ethyl butyrate 1050 1 3 a 3 a 4 a 4 a
ethyl -2-methyl butyrate 1067 0.1 23 a 23 a 24 a 20 b
butyl acetate 1088 66 2 a 1 a 2 a 1 a
butyl -2-methyl acetate 1135 5 25 a 19 b 26 a 24 a
propyl butyrate 1137 18 <1 a <1 a <1 a <1 a
butyl propionate 1153 25 <1 a <1 a <1 a <1 a
pentyl acetate 1183 43 <1 a <1 a <1 a <1 a
butyl butyrate 1225 100 <1 a <1 a <1 a <1 a
butyl-2-methyl butyrate 1236 17 <1 a <1 a <1 a <1 a
ethyl hexanoate 1238 1 -- -- -- --
hexyl acetate 1278 2 69 a 22 b 24 b 6 c
hexyl propionate 1349 8 <1 a b <1 a -__ b
hexyl butyrate 1432 250 <1 a <1 a <1 a <1 a
hexyl -2-methyl butyrate 1443 22 <1 a <1 a <1 a <1 a
Aldehydes
hexanal 1098 5 41 a 27 b 44 a 0 c
2-(E)-hexenal 1227 17 14 a 8 b 11 c 0 d
benzaldehyde 1546 350 <1 a <1 a <1 a <1 a
Alcohols
2-methyl-1-butanol 1214 300 <1 a <1 a <1 a <1 a
1-hexanol 1366 500 <1 a <1 a <1 a 2 b
1-octanol 1568 130 __a __a __a <1 b
(Z)-2-hexen-1-ol 1423 70 <1 a <1 a <1 a <1 a
Others
6-methyl-5-hepten-2-one 1348 50 <1 a <1 a <1 a <1 a
a OT = odor threshold values in water from literature, b compounds identified based on
RI on DB-Wax and DB-5 column using standards, control refer to fresh unpasteurized
cider maintained at 0 oC for 4 weeks









CHAPTER 5
COMPARISON OF THERMAL AND NON-THERMAL TECHNIQUES ON GRAPEFRUIT
JUICE STORAGE QUALITY UNDER EQUIVALENT PROCESS CONDITIONS

Introduction

Increased consumer demand for fresh-like products with minimum flavor and

nutritional losses has paved the way for alternatives to heat processing. PEF treatment

as a non-thermal technique for pasteurization of juices has been studied extensively in

recent years (Jin and Zhang 1999; Min et al. 2003b; Yeom et al. 2000b). PEF

processed juice at a commercial scale had a microbial shelf life of 196 days at 4 oC with

a higher sensory preference compared to heat pasteurized juice (Min et al. 2003a).

Studies on UV pasteurization have shown extension of shelf life of apple juice and

orange juice by 7 and 5 days, respectively, with minimal loss of sensory quality

(Donahue et al. 2004; Tran and Farid 2004b). Radio frequency electric field is a recently

developed non-thermal technique that is not FDA approved. The effect of RFEF

treatment on microbial quality of apple and orange juice has been reported in literature

(Geveke et al. 2007; Geveke and Brunkhorst 2004). Geveke and others (2004)

achieved a 3.3 log reduction in E. coil in RFEF treated orange juice relative to untreated

juice.

There are several studies in the literature comparing the effect of thermal and non-

thermal treatments on juice quality (Yeom et al. 2000b; Tandon et al. 2003; Sanchez-

Vega et al. 2009). However, most often the conditions used for thermal processing are

severe in terms of temperature and time of exposure to heat. Thus, shelf life studies

comparing the effect of non-thermal to thermal techniques on the acceptability of juice

using conditions that do not achieve the same reduction of the target microorganism

would result in incorrect conclusions (Aguillar-Rosas et al. 2007; Yeom et al. 2000b;









Rivas et al. 2006). Therefore, for a fair comparison of the effects on quality of juice, both

thermal and non-thermal processes must achieve an equivalent reduction in

microorganism levels. The current work focuses on comparing the effect of thermal and

non-thermal processing techniques on grapefruit juice quality, where each treatment is

optimized to achieve approximately equivalent 5 log reductions in E. coil K12

population.

Materials and Methods

Determination of Equivalent Treatment Conditions

Pasteurized grapefruit juice was inoculated with E. coli K12 (ATCC 23716)

obtained from the American Type Culture Collection (ATCC) (Manassas, VA). The

bacterium was maintained on Tryptic Soy Agar (Remel, Lenexa, KS, USA) at 4 oC. Prior

to inoculation of product, the organism was cultured in Tryptic Soy Broth (Remel) with

shaking at 37 oC for 16-18 h. Juice was inoculated from the stationary phase culture to

give approximately 7 log CFU/mL population.

Heat treatment

Inoculated grapefruit juice was heat pasteurized using a miniature scale HTST

processing system (model FT74-30-Mklll-33-34, Armfield, Jackson, NJ, USA) described

in chapter 3. Juice was pumped through the system at a flow rate of 15 L/hr, which

translated to a hold time of 1.3s. Microbial assays were performed at holding tube outlet

temperatures of 60, 62, 64, 66, 68 and 70 oC.

Ultraviolet treatment

The UV apparatus used for pasteurization of juice is described in chapter 3. The

experimental system included a feed tank, a peristaltic pump and four UV lamps of the

same dimensions connected in a series. Juice was pumped through the tubing at a flow









rate of 28.8 L/hr, which translated to a treatment time of 14.75s per bulb. Microbial

assays were conducted using 1, 2, 3 and 4 UV lamps, which corresponds to UV

treatment times of 14.75, 29.57, 44.25 and 59s.

Pulsed electric field treatment

A bench scale continuous PEF system (Valappil et al. 2009) at USDA, Wyndmoor,

PA (as described in chapter 3) was used to treat the inoculated grapefruit juice. The

juice was pumped through the system at a flow rate of 7.2 L/hr. The square wave pulse

duration was set at 2.5 ps and the electric field strengths tested were 5, 10, 13, 17, 21

and 23 kV/cm. Grapefruit juice sequentially flowed through all the treatment chambers

via steel coils immersed in a water bath set at 30 oC.

Radio frequency electric field treatment

The RFEF system used in the previous study has been previously described by

Geveke and others (2007). The system was equipped with an 80 kW RF power supply

(Ameritherm, Scottsville, NY model L-80) interfaced with a custom-designed network

that enabled RF energy to be applied to a resistive load (Ameritherm) over a frequency

range of 21.1 to 40.1 kHz. The RFEF power supply was connected to a series of

treatment chambers. Grapefruit juice was supplied into the RFEF system from a feed

tank using a progressing cavity pump. The processing parameters were set at a

frequency of 20 kHz, a flow rate of 33 L/hr and field strength of 15kV/cm. The juice was

quickly cooled to less than 25 C after exiting the treatment chambers using a stainless

steel heat exchanger sample cooler (Madden manufacturing, Elkhart, IN, model

SC0004).









Shelf Life Study

150 L of unpasteurized red grapefruit juice with no additives frozen in 5 gallon

buckets and packed in styrofoam boxes with dry ice layers were shipped from a

commercial juice facility in Florida. The juice was received after 2 days of shipping and

stored immediately in a -17 C freezer. Juice was filtered through 2 layers of

cheesecloth under hygienic conditions due to high pulp content. The filtered juice was

pasteurized by UV, PEF, RFEF and thermal techniques as described below.

Grapefruit juice pasteurization

All processing equipment was sanitized by pumping 5% bleach solution through

the system followed by a distilled water rinse. Grapefruit juice was heat pasteurized at

70 oC for 1.3s. Prior to UV pasteurization, juice was preheated to 50 oC and then

exposed to UV lamps for 44.25s. PEF processing was performed at an electric field

strength of 20 kV/cm, 2.5 ps pulse duration and total treatment time of 150 ps. RFEF

processing was performed at 20 kHz frequency, 15kV/cm field strength, an outlet

temperature 55 oC and a treatment time of 170 ps.

Packaging and storage

Processed grapefruit juice was collected directly into sterile 1 L media glass bottles

(Corning Inc, Corning, NY) inside a sanitary laminar hood equipped with a HEPA air

filter (Forma Scientific Inc., Marietta, OH). The hood was sanitized by UV lighting at

254nm for 30 min before use and then wiped with 100% alcohol. The quality of

grapefruit juice stored at 4 oC was evaluated every week for 4 weeks (0, 1, 2, 3 and 4)

on the basis of microbiology, pectin methyl esterase activity (PME), non-enzymatic

browning (NEB), ascorbic acid content (AA) and physical properties (pH, Brix and









color). All assays were performed in triplicate. Microbial assays were performed in

duplicate.

Microbial assay

Microbial tests were conducted as described in chapter 3. One mL aliquots of juice

were pour plated with Tryptic Soy Agar (Remel, Lenexa, KS, USA) and incubated at 37

C for 24 h. The E. coli K12 population was expressed as CFU/mL of grapefruit juice.

Data for each replicate were normalized against the control and plotted as the log

reduction versus temperature, time, treatment cycle or electric field strength.

For the storage study, the total aerobic plate count (TPC) and yeast and mold

count (YMC) were determined using plate count agar (PCA) and yeast and mold (YM)

pertrifilms. After appropriate dilutions (if needed), samples (1 mL) were then pour plated

with PCA or pertrifilm. The PCA plates were incubated at 37 C for 24 hrs, while YM

plates were incubated at room temperature for 5 days before counting. All samples

were analyzed in duplicate and two replicates of each dilution were prepared and

plated.

Pectin methyl esterase activity assay

PME activity was determined according to a modified method described by

Sampedro and others (2009).Two milliliters of juice sample was added to a 30mL

mixture of 0.35% citrus pectin solution and 125mM NaCI solution. The pH was first

adjusted to 7.5 using 0.1M NaOH (pre-titration). The hydrolysis at 22 C was maintained

at pH 7.5 by adding 0.1M NaOH solution using automated pH-stat titrator (Mettler

Toledo T 70 Titrator, Schwerzenbach, Switzerland). After the initial 30s, the

consumption of NaOH was recorded every 1 s for a 5-20 min reaction period. The slope









(dV/dt) from linear plot between volume of NaOH consumed (dV) vs. time (dt) is used to

calculate PME activity using following formula (Yeom and others 2000a):

PME(units /mL) = slope (0. 1MNaOH) (1000) (5-1)
2mLsample

Percentage of residual PME activity was calculated in relation to the activity of the

untreated sample.

Ascorbic acid assay

The ascorbic acid (Vitamin C) content in grapefruit juice was assayed by the HPLC

method by Geveke and others (2007). Grapefruit juice was centrifuged at 10,000g for

10 min at 5 C in a Sorvall RC-2 refrigerated centrifuge (Kendro Laboratory Products,

Newtown, CT). An aliquot of 0.5ml of supernatant was mixed with 5% meta-phosphoric

acid. The solution was filtered through a 0.45 pm Acrodisc LC 13 PVDF syringe filter

(Gelman Sciences, Ann Arbor, MI). The filtered material was analyzed using a HP Ti-

series 1050 HPLC system (Agilent Technologies, Palo Alto, CA) with a photodiode array

detector. An Aminex HPX-87H organic acids column (300 x 7.8 mm) fitted with a

microguard cation H+ was used for separation. The mobile phase of 5mM sulfuric acid

was passed through the column at a flow rate of 0.5 ml/min. Column temperature was

maintained at 30 oC using a column heater (Bio-Rad Laboratories, Hercules, CA).

Ascorbic acid was monitored at 245nm. A calibration curve was prepared by external

standard method using standard ascorbic acid solutions in a range from 100 -1000

pg/mL in water.

Non enzymatic browning

Browning in grapefruit juice was assayed using the method by Fan and Thayer

(2002). Juice was centrifuged at 10,000g for 10min at 5 oC. An aliquot of 2ml of









supernatant was mixed with 2ml of 100% ethanol. The solution was vortexed and left at

room temperature for 1 hr. The mixture was centrifuged again and the absorbance of

supernatant was measured at 420nm using a spectrophotometer (Shimadzu UV-1601,

Shimadzu Scientific Instruments, Columbia, MD).

pH

The pH meter used for all analysis was an Orion 420A+ (Thermo Electron Corp.,

Beverly, MA). The pH meter was calibrated at pH 4.0 and pH 7.0 using standard

solutions on each day of analysis.

Brix

A Bausch & Lomb Abbe refractometer (B&L Corp., Rochester, NY) was used. To

measure Brix of juice sample, a drop of sample was placed on the refractometer and

the corresponding refractive index was recorded, which gave the measure of soluble

solids in juice.

Color

Color was measured in CIE L* a* b* 3-dimesional color space using ColorQuest

XE spectrophotometer (Hunter Associates Lab, Reston, VA) as described in chapter 3.

The lamp was allowed to warm up for 30 min prior to measurement. The system was

calibrated using black and white standard tiles on each day of analysis. Forty mL of

juice sample in a 20 mm path-length glass cell was used for measuring color. The effect

of processing methods on color of juice was represented using total color difference

(dE) calculated for all the samples using the following equation 2:

dE = (L -Lo)2 +(a ao)2 +(b-bo)2 (5-2)









where L = lightness of treated sample at time t; LO = lightness of untreated sample

at day 0; a = redness of treated sample at time t; aO = redness of untreated sample at

day 0; b = yellowness of treated sample at time t; and bO = yellowness of untreated

sample at day 0.

Statistical Analysis

All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc.,

Raleigh, N.C., USA). Statistical significance of the differences among the treatments

was tested using the analysis of variance (ANOVA) at the significance level of a = 0.05.

The effect of storage and processing techniques were compared using Duncan's

multiple comparison test (a = 0.05).

Results and Discussion

Effect of Treatments on Microbial Inactivation

Pasteurized grapefruit juice was inoculated with E. coli K12 to give approximately

7 log CFU/ml population. The initial microbial count in pasteurized juice was below 40

CFU/mL, indicating a very low microbial load before inoculation. The juice was treated

by thermal, UV, PEF and RFEF techniques to achieve an approximately equivalent 5

log reduction in inoculated microbial levels.

The inlet and outlet temperatures recorded during thermal treatment were between

16- 18 C due to a built-in cooling section. The inoculated E. coli K12 population

decreased from 5.6 logs to 2.09 logs as temperature increased from 60 to 70 C (Fig 5-

1). Treatment of juice with temperatures above 62 C resulted in more than 4 log

reduction in population. Thermal treatment at 70 C for 1.3 s was selected for microbial

stability studies as it resulted in a population reduction by 4.91 logs, which was close to

the target 5 log reduction.









For UV treatment, inoculated juice was passed through 1, 2, 3, and 4 UV bulbs

connected in series at a flow rate of 28.8L/hr. Based on flow rate, the time of exposure

to UV light translates to 14.75s, 29.57s, 44.25s and 59s. The inlet temperature was

recorded at 50 C (pre-treatment temp) and outlet temperatures recorded were from 48-

50 C. The microbial population reduced with increasing number of UV bulbs (Fig 5-2).

UV treatment using three bulbs (44.25 s) reduced the microbial population by 5.05 logs.

These conditions were used for microbial stability studies.

The effect of PEF processing on E. coli K12 inactivation at an electric field strength

from 15 to 22 kV/cm with a pulse duration of 2.5 ps and a mean total treatment time of

150 ps is shown in Figure 5-3. The inlet and outlet temperatures of the filtered juices

were between 20- 21 C and 34.6-50 C, respectively, during the length of treatment. An

electric field strength at 20kV/m gave a 5.23 log reduction in E. coli population. Yeom

and others (2000b) found a 7 log reduction in the inoculated microbial population of

unfiltered orange juice treated with PEF at 35kV/cm for 59 ps. The authors showed that

higher electric field strength and longer treatment times decreased microbial levels.

RFEF is a relatively new non-thermal technique that is not yet FDA approved for

commercial use. It is similar to PEF except that the high voltage is applied continuously

using an AC generator. Previous work by Geveke and others (2007) has shown a 2.1

log reduction in inoculated E. coil K12 population in orange juice at conditions of 21.1

kHz frequency, 15kV/cm electric field strength, an outlet temperature of 60 C and

treatment time of 270 ps. In grapefruit juice processed at 20 kHz frequency, 15 kV/cm

field strength, an outlet temperature of 55 C and treatment time of 170 ps gave 2.85 log


100









reduction in E. coli K12 population (Fig 5-4). After 2 treatment cycles, 5.26 log reduction

was achieved.

Effect of Treatments on Microbial Stability During Storage

The effect of treatments on total aerobic count and yeast and mold count in

grapefruit juice during 4 weeks of storage at 4 C are shown in Fig 5-5 and Table 5-1.

The initial TAC in untreated grapefruit juice (control) was 1.57 log cfu/mL. After 4 weeks

of storage, the microbial population reached 3.52 log cfu/mL. The initial TAC for all

treated samples reduced to < 5 cfu/mL at week 0. Thermal and RFEF treated juice

maintained the population below 1 log cfu/mL for 3 weeks of storage. However, in

RFEF treated juice the microbial population increased to 3.34 log cfu/mL after 4 weeks

of storage. In UV and PEF juice, the microbial population increased after 2 weeks of

storage and reached 3.33 and 3.51 log cfu/mL, respectively after 4 weeks of storage.

The increase in microbial count during storage could be due to two reasons: 1)

ineffective inactivation of microbial spores and their subsequent growth during storage,

or 2) incomplete inactivation or injury to microbial cells and later recovery during

storage. An increase in microbial count has been previously reported in PEF treated

tomato juice due to incomplete inactivation of microbial spores (Min et al. 2003b).

Because RFEF works on a similar principle of microbial inactivation as PEF, it may also

be ineffective in inactivation of spores. UV treatment has been shown to cause only

injury to cells in cider due to lower penetration power. Tran and others (2004b) also

found an increase in aerobic count from 2.47 to 4.33 log cfu/mL after 12 days of storage

in UV treated orange juice.

The initial YMC in control juice was 2.46 log cfu/mL, and it increased to 3.90 log

cfu/mL by the end of 2 weeks storage. After 2 weeks, the mold growth was uncountable


101









due to high growth, and the colonies appeared too diffused on the petriflim. In thermal

and RFEF no Y&M was detected at week 0, indicating effective inactivation of Y&M. In

thermal samples, the Y&M count reached 3.33 log cfu/mL after 4 weeks of storage. In

RFEF, PEF and UV, the mold growth was too high to be counted after 2 weeks of

storage. Similar to total aerobic count, the reason for the increased Y&M growth during

storage could be due either to sublethal injury to cells or resistance of spores to different

treatments. Yeast and mold growth are the most common spoilage agents in

refrigerated citrus juices (Wyatt and Parish 1995). They are also more tolerant to high

temperatures compared to bacteria and are therefore difficult to inactivate. Mold

ascospores like bacterial spores are known to be more resistant to PEF treatment

(Grahl and MarkI 1996).

The results from microbial stability studies show that thermally treated juice had

good microbial quality for 4 weeks of storage. Thermal treatment is more efficient in

limiting bacterial and yeast and mold growth compared to non-thermal treatment. RFEF

treated juice had a shelf life of 3 weeks, whereas PEF and UV treated juice spoiled after

1 week of storage. It could be concluded that heat treatment is more effective at

microbial inactivation than non-thermal techniques at equivalent process conditions.

Effect of Treatments on Pectin Methyl Esterase Activity

The effect of heat, PEF, RFEF and UV treatment on PME activity is shown in

Figure 5-6. PME is primarily responsible for the clarification of citrus juice cloud. PME

causes demethylation of carboxyl groups of pectin. These unprotected COOH groups

react with calcium ions in juice and form pectin aggregates which precipitate out of juice

(Wolfgang, 1990). Previous studies on heat pasteurization of orange juice have shown

90-100% inactivation in PME activity at temperatures higher than 90 C for 20-60 s


102









(Elez-Martinez et al. 2006; Rivas et al. 2006). Thermal treatment at 70 C for 1.3 s

decreased the PME activity in grapefruit juice by 80.471.99%. Tran and others (2004b)

reported a 70% PME inactivation in orange juice treated at 70 C for 2s. RFEF, PEF and

UV treatments decreased the PME activity by 81.663.10%, 59.172.64% and

48.523.62%, respectively. Studies on the effect of PEF on PME activity by Yeom and

others (2000a) showed 88% PME inactivation in PEF treated orange juice at 35kV/cm

for 59 ps and a 60 C outlet temperature. Rivas and others (2006) found 75.6% PME

inactivation at 25kV/cm at 60 C for 280 ps total treatment time. Therefore, it is not

surprising to find a lower inactivation of PME at the milder treatment conditions used in

this study (22kV/cm, 46 C for 150 ps). UV treatment reduced PME activity by

48.523.62% in grapefruit juice. Tran and others (2004b) found only 5% reduction in

PME activity in orange juice at high UV dose at ambient temperatures. The

pretreatment of juice to 50 C could possibly have an additive effect on the inactivation

of PME in grapefruit juice. RFEF treatment gave the highest PME inactivation compared

to other treatments. It is possible that the electric strength, in combination with an output

temperature of 55 C, resulted in increased inactivation. However, there are no previous

data on RFEF effect on citrus PME for comparison.

During storage, RFEF and thermally treated juices maintained residual PME

activity during 4 weeks of storage, indicating irreversible inactivation of PME. PME exist

as isozymes in citrus juices with different degrees of thermostability. The thermolabile

enzyme is quickly inactivated, whereas the thermostable enzyme is gradually

inactivated by exposure to high temperatures for extended periods of time. The residual


103









PME activity observed in juices is mostly due to the presence of a thermostable

enzyme.

A decrease in activity during storage was observed in PEF and UV treated juice

(Figure 5-6). In the control juice, the PME activity reduced to 65% after 4 week of

storage. Since alkaline is optimum pH for PME, a loss in activity during storage under

acidic conditions is expected (McFeeters RF 1983). Because grapefruit juice is acidic in

nature, a loss in PME activity during storage is expected. An exponential decay in PME

activity in untreated orange juice during storage has been reported by Elez and others

(2006).

Effect of Treatments on Non-enzymatic Browning

The effect of thermal and non-thermal treatments on the brown color of grapefruit

juice during 4 weeks of storage at 4 C is given in Figure 5-7. All treated juice showed a

significant increase (p<0.05) in the browning index compared to the control value of

0.069 at week 0. The browning index values at week 0 for thermal, RFEF, UV and PEF

were 0.0835, 0.080, 0.073 and 0.072, respectively. Browning in citrus juice is mainly

due to Maillard reactions between sugars, amino acids and ascorbic acid degradation

products (Clegg 1964). Browning is commonly observed in heat treated citrus juice and

is affected by storage temperature and time (Nagy et al. 1990). The Maillard reaction

between reducing sugars and amino acids results in formation of melanoids, which give

the brown color to juice. However, in citrus juices the importance of Maillard-type

browning is minor due the acidity of these juices, which is not favorable for Maillard-type

reactions. A significant increase (p<0.05) in brown color was noted in all treated juice

samples during 4 weeks of storage compared to initial week 0 values. The browning

index after 4 weeks for thermal, RFEF, UV, PEF and control were 0.097, 0.094, 0.086,


104









0.083 and 0.083, respectively. The browning index for thermal and RFEF treated juices

are significantly higher (p<0.05) compared to the control, PEF and UV treated juices.

Effect of Treatments on Ascorbic Acid Degradation

The initial concentration of ascorbic acid in grapefruit juice was measured at

69.861.92 mg/100mL of juice. After pasteurization, a significant loss (p<0.05) in

ascorbic content was noted in all treated samples (Figure 5-8). Thermal processing

reduced the ascorbic acid content by 29.19% in grapefruit juice. RFEF, PEF and UV

treatment showed 26.33%, 16.77% and 19.72% loss, respectively, in ascorbic acid

content at week 0. Ascorbic acid is a sensitive biomolecule, and therefore treatment

temperature and oxygen availability affects its rate of degradation (Sadler and others

1992; Lee and Coates 1999). Since all treated juice samples were exposed to

temperatures above ambient, a decrease in ascorbic acid content is expected. During

storage, a further decrease was noted in all treated samples. After 4 weeks of storage,

thermally treated samples had the highest ascorbic acid loss (p<0.05) compared to non-

thermally treated juice samples. Ascorbic acid undergoes oxidative degradation to form

dehydroascorbic acid, which further reacts with amino acids to form brown color

compounds. Lee and Nagy (1988) reported a high correlation between the percentage

loss of ascorbic acid and an increase in browning in grapefruit juices. To confirm

whether similar correlation exists in present study, a linear regression plot of ascorbic

acid content versus browning index is given in Figure 5-9. A negative correlation of 0.97

and 0.92, respectively were found for RFEF and thermally treated juices. The high

correlation coefficients indicate that degradation of ascorbic acid is a probable

explanation for the increase in brown color in treated samples during storage. Untreated

grapefruit juice had the highest ascorbic acid content compared to treated juice samples


105









after 4 weeks of storage. Sadler and others (1992) also found greater ascorbic acid

retention in untreated orange juice compared to thermally treated juice during a 50-day

storage period. It was proposed that increased microbial population in untreated juice

may have lowered the dissolved oxygen content and prevented AA oxidation.

Effect of Treatments on Color

The effect of different treatments on color of grapefruit change was determined

using Hunter L*, a* and b* values during 4 weeks of storage at 4 C. The change during

storage on L* and b* values are shown in Fig. 5-10 and 5-11, respectively. The L*

values increased significantly (p<0.05) for thermal and RFEF pasteurized juices

compared to control juice at week 0. This is contradictory to the browning index value,

where an increase in browning was noted for thermal and RFEF samples. Therefore,

these results need to be considered with caution, as they could be due to analytical

error or to bottle-to-bottle sampling variation. No significant change (p<0.05) in L* was

noted in PEF and UV treated juice compared to control at week 0. A significant increase

(p<0.05) in b* values for all treated samples were noted at week 0, indicating a

yellowing of samples. However, during 4 weeks of storage, the b* values significantly

decreased for thermal and RFEF juice. The decrease in b* values could possibly be due

to increased browning in grapefruit juice. The linear regression plot between b* versus

the browning index gave a correlation coefficient of 0.94 and 0.91 for thermal and RFEF

treated juices, respectively (Figure 5-12). These values indicate a strong relationship

between browning and decrease in b* values in stored RFEF and thermal samples.

Cortes and others (2008) reported a decrease in b value during storage in grapefruit

juice. No significant (p<0.05) effect of treatment was observed for a* values during

storage (data not shown).


106









Total color difference values (dE) were calculated to determine the magnitude of

color difference between treated and control grapefruit juice (Figure 5-13). A delta value

of two or higher must be present in order for noticeable visual difference to be observed

(Lee and Coates 1999). In the present study, calculated delta values were less than 2

for all treated juices throughout the storage period. Even though L* and b* value

changed during storage, visually no noticeable differences were found in treated

grapefruit juices compared to control juice.

Effect of Treatments on pH, Brix and Total Acidity

Measured pH values in control, thermal, UV, RFEF and PEF treated grapefruit

juice are given in Table 5-2. The pH values were not significantly (p<0.05) affected by

either processing or storage for treated juice compared to control juice.

Brix value for control grapefruit juice was measured at 10.42. No significant

differences (p<0.05) in Brix values were noted at week 0 in treated samples. During

storage, a significant decrease (p<0.05) was noted in PEF and UV after 4 weeks of

storage. This decrease could be attributed to the utilization of sugars during

fermentation by microorganism. A similar change in Brix due to microbial growth has

been reported by many authors (Yeom et al. 2000b; Min et al. 2003a).

Total titratable acidity for control and treated juice was measured and expressed

as gm citric acid equivalents/100mL of grapefruit juice. Titratable acidity values were not

significantly (p<0.05) affected by either processing or storage for treated juice compared

to control juice.

Conclusion

The effect of thermal and non-thermal pasteurization techniques on the quality of

red grapefruit juice were compared to untreated juice for 4 weeks of storage at 4 oC.


107









The treatment conditions were optimized to achieve equivalent 5 log reductions in the

inoculated E. coli population. Thermal treatment was more effective compared to non-

thermal treatment in terms of microbial stability at after 4 weeks storage. However,

RFEF treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with

significantly (p<0.05) higher Vitamin C content and equivalent PME inactivation

compared to thermally treated juice. PEF and UV treatments maintained good microbial

quality for 1 week with significantly (p<0.05) higher vitamin C content, as well as less

browning, compared to thermal treatment. The results from the study showed that at

equivalent processing conditions, non-thermally treated juice had a shorter shelf life but

higher vitamin C content and better physical qualities compared to thermally treated

juice.


108

















I l'


0.00 60.00 62.00 64.00 66.00 68.00 70.00
temperature (C)

Figure 5-1. Thermal inactivation of inoculate E. coli K12 in grapefruit juice


I.


0 1 2 3 4
no. of bulbs

Figure 5-2. UV Inactivation of inoculated E. coli K12 in grapefruit juice


109


6
,-I
E
4 4
0
2

n


8

6
-J
E
I 4

2

0

























15 17 20 22
electric field strength (kV/cm)


Figure 5-3. PEF inactivation of E. coli K12 in grapefruit juice


0 1
no. of treatment cycle


Figure 5-4. RFEF inactivation of inoculated E. coli K12 in grapefruit juice














110













|-o-thermal PEF -A-RF -*- UV --- control


5 -
-J
E

'B -







0 1 2 3 4
storage time (weeks)


Figure 5-5. Total aerobic plate count for control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 C for 4 weeks


Table 5-1. Yeast & mold count in control, thermal, UV, PEF and RFEF treated grapefruit
juice stored at 4 C for 4 weeks
Week Control Thermal PEF RFEF UV
0 2.460.26 0 1.360.18 0 1.760.52
1 3.900.18 0 2.520.42 1.270.38 3.050.27
2 mold 0 mold mold Mold
3 mold 2.240.36 mold mold Mold
4 mold 3.330.24 mold mold mold


111










-u-PEF -x-RF -A-UV


100

80

60

40

20

0


I



I


storage time (weeks)


Figure 5-6. Change in PME activity in control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 C for 4 weeks


-o--control -*-thermal -x-RFEF -A--UV -m--PEF


0.1 -


0.09 -

0.08 -

0.07 -

0.06 -

0.05


storage time (weeks)


Figure 5-7. Change in browning index of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 oC for 4 weeks.


112













-x-RF -A-UV -*-thermal ----PEF -o--control


storage time (weeks)


Figure 5-8. Change in ascorbic acid content of control, thermal, UV, PEF and RFEF
treated grapefruit juice stored at 4 C for 4 weeks


x RFEF o thermal


S60O


40


S20 -
I-
0
o


0.08


R2= 0.9711


R = 0.9187


0.085 0.09 0.095


browning index





Figure 5-9. Linear regression plot of ascorbic acid vs. browning index for thermal and
RFEF treated grapefruit juice


113


I-










-*- thermal --s- PEF -A--UV -x-RFEF -0--control


29 -

28.5 -

28 1

27.5 -

27 -
0 1 2 3 4
storage time (weeks)


Figure 5-10. Change in color L* value of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 C for 4 weeks


114














-*-thermal --m-PEF -A--UV -x-RFEF -o- control


-3
I F
I


storage time (weeks)


Figure 5-11. Change in color b* values of control, thermal, UV, PEF and RFEF treated
grapefruit juice stored at 4 C for 4 weeks


S thermal RFEF


0.12 -

0.1 -

E 0.08 -
O
< 0.06 -

, 0.04 -

0.02 -

0 -


R2 = 0.9107


4.2 4.4 4.6 4.8


Figure 5-12. Linear regression plot of brown color vs. color b* values for thermal and
RFEF treated grapefruit juice


115










-*-thermal --- PEF -- UV -x- RFEF -o--control


2.0 -

1.5 J






0.0 ,
0 1 2 3 4
storage time (weeks)


Figure 5-13. Change in total color difference (dE) in control, thermal, UV, PEF and
RFEF treated grapefruit juice stored at 4 C for 4 weeks


116









Table 5-2. pH, Brix and total acidity (TA) values for control, thermal, UV, PEF and RFEF


treated grapefruit juice stored at 4 C for


4 weeks


Weeks Control Thermal PEF RFEF UV

0 3.53 a 3.52 a 3.51 a 3.51 a 3.51 a
1 3.51 a 3.50 a 3.50 a 3.52 a 3.51 a
pH 2 3.53 a 3.52 a 3.50 a 3.52 a 3.50 a
3 3.52 a 3.51 a 3.51 a 3.53 a 3.52 a
4 3.53 a 3.50 a 3.52 a 3.53 a 3.51 a
0 10.42 a 10.45 a 10.45 a 10.40 a 10.40 a
1 10.41 a 10.40 a 10.43a 10.42a 10.42a
Brix 2 10.40a 10.48a 10.10 b 10.41 a 10.30a
3 9.91 b 10.50a 10.00 b 10.40a 10.00 b
4 9.75c 10.40a 10.05 a 10.41 a 9.90b
0 1.20a 1.31 a 1.10a 1.13a 1.17a
1 1.22 a 1.26 a 1.2 a 1.25 a 1.23 a
TA 2 1.20a 1.30a 1.15a 1.15a 1.18a
3 1.28 a 1.28 a 1.22 a 1.20 a 1.20 a
4 1.20 a 1.25a 1.11 a 1.16a 1.19a


117









CHAPTER 6
IMPACT OF THERMAL AND NON-THERMAL PROCESSING TECHNOLOGIES ON
AROMA OF RED GRAPEFRUIT JUICE: GC-OLFATOMETRIC COMPARSION

Introduction

Non-thermal techniques like pulsed electric field (PEF), ultraviolet (UV) and high

pressure (HPP) have been examined in the past for their efficiency in extending shelf

life of juice while minimizing nutritional and quality loss. Comparative studies on the

effect of PEF and thermal processing on orange juice has shown reduced loss of flavor,

color, and vitamin C in PEF juice compared to thermal processing (Ayhan et al. 2001).

However, there is only a single literature reference on PEF processed grapefruit juice.

The authors (Cserhalmi et al. 2006) investigated the effect of PEF treatment on physical

properties like pH, Brix, color, non-enzymatic browning and flavor components. No

significant change in any parameter's post processing compared to fresh untreated juice

was observed. The favor analysis was limited to 8 volatiles with no supporting sensory

data.

RFEF, a recently developed non-thermal technique (Geveke 2005), has not been

FDA approved yet. Few studies have been conducted in citrus juice on microbial

inactivation kinetics or on ascorbic acid content and color of juice for non-thermal

treatments (Geveke et al. 2007).

UV is commercially used for water disinfection and apple cider pasteurization.

Studies on UV treated orange juice is limited due to low transmittance of UV light

through the high pulp juice (Tran and Farid 2004b). UV irradiation of unclarified orange

juice extended the shelf life by 5 days with 17% loss in ascorbic acid at 40C.

No data on UV or RFEF treated grapefruit juice has been found in the literature.

Even though the major motivation for non-thermal processing technologies is minimal


118









change to organoleptic properties, it is surprising to note that little research has been

conducted in this area.

Quantitative, as well as qualitative, studies on volatile constituents of grapefruit

juice and essence have been extensively studied by many groups (Lin and Rouseff

2001; Coleman et al. 1972; Buettner and Schieberle 1999). Many odor active volatiles

important to grapefruit aroma are present in low levels and are not usually detected by

mass spectrometry or FID. GCO is a bioassay that measures human response to

odorants separated by GC and is used to detect aroma active compounds. The present

study focuses on comparing the impact of thermal and non-thermal pasteurization

techniques on aroma active volatiles of fresh unpasteurized grapefruit juice during 4

weeks of storage at 4 oC using GCO. As discussed in chapter 5, the non-thermal and

thermal treatments were optimized to achieve equivalent 5 log reductions in

microorganisms for fair comparison.

Materials and Methods

Standard compounds ethyl propionate, ethyl 2-methyl propionate, methyl butyrate, ethyl

butyrate, ethyl 2-methyl butyrate, hexanal, b-pinene, b-myrcene, limonene, 2-methyl

butanol, ethyl hexanoate, p-cymene, octanal, 2-methyl 3-furanthiol, nonanal, ethyl

octanoate, methional, decanal, linalool, geranial, geraniol, a-ionone, g-decalactone, b-

damascenone and 2-methoxy 4-vinyl phenol were purchased from Aldrich (St. Louis,

MO). Natural 4-mercapto-4-methylpentan-2-one (1% in PG) was obtained from Oxford

Chemicals (Hartlepool, UK).

Grapefruit Juice

One hundred and fifty litres of unpasteurized red grapefruit juice with no additives

was procured from a commercial Florida processor. Shipping, storage and initial


119









treatment conditions have been described in chapter 5. Juice was filtered through 2

layers of cheesecloth before processing by PEF, RFEF, UV and thermal treatments.

Grapefruit Juice Processing

Preliminary experiments were performed to determine equivalent processing

conditions using heat, pulse electric field (PEF) radiofrequency electric field (RFEF) and

ultraviolet radiation (UV) for grapefruit juice. Juice was inoculated with E. coli K12

(ATCC 23716) from a stationary phase culture to give an approximately 7 log CFU/mL

population. Microbial assays were conducted for juice heated from 60-72 oC with UV

exposure times of 6-25s at 50 oC, 15-22 kV/cm electric field strengths for PEF treatment

and 0-2cycles at 20kHz frequency and 15kV/cm for RFEF treatment. Final optimized

processing conditions resulted in approximately 5 log reductions of E. coil K12 and were

used for the remainder of the study.

Heat treatment

Grapefruit juice was heat pasteurized using a miniature scale HTST processing

system (Armfield, Jackson, NJ, FT74-30-Mklll-33-34) explained previously in chapter 5.

Non-thermal juice treatments

The UV, PEF and RFEF juice treatment conditions have been described in chapter

5.

Packaging and storage

Processed grapefruit juice was collected directly from the processing unit outlet

into sterile 1L media glass bottles (Corning Inc, Corning, NY) inside a sanitary laminar

hood equipped with a HEPA air filter(Forma Scientific Inc., Marietta, OH). The hood was

sanitized by UV lighting at 254nm for 30min before use and then wiped with 100%

alcohol. The bottled juice was stored at 4 oC for 4 weeks. Volatile analysis of fresh


120









unpasteurized juice was performed at week 0. For all pasteurized juice samples,

volatiles analysis and sensory analysis were conducted at week 0 and week 4 (the end

of storage study). The reference sample used for week 4 sensory evaluation was

untreated grapefruit stored at -17 C and thawed overnight at 4 C one day before

analysis.

Extraction of Grapefruit Juice Volatiles

Aliquots (25 ml) of grapefruit juice were placed in 40 ml glass vials with screw caps

containing Teflon-coated septa. Tetradecane (1 pL of 1000ppm) was added as internal

standard into the juice before equilibration. The juice was equilibrated for 15 min at 40

C with stirring. A 2 cm-50/30pm, DVB/ CarboxenTM/PDMS StableflexTM

(Supelco,Bellefonte, PA) SPME fiber was exposed in the equilibrated headspace for 30

min at 40 C. The fiber was desorbed for 5 min in the GC injection port at 250 C. All

samples were analyzed in quadruplicate.

Volatile Analysis of Grapefruit Juice Volatiles

A GC/MS/O instrument (6890N GC, 5973N MS, Agilent Technologies, Santa

Carla, CA) and olfactory detector (Gerstel, Baltimore, MD) were used for separation and

analysis of volatiles. The GC effluent was split between MS/O in a ratio of 1:3.

Samples were run separately on a polar DB-Wax and non-polar DB-5 column with

identical dimensions (30 m x 0.32 mm x 0.5 pm from J&W Scientific, Folsom, CA). The

column oven temperature was programmed between 40 C to 250 C at 7 C/min with a

5 min hold at 250 C. The injector and detector temperatures were set at 250 oC.

Mass spectrometry conditions were as follows: transfer line temperature at 275 oC,

mass range 30 to 300 amu, scan rate 5.10 scan/s and ionization energy 70 eV. Helium

was used as the carrier gas at a flow rate of 2 mL/min. Mass spectral matches were


121









compared with NIST 2002 standard spectra. Authentic standards were used for

confirmation. Alkane linear index values were determined on both columns.

GCO analysis was performed using two trained sniffers, both non-smokers

between ages of 30-35 yrs. The panelists were trained in a similar manner as reported

by Bazemore and others (1999) using standard solutions of varying concentration of 10

compounds usually found in citrus juices: ethyl butyrate, octanal, hexanal, linalool, a-

pinene, limonene, geranial, neral, citronellal and a-terpineol. The panelists were trained

on intensity rating, optimum positioning and breathing techniques. The panelist seated

at the sniffing port rated the intensity of volatility on a 4-point intensity scale using an

olfactory pad (Gerstel, Baltimore, MD) and the aroma description was audio recorded.

The aroma intensity scale ranged from 0= no aroma perceived to 4 = strong aroma

perceived. The ODP output was integrated into the MSD Chemstation software (Agilent

MSD Productivity Chemstation version D.02) to achieve TIC chromatogram and

aromagram simultaneously. Samples were sniffed two times by each assessor,

resulting in four aromagrams. The results from 4 aromagrams were averaged for each

sample. Odor active compounds producing intensity responses at the same retention

time at least 50% times were selected. The odor active compounds were identified

based on sensory descriptor, retention index calculated, comparing RI and descriptor

from two columns and authentic standards.

Sensory Evaluation

Screening and training of panelists

The screening for panelists was performed using two different commercially

pasteurized grapefruit juices and fresh squeezed grapefruit juice. A total of 40 panelists

were screened for their ability to differentiate between the samples in two sessions.


122









Panelists who were consistently correct in differentiating juice samples were selected

for further training. Twenty-five panelists were selected after the initial screening. The

panel consisted of 12 males and 13 females in an age group between 25-45 yrs.

Selected panelists were further trained for 1- 2 hours using the same juice samples.

They were trained to use verbal terminologies or descriptors to differentiate between

samples and to use scaling procedures to determine the degree of difference between

the samples.

Test method

A difference from control type of sensory evaluation (Meilgaard and others 1999)

was employed to orthonasally detect differences in aroma between unpasteurized and

pasteurized grapefruit juice. All tests were conducted once in a sensory panel facility at

Eastern Regional Research Center (Wyndmoor, PA) with 6 booths equipped with

computers. The sensory analysis was designed and conducted using Compusense five

(Compusense Inc., Ontario, CA). A sample sensory ballot is shown in Appendix D.

Samples were prepared by pouring 40 mL of juice into 100 mL glass bottles with airtight

plastic caps and stored in boxes at 4 C overnight. On the day of testing, the bottles

were taken out 1 hr before the testing. Juice temperature was measured at 10-12 C

during testing. Testing was performed under red light so that color and other visible

differences were masked from panelists. The control sample given to panelists was

fresh untreated grapefruit juice and labeled as "R." The experimental samples consisted

of the 4 treated juice samples (thermal, UV, PEF & RFEF). Each panelist received 1 R

sample and 5 coded samples corresponding to the 4 experimental samples and 1 blind

control (unpasteurized). Panelists were required to specify the degree of difference on a


123









scale of 0-10, 0 = no difference and 10 = extremely large difference. Panelists were also

asked to give verbal descriptions of the difference between samples.

Statistical Analysis

Sensory data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc.,

Raleigh, N.C., USA). The tests for statistical significance were performed by analysis of

variance (ANOVA) at a significance level of a = 0.05. To determine which means are

significantly different from one another, Tukey's test (a = 0.05) was applied.

Results and Discussion

Effect of Treatment and Storage on Grapefruit Juice Volatiles

The volatiles in grapefruit juice were quantified using an internal standard

(tetradecane) by GC/MS. A total ion chromatogram (TIC) of volatiles from untreated

grapefruit juice is shown in Appendix Figure C-1. The volatile concentrations for

untreated and treated grapefruit at week 0 and week 4 are given in Appendix Tables C-

1 and C-2, respectively. The percentage loss in volatiles was calculated for thermally

and non-thermally treated grapefruit juice compared to untreated juice during storage

(Table 6-1). Concentrations of all volatiles except a-terpineol and linalool were

diminished in treated samples at week 0. P-cymenene lost more than 50% and

nootkatone lost more than 40% during the same time period. After 4 weeks storage,

hexanal and ethyl hexanoate were completely lost and decanal and octanal lost more

than 80% in all treated samples. The loss in aldehydes during storage occurs due to

their conversion into corresponding alcohols (Petersen and others 1998). Octanol levels

increased (2-14%) in all treated juice samples. Alpa-terpineol increased by more than

80% in all treated samples. Alpha-terpineol is formed in stored orange juice by acid-

catalyzed hydration of limonene and linalool (Petersen and others 1998). In the present


124









study, a 15-30% loss in limonene and linalool content was noted in treated samples.

The results from week 0 and week 4 analysis suggest that the volatiles of grapefruit

juice are more strongly affected by storage rather than treatment type.

Gas Chromatography Olfactometry Profile of Grapefruit Juice

A total of 38 aroma active components were detected in the headspace of fresh

untreated and treated grapefruit juice, as listed in Table 6-2. Odorants were identified

based on retention indices on two dissimilar columns, odor description and GCO data

from standard compounds (except for compounds 24, 26 and 32). These compounds

were identified based on retention index and odor descriptors. Nine odorants (1, 2, 19,

21, 28, 33, 34, 36 and 38) remain unknown. The odorants in GFJ could be classified

into 5 groups based on similar aroma notes comparable to those of Lin and others

(2002b): fruity/sweet, green, citrus/grapefruit, floral/musky/fragrant, terpeny/piney and

cooked/ roasted/catty.

Sweet/fruity. The sweet/fruity category had the highest number of odorants (11

odorants). The majority were identified as esters: ethyl propionate, methyl butyrate,

ethyl butyrate, ethyl 2-methyl butyrate, ethyl 3-methyl propionate, ethyl hexanoate and

ethyl octanoate. Ethyl octanoate is one of the odorants with the highest aroma value in

the present study. Other compounds belonging to this group were b-damascenone (30),

g-decalactone (35) and two unknowns (33, 34).

Fresh/citrus. Six odorants belonged to the fresh/citrus group. They include

aldehydes (15, 17, 23, 27), alcohol (30) and terpene hydrocarbon (11). Aldehydes such

as octanal, nonanal, decanal and geranial are common to citrus fruits like orange,

grapefruit, and lemon (Allegrone and others 2006; Lin et al. 2002b). Limonene with


125









citrus aroma has been shown to be an important ingredient in orange juice by omission

studies in model flavor mixtures (Buettner and Schieberle 2001).

Cooked/roasted/catty. The group includes the 4 sulfur compounds: 2-methyl 3-

furnathiol (MFT), methional, bis (2-methyl 3-furfuryl) disulfide (MFT-MFT), 4-mercaptone

4-methyl 2-pentanone (4-MMP) and two unknowns (19, 38). The meaty odorant MFT is

a product of thiamine degradation under acidic conditions. It has low odor threshold at

0.007ppm in water. It can also be produced by Maillard reaction between cysteine and

pentose sugar and is often found in heat treated citrus juices (Perez-Cacho and Rouseff

2008). It readily forms a dimer, MFT-MFT, which also has a meaty odor. Methional has

a cooked potato odor and is reported in many fresh fruits like orange, tomato and

grapefruit (Perez-Cacho and others 2008). Its biosynthetic pathway in plants is

unknown, but it can be formed in processed juices by Strecker degradation from the

amino acid methionine during thermal treatments and subsequent storage. Methional

and 2-methyl 3-furanthiol are reported as possible off flavors in orange juice (Bezman

and others 2001). 4-MMP is significant for characteristic grapefruit aroma. It has very

low odor threshold of 0.1 ng/L in water (Buettner and Schieberle 2001). It is reported to

possess a grapefruit aroma at very low concentrations and a catty sulfurous odor at

higher concentrations.

Green/fresh. The green/fresh odor of grapefruit juice is imparted by odorants

hexanal, p-cymene, decanal and 4, 5-epoxy (E)-2-decenal and one unknown (22).

Odorant 4, 5-epoxy (E)-2- decenal has a green metallic odor and is a key ingredient in

fresh grapefruit juice (Buettner and Schieberle 2001).


126









Floral/fragrant. Linalool, 4-vinyl guaiacol, a-ionone and 2 unknowns (1, 28) have

a floral fragrant aroma. Linalool, with its intense floral aroma, is another component that

is commonly found in citrus fruits. Para-viny guaiacol is formed from ferulic acid present

in citrus peels (Perez-Cacho and Rouseff 2008). The presence of this compound is

usually an indicator of thermally abused or aged juice. Alpha-ionone is formed from their

carotenoid precursors in orange juice (Mahattanatawee and others 2005).

Terpeny. Only two odorants, b-pinene and b-myrcene, are responsible for the

terpeny odor in grapefruit juice.

In fresh untreated GFJ, 24 odor active compounds were perceived. GCO analysis

revealed ethyl octanoate and octanal as having the highest aroma intensities, followed

by ethyl butyrate, ethyl hexanoate, methional and decanal. The results are different from

Buettner and others (2001) who reported high FD values for ethyl butyrate, (Z)-3-

hexenal, 1-hepten-3-one, 4-mercapto-4-methyl 2-pentanone, 1-p-menthene -8-thiol, 4,5-

epoxy-(E)-2-decenal and wine lactone in fresh white Marsh grapefruit juice by aroma

extract dilution analysis Except for ethyl butyrate and 4, 5-epoxy-(E)-2-decenal, other

volatiles were not detected in fresh juice in our study. Lin and others (2001) also found

qualitative, as well as odor intensity, differences for the majority of odor active

compounds when compared to results from Buettner and others (2001). Possible

reasons for the differences could be due to variety, fruit quality, juice extraction

methods, storage time and temperature, volatile extraction method and GCO analysis

methods. The grapefruit variety used by Buettner and others (2001) was white Marsh,

whereas in the present study red grapefruit was used. Secondly, the extraction method

used by Buettner and others (2001) was liquid extraction, compared to static headspace


127









SPME used in this work. The quantitative and qualitative volatile profile depends on the

extraction method used. Rouseff and others (2001) showed that the terpenes limonene,

a-pinene and myrcene comprised 86% of the total FID area by SPME compared to 24%

from liquid extraction of the same orange juice. Low vapor pressure compounds are not

readily extracted by SPME. This could be the reason for absence of wine lactone in our

study. Reconstitution of odorants in a model grapefruit juice flavor mixture by Buettner

and others (2001) showed that omission of 4-MMP resulted in a more orange-like odor

rather than grapefruit odor. The authors concluded that 4-MMP is important for typical

grapefruit juice odor. Surprisingly, in our studies the volatile was found only in UV and

thermally treated juice (but not in fresh juice). The possible reasons for the absence of

4-MMP in our studies could be the different extraction methods used. The concentration

of 4-MMP in juice is very low, ranging from 0.8-1 ng/L (Buettner and others, 2001).

Possibly, the HS-SPME used in the present study may not be as efficient as solvent

extraction of 4-MMP at such low levels. Sulfur compound, 1-p-mentha-8-thiol with a

typical grapefruit like odor is reported as a character impact odorant in grapefruit juice.

However, the significance of this compound to grapefruit juice aroma is debatable, since

Lin and others (Lin et al. 2002b) reported the presence of this thiol only in concentrated

juice and not in the original juice. They proposed that the compound was possibly a

reaction product of limonene or a-pinene with hydrogen sulfide in thermally abused

juices, and therefore may not be present in fresh untreated juice.

Gas Chromatography Olfactometry Profile Comparison of Fresh Untreated and
Treated Juice at Week 0

The total number of odorants perceived in fresh, UV, thermal, PEF and RFEF

treated GFJ were 24, 27, 23, 18 and 20, respectively (Table 6-2). A comparative profile


128









based on the six odor categories for treated and untreated juice is plotted as a spider

web diagram in Figure 6-1. The panel average aroma intensities for odorants belonging

to same odor category are added to get the total odor intensity for that particular group

(Table 6-3). It is evident from Figure 6-1 that the aroma profile of fresh juice was

distinctly different from all treated juices. In general, a decrease in fruity/sweet and

citrus/fresh notes is observed for treated juices compared to fresh. Cooked/meaty/catty

odor increased in thermal and UV juice considerably. Terpeny odor was the least

affected by pasteurization techniques. The impact of treatments on odorants belonging

to the six aroma categories are discussed in detail below.

Thermal treatment

The total odor intensity for fruity/sweet, citrus/fresh and green/metallic decreased

in thermally treated juice by 4.7, 3.4 and 2.2 points, respectively, compared to fresh

juice. In the fruity/sweet category, esters like ethyl butyrate, methyl butyrate and ethyl 2-

mehtyl butyrate had lower a intensity, whereas ethyl propionate was completely absent.

Nonanal, geraniol and geranial belonging to the fresh/citrus group were absent in

thermal samples. The green metallic odorant 4, 5-epoxy (E)-2-decenal, important for

grapefruit odor, was also missing in thermally processed juice. The results indicate the

instability or degradation of the above compounds in the presence of heat. The sweet

odorant b-damascenone was detected in thermal juice but not in untreated juice. The

compound has been reported in reconstituted grapefruit juice, which is thermally

processed (Lin and others 2002b). They proposed that there is a release of

gylcosydically bound b-damascenone by acid hydrolysis during heating. A

cooked/meaty/catty odor was increased in thermal juice by 4.8 points compared to fresh

juice due to the formation of MFT, MFT-MFT, 4-MMP and an unknown (19). The meaty


129









odorant MFT could be formed either by Maillard reaction between cysteine and pentose

or from thiamin degradation. The level of cysteine present in red grapefruit juice is 15.3

pmol/L, and pentoses are present in ample amounts in grapefruit juice (Kusmierek and

Bald 2008) Rouseff and others, 2001). Formation of MFT via the Maillard reaction

during heat treatment in grapefruit juice is possible. MFT dimerizes in juice to form the

meaty odorant bis (2-methyl-3-furfurl) disulfide. 4-MMP is present in grapes as a

cysteine conjugate that is released during fermentation by yeast enzymes. Thermal

hydrolysis could result in the release of 4-MMP from such a precursor in heat treated

juice.

Ultraviolet treatment

No major change in sweet/fruity intensity was noted in UV treated juice. A

decrease in citrus/fresh and green/metallic odor by 3 and 1.2 points, respectively was

noted due to the absence of odorants (Z)-2-nonenal, geranial, geraniol and 4,5-epoxy

(E)-2-decenal. Beta-damascenone was also detected in UV treated juice. Thermal

degradation of neoxanthin in a model system containing peroxy acetic acid produced (3-

damascenone. However, the reaction temperatures used for this synthesis were high

(60-90 C) and lasted for extended periods of time (Bezman et al. 2001). The

mechanism of formation and release of P3-damascenone in UV treated samples is not

known.

An increase in cooked/catty odor by 4.2 points was observed in UV treated juice.

The catty odor of 4-MMP was more intense in UV treated juice than in thermally treated

juice. It is surprising to find the meaty odorant MFT-MFT in UV juice because of the

absence of its monomer, MFT. The odor of MFT-MFT is 8.9x10-11 mM in water, whereas

the odor threshold for MFT is 6.14x10-8 mM in water (Dreher and others, 2003).


130









Because the odor threshold of the dimer is lower than the monomer, it is possible that

the concentration of 2-methyl 3-furnathiol may be present below odor detection limits in

UV juice.

Pulsed electric field treatment

Similar to thermal treatment, a decrease in fruity/sweet as well as citrus/fresh odor

was noted in PEF juice. The compound 4, 5-epoxy (E)-2-decenal was also missing in

PEF treated juice. The increase in cooked/catty odor was less compared to that of

thermal and UV treated juice.

Radio frequency electric field treatment

RFEF treated grapefruit had an odor profile similar to PEF juice. This is expected

because both pasteurization techniques use electric field strength for inactivation of

microorganisms. The aroma profile had lower sweet/fruity and citrus characters

compared to fresh juice and juice with a slightly higher cooked/meaty odor.

Gas Chromatography Olfactometry Comparison of Fresh Untreated and Treated
Juice at Week 4

After 4 weeks of storage, the total number of odorants decreased to 14, 13, 12 and

15, respectively for thermal, PEF, RFEF and UV treated juice (Table 6-4). 14 total

odorants were missing in week 4 treated samples compared to untreated juice analyzed

at week 0. Guaiacol, with a medicinal odor, was detected only in UV treated samples

due to microbial contamination. This compound is formed by spoilage microorganisms

like Alicyclobacillus sp. in stored juice (Gocmen D 2005). However, it was not detected

in PEF and RFEF samples, even though both these samples had microbial

contamination. A possible reason for this discrepancy could be bottle-to-bottle variation

in microbial levels during sampling.


131









Sensory Evaluation

A difference from control type of sensory analysis was performed on grapefruit

juice aromas at week 0 and week 4 (Appendix D-1 and D-2). This type of sensory

analysis tells us whether a difference exits between the samples and tells us the

magnitude of difference between two samples. The data recorded by 25 panelists for

differences from the control test between pasteurized and unpasteurized grapefruit juice

at week 0 is given in Appendix D-1. Statistical significance between samples was

determined using an ANOVA test at 95% confidence levels through an F test. Table 6-5

shows that the Fcal value is greater than the Fcrit value. This indicates a statistical

difference between sample means at week 0.

Tukey's test is a single-step multiple comparison procedure. It is performed in

conjunction with ANOVA to find if there is significant difference between treatment

means. The HSD (honestly significant difference) value calculated was 1.58 for week 0

mean values. An HSD value higher than the difference between two sample means

indicates a significant difference between two treatment means. It is evident from Table

6-6 that the difference in mean values between treatments is lower than the HSD value.

The mean values for treated juice are significantly (p<0.05) different from each other

because the calculated HSD is greater than the treatment differences. The difference in

means is highest between UV and fresh untreated juice. The general comments from

panelists for UV treated juice were that the juice had a rotten or spoiled odor. Because

no microbial spoilage was noted in UV samples at week 0, the sensory comments are

possibly a reflection of increased 4-MMP levels in UV samples. Thermally treated juice

was described as having a cooked odor. The presence of 2-methyl 3-furnathiol and bis

(2-methyl-3-furfuryl) disulfide would give a cooked note to the juice. According to


132









panelists' comments, both PEF and RFEF juice had weaker/less fresh aromas

compared to untreated juice

The ANOVA results for week 4 sensory analyses (Table 6-7) indicate a significant

difference (a = 0.05) between sample means. The HSD value calculated was 1.85.

Table 6-8 shows that the difference in means between PEF and thermal samples are

higher than the HSD value, indicating a significant difference (p<0.05) between the two

samples. No significant difference (p<0.05) in sample means was noted between

RFEF, UV and thermally treated juices. Panelists commented that all treated juice

samples except thermal had a fermented, rotten odor. This is due to microbial spoilage

detected in PEF, RFEF and UV samples after 4 weeks of storage (chapter 5). Thermal

samples had a cooked odor.

Conclusion

The results from this study indicate that thermal as well as non-thermal

pasteurization techniques affected the aroma profile of fresh grapefruit juice. The effects

of both thermal and non-thermal treatments on juice aroma were characterized by

losses in desirable fruity and citrus odorants, along with an increase in undesirable

cooked/catty odorants. Aroma of treated juices differed significantly (p<0.05) from fresh

juice by sensory evaluation, further confirming GCO results. Further sensory studies,

such as preference evaluation, need to be conducted to understand consumer

preferences between thermally or non-thermally treated grapefruit juice.


133









fruity/sweet


cooked/meaty/catty






green/metallic


floral/fragrant


terpeny


- fresh
- UV
- thermal
PEF
- RFEF


fresh/citrus
Figure 6-1. Comparative aroma profile for unpasteurized and pasteurized grapefruit
juice at week 0


134









Table 6-1 .Change in GC-MS grapefruit juice volatiles during 4 weeks of storage at 4 C
%loss %loss
Compound weekO week
RI Identified PEF RFEF UV Thermal PEF RFEF UV Thermal
888 ethyl acetate 9.88 13.64 16.54 10.50 41.59 29.87 26.68 37.17
1037 a-pinene 0.84 3.94 3.72 1.43 15.28 38.81 20.24 32.90
1049 ethyl butyrate 19.69 33.23 28.59 30.62 45.90 53.40 88.66 53.75
1098 Hexanal 1.70 9.91 4.58 2.69 100.00 100.00 100.00 100.00
1174 b-myrcene 3.47 6.39 7.40 7.55 8.55 15.92 20.42 31.76
1219 Limonene 4.59 8.20 13.34 12.01 14.38 26.99 25.09 24.77
1243 ethyl hexanaote 21.05 20.53 29.09 18.55 100.00 100.00 100.00 100.00
1260 3-carene 18.02 26.74 27.10 14.34 82.91 31.74 57.64 58.31
1285 p-cymene 35.71 29.30 25.45 25.98 44.67 40.00 53.48 55.83
1299 Octanal 6.55 13.58 17.16 5.20 94.72 95.54 95.38 94.39
1400 Nonanal 6.01 23.40 14.78 23.17 40.85 69.26 55.48 71.35
1436 ethyl octanoate 15.49 32.89 4.61 13.11 19.81 29.21 24.15 22.71
1447 p-cymenene 54.37 57.67 67.79 51.16 75.91 72.78 76.10 78.63

1501 Decanal 4.74 2.18 8.75 3.35 80.84 56.23 83.77 87.71
1506 a-copaene 1.74 29.81 16.12 14.29 29.32 41.45 46.41 57.69
1537 Linalool 14.33 10.66 13.74 13.52 14.86 30.30 27.01 23.88
1549 1-octanol 0.57 -1.98 -4.42 -5.53 -2.14 -6.00 -6.11 -13.78
1608 b-caryophyllene 0.14 1.31 1.48 0.91 5.16 2.17 4.39 10.15
1670 a-caryophyllene 2.16 2.00 1.73 2.53 19.37 10.88 3.84 11.95
1709 Valencene 11.35 32.55 19.00 12.55 65.39 67.03 62.01 63.28
1721 a-selinene 0.38 3.52 3.16 2.02 4.46 2.11 11.12 11.44
1728 a-terpineol -13.97 -3.35 -4.91 -8.43 -80.87 -99.71 -98.34 -85.67
1735 d-cadinene 13.71 27.72 10.97 23.95 31.93 34.74 31.93 34.74
1747 a-panasinsen 7.81 9.19 5.21 11.94 21.89 28.72 25.21 31.97
2677 Nootkatone 39.35 46.81 47.98 47.65 48.10 53.52 56.80 55.97


135









Table 6-2. GCO analysis results of pasteurized and unpasteurized grapefruit juice
No DB- DB- descriptor Compound untre UV Ther PEF RFEF
5 Wax ated mal
P t1 I fliral Imronn I InL-nrwnAi I


823
932
1018
1033
1046
1058
1091
1117
1172
1217
1227
1241
1285
1296
1326
1372
1400

1428
1434
1448
1460
1504
1517
1535
1661


I ll ..I I4 11 I% 1 l i y
bad off
ethanol
fruity ethanol
Sweet
fruity sweet
fruity strawberry
green grass
Piney
musty terpene
fresh citrusy minty
sweet fruity
sweet fruity
green apple skin
fresh citrus
meaty roasted
green citrus
sulfur catty

cooked unpleasant
Fruity
grass musky
cooked potato
citrus green
fatty green
floral fresh
cooked meaty


\^Iil III 1/VVl I
Unknown
ethyl propanoate
ethyl 2-methyl propionate
methyl butyrate
ethyl butyrate
ethyl 2-methyl butyrate
Hexanal
b-pinene
b-myrcene
Limonene
2-methyl butanol
ethyl hexanoate
p-cymene
Octanal
2-methyl 3-furanthiol
Nonanal
4-mercapto 4-methylpentan-2-
one
Unknown
ethyl octanoate
Unknown
Methional
Decanal
(Z)-2-nonenal*
Linalool
bis (2-methyl-3-furfuryl) disulfide*


912
782
803



974
1022
1030
979

985
856

961


1177

889
1190

1081


1.5

2.0
1.5

1.5
1.5


136


0.5
1.5 1.0
2.3 2.0
2.5 2.5
2.5 2.5
2.0 1.0
1.0 1.0
1.0 1.0
1.3 1.0
1.3 1.0
0.8
2.5 2.3
0.8
3.0 2.5

1.5 1.0
2.5


1.0 0.8









Table


6-2. Continued


No. DB-5 DB-Wax descriptor Compound untrea UV therm PEF RFEF
ted al

27 1749 sweet citrus geranial 0.8 -
28 1789 floral unknown 1.5
29 1822 sweet b-damascenone 0.5 0.5 -
30 1840 citrusy floral geraniol 0.5 -
31 1873 floral sweet a-ionone 1.5
32 1985 green metallic 4,5-epoxy (E)-2-decenal* 1.0 -
33 2013 sweet fruity unknown 0.5
34 2055 fresh fruity unknown 0.8 -
35 1489 2069 buttery fragrant peachy g-decalactone 1.5 1.8 1.8 1.0 1.5
36 2163 sweet ethanolic unknown 1.8 -
37 2196 2187 musky cologne 4-vinyl guaiacol 1.8 2.0 1.5 1.8 2.0
38 2240 bad solvent unknown 1.0 -
* Odorants identified based on odor descriptor and retention indices with reported data due to unavailability of data









Table 6-3. Calculated total aroma intensity values for pasteurized and unpasteurized
grapefruit juice belonging to five odor categories
Aroma intensity
Odor category Compounds fresh UV their PEF RFE
mal F
fr iuf scAi t e+h\/ I ro ann at _1 n


11'lLy \J V //


linalool
unknown
a-ionone
4-vinyl guaiacol
total intensity

b-pinene
b-myrcene
total intensity


ethyl 2-methyl propionate
methyl butyrate
ethyl butyrate
ethyl 2-methyl butyrate
2-methyl butanol
ethyl hexanoate
b-damascenone
unknown
unknown
g-decalactone
unknown
total intensity


limonene
octanal
nonanal
geranial
decanal
geraniol
total intensity
hexanal
p-cymene
unknown
(Z)-2-nonenal
4,5-epoxy (E)-2-decenal
total intensity


I J I .w
2.3 2.0
2.5 2.5
2.5 2.5
2.0 1.0
0.8
2.5 2.3
0.5
0.5
0.8
1.5 1.8
1.8
5.5 16.6


1.3
1.5
2.5
1.3

2.0
0.5


1.8

10.8


1.8 1.5
1.0 1.5
2.3 1.5
1.0 0.8

2.5 2.0




1.0 1.5

9.6 8.8


*


1.3 1.3 1.0
2.3 1.8 2.0
0.5 1.0 0.8

1.5 1.5 1.8

5.6 5.5 5.6
0.8 1.0 1.0

0.8


1.0 1.8


1.5 2.0 1.0 1.5
1.5

2.0 1.5 1.8 2.0
3.5 3.5 2.8 5.0


0.5
1.8
2.3


1.0
1.3
2.3


138


fresh/citrus







green/metallic







floral/fragrant


Terpeny


1









Table 6-3. Continued
Aroma intensity
Odor category Compounds fresh UV their PEF RFE
mal F
cooked/meaty/cat 2-methyl 3-furanthiol 1.5
ty unknown 0.5 -
4-mercapto 4-methyl pentan-2- 2.5 1.0 -
one
methional 2.3 2.0 2.5 2.3 2.0
bis (2-methyl-3-furfuryl) disulfide 1.5 1.3 1.5 1.3
unknown 0.8 -
total intensity 2.3 6.5 7.1 3.8 3.3


139









Table 6-4 GCO comparison of pasteurized grapefruit juice at week 4 to unpasteurized grapefruit juice at week 0
No DB- DB- descriptor compound untre UV therm PEF RFEF
5 Wax ated al


I QV93


912
782
803



974
1022
1030
979
985
856

961


1018
1033
1046
1058
1091
1117
1172
1217
1227
1241
1296
1326
1372
1400


1177 1434
889 1460
1190 1504
1517
1081 1535
1664
1661
1720
1749
1822
1840
1445 1875


Ethanol
fruity ethanol
Sweet
fruity sweet
fruity strawberry
green grass
Piney
musty terpene
fresh citrusy minty
sweet fruity
sweet fruity
fresh citrus
meaty roasted
green citrus
sulfur catty

Fruity
cooked potato
citrus green
fatty green
floral fresh
stinky, rotten
cooked meaty
grassy floral
sweet citrus
sweet
citrusy floral
medicinal


hte l ro anoate 1 5


ethyl 2-methyl propionate
methyl butyrate
ethyl butyrate
ethyl 2-methyl butyrate
Hexanal
b-pinene
b-myrcene
Limonene
2-methyl butanol
ethyl hexanoate
octanal
2-methyl 3-furanthiol
Nonanal
4-mercapto 4-methylpentan-2-
one
ethyl octanoate
methional
decanal
(Z)-2-nonenal*
linalool
unknown
bis (2-methyl-3-furfuryl) disulfide*
unknown
geranial
b-damascenone
geraniol
guaiacol


I .
2.3
2.5
2.5
2.0
1.0
1.0
1.3
1.3
0.8
2.5
3.0

1.5


3.0
2.3
2.0
1.0
1.8


0.8

0.5


1.0 1.5
1.0

1.0 0.5


1.0
2.0
1.5

1.5
1.0
1.5


1.0
2.5
1.5

2.0

1.3


0.5

0.5


140









Table 6-4. Continued
No. DB-5 DB- descriptor compound untreated UV PEF RFEF
Wax thermal
28 1873 floral sweet a-ionone 1.5 -
29 1985 green metallic 4,5-epoxy (E)-2-decenal* 1.0 -
30 2055 fresh fruity unknown 0.8 -
31 1489 2069 buttery peachy g-decalactone 1.5 1.0 1.5 1.0 1.5
32 2196 2187 musky cologne 4-vinyl guaiacol 1.8 2.5 2.5 2.3 2.0
33 1358 2647 dirty musty unknown 0.8 -
* Odorants identified based on odor descriptor and retention indices with reported data due to unavailability of standards









Table 6-5. Results of ANOVA for mean values from difference from control test
ANOVA
Source of Variation SS df MS F P-value F crit
Between Treatments 249.57 4 62.39 23.3 3E-14 2.447
Within panelists 321.76 120 2.681
Total 571.33 124




Table 6-6. Difference in means between pasteurized and unpasteurized grapefruit juice
Mean values control PEF RF thermal UV
Mean values 1.32 3.24 3.36 3.64 4.60
Control 1.32 1.92 2.04 2.32 3.28
PEF 3.24 0.12 0.40 1.36
RF 3.36 0.28 1.24
Thermal 3.64 0.96
UV 4.60


Table 6-7. Results of ANOVA of mean values from difference control test at week 4
Source of Variation SS df MS F P-value F crit
Between treatments 414.38 4 103.59 18.61 1.05E-11 2.45
Within panelists 612.34 110 5.56

Total 1026.73 114


Table 6-8. Difference
at week 4


in means between pasteurized and unpasteurized grapefruit juice


control PEF RF thermal UV
mean
values 1.96 7.13 6.43 5.08 6.82
Control 1.96 5.17 4.47 3.12 4.86
PEF 7.13 0.70 2.05 0.31
RF 6.43 1.35 0.39
Thermal 5.08 1.74
UV 6.82


142









CHAPTER 7
CONCLUSION

Non-thermal processing has been extensively researched over the past few years

as an alternative to heat pasteurization. However, non-thermal processes are often

compared to thermal processes using conditions that did not achieve the same

reduction in concerned microorganismss. This made a fair comparison of their effects

on juice quality impossible. Therefore, the objective of this study was to compare the

quality of apple cider and grapefruit juice treated by thermal and non-thermal techniques

where treatment conditions were carefully selected to achieve a similar reduction in E.

coil k12 population. The non-thermal techniques selected for the study were PEF, RFEF

and UV. The quality attributes studied were aroma, sensory, microbial, physical,

enzymatic and nutritional.

In apple cider, microbial inactivation by PEF technique was comparable to heat

pasteurization. UV treated cider had a shelf life of only 2 weeks. Other attributes like

aroma volatiles, color and sensory quality was better preserved in PEF treated cider

compared to thermally and UV treated cider. Comparing the three techniques, PEF

shows the best promise as a future pasteurization technique for the preservation of

apple cider.

In grapefruit juice, the thermal treatment was more effective when compared to

non-thermal treatments in terms of microbial stability after 4 weeks storage. The

sensory quality of juice was affected by all treatments. UV treated grapefruit juice

differed the most compared to the control juice, followed by thermal, RFEF and PEF,

respectively. The impact of treatments on odorants was highlighted by a loss of

desirable fruity and citrus odorants, along with an increase in undesirable cooked or


143









catty odorants. RFEF treatment ensured grapefruit juice microbial safety for 3 weeks of

storage with higher Vitamin C content and equivalent PME inactivation compared to

thermally treated juice. PEF and UV treatments maintained good microbial quality for

only 1 week. Non-thermally treated juice had a shorter shelf life but better nutritional and

physical qualities compared to thermally treated juice.

The current study has shown that the effects of thermal and non-thermal

treatments on juice quality are not the same at equivalent process conditions. The

thermal technique consistently showed good microbial quality during storage compared

to non-thermal techniques. However, organoleptic and nutritional qualities of juice were

better preserved in non-thermally treated juice than in thermally treated juice. The

efficacy of non-thermal treatment in extending shelf life also depends on the type of

food product. Though it is difficult for any non-thermal technique to completely replace

thermal process, some of them might find niche applications for certain products that

are sensitive to heat.

For commercial success, non-thermal technologies should show cost

effectiveness and pathogen control comparable to thermal treatment. To achieve this

goal, combinations of these techniques with traditional or emerging food reservations

techniques where a synergistic effect in terms of improving food quality and safety is

achieved needs to be explored. This combination of new and traditional techniques is

commonly called "hurdle technology." Moreover, it is necessary to find the niche

products for applications of these technologies by conducting more detailed research on

nutritional and organoleptic properties of the product. In the future, consumer demands


144









for fresh and minimally processed food would definitely help in the acceptance and

commercialization of non-thermal processing techniques by the food industry.


145








APPENDIX A
CHANGE IN APPLE CIDER APPEARANCE DURING STORAGE


Control





PEF


Thermal


v771N


Week 0


P ssM


Week 2






Week 4


SO pp 1=


Figure A-1. Change in appearance in treated and untreated apple cider during 4 weeks
of storage at 40C


146


*=-------*









APPENDIX B
CALIBRATION TABLE FOR APPLE CIDER


Table B-1. Regression equation for standard compounds
Compound Regression equation
hexyl hexanoate y = 139805x 26161
p-allyl anisole y = 57923x + 46022
butyl propionate y = 105603x + 65342
2-methyl hexyl butyrate y = 70481x 40381
1 -octanol y = 10696x 25739
6-methyl hepten-5-en-2-one y = 759936x + 10268
pentyl acetate y = 184623x + 56243
2-hexen-1-ol, acetate y = 54292x + 11218
2-hexen-1 ol (z) y = 83730x + 51963
ethyl propionate y = 316024x 592703
ethyl butyrate y = 23796x + 144461
ethyl hexanoate y = 121026x 32819
Farnesene y = 5E+08x + 24019
butyl 2-methyl butyrate y = 106763x + 15192
hexyl acetate y = 43260x + 544732
2methyl butyl acetate y = 29856x + 250422
2methyl butanol y = 1820.2x + 28272
1-hexanol y = 12069x + 111609
butyl acetate y = 16391x + 184478
Hexanal y = 12165x + 140960
2-methyl ethyl butyrate y = 79357x 147057
2-e-hexenal y = 11392x 226626
propyl butyrate y = 107921x 408031
Benzaldehyde y = 51164x + 361796
butyl butyrate y = 92039x 52362
hexyl propionate y = 86064x 117617
propyl hexanoate y = 125122x 319336
hexyl butyrate y = 71844x 38727
methyl butyrate y = 21678x 51380


VOLATILES


Correlation
0.9769
0.9825
0.9703
0.9604
0.9949
0.9851
0.8015
0.9784
0.9921
0.8861
0.9619
0.998
0.9057
0.9679
0.9839
0.97
0.9183
0.9967
0.9514
0.9609
0.9332
0.9395
0.9754
0.9422
0.9639
0.9467
0.8724
0.9595
0.8882


147


Base ion
117
148
57
103
56
43
70
67
57
57
71
88
93
103
56
70
57
56
56
56
102
69
71
106
71
57
99
71
74









APPENDIX C
GRAPEFRUIT JUICE VOLATILE CONCENTRATION


148









Table C-1.Volatile concentration (pg/L) in untreated (control) and treated grapefruit juice at weekO


RT
4.45
6.50
6.70
7.56
9.06
9.97
10.46
10.82
11.33
11.61
13.61
14.29
14.51
15.52
15.63
16.20
16.44
17.44
17.55
18.77
19.54
19.77
20.05
20.31
30.56


LRI
888
1037
1049
1098
1174
1219
1243
1260
1285
1299
1400
1436
1447
1501
1506
1537
1549
1602
1608
1670
1709
1721
1735
1747
2677


compound
identified
ethyl acetate
a-pinene
ethyl butyrate
Hexanal
b-myrcene
Limonene
ethyl hexanaote
3-carene
p-cymene
Octanal
Nonanal
ethyl octanoate
dimethyl styrene
Decanal
a-copaene
Linalool
1 -octanol
terpinen-4-ol
b-caryophyllene
a-caryophyllene
Valencene
a-selinene
d-cadinene
a-panasinsen
Nootkatone


control
6.490.73
1.360.09
9.470.34
2.980.06
129.014.73
2309.5021.47
32.565.03
15.542.83
15.630.92
78.872.84
8.871.47
1.350.06
5.150.46
16.322.82
6.031.10
1.790.22
9.892.91
2.920.61
135.994.83
14.942.58
3.701.19
4.071.38
4.632.37
1.450.03
6.132.82


PEF
5.850.29
1.350.12
7.601.32
2.930.05
124.403.68
2203.5835.92
25.713.18
12.742.25
10.052.63
73.703.78
8.332.16
1.140.02
2.350.27
15.552.32
5.922.17
1.570.14
9.832.34
3.331.80
135.795.20
14.621.79
3.281.03
4.081.22
4.462.18
1.340.06
3.721.38


RFEF
5.611.25
1.300.03
6.322.17
2.090.08
120.764.23
2120.1123.28
25.883.73
11.382.04
11.051.27
68.222.69
6.791.30
0.910.03
2.181.02
15.973.26
4.232.38
1.810.44
9.692.82
3.021.99
86.61 4.27
15.243.23
2.491.17
3.111.83
3.342.08
1.320.70
3.262.68


149


5.422.03
1.170.21
6.762.74
2.841.02
119.464.29
2001.3942.83
23.093.25
11.335.27
11.662.99
65.345.24
7.561.47
1.290.93
1.661.11
14.892.46
5.052.25
1.890.29
10.322.47
2.971.72
133.974.88
14.694.16
4.031.81
4.602.38
4.121.32
1.381.28
3.192.01


Thermal
6.402.47
1.341.04
6.572.46
2.902.12
119.275.79
2032.1228.71
26.482.08
13.312.56
11.572.18
74.774.43
6.813.23
1.180.07
2.511.16
15.783.52
5.172.76
1.860.23
10.433.73
3.172.01
134.756.23
14.574.29
3.792.08
4.882.74
3.521.72
1.280.84
3.212.22









Table C-2. Volatile concentration (pg/L) in untreated (control week 0) and treated grapefruit juice at week 4

RT LRI compound identified Control PEF RFEF UV thermal


4.45
6.50
6.70
7.56
9.06
9.97
10.46
10.82
11.33
11.61
13.61
14.29
14.51
15.52
15.63
16.20
16.44
17.44
17.55
18.77
19.54
19.77
20.05
20.31
30.56


888
1037
1049
1098
1174
1219
1243
1260
1285
1299
1400
1436
1447
1501
1506
1537
1549
1602
1608
1670
1709
1721
1735
1747
2677


ethyl acetate
a-pinene
ethyl butyrate
Hexanal
b-myrcene
limonene
ethyl hexanaote
3-carene
p-cymene
octanal
nonanal
ethyl octanoate
dimethyl styrene
decanal
a-copaene
linalool
1 -octanol
terpinen-4-ol
b-caryophyllene
a-caryophyllene
valencene
a-selinene
d-cadinene
a-panasinsen
nootkatone


6.490.73
1.360.09
9.470.34
2.980.06
129.014.73
2309.5021.47
32.565.03
15.542.83
15.630.92
78.872.84
8.871.47
1.350.06
5.150.46
16.322.82
6.031.10
1.790.22
9.892.91
2.920.61
135.994.83
14.942.58
3.701.19
4.071.38
4.632.37
1.450.03
6.132.82


3.791.28
1.150.03
15.122.37
Nd
117.985.23
1977.4853.38
Nd
2.662.12
8.653.42
4.162.78
5.253.15
1.090.29
1.240.28
3.133.02
4.262.44
1.530.09
9.673.10
5.282.43
143.004.82
12.053.84
1.280.89
3.482.46
3.151.82
1.141.03
3.183.20


4.552.25
0.830.04
4.411.48
Nd
108.473.49
1686.2730.28
Nd
10.712.43
9.382.83
3.521.67
2.731.38
0.961.31
1.400.72
7.142.89
3.531.21
1.140.70
10.482.56
5.833.83
134.396.88
16.573.74
1.221.04
3.982.79
3.023.38
7.692.45
2.853.52


150


4.761.29
1.080.88
1.070.27
Nd
102.676.38
1730.1129.47
Nd
6.582.71
7.274.29
3.641.93
3.952.10
1.030.03
1.231.12
2.651.37
3.232.63
1.310.37
10.492.89
5.792.71
130.0212.32
14.374.20
1.401.41
3.212.63
3.152.85
1.090.67
2.653.49


4.080.84
0.910.07
4.381.81
Nd
88.043.67
1737.3951.84
nd
6.482.48
6.913.89
4.432.50
2.541.37
1.050.79
1.100.09
2.011.18
2.552.45
1.731.08
15.205.27
5.422.09
108.584.76
13.162.94
1.360.65
2.792.36
3.022.89
0.991.01
2.702.73









































TIC GFJ_UV1(VVEEK4) D\data ms


3



L2


6
5
7
8


9
3






4


L\


Figure C-1. TIC of grapefruit volatiles extracted by HS-SPME A. untreated juice (week
0) B. UV treated juice (week 4). Separation on DB-Wax column. Highest
change in peak nos. 4 = hexanal, 7 = ethyl hexanoate, 10 = octanal, 14 =
decanal.


151


Abundance


18 19


3000Time
20000
10000
Time o i


Abundance


Time


5 6









APPENDIX D
GRAPEFRUIT JUICE SENSORY RESULTS


152


Table D-1. Difference from control data between hidden control (unpasteurized juice)
and pasteurized juices (thermal, PEF, RFEF and UV) at week 0
Panelist thermal PEF RF UV control
1 9 2 2 6 1
2 5 4 2 7 2
3 6 3 3 8 3
4 4 2 2 6 0
5 5 4 4 8 2
6 2 4 2 4 0
7 6 2 2 9 0
8 4 3 5 7 3
9 3 3 2 5 1
10 4 2 5 5 0
11 6 2 3 6 0
12 3 2 4 3 1
13 5 4 6 9 2
14 3 3 4 8 0
15 2 2 3 3 2
16 3 4 2 5 2
17 4 2 3 5 1
18 6 2 3 3 0
19 4 3 3 7 3
20 4 3 4 9 2
21 2 4 4 4 2
22 5 6 6 6 2
23 3 3 3 7 1
24 4 4 2 4 2
25 5 6 3 2 1
Sum 107 79 82 146 33
Average 3.64 3.24 3.36 4.6 1.32
Variance 4.407 4.523 2.323 8.083 1.060









Table D-2. Difference from control data between hidden control (unpasteurized fresh
uice ) and pasteurized juices (thermal, PEF, RFEF and UV) at week 4

Panelist PEF RF thermal UV control
1 7 5 0 8 1
2 4 7 7 5 1
3 8 3 4 7 1
4 10 10 10 10 4
5 10 9 2 9 0
6 8 10 5 5 1
7 8 7 6 8 2
8 8 9 7 4 4
9 9 6 9 9 3
10 9 9 7 9 1
11 10 10 8 10 6
12 6 4 1 5 1
13 4 6 6 4 3
14 10 10 9 5 0
15 6 3 3 9 2
16 7 1 8 8 3
17 7 7 6 4 3
18 4 4 2 4 2
19 3 6 5 3 3
20 9 4 4 8 2
21 5 7 5 8 2
22 8 5 3 5 0
23 4 6 0 10 0
24 8 6 5 6 3
25 6 7 5 8 1
sum 178.00 161.00 127.00 171.00 49.00
average 7.12 6.44 5.08 6.84 1.96
variance 5.02 6.71 8.35 5.43 2.31


153








APPENDIX E
SAMPLE SENSORY BALLOT FOR DIFFERENCE FROM CONTROL TEST

Difference from control test


Date:

Test#


INSTRUCTIONS


Please DO NOT taste the samples. Evaluate the samples marked "control" first. Open
the vial and sniff headspace of the vial and remember the odor of the sample. Wait 10
seconds and then sniff the sample marked with three digit code. Assess the overall
sensory difference between the two samples using scale below. Mark the scale to
indicate the size of the overall difference.


Scale
0


Mark to indicate difference


No Difference


2
3
4
5
6
7
8
9
10 Extremely different


Comments: please give verbal descriptors for the odor


154


Name


Type of sample









LIST OF REFERENCES


Aaby K, Haffner K & Skrede G. 2002. Aroma quality of Gravenstein apples influenced
by regular and controlled atmosphere storage. Lebens Wiss Technol 35(3):254-
259.
Adler E, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJP & Zuker CS. 2000. A novel
family of mammalian taste receptors. Cell 100(6):693-702.

AFDO. 2001. AFDO requirements and recommendations for apple cider processing
operations. J. Assoc. Food Drug Office 65(1):40-58.

Aguillar-Rosas SF, Ballinas-Casarrubias ML, Nevarez-Moorillon GV, Martin-Belloso 0 &
Ortega-Rivas E. 2007. Thermal and pulsed electric fields pasteurization of apple
juice: Effects on physicochemical properties and flavour compounds. J. Food
Eng. 83(1):41-46.

Allegrone G, Belliardo F & Cabella P. 2006. Comparison of volatile concentrations in
hand-squeezed juices of four different lemon varieties. J. Agric. Food Chem.
54(5): 1844-1848.

AMS. 1983. http://www.ams.usda.gov/standards. Accessed 16th Nov. 2008

Ananthakumar A, Variyar PS & Sharma A. 2006. Estimation of aroma glycosides of
nutmeg and their changes during radiation processing. J. Chromatogr.
1108(2):252-257.

Araneda RC, Kini AD & Firestein S. 2000. The molecular receptive range of an odorant
receptor. Nat. Neurosci. 3(12): 1248-1255.

Ayhan Z, Yeom HW, Zhang QH & Min DB. 2001. Flavor, color, and vitamin C retention
of pulsed electric field processed orange juice in different packaging materials. J.
Agric. Food Chem. 49(2):669-674.

Ayhan Z, Zhang QH & Min DB. 2002. Effects of pulsed electric field processing and
storage on the quality and stability of single-strength orange juice. J. Food Prot.
65(10):1623-1627.

Bazemore R, Goodner K & Rouseff R. 1999. Volatiles from unpasteurized and
excessively heated orange juice analyzed with solid phase microextraction and
GC-olfactometry. J. Food Sci. 64(5):800-803.

Berry RE, Wagner CJ & Moshonas MG. 1967. Flavor studies of nootkatone in grapefruit
juice J. Food Sci. 32(1):75-&.

Besser RE, Lett SM, Weber JT, Doyle MP, Barrett TJ, Wells JG & Griffin PM. 1993a. An
outbreak of diarrhea and hemolytic uremic syndrome from Escherichia-coli 0157-
H7 in fresh-pressed apple cider. J. Am. Med. Assoc. 269(17):2217-2220.


155










Bezman Y, Rouseff RL & Naim M. 2001. 2-methyl-3-furanthiol and methional are
possible off-flavors in stored orange juice: Aroma-similarity, NIF/SNIF GC-O, and
GC analyses. J. Agric. Food Chem. 49(11):5425-5432.

Biasioli F, Gasperi F, Aprea E, Colato L, Boscaini E & Mark TD. 2003. Fingerprinting
mass spectrometry by PTR-MS: heat treatment vs. pressure treatment of red
orange juice a case study. Int J Mass Spect 223(1-3):343-353.

Boff JM, Truong TT, Min DB & Shellhammer TH. 2003. Effect of thermal processing and
carbon dioxide-assisted high-pressure processing on pectin methylesterase and
chemical changes in orange juice. J. Food Sci. 68(4):1179-1184.

Braddock RJ. 1999. Single strength orange juices and concentrate. New York: Wiley-
Interscience, p 53-83

Buck L & Axel R. 1991. A novel multigene family may encode odorant receptors a
molecular-basis for odor recognition. Cell 65(1): 175-187.

Buettner A & Schieberle P. 1999. Characterization of the most odor-active volatiles in
fresh, hand-squeezed juice of grapefruit (Citrus paradisi Macfayden). J. Agric.
Food Chem. 47(12):5189-5193.

Buettner A & Schieberle P. 2001. Evaluation of key aroma compounds in hand-
squeezed grapefruit juice (Citrus paradisi Macfayden) by quantitation and flavor
reconstitution experiments. J. Agric. Food Chem. 49(3):1358-1363.

Bult JHF, Schifferstein HNJ, Roozen JP, Boronat ED, Voragen AGJ & Kroeze JHA.
2002. Sensory evaluation of character impact components in an apple model
mixture. Chem. Senses 27(6):485-494.

Buttery RG, Turnbaugh JG & Ling LC. 1988. Contributions of volatiles to rice aroma. J.
Agric. Food Chem. 36(5):1006-1009.

Cadwallader KR & Xu Y. 1994. Analysis of volatile components in fresh grapefruit juice
by purge-and-trap gas-chromatography. J. Agric. Food Chem. 42(3):782-784.

Casas E, Valderrama MJ & Peinado JM. 1999. Sorbate detoxification by spoilage
yeasts isolated from marzipan products. Food Technol. Biotechnol. 37(2):87-91.

Castro AJ SB, Barbosa-Canovas, Zhang QH. 2001. Pulsed electric field modification of
milk alkaline phosphatase activity. Lancaster, PA: Technomic Publishing
Company, Inc.

CFSAN. 2004. Juice HACCP Hazards and Controls Guidance. p.
http://www.cfsan.fda.gov/~dms/juicgul0.html. Accessed 17th Nov. 2008


156










Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng LX, Guo W, Zuker CS & Ryba
NJP. 2000. T2Rs function as bitter taste receptors. Cell 100(6):703-711.

Chang JCH, Ossoff SF, Lobe DC, Dorfman MH, Dumais CM, Quails RG & Johnson JD.
1985. UV inactivation of pathogenic and indicator microorganisms. Appl. Environ.
Microbiol. 49(6):1361-1365.

Charles-Rodriguez AV, Nevarez-Moorillon GV, Zhang QH & Ortega-Rivas E. 2007.
Comparison of thermal processing and pulsed electric fields treatment in
pasteurization of apple juice. Food Bioprod. Process. 85(C2):93-97.

Choi LH & Nielsen SS. 2005. The effects of thermal and nonthermal processing
methods on apple cider quality and consumer acceptability. J. Food Qual.
28(1):13-29.

Clegg KM. 1964. Non-enzymatic browning of lemon juice. J. Sci. Food Agric.
15(12):878-&.

Coleman RL, Shaw PE & Lund ED. 1972. Analysis of grapefruit essence and aroma oils
J. Agric. Food Chem. 20(1):100.

Cortes C, Esteve MJ & Frigola A. 2008. Color of orange juice treated by High Intensity
Pulsed Electric Fields during refrigerated storage and comparison with
pasteurized juice. Food Control 19(2):151-158.

Crupi F & Rispoli G. 2002. Citrus Juice Technology. NY, USA: Taylor & Francis.

Cserhalmi Z, Sass-Kiss A, Toth-Markus M & Lechner N. 2006. Study of pulsed electric
field treated citrus juices. Innov. Food Sci. Emerg. Technol. 7(1-2):49-54.

Cunningham DG, Acree TE, Barnard J, Butts RM & Braell PA. 1986. Charm analysis of
apple volatiles. Food Chem. 19(2): 137-147.

da Silva MAAP & Lundahl DS. 1994. The capabilities and psychophysics of Osme: a
new GC-Olfactometry Technique. Elsevier: New York.p 191-209

David JRD, Graves RH & Carlson VR. 1996. Aseptic processing and packaging of food:
a food industry perspective. Boca Raton, Florida: CRC Press LLC.p 14

Deak T & Beuchat LR. 1993. Yeasts associated with fruit juice concentrates. J. Food
Prot. 56(9):777-782.

Demole E & Enggist P. 1983. Further investigation of grapefruit juice flavor components
(citrus-paradisi macfayden) valencane-type and eudesmane-type sesquiterpene
ketones. Helv. Chim. Acta 66(5):1381-1391.


157










Demole E, Enggist P & Ohloff G. 1982. 1-Para-menthene-8-thiol a powerful flavor
impact constituent of grapefruit juice (citrus-paradisi macfayden). Helv. Chim.
Acta 65(6): 1785-1794.

Dever MC, Cliff M & Lau OL. 1992. Maturity and ripening effects on chemical and
sensory properties of apple juice. J. Sci. Food Agric. 60(3):355-360.

Dimick PS & Hoskin JC. 1983. Review of apple flavor state of the art. Crit. Rev. Food
Sci. 18(4):387-409.

Dixon J & Hewett EW. 2000. Factors affecting apple aroma/flavour volatile
concentration: a review. New. Zeal. J. Crop Hort. Sci. 28(3):155-173.


Donahue DW, Canitez N & Bushway AA. 2004. Uv inactivation of E-coli 0157 : H7 in
apple cider: Quality, sensory and shelf-life analysis. J. Food Process. Pres..
28(5):368-387.

Dreher JG, Rouseff RL & Naim M. 2003. GC-olfactometric characterization of aroma
volatiles from the thermal degradation of thiamin in model orange juice. J. Agric.
Food Chem. 51(10):3097-3102.

Duffy S, Churey J, Worobo RW & Schaffner DW. 2000. Analysis and modeling of the
variability associated with UV inactivation of Escherichia coli in apple cider. J.
Food Prot. 63(11):1587-1590.

Durr P & Schobinger U. 1981. The contribution of some volatiles to the sensory quality
of apple and orange juice odour. In: walter de Gruyter & Co, B. N. Y., editor).
Flavour '81 Berlin: Walter de Grutyer & Co. p 179-86

Elez-Martinez P, Soliva-Fortuny RC & Martin-Belloso 0. 2006. Comparative study on
shelf life of orange juice processed by high intensity pulsed electric fields or heat
treatment. Eur. Food Res. Technol. 222(3-4):321-329.

Evrendilek GA, Jin ZT, Ruhlman KT, Qiu X, Zhang QH & Ritcher ER. 2000a. Microbial
safety and shelf-life of apple juice and cider processed by bench and pilot scale
PEF systems. Innov. Food Sci. Emerg. Technol. 1(1):77-86.

Evrendilek GA, Jin ZT, Ruhlman KT, Qiu X, Zhang QH & Ritcher ER. 2000b. Microbial
safety and shelf-life of apple juice and cider processed by bench and pilot scale
PEF systems.Innov. Food Sci. Emerg. 1: 77-86

Evrendilek GA, Dantzer WR, Streaker CB, Ratanatriwong P & Zhang QH. 2001. Shelf-
life evaluations of liquid foods treated by pilot plant pulsed electric field system. J
Food Process Pres 25(4):283-297.


158










Fan XT & Gates RA. 2001. Degradation of monoterpenes in orange juice by gamma
radiation. J. Agric. Food Chem. 49(5):2422-2426.

Fan XT & Geveke DJ. 2007. Furan formation in sugar solution and apple cider upon
ultraviolet treatment. J. Agric. Food Chem. 55(19):7816-7821.

Fan XT & Thayer DW. 2002. Nutritional quality of irradiated orange juice. J. Food
Process. Pres. 26(3):195-211.

FDA. 2001. FDA Publishes Final Rule to Increase Safety of Fruit and Vegetable Juices.
http://www.cfsan.fda.gov/~lrd/hhsjuic4.htmlFDA/CFSAN Web site. Accessed 4th
October, 2009.

Flath RA, Black DR, Guadagni DG, McFadden WH & Schultz TH. 1967a. Identification
and organoleptic evaluation of compounds in delicious apple essence
J. Agric. Food Chem. 15(1):29-&.

Fuhrmann E & Grosch W. 2002. Character impact odorants of the apple cultivars Elstar
and Cox Orange. Nahrung 46(3):187-193.

Garcia D, Hassani M, Manas P, Condon S & Pagan R. 2005. Inactivation of Escherichia
coli 0157 : H7 during the storage under refrigeration of apple juice treated by
pulsed electric fields. J. Food Saf. 25(1):30-42.

Garruti DS, Franco MRB, da Silva M, Janzantti NS & Alves GL. 2003. Evaluation of
volatile flavour compounds from cashew apple (Anacardium occidentale L) juice
by the Osme gas chromatography/olfactometry technique. J. Sci. Food Agric.
83(14): 1455-1462.

Genovese DB, Elustondo MP & Lozano JE. 1997. Color and cloud stabilization in
cloudy apple juice by steam heating during crushing. J. Food Sci. 62(6): 1171-
1175.

Geveke DJ. 2005. UV inactivation of bacteria in apple cider. J. Food Prot. 68(8):1739-
1742.

Geveke DJ. 2008. UV Inactivation of E-coli in Liquid Egg White. Food Bioproc. Technol.
1 (2):201-206.

Geveke DJ & Brunkhorst C. 2004. Inactivation of Escherichia coli in apple juice by radio
frequency electric fields. J. Food Sci. 69(3):E134-E138.

Geveke DJ & Brunkhorst C. 2008. Radio frequency electric fields inactivation of
Escherichia coli in apple cider. J. Food Eng. 85(2):215-221.


159









Geveke DJ, Brunkhorst C & Fan XT. 2007. Radio frequency electric fields processing of
orange juice. Inn. Food Sci. Emerg.Technol. 8(4):549-554.

Girard B & Lau OL. 1995. Effect of maturity and storage on quality and volatile
production of 'Jonagold' apples. Food Res. Int. 28(5):465-471.

Girard B & Tv. 1996. Retention index calculation using Kovats constant model for linear
temperature-programmed gas chromatography. J. Chromatogr. 721(2):279-288.

Gocmen D EA, Williams T, Parish M, Rouseff RL. 2005. Identification of medicinal off-
flavours generated by Alicyclobacillus species in orange juice using GC-
olfactometry and GC-MS. Lett. Appl. Microbiol. 40(3):172-177.

Grahl T & MarkI H. 1996. Killing of microorganisms by pulsed electric fields. Appl.
Microbiol. Biotechnol. 45(1-2): 148-157.

Guerrero-Beltran JA, Welti-Chanes J & Barbosa-Canovas GV. 2009. Ultraviolet-c light
processing of grape, cranberry and grapefruit juices to inactivate saccharomyces
cerevisiae. J. Food Process Eng. 32(6):916-932.

Guiavarc'h Y, Segovia 0, Hendrickx M & Van Loey A. 2005. Purification,
characterization, thermal and high-pressure inactivation of a pectin
methylesterase from white grapefruit (Citrus paradisi). Innov Food Sci Emerg
Technol 6(4):363-371.

Hatt H. 2000. Follow your nose: Mechanisms of signal transduction. Zool.-Anal.
Complex Syst. 102(2-3):120-126.

Heinz V, Toepfl S & Knorr D. 2003. Impact of temperature on lethality and energy
efficiency of apple juice pasteurization by pulsed electric fields treatment.
Inn.Food Sci.Emerg. Technol. 4(2):167-175.

Hoyer 0. 1988. Testing performance and monitoring of UV systems for drinking water
disinfection. Water Supply Pap. U.S. Geol. Surv. 16(1-2):424-429.

Hulsheger H, Potel J & Niemann EG. 1983. Electric-field effects on bacteria and yeast-
cells. Radiat. Environ. Biophys. 22(2):149-162.

Jella P & Rouseff R. 1997. Comparison of extraction methods of volatile aroma
compounds in processed grapefruit juice. Abstracts of Papers of the American
Chemical Society 214:78-AGFD.

Jella P, Rouseff R, Goodner K & Widmer W. 1998. Determination of key flavor
components in methylene chloride extracts from processed grapefruit juice. J
Agric. Food Chem. 46 (1)p. 242-247.


160









Jeyamkondan S, Jayas DS & Holley RA. 1999. Pulsed electric field processing of foods:
A review. J. Food Prot. 62(9):1088-1096.

Jia MY, Zhang QH & Min DB. 1999. Pulsed electric field processing effects on flavor
compounds and microorganisms of orange juice. Food Chem. 65(4):445-451.

Jin ZT & Zhang QH. 1999. Pulsed electric field inactivation of microorganisms and
preservation of quality of cranberry juice. J. Food Process. Pres. 23(6):481-497.

Kale PN & Adsule PG. 1995. Citrus. Handbook of fruit science and
technology: production, composition, storage and processing. New York: Marcel
Dekker, Inc. p. 56.

Kataoka H, Lord HL & Pawliszyn J. 2000. Applications of solid-phase microextraction in
food analysis. J. Chromatogr. 880(1-2):35-62.

Kato T, Shimoda M, Suzuki J, Kawaraya A, Igura N & Hayakawa I. 2003. Changes in
the odors of squeezed apple juice during thermal processing. Food Res. Int.
36(8):777-785.

Keyser M, Muller IA, Cilliers FP, Nel W & Gouws PA. 2008. Ultraviolet radiation as a
non-thermal treatment for the inactivation of microorganisms in fruit juice. Innov
Food Sci Emerg Technol 9(3):348-354.

Komthong P, Katoh T, Igura N & Shimoda M. 2006. Changes in the odours of apple
Juice during enzymatic browning. Food Qual. Pref. 17(6):497-504.

Koutchma T. 2008. UV light for processing foods. Ozone-Sci Eng 30(1):93-98.

Koutchma T. 2009. Advances in Ultraviolet Light Technology for Non-thermal
Processing of Liquid Foods. Food Bioprocess Technol. 2(2):138--155.

Koutchma T, Paris B & Patazca E. 2007. Validation of UV coiled tube reactor for fresh
juices. J. Environ. Eng. Sci. 6(3):319-328.

Kozempel M, McAloon A & Yee W. 1998. The cost of pasteurizing apple cider. Food
Technol. 52(1):50-52.

Krautwurst D, Yau KW & Reed RR. 1998. Identification of ligands for olfactory receptors
by functional expression of a receptor library. Cell 95(7):917-926.

Kusmiere KK & Bald E. 2008. Reduced and total glutathione and cysteine profiles of
citrus fruit juices using liquid chromatography. Food Chem. 106(1):340-344.

Laing DG & Jinks A. 1996. Flavour perception mechanisms. Trends Food Sci. Technol.
7(12):387-389.


161










Lancet D, Benarie N, Cohen S, Gat U, Grossisseroff R, Hornsaban S, Khen M, Lehrach
H, Natochin M, North M, Seidemann E & Walker N. 1993. Olfactory receptors -
transduction, diversity, human psychophysics and genome analysis CIBA Found.
Symp. 179:131-146.

Lee HS & Coates GA. 1999. Thermal pasteurization effects on color of red grapefruit
juices. J. Food Sci. 64(4):663-666.

Lee HS & Nagy S. 1988. Quality changes and nonenzymatic browning intermediates in
grapefruit juice during storage. J. Food Sci. 53(1):168-&.

Liang ZW, Cheng Z & Mittal GS. 2006. Inactivation of spoilage microorganisms in apple
cider using a continuous flow pulsed electric field system. Food Sci Technol
39(4):351-357.

Liang ZW, Mittal GS & Griffiths MW. 2002. Inactivation of Salmonella typhimurium in
orange juice containing antimicrobial agents by pulsed electric field. J. Food Prot.
65(7): 1081-1087.

Lin J, Jella P & Rouseff RL. 2002a. Gas chromatography-olfactometry and
chemiluminescence characterization of grapefruit juice volatile sulfur compounds.
Heteroatomic Aroma Compounds. ACS Sym. Sr.p. 110-120.

Lin JM & Rouseff RL. 2001 a. Characterization of aroma-impact compounds in cold-
pressed grapefruit oil using time-intensity GC-olfactometry and GC-MS. Flavour
Frag. J. 16(6):457-463.

Lin JM, Rouseff RL, Barros S & Haim M. 2002b. Aroma composition changes in early
season grapefruit juice produced from thermal concentration. J. Agric. Food
Chem. 50(4):813-819.

Lin JM, Rouseff RL & Barros SM. 2001 b. Aroma impact and volatile component
changes in early season grapefruit juice produced from thermal concentration.
Abs. Papers Am. Chem. Soc. 222:U31-U31.

Lindemann B. 1996. Taste reception. Physiol Rev 76(3):718-766.

Linforth R, Martin F, Carey M, Davidson J & AJ T. 2002. Retronasal transport of aroma
compounds. J. Agric. Food Chem. 50(5):1111-1117.

Lu YR & Foo Y. 1998. Constitution of some chemical components of apple seed. Food
Chem. 61(1-2):29-33.

Macleod WD & Buigues NM. 1964. Sesquiterpenes .1. nootkatone new grapefruit flavor
constituent J. Food Sci. 29(5):565-&.


162










Mahattanatawee K, Rouseff R, Valim MF & Naim M. 2005. Identification and aroma
impact of norisoprenoids in orange juice. J. Agric. Food Chem. 53(2):393-397.

Malnic B, Hirono J, Sato T & Buck LB. 1999. Combinatorial receptor codes for odors.
Cell 96(5):713-723.
Marcotte M, Stewart B & Fustier P. 1998. Abused thermal treatment impact on
degradation products of chilled pasteurized orange juice. J. Agric. Food Chem.
46(5):1991-1996.

Matich AJ, Rowan DD & Banks NH. 1996. Solid phase microextraction for quantitative
headspace sampling of apple volatiles. Anal Chem 68(23):4114-4118.

McDonald CJ, Lloyd SW, Vitale MA, Petersson K & Innings F. 2000. Effects of pulsed
electric fields on microorganisms in orange juice using electric field strengths of
30 and 50 kV/cm. J. Food Sci. 65(6):984-989.

McFeeters RF FHaTR. 1983. Effect of heating and acid storage on pectinesterase
activity in cucumber slices. Abs. Papers Am. Chem. Soc. 186:131.

McLellan MR & Padilla-Zakour 01. 2004. Juice Processing. Boca Raton, Fl: CRC Press
LLC. p 77

Mehinagic E, Royer G, Symoneaux R, Jourjon F & Prost C. 2006. Characterization of
odor-active volatiles in apples: Influence of cultivars and maturity stage. J. Agric.
Food Chem. 54(7):2678-2687.

Meilgaard M, Civille GV & Carr BT. 1999. Sensory Evaluation Techniques,2 ed. Boca
Raton, Fl: CRC Press.

Meyerhof W, Batram C, Kuhn C, Brockhoff A, Chudoba E, Bufe B, Appendino G &
Behrens M. 2010. The Molecular Receptive Ranges of Human TAS2R Bitter
Taste Receptors. Chem. Senses 35(2):157-170.

Min S, Jin ZT, Min SK, Yeom H & Zhang QH. 2003a. Commercial-scale pulsed electric
field processing of orange juice. J. Food Sci. 68(4):1265-1271.

Min S, Jin ZT & Zhang QH. 2003b. Commercial scale pulsed electric field processing of
tomato juice. J. Agric. Food Chem. 51(11):3338-3344.

Min S & Zhang QH. 2003. Effects of commercial-scale pulsed electric field processing
on flavor and color of tomato juice. J. Food Sci. 68(5):1600-1606.

Moshonas MG. 1971. Analysis of carbonyl flavor constituents from grapefruit oil. J.
Agric. Food Chem. 19(4):769-&.


163









Moshonas MG & Shaw PE. 2000. Changes in volatile flavor constituents in pasteurized
orange juice during storage. J. Food Qual. 23(1 ):61-71.

Mueller KL, Hoon MA, Erlenbach I, Chandrashekar J, Zuker CS & Ryba NJP. 2005. The
receptors and coding logic for bitter taste. Nature 434(7030):225-229.

Murakami EG, Jackson L, Madsen K & Schickedanz B. 2006. Factors affecting the
ultraviolet inactivation of Escherichia coli K12 in apple juice and a model system.
J. Food Process Eng. 29(1):53-71.

Nagy S, Lee H, Rouseff RL & Lin JCC. 1990. Nonenzymic browning of commercially
canned and bottled grapefruit juice. J. Agric. Food Chem. 38(2):343-346.

Nelson G, Chandrashekar J, Hoon MA, Feng LX, Zhao G, Ryba NJP & Zuker CS. 2002.
An amino-acid taste receptor. Nature 416(6877):199-202.

Nelson G, Hoon MA, Chandrashekar J, Zhang YF, Ryba NJP & Zuker CS. 2001.
Mammalian sweet taste receptors. Cell 106(3):381-390.

Nijssen LM, Visscher CA, Maarse H & Willemsen L. 1996. Volatile Compounds in
Foods, Qualitative and Quantitative Data,7th ed.: Zeist, The Netherlands, TNO
Nutrition and Food Research.

Nunez AJ, Bemelmans JMH & Maarse H. 1984. Isolation methods for the volatile
components of grapefruit juice distillation and solvent-extraction methods.
Chromatographia 18(3): 153-158.

Nunez AJ, Maarse H & Bemelmans JMH. 1985. Volatile flavor components of grapefruit
juice (citrus-paradisi macfadyen). J. Sci. Food Agric. 36(8):757-763.

Paillard NMM & Rouri 0. 1984. Hexanal and 2-hexenal productions by mashed apple
tissues. Lebensm. Wiss. Technol. 17(6):345-350.

Parish ME. 1997. Public health and nonpasteurized fruit juices. Crit. Rev. Microbiol.
23(2): 109-119.

Peleg M. 1995. A model of microbial survival after exposure to pulsed electric-fields. J.
Sci. Food Agric. 67(1):93-99.

Perez-Cacho PR & Rouseff R. 2008. Processing and Storage Effects on Orange Juice
Aroma: A Review. J. Agric. Food Chem. 56(21):9785-9796.

Petersen MA, Tonder D & Poll L. 1998. Comparison of normal and accelerated storage
of commercial orange juice Changes in flavour and content of volatile
compounds. Food Qual. Pref. 9(1-2):43-51.


164









Pino J, Torricella R & Orsi F. 1986. Correlation between sensory and gas-
chromatographic measurements on grapefruit juice volatiles. Acta Aliment.
15(3):237-246.

Plotto A, McDaniel MR & Mattheis JP. 1999. Characterization of 'Gala' apple aroma and
flavor: Differences between controlled atmosphere and air storage. J. Am. Soc.
Hort. Sci. 124(4):416-423.

Plotto A, McDaniel MR & Mattheis JP. 2000. Characterization of changes in 'Gala' apple
aroma during storage using osme analysis, a gas chromatography-olfactometry
technique. J. Am. Soc. Hort. Sci. 125(6):714-722.

Pothakamury UR, Vega H, Zhang QH, BarbosaCanovas GV & Swanson BG. 1996.
Effect of growth stage and processing temperature on the inactivation of E-coli by
pulsed electric fields. J. Food Prot. 59(11):1167-1171.

Prescott J, Allen S & Stephens L. 1993. Interactions between oral chemical irritation,
taste and temperature. Chem. Senses 18(4):389-404.

Prescott J & Stevenson RJ. 1995. Pungency in food perception and preference. Food
Rev. Int. 11(4):665-698.

Qin BL, Barbosa-Canovas GV, Swanson BG, Pedrow PD & Olsen RG. 1998.
Inactivating microorganisms using a pulsed electric field continuous treatment
system. IEEE T Ind. Appl. 34(1) p. 43-50.

Qin BL, Chang FJ, BarbosaCanovas GV & Swanson BG. 1995. Nonthermal inactivation
of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Food
Sci.Technol. 28(6):564-568.

Qin BL, Pothakamury UR, BarbosaCanovas GV & Swanson BG. 1996. Nonthermal
pasteurization of liquid foods using high-intensity pulsed electric fields. Crit. Rev.
Food Sci. Nutr. 36(6):603-627.

Qiu X, Sharma S, Tuhela L, Jia M & Zhang QH. 1998. An integrated PEF pilot plant for
continuous nonthermal pasteurization of fresh orange juice. Trans. ASAE
41(4): 1069-1074.

Raso J, Palop A, Pagan R & Condon S. 1998. Inactivation of Bacillus subtilis spores by
combining ultrasonic waves under pressure and mild heat treatment. J. Appl.
Microbiol. 85(5):849-854.

Rivas A, Rodrigo D, Martinez A, Barbosa-Canovas GV & Rodrigo M. 2006. Effect of
PEF and heat pasteurization on the physical-chemical characteristics of blended
orange and carrot juice. Lwt-Food Sci. Technol. 39(10): 1163-1170.


165









Rouseff R, Bazemore R, Goodner K & Naim M. 2001. GC-olfactometry with solid phase
microextraction of aroma volatiles from heated and unheated orange juice.
Headspace Analysis of Foods and Flavors. p. 101-112.

Rouseff R, Lin J & Valim F. 2003. Combined GC-O/GC-PFPD detection of sulfur aroma
impact compounds in citrus juice. Flavour research at dawn of twenty-first
century; proceeding of the 10th Weurman flavour research symposium:476-481.

Sadler GD, parish ME & Wicker L. 1992. Microbial, enzymatic and chemical changes
during storage of fresh and processed orange juice. J. Food Sci. 57 (5): 1187-91.

Saguy I, Kopelmani J & Mizrahi S. 1978. Simulation of ascorbic acid stability during heat
processing and concentration of grapefruit juice J. Food Process Eng. 2(3):213 -
225.

Sale AJH & Hamilton WA. 1967. Effects of high electric fields on microorganisms .I.
Killing of bacteria and yeasts. Biochim. Et Biophys. Acta 148(3):781-&.

Sampedro F, Geveke DJ, Fan X, Rodrigo D & Zhang QH. 2009. Shelf-Life Study of an
Orange Juice-Milk Based Beverage after PEF and Thermal Processing. J. Food
Sci. 74(2):S107-S112.

Sanchez-Vega R, Mujica-Paz H, Marquez-Melendez R, Ngadi MO & Ortega-Rivas E.
2009. Enzyme inactivation on apple juice treated by ultrapasteurization and
pulsed electric fields technology. J. Food Process. Preserv. 33(4):486-499.

Schamp N, De Pooter H & Dirinck P. 1988. Aroma development in ripening fruits. In:
Flavor chemistry: Trends and developments. Abs. Pap. Chem. Cong. North
Amer. 3(1):AGFD 6: 23-34.

Schreier P, Drawert F, Steiger G & Mick W. 1978. Effect of enzyme treatment of apple
pulp with a commercial pectinase and cellulase on volatiles of juice. J. Food Sci.
43(6): 1797-1800.

Sentandreu E, Carobonell L, Rodrigo D & Carbonell JV. 2006. Pulsed electric field
versus thermal treatment: Equivalent processes to obtain equally acceptable
citrus juices. J. Food. Prot. 60(8): 2106-8.

Shaw PE, Ammons JM & Braman RS. 1980. Volatile sulfur-compounds in fresh orange
and grapefruit juices identification, quantitation, and possible importance to
juice flavor J. Agric. Food Chem. 28(4):778-781.

Shaw PE, Moshonas MG, Hearn CJ & Goodner KL. 2000. Volatile constituents in fresh
and processed juices from grapefruit and new grapefruit hybrids. J. Agric. Food
Chem. 48(6):2425-2429.


166









Shaw PE & Wilson CW. 1981. Importance of nootkatone to the aroma of grapefruit oil
and the flavor of grapefruit juice. J. Agric. Food Chem. 29(3):677-679.

Shaw PE & Wilson CW. 1982. Volatile sulfides in headspace gases of fresh and
processed citrus juices. J. Agric. Food Chem. 30(4):685-688.

Shewfelt RL. 1986. Flavor and color of fruits as affected by processing. In: Luh, J. G. W.
a. B. S., editor). Commercial Fruit Processing. Westport, CT: Avi Publishing Co.
p. 481-529.

Sommer R, Haider T, Cabaj A, Pribil W & Lhotsky M. 1998. Time dose reciprocity in UV
disinfection of water. Water Sci Technol 38(12): 145-150.

Stevens SS. 1964. The psychophysics of sensory function. Sensory Comm.:1-33.

Streif J & Bangerth F. 1988. Production of volatile aroma substances by golden
delicious apple fruits after storage for various times in different co2 and o-2
concentrations. J. Hortic. Sci. 63(2): 193-199.

Su MS & Chien PJ. 2010. Aroma impact components of rabbiteye blueberry (Vaccinium
ashei) vinegars. Food Chem. 119(3):923-928.

Su SK & Wiley RC. 1998. Changes in apple juice flavor compounds poring processing.
J. Food Sci. 63(4):688-691.

Sumitani H, Suekane S, Nakatani A & Tatsuka K. 1994. Changes in composition of
volatile compounds in high-pressure treated peach. J. Agric. Food Chem.
42(3):785-790.

Takeoka GR, Flath RA, Mon TR, Teranishi R & Guentert M. 1990. Volatile constituents
of apricot (prunus-armeniaca). J. Agric. Food Chem. 38(2):471-477.

Tandon K, Worobo RW, Churey JJ & Padilla-Zakour 01. 2003. Storage quality of
pasteurized and UV treated apple cider. J. Food Process. Preserv. 27(1):21-35.

Tatum JH, Nagy S & Berry RE. 1975. Degradation products formed in canned single-
strength orange juice during storage. J. Food Sci. 40(4):707-709.

Tominaga T, des Gachons CP & Dubourdieu D. 1998. A new type of flavor precursors
in Vitis vinifera L cv Sauvignon blanc: S-cysteine conjugates. J. Agric. Food
Chem. 46(12):5215-5219.

Tran MTT & Farid M. 2004. Ultraviolet treatment of orange juice. Innov.Food Sci.
Emerg. Technol. 5(4):495-502.


167









Ukuku DO, Geveke DJ, Cooke P & Zhang HQ. 2008. Membrane damage and viability
loss of Escherichia coli K-12 in apple juice treated with radio frequency electric
field. J. Food Prot. 71(4):684-690.

Uljas HE, Schaffner DW, Duffy S, Zhao LH & Ingham SC. 2001. Modeling of combined
processing steps for reducing Escherichia coli 0157 : H7 populations in apple
cider. Appl. Environ. Microbiol. 67(1):133-141.

Valappil ZA, Fan XT, Zhang HQ & Rouseff RL. 2009. Impact of Thermal and
Nonthermal Processing Technologies on Unfermented Apple Cider Aroma
Volatiles. J. Agric. Food Chem. 57(3):924-929.

VegaMercado H, Pothakamury UR, Chang FJ, BarbosaCanovas GV & Swanson BG.
1996. Inactivation of Escherichia coli by combining pH, ionic strength and pulsed
electric fields hurdles. Food Res. Int. 29(2):117-121.

Vikram VB, Ramesh MN & Prapulla SG. 2005. Thermal degradation kinetics of nutrients
in orange juice heated by electromagnetic and conventional methods. J. Food
Eng. 69(1):31-40.

Wicker L. 2004. Frozen citrus juices. in Hui YH, editor. Handbook of Frozen Foods. New
York, Marcel Dekker Inc; p483-498.

Wolfgan A. 1990. Enzymes in industry: production and application. Germany-Wiley
Weiheim-VCH. P 161-165.

Wouters PC, Alvarez I & Raso J. 2001. Critical factors determining inactivation kinetics
by pulsed electric field food processing. Trends Food Sci. Technol. 12(3-4): 112-
121.

Wyatt MK & Parish ME. 1995. Spore germination of citrus juice-related fungi at low-
temperatures. Food Microbiol. 12(3):237-243.

Yeom HW, Evrendilek GA, Jin ZT & Zhang QH. 2004. Processing of yogurt-based
products with pulsed electric fields: Microbial, sensory and physical evaluations.
J. Food Process. Preserv. 28(3):161-178.

Yeom HW, Streaker CB, Zhang QH & Min DB. 2000a. Effects of pulsed electric fields on
the activities of microorganisms and pectin methyl esterase in orange juice. J.
Food Sci. 65(8):1359-1363.

Yeom HW, Streaker CB, Zhang QH & Min DB. 2000b. Effects of pulsed electric fields on
the quality of orange juice and comparison with heat pasteurization. J. Agric.
Food Chem. 48(10):4597-4605.


168









Yoo ZW, Kim NS & Lee DS. 2004. Comparative analyses of the flavors from Hallabong
(Citrus sphaerocarpa) with lemon, orange and grapefruit by SPTE and HS-SPME
combined with GC-MS. B. Korean Chem. Soc. 25(2):271-279.

Young H, Gilbert JM, Murray SH & Ball RD. 1996. Causal effects of aroma compounds
on Royal Gala apple flavours. J. Sci. Food Agric. 71(3):329-336.

Young JC, Chu CLG, Lu XW & Zhu HH. 2004. Ester variability in apple varieties as
determined by solid-phase microextraction and gas chromatography-mass
spectrometry. J. Agric. Food Chem. 52(26):8086-8093.

Zhang Q, Monsalve-Gonzalez A, Qin B, Barbosa-Canovas GV & Swanson BG. 1994.
Inactivation of Saccharomyces cerevisiae by square wave and exponential-decay
pulsed. J. Food Proc. Eng. 17:469-478.

Zhang YF, Hoon MA, Chandrashekar J, Mueller KL, Cook B, Wu DQ, Zuker CS & Ryba
NJP. 2003. Coding of sweet, bitter, and umami tastes: Different receptor cells
sharing similar signaling pathways. Cell 112(3):293-301.

Zozulya S, Echeverri F & Nguyen T. 2001. The human olfactory receptor repertoire.
Genome Biol 2(6):Research0018.


169









BIOGRAPHICAL SKETCH

Zareena Azhu Valappil grew up in Delhi, India. She attended Delhi University and

graduated in 1996 with an undergraduate degree in biological sciences. She received

her master's degree in biochemistry from Hamdard University in 1998. After earning her

master's degree, she joined the Bhabha Atomic Research Center as a Scientific Officer

and worked there for a period of 6 years. She was awarded the prestigious Graduate

Alumni Fellowship to pursue a doctoral degree in food science at the University of

Florida in 2005. In 2006, she moved from Gainesville, FL to the United States

Department of Agriculture in Wyndmoor, Pennsylvania to conduct her doctoral research.

In 2008 she accepted a position at Takasago International Corporation in Rockleigh,

New Jersey. She completed her PhD in 2010.


170





PAGE 1

1 IMPACT OF THERMAL AND NON THERMAL PROCESSING TECHNIQUES ON APPLE AND GRAPEFRUIT JUICE QUALITY By ZAREENA AZHU VALAPPIL 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 2010

PAGE 2

2 2010 Zareena Azhu Valappil

PAGE 3

3 To my daughter Suha and my husband Niyas

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Russell Rouseff for his guidan ce and unending support throughout the course of my research work. It would not have been possible to conduct my research at the U nited S tates D epartment of A griculture without his help and encouragement. I would also like to thank my committee members : Dr M. Marshall, Dr. B. Ache Dr. W. Yang and Dr. H. Zhang. I would also like to thank my previous committee members Dr. M. Balaban and Dr. S. Talcott who have left University of Florida. My specials thanks to Dr. Fan, Dr. Geveke and Dr. Zhang at USDA for helping me successfully conduct my research at USDA I would also like to thank my lab mates Kim, Jen, Kym, Glen and Tim for their constant help and support and for making my life easier and enjoyable at USDA. A special thanks to Kim for all her help in th e lab and participating in GCO and sensory work. I also want to thank my Mom and Dad for their love and guidance throughout my life. Finally I would like to thank my husband Niyas and daughter Suha for supporting and helping me achieve my goals. Lastly I want to thank God for everything

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTERS 1 INTRODUCTION ................................ ................................ ................................ .... 15 2 L ITERATURE REVIEW ................................ ................................ .......................... 18 Flavor Perception ................................ ................................ ................................ .... 18 Fruit Flavors ................................ ................................ ................................ ............ 22 Apple Juice/Cider Aroma ................................ ................................ .................. 22 Grapefruit Juice Aroma ................................ ................................ .................... 25 Volatile Extraction ................................ ................................ ................................ ... 28 Gas Chromatography Olfactometry ................................ ................................ ........ 30 Processing ................................ ................................ ................................ .............. 31 Thermal Pasteurization ................................ ................................ .................... 32 Citrus juice thermal processing ................................ ................................ .. 33 Apple juice thermal processing ................................ ................................ .. 33 Effect of Thermal Pasteurization on Juice Flavor ................................ ............. 34 Non Thermal Processing Methods ................................ ................................ ... 36 Pulsed electric field ................................ ................................ .................... 38 Radio frequency electric field ................................ ................................ ..... 45 Ultraviolet radiation ................................ ................................ .................... 46 3 COMPARISON OF THERMAL AND NON THERMAL TECHNIQUES ON APPLE CIDE R STORAGE QUALITY UNDER EQUIVALENT PROCESS CONDITIONS ................................ ................................ ................................ ......... 55 Introduction ................................ ................................ ................................ ............. 55 Materials and Methods ................................ ................................ ............................ 56 Determination of Processing Conditions That Achieve 6 log Reduction of E. coli K12 ................................ ................................ ................................ ......... 56 Ultraviolet process ................................ ................................ ..................... 56 Pulsed electric field process ................................ ................................ ....... 57 Thermal process ................................ ................................ ........................ 58 Shelf Life of Processed Apple Cider ................................ ................................ 58 Processing and packaging ................................ ................................ ......... 58 Storage ................................ ................................ ................................ ...... 59

PAGE 6

6 Microbial tests ................................ ................................ ............................ 59 pH ................................ ................................ ................................ .............. 60 Brix ................................ ................................ ................................ ............. 60 Color ................................ ................................ ................................ .......... 60 Stati stical Analysis ................................ ................................ ............................ 61 Results and Discussion ................................ ................................ ........................... 61 Optimization of Equivalent Processing Conditions ................................ ........... 61 Effect of Thermal and Non Thermal Processing on Microbial Stability During Storage of Apple Cider ................................ ................................ .................. 63 Effect of Thermal and Non Thermal Processing on Appearance of Apple Cider During Storage ................................ ................................ .................... 65 Effect of Thermal and Non thermal Processing on the pH and Brix of Apple Cider During Storage ................................ ................................ .................... 65 Effect of Thermal and Non Thermal Processing on Color of Apple Cider During Storage ................................ ................................ .............................. 66 Conclusion ................................ ................................ ................................ .............. 68 4 IMPACT OF THERMAL AND NON THERMAL PROCESSING TECHNOLOGIES ON APPLE CIDER AROMA ................................ ...................... 74 Introduction ................................ ................................ ................................ ............. 74 Materials and Methods ................................ ................................ ............................ 74 Apple Cider ................................ ................................ ................................ ....... 75 Apple Cider Processing ................................ ................................ .................... 75 Heat treatment ................................ ................................ ........................... 76 Ultraviolet treatment ................................ ................................ ................... 76 Pulsed electric field treatment ................................ ................................ .... 76 Packaging and Storage ................................ ................................ .................... 77 Microbial Stability ................................ ................................ ............................. 77 Volatile Extraction ................................ ................................ ............................. 78 Gas Chromatography Mass Spect rometry Analysis ................................ ......... 78 Quantification of Apple Cider Volatiles ................................ ............................. 79 Sensory Evaluation ................................ ................................ .......................... 79 Statistical Analysis ................................ ................................ ............................ 80 Results and Discussion ................................ ................................ ........................... 81 Microbial Stability During Storage ................................ ................................ .... 81 Volatile Composition ................................ ................................ ......................... 81 Odor Activity Values ................................ ................................ ......................... 82 Effect of Treatment and Storage on Volatiles ................................ ................... 83 Effect of Treatment and Storage on Odor Activity Values of Volatiles .............. 85 Aroma Sensory Studies ................................ ................................ .................... 86 Conclusion ................................ ................................ ................................ .............. 87 5 COMPARISON OF THERMAL AND NON THERMAL TECHNIQUES ON GRAPEFRUIT JUICE STORAGE QUALITY UNDER EQUIVALENT PROCESS CONDITIONS ................................ ................................ ................................ ......... 92

PAGE 7

7 Introduction ................................ ................................ ................................ ............. 92 Materials and Methods ................................ ................................ ............................ 93 Determination of Equivalent T reatment Conditions ................................ .......... 93 Heat treatment ................................ ................................ ........................... 93 Ultraviolet treatment ................................ ................................ ................... 93 Pulsed electric field treatment ................................ ................................ .... 94 Radio frequency electric field treatment ................................ ..................... 94 Shelf Life Study ................................ ................................ ................................ 95 Grapefruit juice pasteurization ................................ ................................ ... 95 Packaging and storage ................................ ................................ .............. 95 Microbial assay ................................ ................................ .......................... 96 Pectin methyl esterase activity assay ................................ ........................ 96 Ascorbic acid assay ................................ ................................ ................... 97 Non enzymati c browning ................................ ................................ ............ 97 pH ................................ ................................ ................................ .............. 98 Brix ................................ ................................ ................................ ............. 98 Color ................................ ................................ ................................ .......... 98 Statistical Analysis ................................ ................................ ............................ 99 Results and Discussion ................................ ................................ ........................... 99 Effect of Treatments on Microbial Inactiv ation ................................ .................. 99 Effect of Treatments on Microbial Stability During Storage ............................ 101 Effect of Treatments on Pectin Methyl Esterase Activity ................................ 102 Effect of Treatments on Non enzymatic Browning ................................ ......... 104 Effect of Treatments on Ascorbic Acid Degradation ................................ ....... 105 Effect of Treatments on Color ................................ ................................ ......... 106 Effect of Treatments on pH, Brix and Total Acidity ................................ ......... 107 Conclusion ................................ ................................ ................................ ............ 107 6 IMPACT OF THERMAL AND NON THERMAL PROCESSING TECHNOLOGIES ON AROMA OF RED GRAPEFRUIT JUICE: GC OLFATOMETRIC COMPARSION ................................ ................................ ........ 118 Introduction ................................ ................................ ................................ ........... 118 Materials and Methods ................................ ................................ .......................... 119 Grapefruit Juice ................................ ................................ .............................. 119 Grapefruit Juice Processing ................................ ................................ ........... 120 Heat treatment ................................ ................................ ......................... 120 Non thermal juice treatments ................................ ................................ .. 120 Packaging and storage ................................ ................................ ............ 1 20 Extraction of Grapefruit Juice Volatiles ................................ ........................... 121 Volatile Analysis of Grapefruit Juice Volatiles ................................ ................. 121 Sensory Evaluation ................................ ................................ ........................ 122 Screening and training of panelists ................................ .......................... 122 Test method ................................ ................................ ............................. 123 Statistical Analysis ................................ ................................ .......................... 124 Results and Discussion ................................ ................................ ......................... 124 Effect of Treatment and Storage on Grapefruit Juice Volatiles ....................... 124

PAGE 8

8 Gas Chromatography Olfactometry Profile of Grapefruit Juice ....................... 125 Gas Chromatography Olfactometry Profile Comparison of Fresh Untreated and Treated Juice at Week 0 ................................ ................................ ...... 128 Thermal treatment ................................ ................................ .................... 129 Ultraviolet treatment ................................ ................................ ................. 130 Pulsed electric field treatment ................................ ................................ .. 131 Radio frequency electric field treatment ................................ ................... 131 Gas Chromatography Olfactometry Comparison of Fresh Untreated and Treated Juice at Week 4 ................................ ................................ ............. 131 Sensory Evaluation ................................ ................................ ........................ 132 Conclusion ................................ ................................ ................................ ............ 133 7 CONCLUSION ................................ ................................ ................................ ...... 143 APPENDIX A CHANGE IN APPLE CI DER APPEARANCE DURING STORAGE ...................... 146 B CALIBRATION TABLE FOR APPLE CIDER VOLATILES ................................ .... 147 C GRAPEFRUIT JUICE VOLATILE CONCENTRATION ................................ ......... 148 D GRAPEFRUIT JUICE SENSORY RESULTS ................................ ....................... 152 E SAMPLE SENSORY BALLOT FOR DIFFERENCE FROM CONTROL TEST ...... 154 LIST OF REFERENCES ................................ ................................ ............................. 155 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 170

PAGE 9

9 LIST OF TABLES Table page 2 1 Time temperature parameters for PE inactivation in citrus juices .................... 52 3 1 Change in pH and Brix values for fresh*, thermal, UV and PEF treated apple cider stored at 4 C for 4 weeks ................................ ................................ .......... 72 3 2 Change in Hunter color values** for fresh*, thermal, UV and PEF treated apple cider stored at 4 C for 4 weeks ................................ ................................ 73 4 1 Effect of thermal and non thermal treatments on apple cider volatiles compared to fresh untreated cider after 4 weeks of storage at 4 C ................... 89 4 2 Effect of thermal and non ther mal treatments on odor activity values (OAV) of apple cider volatiles after 4 weeks of storage at 4 C ................................ ......... 91 5 1 Yeast & mold count in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ................................ ........ 111 5 2 pH, Brix and total acidity (TA) values for control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ............ 117 6 2 GCO analysis results of pasteurized and unpasteurized grapefruit juice .......... 136 6 3 Calculated total aroma intensity values for pasteurized and unpasteu rized grapefruit juice belonging to five odor categories ................................ ............. 138 6 4 GCO comparison of pasteurized grapefruit juice at week 4 to unpasteurized grapefruit juice at week 0 ................................ ................................ .................. 140 6 5 Results of ANOVA for mean values from difference from control test .............. 142 6 6 Difference in means between pasteurized and unpasteurized grapefruit ju ice 142 6 7 Results of ANOVA of mean values from difference control test at week 4 ....... 142 6 8 Difference in means between pasteuriz ed and unpasteurized grapefruit juice at week 4 ................................ ................................ ................................ .......... 142 B 1 Regression equation for standard compounds ................................ ................. 147 C 1 Volatile concentratio n ( g/L) in untreated (control) and treated grapefruit juice at week0 ................................ ................................ ................................ ........... 149 C 2 Volatile concentration ( g/L) in untreated (control week 0) and treated grapefruit juice at week 4 ................................ ................................ .................. 150

PAGE 10

10 D 1 Difference from control data between hidden control (unpasteurized juice) and pasteurized juices (thermal, PEF, RFEF and UV) at week 0 ..................... 152 D 2 Difference from control data between hidden control (unpasteurized fresh j uice ) and pasteurized juices (thermal, PEF, RFEF and UV) at ....................... 153

PAGE 11

11 LIST OF FIGURES Figure page 2 1 Key odorants of grapefruit and apple ................................ ................................ .. 52 2 2 Schematic diagram of pulsed electric field processing unit. (Jia and other, 1999) ................................ ................................ ................................ .................. 53 2 3 Schematic diagram of radio frequency electric field processing unit (Geveke and other, 2007) ................................ ................................ ................................ 53 3 1 UV inactivation of inoculated E. coli K12 in apple cider at different treatment times. Treatment conditions: wavelength 254 nm, outlet temperature < 15 C ... 69 3 2 PEF inactivation of inoculated E. coli K12 in apple cider at different electric field str engths. Treatment conditions: pulse duration of 2.5s, total treatment time of150 s, outlet temperature 49 51 C. ................................ .................... 69 3 3 Thermal inactivation of inoculated E. coli K12 in apple cider at dif ferent temperatures. Treatment condition: 1.3 s hold time, outlet temperature <15 C. ................................ ................................ ................................ ...................... 70 3 4 Total aerobic plate count for control, thermal, UV and PEF treated apple cider samples stored at 4 c for 4 weeks. Vertical bar indicates standard deviation (n=4) ................................ ................................ ................................ .... 70 3 5 Yeast and mold count in control, thermal, UV and PEF treated apple cider samples stored at 4 c for 4 weeks. Vertical bar indicates standard deviation (n=4) ................................ ................................ ................................ ................... 71 3 6 Change in total color difference (de) in thermal, UV and PEF treated apple cider during storage. Vertical bar indicates standard deviation (n=3) ................. 71 4 1 Effect of treatment and storage (after 4weeks) on major volatiles of apple cider 1 = butyl acetate, 2 = hexanal, 3 = 2 methyl butyl acetate, 4 = 2 (E) hexanal, 5 = hexyl acetate, 6 = benzal dehyde, 7 = 1 hexanol, a,b,c,d = different letters for each volatile indicate significant difference(p<0.05), control refers to fresh unpasteurized cider maintained at 0 C for 4 weeks ........ 88 5 1 Thermal inactivation of inoculate E. coli K12 in grapefruit juice ........................ 109 5 2 UV Inactivation of inoculated E. coli K12 in grapefruit juice .............................. 109 5 4 RFEF inactivation of inoculated E. coli K12 in grapefruit juice .......................... 110 5 5 Total aerobic plate count for control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ........................ 111

PAGE 12

12 5 6 Change in PME activity in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ........................ 112 5 7 Change in browning index of control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks. ................................ ....................... 112 5 8 Change in ascorbic acid content of control, thermal, U V, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ............ 113 5 9 Linear regression plot of ascorbic acid vs. browning index for thermal and RFEF treated grapefruit juice ................................ ................................ ............ 113 5 10 Change in color L* value of control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ........................ 114 5 11 Change in color b* values of control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ........................ 115 5 12 Linear regression plot of brown color vs. color b* values for thermal and RFEF treated grapefruit juice ................................ ................................ ............ 115 5 13 Change in total color difference (dE) in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks ................................ ............ 116 6 1 Comparative aroma profile for unpasteurized and pasteurized grapefruit juice at week 0 ................................ ................................ ................................ .......... 134 A 1 Change in appearance in treated and untreated app le cider during 4 weeks of storage at 4 C ................................ ................................ ............................... 146 C 1 TIC of grapefruit volatiles extracted by HS SPME A. untreated juice (week 0) B. UV treated juice (week 4). Separation on DB Wax column. Highes t change in peak nos. 4 = hexanal, 7 = ethyl hexanoate, 10 = octanal, 14 = decanal. ................................ ................................ ................................ ............ 151

PAGE 13

13 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 IMPACT OF THERMAL AND NON THERMAL PROCESSING TECHNIQUES ON APPLE AND GRAPEFRUIT JUICE QUALITY By Zareena Azhu Valappil August 2010 Chair: Russell Rouseff Major: Food Science and Human Nutrition Increa sed consumer demand for fresh like products with minimum flavor and nutritional losses has paved the way for alternatives to heat processing. The impact of non thermal techniques including pulsed electric field (PEF), radio frequency electric field (RFEF) or ultraviolet irradiation (UV) and thermal pasteurization on quality of fruit juices after 4 weeks of storage at 4C were studied in this project All treatments were optimized to achieve equivalent five log reduction in E. coli K12 levels. The effect of PEF, UV and thermal treatments on apple cider quality was investigated based on microbial, color, physical properties volatile and sensory analysis PEF and thermally processed cider maintained good microbial quality during 4 weeks of storage but UV tre ated cider fermented after 2 weeks. Thermal and UV pasteurized cider color faded significantly (p<0.05) during storage CIE L* (lightness) and b* (yellow) values increased compared to PEF cider. Significant differences (p<0.05) in the level of key cider od orants were observed between treated apple ciders after 4 weeks of storage. Thermal samples lost 30% and PEF cider lost less than 2% of the total ester and aldehydes during storage. In UV cider, hexanal and 2 (E) hexenal were completely lost after 4 weeks of storage. Microbial spoilage in UV cider was

PAGE 14

14 chemically confirmed by the detection of the microbial metabolite 1 ,3 pentadiene. Triangle sensory analysis indicated a significant difference (p<0.05) in aroma between treatments after 4 weeks storage. PEF t reated cider was preferred over thermal ly treated cider b y 91% of the sensory panel. PEF treated apple cider had a longer shelf life than UV treated cider and a better aroma and color than the thermally processed sample. The effect of PEF, RFEF, UV and the rmal techniques on the quality of red grapefruit juice were investigated based on microbial, color, physical properties, vitamin C content, non enzymatic browning, pectin esterase inactivation, aroma volatiles and sensory analysis Thermal treatment was mo re effective compared to non thermal treatment in terms of microbial stability after 4 weeks storage. However, RFEF treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with significantly (p<0.05) higher Vitamin C content and equiv alent PME inactivation compared to thermally treated juice. PEF and UV treatments maintained good microbial quality for 1 week with significantly (p<0.05) higher Vitamin C content as well as better color preservation compared to thermal. Thermal as well as non thermal pasteurization affected the aroma profile of fresh grapefruit juice considerably. The effects of treatments on juice aroma were highlighted by loss in desirable fruity and citrus odorants along with an increase in undesirable cooked/catty odorants. Aroma of treated juice differed significantly (p<0.05) from fresh juice by sensory evaluation. Non thermally treated juice had a shorter shelf life but higher vitamin C content and superior physical qualities compared to thermally treated juice.

PAGE 15

15 CHAPTER 1 INTRODUCTION Public perception of juices as a healthy natural source of nutrients and heightened public interest in health issues ha ve led to increased fruit juice consumption worldwide However, in rece nt years many outbreaks of food borne il lness have been reported after consumption of unpasteurized juice due to the presence of microorganisms like Salmonella sp ., Escherichia coli O157:H7 and Cryptosporidium parvum (Besser and others ) The 1999 Salmonella outbreak in the United States was caused by consumption of unpasteurized orange juice. Under the federal Juice HACCP rule published in 2001, juice processors must implement treatments to reduce populati on s of the most resistant microorganism of public health significance that is likely to occur in the juice in question. At present E. coli O157:H7 and Cryptosporidium p arvum are accepted as the pertinent organisms for apple juice and Salmonella are the accepted pertinent organisms in citrus juice Thermal processing is the most commonly used technique to reduce spoilage and pathogenic microorganisms in fruit juice s (Choi and Nielsen 2005) Unfortunately, thermal pasteurizatio n can produce undesirable quality changes like loss of color and flavor in addition to reducing the nutritional quality of juice (Rouseff and others 2001; Vikram and others 2005) Recently, non thermal processing alternatives lik e high pressure, dense phase CO 2 pulse electric fields, oscillating magnetic fields, pulsed high intensity light and ultraviolet irradiation ha ve been examined for their efficacy in extending shelf life and enhancing juice, pulp or cider microbial safety while minimizing

PAGE 16

16 quality and nutritional losses (Cserhalmi and others 2006; Tandon and others 2003; Dona hue and others 2004) Even though there are many non thermal processing techniques available for the pasteurization of juices, a lot more research is required to make them commercially successful. Particularly, studies about the effects of non thermal te chniques on quality and consumer acceptability compared to the effects of thermal pasteurization are required. Most often the conditions used for thermal processing are severe in terms of temperature and time of exposure to heat. Thus shelf life studies c omparing the effect s of non thermal to thermal techniques on acceptability of juice using conditions that do not achieve the same reduction of a concerned microorganism would result in incorrect conclusions (Rivas an d others 2006; Aguillar Rosas and others 2007; Yeom and others 2000b) Although a large amount of microbial information concerning thermal and non thermal processed fruit juice can be found in the literature, little information exists on the effects of the se processes on the flavor of juice. A major motivation for non thermal processing technologies is a minimal change to organoleptic properties Therefore an in depth analysis of the effect of the above processes on the flavor profile of juice and its relat ion to sensory quality is necessary. A recent study by Sentandreu and others (2006) compared the sensory acceptability of citrus juices (Valencia, Clementine and Ortanique) pasteurized by pulsed electric field (PEF) and heat treatment Each treatment was optimized to achieve comparable 90% pectin methyl esterase (PME) inactivation A s imple ranking test revealed no significant difference in sensory acceptability between PEF (25kV/cm for 330 s) and heat (85 C for 10s) treated juices

PAGE 17

17 I t is hypothesized that p rocessing of juices by thermal and non thermal techniques at equivalent microbial inactivation conditions would have comparable effect s on juice quality. The main objectives of this st u dy were to : a) d etermine optimal conditions for apple cider and red grapefruit juice pasteurization by thermal and non ther mal (PEF, RFEF & UV) techniques b) c ompare the impact of thermal and non thermal pasteurization techniques on quality of apple cider and r ed grape fruit juice c) s tudy the change in quality of apple cider and red grapefruit juice during 4 weeks storage at 4 C

PAGE 18

18 CHAPTER 2 LITERATURE REVIEW Flavor Perception Flavor is one of the major influences on consumer purchases and consumption. It is an ama lgamation of aromatic character, gustatory stimuli and trigeminal sensations. The aromatic character is imparted by a complex mixture of volatiles whereas taste and trigeminal components are mostly non volatile, hydrophobic molecules soluble in saliva (Laing and Jinks 1996) Aroma is sensed in the nasal cavity and encompasses most of what is considered fla vor. Odorants first interact with olfactory sensory neurons located in the olfactory epithelium lining the nasal cavity. Conformational changes in the receptor due to binding of odorant results in a cascade of biochemical changes leading to transmission of signals to the olfactory bulb These signals are further relayed to other brain areas where the information is processed to given an integrated flavor profile (Hatt 2000; Lancet and others 1993) A major breakthr ough with regards to odorant and receptor specificity came from the Nobel Prize winning work of Buck and Axel (Buck and Axel 1991) The three major points they presented were : a) each odor receptor can recognize multiple odorants b) each odorant can be detected by multiple receptors and c) the olfactory system uses a combinatorial receptor coding scheme to encode odor identity. The receptor specificity for cert ain molecular groups is evident in the case of the rat 17 odor receptor. This receptor binds to seven to nine carbon aliphatic aldehydes with maximum affinity to octanal and lesser interaction with molecule like heptanal (Krautwurst and others 1998) However, with substitution of the aldehyde group with any other functional group, this affinity is completely lost (Araneda and others 2000) The functional grou p of compound s appears to be significant for recognition of odorants.

PAGE 19

19 On the other hand, each odorant can be recognized by multiple receptors and thus stimulate several receptors at same time. It is predicated that those odor receptors which interact with the same odorant would have a similar protein sequence. For example, highly related odor receptors S6, S50 and S79 interact with odorant nonanedioic acid (Malnic and others 1999) Each odorant thus activates a unique combination of olfactory cells and send a unique pattern of neuronal activity to the olfactory bulb. The signal is then relayed to brain to encode the odor i dentity. Humans have about 350 different odor receptors ; hence the olfactory system is well adapted to recognize an endless list of odorants and to discriminate them (Zozulya and others 2001) Gustatory stimuli ( or taste ) is the perception of sweet, sour, salty, bitter, and umami sensations in the mouth. Umami is the characteristic taste imparted by glu ta nucleoti des like inosine and guanine. Taste cells are clustered in taste buds on the tongue, palate and pharynx The tastants interact either with ion channels (salt y sour) or specific G protein coupled receptors (bitter, sweet, umami) on the taste cells and tran sducts the signal to brain. Generally, chemicals that elicit sour and salty tastes are ionic in nature and are associated with high concentrations of H + and Na + respectively (Lindemann 1996) Ions enter the cell via selective membrane channels resulting i n depolarization of cell. This causes the release of neurotransmitters between taste cell s and taste neuron s Bitter, sweet and umami taste components bind to G protein coupled receptors similar to olfactory receptors al though the chemical recognition is not as discriminating as it is in the case of the olfactory system (Zhang and others 2003) Bitter receptors belong exclusively to a class known as the T2R

PAGE 20

20 family (Mueller and others 2005; Chandrashekar and others 2000) Bitter tasting compounds ha ve an important role in survival as they signal the presence of potentially dangerous components. In nature nu merous and biologically diverse bitter compou nds exist such as alkaloids, flavo noids, glycosides, steroids, phenols, N heterocyclic compounds and urea and related compounds. In comparison to the wide range of bitter compounds, there are far fewer T2Rs (~ 25) identified in mammals Studies using functional expression of T2R transcripts in taste receptor cells have shown that each bitter sensing taste cell co expresses the majority of T2R genes (Adler and others 2000) Meyerhof and others (2010) suggested that T2Rs are broadly tuned to perceive a large variety of bitter molecules. When 25 hTAS2Rs (human bitter taste receptors) were challenged with 104 bitter chemicals, they found that 3 of th ese hTAS2Rs were able to detect ~50% of the bitter compounds. However, the ability of bitter receptors to discriminate between the hTAS2Rs was limited (Adler et al. 2000) Thus, quite often many bitter compounds taste the same. Both sweet and umami receptors belong to the family of receptors called T1R (Nelson and others 2001; Nelson and others 2002) Heterodimers of these receptors namely T1R1 T1R3, T1R2 T1R3 or T1R3 function as receptors for sweet taste s These receptors expressed differentially among the taste cells enable binding of a wide range of structurally dissimilar molecules which are perceived as sweet like sweet sugars, sweet proteins and artificial sweeteners (Nelson et al. 2001) Heterodimer T1R1 T1R3 acts as umami taste receptors and binds to monosodium L glutamate and other L amino acids (Nelson et al. 2002) Because this type of heterodimer is common to both umami and sweet taste s there may be a relati onship between these two taste modalities. Compared to the olfactory system, the

PAGE 21

21 taste system is incapable of detailed chemical analysis due to the small number of taste receptors and ion channels. Another system found in olfactory epithelium is the trige minal nerve receptors. The trigeminal nerve extends to second set of nerve endings in areas around the mouth, eyes and nasal cavity. They are responsible for tactile sensations pressure, pain and temperature. Some food components like capsaicin, menthol, flavonoids and alkaloids are responsible for trigeminal sensations like pungency, cooling and astringency. The trigeminal system influences the perception of taste and smell. A study by Prescott and others (1993) showed that capsaicin inhibits the perceived sweetness of sucrose. In another study, the burning sensation produced by capsaicin was influenced by the presence of sodium chloride but not by the presence of sucrose (Prescott and Stevenson 1995) More studies of the role of trigeminal sy stem in flavor perception are required. The perception of aroma is also influenced by whether the odorant is perceived via the orthonasal or retronasal path. Orthonasal refers to when the aroma is taken directly through the nose (Linforth and others 2002) Retronasal refers to the aroma volatiles produced after the food is swallowed and air from the lungs is exhaled through the nasal cavity. In an orthonasal case, the release of aroma compounds from foods depends on the partition coefficient between air phase and food matrix. In retronasal olfaction aroma release depends on the partition coefficient between the water phase (saliva) and the food matrix. The eating process is a dynamic process a nd the release of volatiles from food depends on the different physical and chemical conditions in the mouth like pH, temperature, mastication and enzymes. The rate of release of odorants

PAGE 22

22 is also affected by the degree of interactions between aroma compoun ds and both macro (carbohydrates, proteins and lipids) and micro (acids, salt) food constituents. The retronasal impact is thus a combination of olfaction and gustatory perception Retronasal aroma would most likely leave a lasting impression on the consum er. Fruit Flavors Fruit and fruit juice consumption have increased recent past years due to their perceived health benefits. Because flavor is one of the major influences of consumer purchases and consumption, a significant amount of research in last 30 ye ars has been conducted in area of fruit flavors. The focus of research has been on characterizing the key volatile components that deliver the characteristic flavor unique to particular fruit s Apple Juice/Cider Aroma Apple cider (sometimes called soft c ider ) is the name used in the United States for an unfiltered, unsweetened, non alcoholic beverage pro duced from apples. It is opaque due to the fine apple particles in suspension and may be tarter than conv entional filtered apple juice depending on variet al characteristics of the apples used. In European countries, cider refers to an alcoholic beverage made from the fermented juice of apples. Volatile compounds in fresh apples have been a subject of interest to many investigators. Over 300 volatile compou nds have been detected in apples comprising of alcohols, aldehydes, carboxylic esters, ketones and ethers (Dimick and Hoskin 1983) Esters are present in highest amounts (78 92%) followed by alcohols (6 16%). E ster volatiles in apple s give a fruity odor and are classif ied as ethyl esters, butyric esters, propanoic esters and hexanoic esters. The alcohols mostly are ethyl, butyl and hexyl alcohols.

PAGE 23

23 The volatile composition and content in apple varies according to variety, maturity and storage conditions. M ost of the ch anges in flavor and aroma characteristic s develop after harvest. The aroma volatiles in whole intact fruit s are mostly formed via beta oxidation or the lipoxygenase pathway from fatty acids which are the major precursors of volatiles (Dimick and Hoskin 1983) The str aight chain esters are synthesized via beta oxidation of fatty acids to give acetic, butanoic and hexanoic acids These acids are then reduced to corresponding alcohols and esters using Acyl CoA enzyme (Paillard and Rour i 1984a) As the fruit ripens, the cell wall and membranes becomes more permeable and the lipoxygenase enzyme comes in contact with fatty acids like linoleic and linolenic acid, converting them into C6 and C9 aldehydes (Dever and others 1992) These aldehydes are responsible for the green notes in apple aroma. The C6 and C9 aldehydes formed in homogeni z ed fruit reach their maximum concentration within the first hour after hom o geni z ation. Varietal differences affect volatile profile of apples. Granny Smith and Nico variet ies are characterized by a high concentration of ethyl butyrate and hexanol han apples are characterized by high levels of hexanal and 2 E hexenal (Dixon and Hewett 2000; Schamp and others 1988) In addition to the ripening and varietal effect, storage time and environment also impacts apple flavor (Aaby and others 2002; Plotto and others 1999) Apples are mostly held in controlled atmosphere s during storage to delay ripening and reduce respiration. The reduced oxygen environment induces acetaldehyde and ethanol accumula tion and decreases ester and aldehyde levels. Exposure of apples to low temperatures for more than 3 months decreases volatile concentration (Streif and Bangerth 1988)

PAGE 24

24 One of the earliest studies on sensory contribution of apple volatiles to aroma was by Flath and others (1967b) They performed GC sniffing of Delicious apple essence. In total 56 compounds were iden tified by mass spectrometry Compounds with apple characteristics were listed as ethyl 2 methyl butyrate, hexanal and 2 E hexenal. A study by Durr and others (1981) on 48 samples of commercial apple esse nce showed that the concentration of 2 E hexenal and butyl acetate ha d a correlation of 0.78 and 0.72 respectively to the odor intensity of apple essence. Good apple essence was characterized by high levels of C6 aldehydes and esters and low alcohol leve ls. In R ed D elicious apple s ethyl butyrate, ethyl 2 methyl butyrate, propyl 2 methyl butyrate, hexyl acetate, ethyl hexanaote and 1,3,5 (E,Z) undecatriene were identified as key odorants by GCO analysis (Cunningham and others 1986) In Gala apples stored in a regular atmosphere the key contributors to aroma w ere butyl acetate, hexyl acetate, butyl 2 methyl butyrate, hexyl 2 methyl butyrate and hexyl propanoate (Plotto and others 2000) Its profile change d in a controlled a tmosphere by a decrease in esters hexyl acetate and butyl acetate. Fuhrmann and others (2002) compared GCO data of three ap ple cultivars, Cox Orange, Elstar and Royal Gala to determine the key contributing odorants. They studied the odor profile in the headspace of whole fruit as well as homogenized fruit. The key ester volatiles in whole fruit for different apple varieties w ere ethyl 2 methyl butyrate, ethyl butyrate (Elstar), ethyl 2 methyl propanoate, ethyl butyrate, 2 methyl butanol (Cox) and methyl 2 methyl butyrate, ethyl 2 methyl butyrate, and propyl 2 methyl butyrate (Royal Gala). In homogenized fruit, the FD profile c hanged compared to whole fruit High FD values for compounds hexanal, cis 3 hexenal and hexyl acetate in

PAGE 25

25 Cox and Elstar varieties were noted. The increase in levels of aldehydes was due to oxidation of linoleic acid and linolenic acid by liopxygenase relea sed from crushed cells. Other volalites noted with high FD value s damascenone and p allyl anisole. Beta damascenone has a rosy fragrant odor which adds intensity to the fruity odor of an apple Para allyl anisole with musk herbaceous odor along with its isomer anethole imparts spicy note s to an apple arom a. Mehanigic and others (2006) characterized odor volatiles of three apple cultivars (Fuji, Golden D elicious and Braebu rn) at different maturity stages. They found 15 odorants common to all varieties, the important ones being ethyl butyrate, ethy 2 methyl butyrate, butyl acetate, hexyl acetate and hexanal. The overall quantity of odorants increased during maturation in Fuj i and Golden Delicious apples with more alcohols and esters (butyric & hexanoic) and less aldehydes. The data from various literature reports on different varieties of apples indicate esters like ethyl butyrate (4) ethyl 2 methyl butyrate (5) butyl aceta te (6) and hexyl acetate (7) (Figure 2 1) as key odorants of apple aroma. Grapefruit Juice Aroma Grapefruit juice has a unique flavor due to its sweet tart and bitter taste combined with a characteristic aroma. Q uantitative and qualitative studies on volat ile constituents of grapefruit juice essence and oils have been carried out by many groups (Buettner and Schieberle 1999; Lin and others 2002a; Coleman and others 1972) Moshonas and others (1971) reported a total of 32 compounds in grapefruit essence extracted from canned grapefrui t juice Based on the relative amounts by GC FID, they reported major volatiles as ethanol, acetal, ethyl acetate, ethyl butyrate, isoamyl alcohol, cis and tran linalool oxide, linalool, octanol, alpha terpineol, ethyl 3 hydroxyhexanoate and terpinen

PAGE 26

26 4 o l as major components. In contrast to essence, fresh juice ha d high ethanol content followed by volatiles limonene, beta caryophyllene, ocimene and myrcene. Other compounds found in lower quantities were linalool, citral, terpeniol, phelladrene, copaene and nootkatone (Shaw and others 2000; Nunez and others 198 5) Pino and others (1986) studied the relationship between sensory response and 32 compounds present in grapefruit juice. Some of the important aroma compounds identified were limonene, acetaldehyde, deca nal, nootkatone, ethyl acetate, methyl butyrate and ethyl butyrate. They determined the odor threshold value of the compounds in water and quantified the amount of each compound in juice. The relative flavor contribution of each compound was calculated bas ed on the ratio of amount in juice to odor threshold in water. Ethyl acetate and acetaldehyde had the highest relative threshold value s and nootkatone the lowest. The authors performed a sensory analysis where in panelists scored the odor of juice with fou r different dilutions of compo unds in increasing order. These sensory scores were then related to quantities of compounds from GC data using linear regression analysis. N ootkatone, limonene, decanal, ethyl butyrate and methyl butyrate showed positive corre lation which indicates that they contribute significantly to good grapefruit juice flavor. However, a n increase in concentration of terpineol, cis and trans epoxydihydro linalool had a negative correlation with sensory scores. In another study by Jella and others (1998) a positive correlation in grapefruit juice acceptability with beta caryophyllene levels and negative correlation wi th myrcene and linalool levels were reported. T wo compounds, nootkatone (1) and p 1 menthene 8 thiol (2) (Figure 2 1) have been identified and proposed as character impact components of grapefruit juice aroma

PAGE 27

27 (Macleod and Buigues 1964; Demole and others 1982) The flavor threshold of nootkatone is at 1ppm in water and 6ppm in reconstituted grapefruit juice (Berry and others 1967) N ootkatone at levels more than 6 7ppm impart an unpleasant bitterness to juice The importance of nootkatone to grapefruit juice flavor is debatable as Shaw and others (1981) found that addition of nootkatone to grapefruit juice had very little impact on juice flavor. T hey suggested the presence of other aroma components in juice that are significant to grapefruit flavor. S ulfur compound 1 p menthene 8 thiol is a character impact odorant in grapefruit juice Demole and others (1982) isolated 1 p menthene 8 thiol (1 PMT) and its bicyclic epimer 2, 8 epithio cis p menthane from canned grapefruit juice. They reported 1 PMT as the most potent odorant in nature with a very low odor threshold at 0.1 x 10 9 g/L. It occurs at 200 fold higher concentration in juice implying its significance to grapefruit flavor. The threshold of the 1 PMT isomer was about 10 5 greater than the thiol indicating lesser contribut ion to grapefruit flavor. To investigate other impo rtant odorants in grapefruit juice, further studies by the same authors led to the identification of 15 related sesquiterpenes ketone s in grapefruit juice (Demole and Enggist 1983) One of the se ketone s 8,9 didehydronootkatone was a more intense (lower threshold) grapefruit aroma component than nootkatone Most of the odor active volatiles important to grapefruit aroma are present in very low levels and often not detected by mass spectrometry or FID Numerous GC olfactometry studies have been performed to identify the character impact volatiles in grapefr uit juice. A recent study by Buettner and others (1999) detected 37 odor active volatiles in fresh grapefruit juice. Although acetaldehyde and limonene were the most abundant compounds present in grapefruit juice, they had a minor impact on juice

PAGE 28

28 flavor. The au thors reported high FD values for : ethyl butyrate, 1 p menthene 8 thiol, (Z) 3 hexenal, 4, 5 epoxy (E) 2 decenal, 4 mercapto 4 methyl pentan 2 one (4 MMP) (3) 1 hepten 3 one and wine lactone. The t race component 4 MMP had the highest odor activity value The s ame authors conducted further studies by quantitation reconstitution and omission methods to determine the contribution of above odorants (Buettner and Schieberle 2001) Sensory evaluation of the reconstituted grapefruit aroma model using 20 odor active components in water showed the sulfurous grapefruit like odor quality due to 4 MMP and 1 p menthene 8 thiol to be important Single compound omission of 4 MMP from aroma model s indicated that it had a higher impact on grapefruit odor than 1 p menthene 8 thiol. Other grapefruit juice sulfur compounds reported in other studies included: 4 mercapto 4 methyl pentanol, 3 mercapo hexanol and 3 mercapto hexyl acetate (Lin and others 2002b) Hydro gen sulfide was also reported as a potential contributor to grapefruit juice aroma as it is present at levels above its odor threshold in juice (Shaw and others 1980) Grapefruit juice has 2 4X more sulfur components compared to orange jui ce (Rouseff and others 2003) Volatile Extraction The first step when characterizing odor active chemicals in a complex mixture is to separate them from nonvolatile components. This is accomplished through a variety of techniques such as solvent extraction, headspace concentration or distillation. The results can vary significantly in terms of final composition and flavor c omponent mixture depending on the method used for volatile collection and concentration of aromas. For example, low impact methods of flavor isolation like hydrodistillation and solvent extraction are preferred for concentrating aromas from fruits and blo ssoms as they are

PAGE 29

29 less invasive and use mild temperature s However, these methods might not be suitable for reaction flavors that have already been subjected to heat treatment. The extraction methods used in the past for apple volatiles include: vacuum h ydro distillation, dynamic and static headspace and liquid extraction (Komthong and others 2006a; Matich and others 1996; Mehinagic and others 2003) Young and others (2004) found that butanol was detected at the highest levels in Royal Gala apples by vacuum hydrodistillation while butyl acetate was detected at the highest level s using headspace method s Matich and others (1996) compared solid phase micro extraction (SPME) an d solid phase extraction (SPE) method for Granny Smith apple volatiles. SPME provided a greater adsorption of high molecular components but required long period of time for equilibration and exposure times Various methods like solvent extraction, vacuum distillation, purge and trap, steam distillation extraction (SDE) and SPME have been used for extraction of grapefruit juice volatiles (Nunez and others 1984; Cadwallader and Xu 1994; Yoo and others 2004) SDE meth od was good for extraction of medium and low volatile components but the high temperature used during the procedure combined with the presence of oxygen could alter composition of volatiles (Nunez et al. 1985) Purge and trap extraction eliminates some artifact formation However, the method showed a few drawbacks when used for grapefruit juice volatile analysis. Compounds having lower vapor pressure like oxygenated mono and sesquiterpenes could not be effectively collected and therefore could not be detected. Moreover, sulfur compounds significant for grapefruit juice aroma were also not detected using the above extraction method (Cadwallader and Xu 1994) Solvent extraction is reported to be a better method of

PAGE 30

30 volatile extraction for grapefruit juice but it is cumbersome and loss of volatiles may occur during solvent evaporation (Buett ner and Schieberle 1999; Jella and Rouseff 1997) Reports on SPME extraction of grapefruit volatiles are limited (Yoo et al. 2004) Static h eadspace analysis by solid phase micro extraction (SPME) is a simple, rapid and solventless technique for extraction of volatiles (Kataoka and others 2000) Equilibrium is established among the analyte concentrations above the sample headspace and the polymer coating on the fused silica fiber. The amount of analyte adsorbed by the fiber depends on the thickness of polymer coati ng, the type of fiber coating and the distribution constant for the analyte. In addition to fiber, the transfer of analyte from food matrix to SPME fiber also depends on the matrix composition time and temperature of extraction. Some of the fiber coatings used are polydimethylsiloxane (PDMS), divinylbenzene (DVB), carbowax (CW), carboxen and polyacrylate. Most often a combination of these coatings is used for broad range volatile extraction. Gas Chromatography Olfactometry Even though food aroma is a compl ex mixture of volatiles, only a fraction of these volatiles are odor active Most of the potent odorants are not often detected by instrumental techniques due to very low odor thresholds Human olfaction has superior sensitivity and selectivity to odor com pounds compared to many instrumental systems. The hyphenation of olfactometry to gas chromatography technique is an effective tool for the discrimination of relevant food flavor components enabling the differentiation of a multitude of volatiles into odor active and non odor active categories In GCO, assessors judge the olfactory impressions elicited by the volatile compounds immediately after elution from a GC column in order to associate odor activity with the

PAGE 31

31 eluting compounds. GC/O methods are cla ssif ied into three categories: d ilution methods, time intensity methods and frequency of detection method s Time intensity methods are dynamic and odor intensity is recorded with the time intensity approach for evaluating the significance of odor compounds in the GC effluent. Trained subjects sniffing the GC effluent mixed with humidified air directly record the odor intensity and duration time of each odor active compound while describing its odor quality. The plot of the retention time/index versus odor significance in the flav (Bazemore and others 1999) Higher peaks suggest greater sensory importance of the c ompounds. Time intensity olfactometry has been used to identify key odorants in various fruits like apple s orange s grapefruit s blueberr ies and cashew apple s (Plotto et al. 2000; Lin and Rouseff 2001; Rouseff et al 2001; Su and Chien 2010; Garruti and others 2003) Processing Fresh fruit juices can undergo quality degradation due to microbiological and enzymatic activities as well as chemical reactions. In recent years, there has been increased concern about the safety of unpasteurized fruit juices due to outbreaks of Escherichia coli O157:H7, Salmonella spp. and Cryptosporidium parvum (Besser and others 1993a; Parish 1997) On January 19, 2001, t he FDA published a final ru le in the Federal Register that requires processors of juice to develop and implement Hazard Analysis and Critical Control Point (HACCP) systems for their processing operations (66 FR 6138). HACCP is a systemic preventive process applied in the food indust ry to address food safety by control and analysis of physical, biological and chemical hazards

PAGE 32

32 at various processing stages. Under this rule, juice processors must comply with two requirements: (1) s ubpart A of the rule requires use of HACCP principles an d systems in their operations, and (2) s ubpart B of the rule requires that processors implement treatment(s) to uice by 99.999% or 5 log cycles. d as the most resistant microorganism of public health significance that is likely to occur in the juice. At the present time, Salmonella is generally recognized as the pertinent organism for citrus juices and E. coli O157:H7 and Cryptosporidium parvum for apple juice (FDA 2001) Thermal Pasteurization Thermal treatment has been widely used to inactivate spoilage pathogenic microorganisms and enzymes to extend shelf life of juice products. Initially pasteurization of fruit juices was done at low temperature s for long periods of time (63 C for 30 min). But extended heat treatment lowered the sensory and nutritional qualities of juices (Moshonas and Shaw 2000) Current pasteurization methods in practice are high temperature s short time (HTST) and ultra high temperature (UHT). UHT process is performed at 135 150 C for a few seconds (2 45 s). There are 2 methods: direct heating by steam injection and indirect heat transfer by heat exchanger. The product is aseptically packaged after UHT proce ssing in order to obtain a product with a shelf life of 1 to 2 years at ambient temperatures. UHT destroys all pathogenic microorganisms as well as spores. It is commonly used for milk pasteurization (David

PAGE 33

33 and others 1996) However, UHT causes considerable change to the quality of milk as it renders a heated flavor. HTST or flash pasteurization is the most comm on method used for fruit pasteurization (David et al. 1996) Flash pasteurization is used for perishable beverag es like milk, fruit and vegetable juices, and beer. It is done prior to pouring the beverages into containers in order to kill spoilage microorganisms in an effort to make the products safer and to extend their shelf life. The liquid moves in a controlled, continuous flow through a heat plate exchanger system subjected to temperatures of 72 74 C for about 15 to 30 s. It increases the shelf life by 2 3 weeks under refrigerated conditions. Flash pasteurization destroys all pathogenic microorganisms but doe s not inactivate spores. The time/temperature requirement for pasteurization of juice depends on the initial microbial load, pH of the product and if enzyme inactivation is required. Citrus juice thermal processing I n addition to microbial inactivation citrus juice processing requires pectin esterase (PE) inactivation as this enzyme leads to cloud loss in stored citrus juices. Normally, temperatures above 71 C for few seconds are enough to kill pathogens and spoilage bacteria in orange juice (Kale and Adsule 1995) However, treatment of juice at 90 C for 0.8min to 1min is required for 99% inactivation of PE activity (Wicker 2 004) The food i ndustry has different time and temperature parameters for enzymatic inactivation and destruction of microbial population in citrus juices by heat (Table 2 1) (Crupi and Rispoli 2002) Apple juice thermal processing F lash pasteurization of apple juice involves heating the juice to 71 C for 6s or heat ing cider to an equivalent time /temperature combination (AFDO 2001) However,

PAGE 34

34 the cost of flash pasteurization for small cider producers is unaffordable, and therefore opti ons like the hot fill technique are advantageous whe n the whole packaged product is pasteurized. This reduces the cost of steriliz ing the container. Hot filling is conducted by passing the final product through a heat exchanger and raising the temperature such that the temperature of the juice filling container reaches a recommended temperature of 88 to 95 C. The juice is usually held for at least 3 min before cooling. The hot fill technique is adequate for acidic beverages like apple juice, cranberry juic e and grape juice. Usually a shelf life extension from 9 to 12 months in glass bottles can be achieved by this method (McLellan and Padilla Zakour 2004) Effect of Th ermal Pasteurization on Juice Flavor Conventional juice pasteurization by heat causes significant loss of fresh flavor, color and nutritional content (Braddock 1999) There are few studies regarding the effect of thermal processing on the volatile composition of apple juice. A decrease in ester concen trations due to thermal pasteurization has been found in all these reports (Kato and others 2003; Su and Wiley 1998; Aguillar Rosas et al. 2007) Su and others (1998) reported a decrease in apple juice aroma components iso butyl acetate, ethyl butyrate, ethyl 2 methyl butyrate and hexanal after pasteurization at 85 C for 10 min. Apple juice proce ssed by HTST at 90 C for 30 s resulted in more than 50% loss in hexanal, ethyl acetate, ethyl butyrate, methyl butyrate and acetic acid concentrations (Aguillar Rosas et al. 2007) Kato and others (2003) correlated the change in apple juice volatiles pasteuriz ed at various time temperatures (2 320s; 80 120 C) to sensory data by principal component analysis. They found that the change in volatile concentrations were very complicated with respect to different thermal processing conditions as the odor attributes of processed apple juice were not predictable. Principal component

PAGE 35

35 analysis indicated that treatment temperature affected the volatile concentration more than treatment time. Citrus juice flavors are also sensitive to thermal pasteurization Shaw and oth ers (2000) found a decrease in volatile constituents in heat pasteurized orange juice during a 9 week storage study. Tatum and others (1975) reported occurrence of alpha terpineol, 4 vinyl gu aiacol and Furaneol in thermally treated canned orange juice stored at elevated temperatures PVG was found to be the most detrimental to orange juice flavor as it gave a rotten flavor to the juice. Furanones like Furaneol are formed from Maillard reac tions between amino acids and sugars present in juice and impart a caramel cooked odor quality to juice (Perez Cacho and Rouseff 2008) The acid catalyz ed degradation of limonene under acidic conditions results in formation of compounds like terpineol, cineole, carvone and carveol (Marcotte and others 1998) Alpha terpieno l is considered to be a marker for thermally abused citrus juices. In contrast to orange juice, there are few reports on the thermally induced changes in grapefruit juice aroma (Lin et al. 2002b; Lin and others 2001 ; Lin et al. 2002a) Shaw and others (1982) compared the headspace levels of sulfur compounds in fresh and commercially heat processed grapefruit juice. They reported higher levels of hydrogen sulfide (tentatively identified) in fr esh juice compared to processed juice whereas dimethyl sulfide was detected only in processed juice. In a more recent study, GCO analysis of thermally processed grapefruit juice showed a loss of 6 odor active odorants and formation of meaty, cooked odoran ts 2 methyl 3 furnathiol and 2 acetyl 2 thiazoline (Lin and Rouseff 2001) These compounds negatively affect the flavor of juice A s ulfur volatile quantitation using sulfur chemiluminescence detector showed that pasteurized

PAGE 36

36 grapefruit juice had higher levels of sulfur compounds compared to untrea ted juice (Lin et al. 2002a) Sulfu r containing the amino acids methionine, glutathione and cysteine were proposed as the possible precursors for these sulfur volatile compounds. A comparison of single strength and grapefruit juice reconstituted from concentrate without volatile restorati on showed more than 90% loss of volatile components in the reconstituted juice (Lin et al. 2002b) Olfactometric analysis showed a drastic loss in fresh/citrusy and sulfur/grapefruit characteristic notes. The loss in fresh/ ci trusy notes was attributed to a loss of limonene, octanal, and nonanal whereas the loss in 1, 10 dihrydronootkatone, 3 mercaptohexyl acetate, 3 mercapto hexanol and 4 mercapto 4 methyl pentanol contributed to a decrease in sulfur/grapefruit odor. Thermal instability as well evaporation during heating contributed to flavor loss. In addition to flavor loss, thermal processing also causes degradation of vitamin C. Saguy and others (1978) found 7 12% loss of Vi tamin C content in grapefruit juice pasteurized at 95 C for 1min. Non Thermal Processing Methods Consumer demand for safer and minimally processed food has resulted in investigation of non thermal processing alternatives. The main objective of non therma l preservation methods is to minimize degradation of food quality by limiting heat damage to foods. In addition to preservation of food quality, non thermal process should also achieve equivalent microbial safety level as thermal process es J uices are gra ded in United States based on standards such as Brix, Brix/acid ratio, color and flavor (AMS 1983) Therefore it is important to study the impact of non thermal processing on these quality parameters to determine if they maintain the

PAGE 37

37 Agricultural Marketing Standards (AMS). Citrus juices are consumed for their nutritional benefits (Vitamin C) as well as for their flavor. However, ascorbic acid is heat and oxygen sensitive and undergoes degradation during processing and storage. Thus the effect of n on thermal processing on ascorbic acid content is an important parameter to study Enzymes present in juice cause deterioration in the quality of juice by modification of pectin, degradation of ascorbic acid and browning. They change the flavor, color and texture of juice. In citrus juices it is equally important to maintain the cloud stability equally as flavor and color. About 5% of the cloud is pectin and it has a dominating effect on the behavior of cloud s Pectin methyl esterase enzyme present in ju ice demethylates the pectin molecule causing aggregation of pectin molecules. These aggregates settle out of the juice resulting in juice clarification or cloud loss (Guiavarc'h and others 2005) Thermal pasteurization is the most common method used for PME inactivation. The efficiency of non thermal processing to inactivate PME is a crucial factor in terms of commercial viability. Some of the non thermal techniques extensively studied for juice pasteurization are high pressure (HPP) dense phase CO 2 pulse electric fields (PEF) oscillating magnetic fields, pulsed high intensity light and ultraviolet irradiation (UV) (Elez Martinez and ot hers 2006; Boff and others 2003; Sampedro and others 2009; Donahue et al. 2004; Koutchma 2008) Many reports have been published in the last decade studying the efficacy of non thermal techniques to extend shelf life and enhance the safety of fresh juice while minimizing changes to organoleptic and nutritional qualities of the juice (Grahl and M arkl 1996; Tandon et al. 2003)

PAGE 38

38 Regulatory approval is important for the commercialization of non thermal technologies. Some of the FDA approved technologies for juice processing are PEF, UV and HPP. However, it has to be kept in mind that no single techn ology can be practically applied to all products and within technologies pathogen reduction varies among products treated. Pulsed electric field PEF processing involves treating liquid foods with high voltage pulses in the range of 20 80 kV for microse conds. A high electric field is generated between two electrodes (treatment chamber) which results in a large electrical flux flow through the product. A schematic diagram of a typical bench top PEF unit is given in Figure 2 2 (Jia and others 1999) The apparatus consist s of a high voltage power supply, a pulse generator, treatment chambers and a water bath for temperature cont rol. PEF process achieves both microbial and enzymatic inactivation while minimizing heating of foods and associated detrimental changes to sensory and physical properties of foods. Though there are several theories on microbia l inactivation, the most acce pted is by electroporation. The high voltage pulses break cell membranes of vegetative microorganisms thus altering the membrane permeability This results in protein inactivation and leakage of cellular contents and eventually disruption of microbial cel ls (Sale and Hamilton 1967) The factors that affect the efficiency of microbial inactivation by PEF are product composition microbial characteristics and treatment parameters. Product Parameters Only pumpable food products can be treated by PEF. The critical parameters pertaining to food product s are electrical conductivity, density, viscosity, pH and water activity (Cserhalmi et al. 2006) The food product s should have low electrical conductivity as conductivity increases the difference in conductivity

PAGE 39

39 between medium and micro bial cytoplasm resulting in a weakening of membrane structure (VegaMercado and others 1996) An enhanced efficiency in microbial inactivation in acidic environment was also reported by the same authors. Microbial Characteristics Generally gram positive bacteria are more resistant to PEF than gram negative bacteria (Hulsheger and others 1983) Yeasts and mold show higher sensitivity to PEF than bacteria due most likely to their larger cell size (Qin and others 1996) Wouters and others ( 2001) showed that Lactobacillus species in different sizes or shapes had different membrane permeability to PEF. In addition to the type of microorganism, the growth stage also chang es the sensitivity to PEF. Bacteria and yeasts at their logarithmic stage are more sensitive than those at the stationary or lag growth stage (Pothakamury and others 1996) PEF processing is more efficient at vegetative cell inactivation compared to spores Raso and others (1998) compared the effect of PEF treatment (32 36.5 kV/cm) on vegetative cells and Zygosaccharomyces baili ascospores. PEF treatment decreased the vegetative cell population by 4.5 5 log cycles and ascoproes by 3.5 4 log cycles. Treatment Parameters. The critical process factors for PEF treatment are electric field intensity, treatment time, pulse wave shape, pulse length, number of pulses and temperature. M icrobial inactivation efficiency increase s with an increase in electric field strength above the critical electric field (Qin and others 1998) The critical electric field is the intensity below which no microbial inactivation occurs. Hulseger and others (1983) found a linear relationship between electric field strength and E. coli inactivation Treatment time is another important parameter that affects the efficiency of PEF treatment. It is the product of numb er of pulses and pulse duration. Therefore, an

PAGE 40

40 increase in any of the above two parameters result s in increased microbial inactivation. However, longer pulse width also increases food temperature. Peleg and others (1995) showed that the rate of microbial inactivation by PEF at a constant electric field strength increased with an increase in treatme nt time. The electric field pulse can be applied in the form of exponential decaying, square wave and oscillatory pulses. Zhang and others (1994) compared the effect of square wave, exponentially deca ying and charge reverse pulses on the shelf life of orange juice. Square wave pulse s yielded juice with longest shelf life. PEF treatment of juice can lead to the formation of a shielding layer on electrodes in treatment chambers due to migration of charge d molecules to surface of electrode. This layer reduces the efficiency of PEF treatment. Bipolar pulses prevent the formation of shielding layer as the polarity reversal causes a corresponding change in direction of charged molecules (Evrendilek and Zhang 2005) Therefore, bipolar pulses are used with square wave or exponential pulses to increase efficiency of PEF processing. Moderate temperatures (40 60 C) exhibit synergistic effects with PEF on the i nactivation of organism. At moderate temperatures the membrane fluidity of cell is altered increasing sensitivity to PEF treatment. Heinz and others (Heinz and others 2003) found that E. coli inactivation increased from 1 to 6.5 log cycles with a temperature increase from 32 C to 55 C in inoculated apple juice. However, since application of electric field strength also increases the temperature of product, a proper post treatment cooling is necessary. Microbial stability studies in PEF treated juices. Microbial stability in PEF treated jui ces ha ve been studied in various fruit juices like apple juice, apple cider,

PAGE 41

41 orange juice, cranberry juice, tomato juice, and mango juice as well as in orange milk beverage s (Ayhan and others 2001; Charles Rodriguez and others 2007; Jin and Zhang 1999; Yeom and others 2004; Min and Zhang 2003) There are no reports to date on microbial stability in PEF treated grapefruit juice. The commercial scale processing of orange juice by PEF treatment at 40 kV/cm for 97 s re duced the total aerobic count and yeast and mold count by 6 log cycles (Min and others 2003a) Commerci ally processed PEF juice had a shelf life of 196 days at 4 C. of 7 months at 4 C (Qiu and others 1998) In appl e juice PEF treatment (34 kV/cm, 166 s) using a bench scale system resulted in a 4.5 log reduction in inoculated E. coli O157:H7 levels (Evrendilek and others 2000b) The same juice treated using a pilot scale unit at 35 kV/cm for 94 s and combination of heat treatment at 60 C for 30 s gave a sh elf life of more than 67 days at both 4 C and 22 C. Lately, the synergistic effect of PEF with antimicrobials to achieve improved microbial safety has been a subject of interest. Liang and others (2002) reported a 5.9 log reduction in inoculated Salmonella levels in fresh orange juice at 90 kV/cm, 50 p ulses and temperature at 55 C. Addition of antimicrobials nisin and lysozyme increased the inactivation by 1.37 log s Interestingly they also reported that PEF inactivation was significantly more extensive in pasteurized juices than fresh juice probably due to differences in their composition. This is an important parameter to look into when comparing the effect of varying PEF parameters on microbial inactivation in juices. Similar studies of apple juice also showed an increase in microbial inactivation

PAGE 42

42 by 1 2 log cycles using electric field strength in range 27 33 kV/cm in combination with nisin and lysozyme (Liang and others 2006) Though numerous studies on microbial stability and shelf life of products are present in the literature, there are fewer studies on the effect of PEF technology on different microorganisms. McDonald a nd others (2000) achieved more than 5 log reductions of Leuconostoc mesenteroides E. coli and Listeria innocua in orange juice at 30 kV/cm and 50 kV/cm electric field strength using varying pulses. Inactivation of spores was more difficult as a maximum of 2.5 log reductions of S. cerevisiae ascospores was achievable at 50 kV/cm 50 C. Garcia and others (2005) found that E. coli population s exhibited greater sensitivity to subsequent ho lding of apple juice at refrigerated conditions than immediately after PEF treatment (40kV/cm, 80 pulses) An increase in inac tivation from 0.5 log to 5 log was noted after 3 days of storage under refrigeration. This phenomenon was attributed to increased sensitivity of injured cells to the acidic conditions of apple juice. In summary, due to the numerous critical process factors and different experimental conditions used, definite conclusions about the critical process parameter effect on specific pathogen reductions are difficult to establish. Research that provides conclusive data on the effect of critical PEF factors on microbial inactivation is required for commercialization of the PEF process. Effect of PEF processing on juice quality The effect of P EF treatment on aroma, nutrition, color, sensory and other physiochemical parameters of orange, apple, cranberry and tomato juice s are present in literature (Jin and Zhang 1999; Biasioli and others 2003; Aguillar Ros as et al. 2007; Ayhan and others 2002)

PAGE 43

43 In apple juice, PEF extended the shelf life of fresh juice for up to 56 days with no change in sensory or physiochemical properties compared to fresh ly squeezed juice (Qin and others 1995) Evrendilek and others (2000b) found no significant change in sensory acceptanc juice by paired preference test. Aguillar Rosas and others (2007) reported a significantly higher loss in eight select volatiles (acetic acid, hexanal, butyl hexanoate, ethyl ace tate, ethyl butyrate, methyl butyrate, hexyl acetate, and 1 hexanal) in HTST treated apple The effect of PEF treatment on orange juice has been more extensively studied compar ed to other beverages. Jia and others (1999) noted that the loss of volatile compounds in PEF (30kV/cm fo r 240 s or 480 s) and heat processed (90 0 C for 1min) orange ju ice were greatly influenced by compound type and processing methods. Loss in v pinene, limonene and ethyl butyrate were common in both treatments. However, decanal and octanal were lost only in heat treated samples. The flavor loss by PEF process was mainly due to the vacuum degassing system rather than PEF application. Yeom and others (2000b) investigated the effects of PEF treatment on microorganisms and pectin methyl esterase activity They compared vitamin C, volatile and other attributes of PEF treated juice (35kV/cm, 59 s) to heat pasteurized (94.6 0 C for 30s) orange juice. PEF processing prevented growth of microorganisms at 4, 22 and 37 0 C for 112 days and inactivated 88% of PME activity. PEF treated juice also retained greater amounts of vitamin C and aroma compounds (pinene, myrcene, octanal, limonene and de canal) compared to heat treated samples.

PAGE 44

44 In contrast Ayhan and others (2002) reported a significant increase in hydrocarbons d limonene, alpha pinene, myrcene and valencene for PEF treated single strength orange juice (35kV/cm for 50 s) compared to untreated juice. The y proposed that the hydrophobic flavor compou nds present inside pulp of orange juice become more available during PEF application Because most of the previous PEF studies were performed in laborator ies or pilot plant scale s Min and others (2003a) studied the effect of commercial scale PEF processing (40kV/cm for 97 s ) on microbial stability, ascorbic acid content, flavor comp ounds and other sensory attributes compared to thermally processed (90 C for 90 s) and fresh ly squeezed orange juice. PEF and thermal ly processed juice had similar microbial shelf li ves at 4 C for 196 days PEF processed juice retained more ascorbic aci d, flavor and color compared to thermal ly treated juice. Preference sensory analysis indicated significantly higher sensory attributes of texture, flavor and overall acceptability for PEF treated juice compared to thermal ly processed juice Only one refer ence in the literature on PEF processed grapefruit juice could be found (Cserhalmi et al. 2006) The authors investigated the effect of PEF technology on physical properties, color, non enzymatic browning and flavor components They reported no significant changes in any of the quality parameters post processing compared to fresh untreated juice (Cserhalmi et al. 2006) The aroma analysis in the study was limited to eight select volatiles (terpinen 4 ol, ethyl octanoate, decanal, carvone, geraniol, geranyl acetate, gerany l acetone, and nootkatone) in grapefruit juice. Inactivation of enzymes by PEF Enzyme c onformational change is suggested as the possible mechanism of enzyme inactivation by PEF (Castro AJ 2001) The

PAGE 45

45 authors found that alkaline phospatase molecules tend to associate and aggregate due to polarization created by electric charges. Yeom and others (2002) reported an 83.2 % inactivation of PME in fresh orange juice at 35 kV/cm for 180 ms at 30 C. However, a 90% PME inactivation was achieved at 25 kV/cm when the temperature was increased to 50 C indicating a synergistic effect of heat and PEF treatment on PME inactivation. In apple juice, PEF reduced the PPO activity by 50 70% at treatment condition 38.5kV/cm, 300 pps and 50 C (Sanchez Vega and others 2009) Min and others (2003b) reported 88.1% inactivation in lipoxygenase in tomato juice treated by PEF at 30 kV/cm for 50 s at 50 C. Radio frequency electric field Radio frequency electric fields (RFEF) processing is a relatively new pasteurization technique and is not FDA approved yet. The key equipment components of the process include a radio frequency power supply and a treatment chamber that is capable of applying high electric fields to liquid foods (Figure 2 3) (Geveke and others 2007) The process is similar to the pulsed electric fields process, except that the power supply is continuous using an AC generator rather than pulses, thus reducing the capital cos t. Ukuku and others (2008) worked on E. coli inoculated apple juice to understand the mechanism of RFEF on microbial inactivation. They demonstrated a 4 log reduction in inoculated apple juice at 15kV/ cm entirely due to the non thermal effect of RFEF and n ot due to temperature increase during treatment. Membrane damage in bacterial cell wall leading to leakage of intracellular material was cited as a mechanism of microbial inactivation.

PAGE 46

46 The process parameters affecting RFEF efficiency are electric field st rength, frequency, treatment time, output temperature and number of treatment cycles. Radio frequencies of 15 and 20 kHz were more effective in inactivation of E. coli than 30 70 kHz (Geveke and Brunkhorst 2004) Increasing electric field strength (20 30 kV/cm), treatment time (140 420 s) and outlet temperature (50 60 C) increased inactivation of E. coli in apple cider (Geveke and others 2008) An increase in treatment cycles also increased inactivation of inoculated E. coli K12 at conditions 18kV/cm, 170 the effect of RFEF on physiochemical properties, flavor or color of apple juice are reported in the literature. In orange juice, an electric field strength of 20 kV/cm and outlet temperature of 6 5 C gave a 3.9 log reduction in E. coli K12 population with no significant non enzymatic browning and loss in ascorbic acid content (Geveke et al. 2007) Because RFEF is a relatively new pasteurization technique not much information is available on its effect o f the quality of juices. Ultraviolet radiation UV light in the wavelength 220 300nm exhibits germicidal properties and is used for inactivation of bacteria and viruses. UV light absorbed by the DNA of microorganism caus es cross linking between neighboring pyrimidine nucleoside bases (thymidine and c ytosine) in the same DNA strand and prevents cell replication. UV light has low penetration in juices and other beverages due to the presence of color compounds, organic solutes and suspended particles. Therefore, UV irradiation is limited to microbial ina ctivation of food surfaces, packaging material or processing plants. In recent years, due to many developments in design of treatment chambers by utilization of turbulent flow to form continuous renewed surface, microbial inactivation in juices and other

PAGE 47

47 b everages is enabled. In 2000, the FDA approved UV irradiation as an alternative to thermal treatment for fresh juices. UV light is now commercially used for water disinfection and apple cider pasteurization. A picture of UV processing unit used by Geveke (2008) for pasteurization of liquid egg white s is given in Figure 2 4. The unit consists of a bi pin base, a 30 W germicidal UV bulb, UV transparent tubing and silicon rubber tape. The lamp generated 90% of its energy at 254nm. The product flow s through tubing and the time of exposure to UV light is calculated based on the flow rate. The effectiveness of microbial inactivation by UV process depends on the UV light source, the product composition and microbial characteristics. UV light sources. The different light sources used for UV are mercury lamps, excimer lamps, microwave lamps and broadband pulsed lamps (Koutchma 2009) The correct UV source enhances microbial inactivation by increasing UV penetration i n liquid as well as by employing higher UV intensity from pulsed sources. Mercury lamps are generally used due to their low cost and effective microbial inactivation. Based on the vapor pressure of mercury when lamps are operating, they are categorized as : low pressure lamps, low pressure high output lamps a n d medium pressure lamps. Low pressure mercury lamps are generally used in food processing and are approved by the FDA. In pulsed lamps, alternating current is stored in a capacitor and energy is discha rged in the form of intense emission of light within microseconds This technology has been applied to food surface treatment in fresh produce and meats but it has not yet been established in the field of liquid foods. Product composition. The physical, chemical and optical properties of juices impact the effectiveness of microbial inactivation by UV. The variation in UV absorption

PAGE 48

48 between different juices is due to the difference in their pH, viscosity, soluble solid and suspended solid content. Clear ju ices are better candidates for UV treatment compared to opaque juices as the presence of dissolved solutes in juice causes strong UV attenuation effects (Koutchma and others 2007) C arrot and pineapple juices (opaque juices ) had higher UV absorptivi ty than semi transparent watermelon and apple juice. Ascorbic acid present in juice s also has strong absorbance between 220 300 nm even at very low concentrations. Tran and others (2004a) found a 17% loss in vitamin C content in UV treated orange juice. It is important to study the ascorbic acid content and its destruction during UV treatment in terms of UV dose delivery. Suspended solids present in juice not only attenuate UV dose but also provide a site for aggregation of bacteria to particle surfaces. S uspended solids also cause scattering in light such that microbes far away from the UV light are not effectively inactivated (Murakami and others 2006) Microbial characteristics. The information on the effect of UV on different pathogens is very limited. Factors like microbial species, strain, growth media, and initial microbial load as well as stage of growth affect the efficiency of microbial inactivation by UV. Generally gram po sitive bacteria are more resistant to UV than gram negative bacteria due to difference in cell wall thickness and composition. Bacterial spores, like Bacillus subtilis are very resistant to UV requiring 36mJ/cm 2 for 1 log reduction in water (Chang and others 1985) Mold spores are considered to be more UV resistant than yeast and lactic bacteria They are the major spoilage microorganisms in juices. Microbial inactivation studies in UV treated jui ces. UV light inactivates microorganism s by causing cross linking between neighboring pyrimidine nucleoside in

PAGE 49

49 the same DNA strand thereby preventing cell replication. The extent of cross linking is directly proportional to the UV light exposure. However photo reactivation can occur in UV injured cells when exposed to visible light in blue spectra ( Hoyer 1988) Therefore it is important to store UV treated juice under refrigeration and in dark conditions. UV dose is calculated as a product of e xposure time and intensity. It is assumed that change in either time or intensity should not affect microbial inactivation as long the UV dose used is same. However, recent studies have shown that some E. coli strains do not follow time intensity reciproci ty (Sommer and others 1998) Time intensity reciprocity is crucial for the scale up from pilot to commercial scale S cale up is possible only if time intensity reciprocity exists. Murakami and others (2006) showed that in apple juice inoculated with E. coli K12 the reciprocity occurs within a range of intensities only and the range decreases as UV dose level increases. UV processing of apple juice and apple cider has previously been conducted t o study the inactivation kinetics of microorganisms like E. coli O157:H7 and Listeria innocua (Duffy and others 2000; Geveke 2005) Duffy and others (2000) showe d a 5 log reduction in E. coli in apple cider treated by UV irradiation using a CiderSure pasteurizer (OESCO, Inc., Conway, MA). This unit has 8 UV lamps and the UV light is set to penetrate a (2005) exposed apple cider inoculated with E. coli and Listeria innocua to a UV lamp for varying length s of time. They achieved a 3.4 log reduction in E. coli population at 19 s of UV exposure at 25 C. L. innocua required a longer exposure time of 58 s for 2.5 log reduction. In a different study by the same author s on liquid egg white (Geveke 2008) the effect of exposure time, temperature and pH on E. coli inactivation was investigated. They found an increase in inactivation

PAGE 50

50 with an increase in exposure time (0 160 s) and that an exposure time of 160 s at 50 C reduced the microbial population by 4.3 log. They mentioned a direct dependency between inactivation and temperature but an indirect relationship between pH and inac tivation. UV inactivation was greater at neutral pH than higher pH values. In grapefruit juice inoculated with Saccharomyces cerevisiae a UV dose of 450 kJ/m2 at flow rate of 1.02L/min using a two coupled UV disinfection system resulted in a maximum of 2.4 log reduction (Guerrero Beltran and others 2009) Keyser and others (2008) treated apple juice, guava pineapple juice, mango nectar, strawberry nectar, two different orange juice s and tropical juice using a PureUV pilot scale system. Reduction in inoculated E. coli levels in apple juice from 7.92 log cfu/mL to 7.42 lof cfu/mL at a UV dose of 1377J/L was observed However, in the remaining juice s no inoculation studies were done by the authors. I t was not possible to interpret the effective UV dose required to achieve paste urization in juice as the initial microbial load varied between the juices The authors suggested that UV treatment optimization is required for each new juice product. Effect of UV treatment on juice quality Donahue and others (2004) showed a 5 log reduction in E. coli when exposed to UV light for 8.12 s and energy of 35.1mJ/cm2. UV treated cider had a shelf life 7 days longer than untreated apple cider with no significant sensory difference. Choi and others (2005) found that thermal pasteurized apple cider had better microbial quality compared to UV t reated cider after 21 days of storage. However, UV treated cider preserved better color. Consumer acceptability studies showed no significant difference between UV and untreated cider whereas thermally treated cider had the lowest acceptability scores.

PAGE 51

51 T andon and others (2003) compared UV treated apple cider at dose 14 mJ/cm 2 to hot fill pasteurized cider at 63 C. UV treated cider had a shelf life of 2 weeks compared to 4 weeks for hot fill cider. UV treated cider was less acceptable than hot fill cider by sensory preference test s due to ferment ation after 2 weeks of storage. Studies on UV treated orange juice are limited due to the low transmittance of UV light through the high pulp juice (Tran and Farid 2004b) UV exposure of 73.8m J/cm 2 extended the shelf life of orange juice by 5 days with a 17% loss of ascorbic acid. Vitamin C is a light sensitive component and is degraded by U V light. No PME inactivation was noted by the authors in UV processed juice.

PAGE 52

52 Figure 2 1. Key odorants of grapefruit and apple juice Table 2 1. Time temperature parameters for PE inactivation in citrus juices Orange, mandarin and tangerine juice Grapefruit juice Lemon juice Temperature 90 98 C 85 90 C 75 85 C Time 60 s 30 40 s 30 s

PAGE 53

53 Figure 2 2. Schematic diagram of pulsed electric field processing uni t (Jia et al. 1999) F igure 2 3. Schematic diagram of radio frequency electric field processing unit (Geveke et al. 2007)

PAGE 54

54 Figure 2 4. Picture of Ultraviolet processing unit (Geveke 2008)

PAGE 55

55 CHAPTER 3 COMPARISON OF THERMA L AND NON THERMAL TECHNIQUES O N APPLE CIDER STORAGE QUALIT Y UNDER EQUIVALENT P ROCESS CONDITIONS Introduction Non thermal processing techniques like pulse electric field (PEF) and ultraviol et (UV) treatment have been explored for their efficacy to extend shelf life and enhance the safety of fresh juice while preserving organoleptic and nutritional qualities (Evrendilek and others 2001; Min and Zhang 2003) Studies on the inactivation of microorganisms by PEF processing of orange, apple and tomato juices have show n better preservation of flavor, color and nutrients in com parison to heat pasteuriz ed juices (Min et al. 2003b; Evrendilek and others 2000a; Ayhan et al. 2001) UV processing of apple juice and apple cider has prev iously been conducted to study the inactivation kinetics of microorganisms like E. coli O157:H7 and S. typhimurium (Duffy et al. 2000; Uljas and others 2001) Even though there are many non thermal processing techniques available for the pasteurization of juices, a lot more research is required to make them commercially successful. Particularly, studies about the effects of non thermal techniques on quality and consumer acceptability in comparison to thermal pasteurization are required. Most often the conditions used for thermal processing are severe in terms of temperature and time of exposure to heat. Thus she lf life studies comparing non thermal to thermal techniques on acceptability of juice using conditions that do not achieve the same reduction of a target microorganism would result in incorrect conclusions (Rivas et al. 2006; Aguillar Rosas et al. 2007; Yeom et al. 2000b) F or proper comparison of thermal and non thermal treatment effect s on product quality both processes must achieve similar levels of microbial inactivation. The current work focuses on comparing th e effect of thermal and non

PAGE 56

56 thermal processing techniques on microbial population and overall quality of apple cider to that of untreated cider Each treatment is optimized to achieve approximately 6 log reduction of added E. coli K12, a surrogate of E. co li O157:H7. Materials and Methods Determination of Processing Conditions That Achieve 6 log Reduction of E. coli K12 Pasteurized apple cider (Ziegler Juice Co., Lansdale, PA) inoculated with E. coli K12 (ATCC 23716) was used to stand ardize processing par ameters to achieve a 6 log reduction. E. coli K12 obtained from the American Type Culture Collection (ATCC) (Manassas, VA) was maintained on Tryptic Soy Agar (Remel, Lenexa, KS, USA) at 4 C. It was cultured in Tryptic Soy Broth (Remel) with shaking at 37 C for 16 18 h. Apple cider inoculated from the stationary phase culture gave an approximately 7 log CFU/mL population. Ultraviolet process Pasteurization was performed using a simple apparatus consisting of four low pressure mercury lamps each surrounde d by a coil of UV transparent tubing. The UV apparatus was scaled up from a smaller sized apparatus developed by Geveke (2005). The UV lamp assemblies contained a bi pin base (model S130 120 LPF, Lithonia Lighting, Conyers, GA) and a 30 W bulb (G30T8, Buyl ighting.com, Burnsville, MN) that generated 90% of its energy at a wavelength of 254 nm. Norton Chemfluor 367 tubing (Cole Parmer, Vernon Hills, IL) with an ID of 3.2 mm and a wall thickness of 1.6 mm was wrapped around the entire length of each UV lamp. T he four UV lamps were connected in a series with 14m of tubing wrapped around them. The experimental system included a feed tank, a peristaltic pump and four UV lamps of the same

PAGE 57

57 dimensions connected in a series. Cider was pumped through the tubing at a fl ow rate of 25 L/hr which translated to an exposure time of 17 s per bulb. Microbial assays were conducted using 1, 2 3 and 4 UV lamps corresponding to UV treatment times of 17, 34, 51 and 68 s. Pulsed electric field process A bench scale continuous PEF system (OSU 4F, Ohio State University, Columbus, OH, USA) was used to treat the inoculated apple cider. The system consisted of 6 co field treatment chambers with a diameter of 0.23 cm and a gap distance of 0.29 cm between electrodes connected in a series Applied voltage and current were monitored by a digital real time oscilloscope (Tektronix DS210, Beaverton, OR). The inlet and outlet cider temperatures were continuously monitored via thermocouples. C ider was pumped through the system using a digital ge ar pump (Cole Parmer 75211 30, Vernon Hills, IL) at a flow rate of 7.2 L/hr. The e lectric field strengths tested were 5, 10, 13, 17, 21 and 23 kV/cm at a square wave pulse duration of 2.5 Apple cider sequentiall y flowed through all the treatment chambers via steel coils immersed in a water bath set at 48 C. The mean total treatment time (t) was calculated as 150 s using the following equation 1: (3 1) Where n is the number of treatment chambers (n = 6), T is pulse width ( T = 2.5 is the repetition frequency ( f = 1670 Hz), d is the distance between two electrodes ( d = 0.29 cm), v is the velocity of flow inside treatment chamber ( v = cm/s) which is determ ined by flow rate and the diameter of the treatment chamber (0.23 cm).

PAGE 58

58 Thermal process Inoculated apple cider was heat pasteurized using a miniature scale Armfield HTST processing system (model FT74 30 MkIII 33 34, Jackson, NJ, USA). The system included a feed tank, a peristaltic pump, a plate heat exchanger (comprising a regeneration section, a heating section and a cooling section that mimics industrial scale systems), a holding tube, thermocouples, and an electric powered hot water boiler and pump. A f low rate of 15 L/hr was maintained through the system which translated to a hold time of 1.3 s. Microbial inactivation was studied at holding tube outlet temperatures of 60, 63, 66, 69, 72, 74 and 76 C. Shelf Life of Processed Apple Cider Unpasteurized apple cider procured from Ziegler Juice Company (Lansdale, Pennsylvania) was frozen at 17 C in gallon plastic containers. Samples were thawed at 4 C overnight prior to use Processing and packaging All processing equipment was sanitized by pumping 5% b leach solution through the system, followed by a distilled water rinse. H eat pasteurization was performed at 76 C for 1.3 s. For UV treatment, apple cider was exposed for 51 s at an outlet temperature of 15 C. PEF processing was conducted at an electric field strength of 23 kV/cm, 2.5 s pulse duration, a total treatment time of 150 s and a treatment temperature of 48 C. The treatment conditions were selected based on 6 log reduction of inoculated E. coli K12 as determined in the above experiments. As a control sample, apple cider was passed through the HTST processing system without any heat at room temperature.

PAGE 59

59 Cider was packaged in clean 1 L media bottles inside a sanitary laminar hood after processing. The laminar hood contained a UV lamp and HEPA air filter. The hood was sanitized by a UV lamp at 254 nm for 30 min before use and then wiped with alcohol. Storage Bottled s amples stored at 4 C for four weeks were periodically analyzed for quality including microbiology, pH, Brix and color attrib utes. Microbial analyses were performed every week, while all other analyses were done at 0, 2 and 4 weeks Control apple cider consisted of untreated cider stored at 4 C while fresh apple cider was untreated cider stored at 17 C. Microbial tests Micr obial inactivation and growth were examined by preparing appropriate dilutions of samples with Butterfield's Phosphate Buffer (Hardy Diagnostics, Santa Maria, CA). Duplicate samples (1 mL) were then pour plated with Tryptic Soy Agar (Remel, Lenexa, KS, USA ) and incubated at 37 C for 24 h. Plates with 30 300 colonies were enumerated using a Bantex model 920 man ual colony counter ( Burlingame, CA, USA). The E. coli K12 population was expressed as CFU/mL of apple cider. Data for each replicate were normalized against the control and plotted as the log reduction versus temperature, time or electric field strength. For the shelf life study, total aerobic plate count (TPC) and yeast and mold count (YMC) were determined using plate count agar (PCA) and yeast and mold (YM) pertrifilms. The PCA plates were incubated at 37 C for 24 hrs while YM plates were incubated at room temperature for 5 days before counting. All samples were analyzed in duplicate and two replicates of each dilution were prepared and plated.

PAGE 60

60 p H The pH meter used for all analysis was an Orion 420A+ (Thermo Electron Corp., Beverly, MA). The pH meter was calibrated at pH 4.0 and pH 7.0 using standard solutions on each day of analysis. The pH measurements for all samples were performed in triplicat e. Brix A Bausch & Lomb Abbe refractometer (B&L Corp., Rochester, NY) was used. To measure Brix of juice samples, a drop of the sample was placed on the refractometer and the corresponding refractive index was recorded which gave the measure of soluble solids in juice. All measurements of Brix for each cider sample were performed in triplicate. Color Color was measured in CIE L* a* b* 3 dimesional color space using a ColorQuest XE spectrophotometer (Hunter Associates Lab, Reston, VA) where L designated lightness and measured the relative lightness and darkness of juice with L = 0 the measure of green to red with positive values indicating m ore red and negative values indic atin signalin g more toward yellow and negative value signaling more toward blue. A dual beam xenon lamp was used as light source. The lamp was allowed to warm up for 30 min prior to measure ment. The system was calibrated using black and white standard tiles on each day of analysis. Forty mL of cider sample in a 20 mm path length glass cell were used for measuring color. All measurements of color for each sample were performed in triplicate. The effect of processing methods on the color of apple cider was

PAGE 61

61 represented using total color difference (dE) calculated for all the samples using the following equation (3 2) (Lee and Coates 1999) : (3 2) w here L = lightness of treated sample at time t; L0 = lightness of untreated sample at day 0; a = redness of treated sample at time t; a0 = redness of untreated sample at day 0; b = yellowness of treated sample at time t; and b0 = yellowness of untreated sample at day 0. Statistical Analysis All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc., Ralei gh, N.C., USA). Statistical significance of the differences among the treatments multip also determined statistically. Results and Discussion Optimization of Equivalent Processing Conditions Pasteurized apple cider was inoculated with E. coli K12 to give an approximately 7 log CFU/ml population. The initial microbial count in pasteurized apple cider was below 20 CFU/mL indicating very low microbial load before inoculation. Inactivation of inoculated E. coli K12 in apple cider increased with increasing exposu re time, temperature and electric field strength for UV, PEF and thermal process, respectively (Figures 3 1, 3 2, 3 3).

PAGE 62

62 The population of E. coli K12 decreased with increasing UV treatment time in apple cider (Figure 3 1). UV exposure for 34 s reduced the population by 3.7 logs and lengthening the exposure time to 51 s reduced the population by 6.3 logs. Using the same UV apparatus, Geveke (2005) was able to achieve 4.7 log reduction of the same bacterium in apple cider for a treatment time of 30 s and temperature at 25 C The inlet and outlet temperatures recorded in this study were below 15 C during processing since refrigerated apple cider was inoculated and passed through the UV proces sing unit. Thus no detrimental effect of heat on the inactivation of E. coli K12 could be expected in the UV processed cider. The energy applied was calculated to be 13 J/mL for 51 s of exposure time based on the flow rate and UV lamp wattage UV pasteuri zation energy is less compared to conventional thermal pasteurization energy of 34 J/mL (Kozempel and others 1998) The e ffect of PEF processing on E. coli K12 inactivation at electric field strength from 5 to 27 kV/cm with a pulse d duration of 2.5 s and a mean total treatment time of 150 s is shown in Figure 3 2. Many authors have reported significant synergistic ef fect against microorganisms when PEF is used in conjunction with moderate heat (Heinz et al. 2003) PEF processing was performed with minimal heat of 48 C. The outlet temperatures recorded were between 49 51 C during the length of treatment. The increase in temperature is a consequence of the electric energy supplied to the medium during PEF treatment. The incubation of inoculated cider at 48 C in the absence of PEF treatment did not achieve any reduction in microbial population. Inactivation of the E. coli K12 population increased with an increase in electric field strength from 13 to 23 kV/cm. An e lectric field strength of 23 kV/cm reduced the population in apple cider by

PAGE 63

63 6.1 log cycles. Evrendilek and others (2000b) achieved a 4.5 log reduction in E. coli O157:H7 population in apple juice using a similar bench scale PEF system with treatment conditions of 34 kV/cm electric field strength, 4 s bipolar pulse duration, a total treatment time of 166 s and temperature below 38 C The increased reduction in bacterial population at a lower electric field strength in the current study is evidence of the synergistic effect of PEF with heat on microbial inactivation. Thermal processing of inoculated cider performed at a flow rate of 15 L/hr gave a hold time of 1.3 s. The inlet and outlet product temperatures were recorded continuously and did not increase beyond 15 C due to built in cooling section in the equipment. A significant decrease in E. coli K12 population was noted with increase in temperature beyond 63 C (P < 0.05). Heat treat ment at 76 C for 1.3 s is recommended by the FDA for pasteurization of apple juice of pH 4.0 or less (CFSAN 2004) Treatment of apple cider by heat at the same conditions resulted in 6.0 log reduction of inoculated E. coli K12 population in this study (Figure 3 3). Effect of Thermal and Non Thermal Processing on Microbial Stability During Storage of Apple Cider Fresh unpasteurized apple cider was treated by heat, PEF and UV at cond itions achieving approximately 6 log reduction in E. coli K12 population. The microbial stability of treated apple cider was studied for a storage period of 4 weeks at 4 C. The TPC and YMC of apple cider stored at 4 C for 4 weeks in both treated and untr eated cider are given in Figures 3 4 & 3 5, respectively. The initial TPC for untreated control apple cider was 2.4 log CFU/mL and reached 3.1 log CFU/mL at the end of the storage period. The TPC for thermal, UV and PEF treated cider was reduced to 1.8, 1 .6 and 1.8 log CFU/mL, respectively at week 0.

PAGE 64

64 Thermal and UV processed cider did not show any significant increase (P < 0.05) in log values during storage. However, TPC of PEF processed cider increased to 2.4 log CFU/mL by the end of the 4 week storage p eriod. The increase in microbial population during storage is possibly due to a lower inactivation of spores by PEF and their subsequent growth during storage. Grahl and others (1996) have reported PEF to be ineffective for inactivation of microbial spores. Apple juice/cider is highly susceptible to yeast spoilage due to low pH and high sugar content (Deak and Beuchat 1993) Examination on petrifilm showed yeast as the major microorganism in apple cider. The initial YMC in untreated control cider was around 2 .5 log CFU/mL which steadily increased to 4.7 log CFU/mL by week 2 of storage (Figure 3 5). No significant increase (P < 0.05) in YMC was noticed by week 2 and 3 indicating a stationary phase in the growth cycle of microorganism s However, a decline in Y MC to 3.7 log CFU/mL by week 4 in the control cider was observed, suggesting that the microorganisms were in the death phase of their cycle. Analysis of UV treated cider showed an increase in YMC to 4.0 log CFU/mL after 2 weeks of storage at 4 C. The YMC decreased to 3.5 log by the end of 4 weeks of storage similar to untreated cider. Donahue and others (2004) proposed that the decrease in efficiency of microbial inactivation by UV is due to high turbidity and inadequate mixing of apple cider as it flows through the tubes. However, TPC of UV cider shows effective bacterial inactivation and stability during storage in thi s study. T he inefficient inactivation of yeast and mold is due to their apparent higher resistance to UV radiation compared to bacteria. Yeast and mold have different chemical composition and thicker cell wall s than bacteria which is a major factor in det ermining the relative UV resistance of an

PAGE 65

65 organism (Tran and Farid 2004b) The YMC for PEF and thermally processed cider were <1 log CFU/mL throughout storage indicating effective inactivation of spoilage microorganisms. The PEF pro cess is known to be more effective in yeast inactivation compared to bacteria due to their larger cell size (Jeyamkondan and others 1999) Larger cells are more permeable than smaller cells. Effect of Thermal and Non Thermal Processing on Appearance of Apple Cider During Storage No visual changes were noted in treated apple cider samples compared to untreated cider at week 0. However, after 2 weeks of storage both untreated and UV treated apple cider samples were accompanied by gas formation due to fermentation. Also, v isually both the samples clarified after 2 weeks o f storage (Appendix A 1). Previous studies also showed clarification of juice due to production of fungal enzymes as well as deterioration in quality of apple cider after 2 weeks of storage due to mold and yeast growth (Tandon et al. 2003; Donahue et al. 2004) No clarification was noted in PEF and thermally treated cider during 4 weeks of storage. Effect of Thermal and Non thermal Processing on the pH and Brix of Apple Cider During Storage The changes in pH and Brix for untreated and treated apple c ider during 4 weeks of storage at 4 C are shown in Table 3 1. Untreated apple cider from the same juice lot maintained at 17 C during 4 weeks of storage was used as the fresh sample. The initial pH values were in the range of 3.76 to 3.77 for both trea ted and fresh apple cider with no significant difference (P < 0.05) in mean values. Storage at 4 C for 4 weeks resulted in a slight but not significant (P < 0.05), decrease in pH for UV treated cider. The decrease in pH in UV treated cider could be due t o fermentation in cider as a result of increased yeast and mold growth during storage. PEF and thermal processing

PAGE 66

66 did not significantly alter the pH of apple cider during storage (P < 0.05). An increase in pH in heat pasteurized apple juice (85 C for 27s) due to evaporation of organic acids was reported by Charles Rodriguez and others (2007) compared to PEF treated juice. However, in the present study at equivalent process conditions, the heat treatment condition s used were mild (76 C for 1.3s) and therefore no increase in pH compared to PEF was noted. The result thus confirms the importance of comparison of quality parameters under equivalent process conditions. Brix value which indicates soluble solids content in cider is an important measure of juice quality. UV treated cider during storage showed a significant (P < 0.05) decrease in Brix after 2 weeks of storage corresponding to yeast and mold growth. Microorganisms utilize sugars for growth and therefore could alter the Brix value of juice. Tandon and others (2003) also noted a signif icant decrease in pH and Brix values in UV treated cider after 2 weeks of refrigerated storage due to microbial spoilage. Brix values were neither significantly (P < 0.05) affected by processing nor storage for thermal and PEF treated apple cider, which co rresponds to their good microbial quality. Effect of Thermal and Non Thermal Processing on Color of Apple Cider During Storage The effect of treatment and storage on color of apple cider was determined using Hunter L* a* b* values (Table 3 2). A s ignifica nt (P < 0.05) increase in L* values were noted in treated cider samples compared to fresh cider at week 0. Increase in L* values for pasteurized juice indicates that the juice color became lighter or whiter. Genovese and others (1997) a ttributed the initial increase in L* values in pasteu rized cloudy apple juice to partial precipitation of suspended particles in juice. After 4 weeks of storage, L* and b* values increased significantly ( P < 0.05) for thermal and UV pasteurized ciders.

PAGE 67

67 Noticeable decrease in a* values for all samples indicat es a loss in redness probably due to breakdown of anthocyanins during treatment (Shewfelt 1986) To understand the impact of the L* a* b* value changes on color of apple cider, the total color difference (dE) valu es were calculated which gives the magnitude of color difference between treated and fresh cider ( Lee and Coates 1999) Thermally treated cider had significantly higher dE (P < 0.05) compared to both PEF and UV samples at week 0 (Figure 3 6). Thermal treatments are known to have pronounced effects on color of juice due to degradation of color pigments and Maillard reactions between sugars and amino acids resulting in browning of juice (Min et al. 2003b; Clegg 1964; Lee and Coates 1999) Browning is commonly observed in heat treated citrus juice and is affected by storage temperature and time (Nagy and others 1990) The dE value increased for all treated ciders during 4 weeks of storage indicating loss in overall color compared to fresh cider. The increase in dE from 1.17 to 1.91 in UV cider during storage is possibly due to microbial growth. Donahue and others (2004) attributed the color change in UV treated cider to by products formed during fermentation by yeast and mold growth. The dE value for PEF treated cider did not show any significant (P < 0.05) increase during storage. In comparison to UV and thermally treated samples, PEF processed cider showed maximum color st ability during 4 weeks of storage. Color was better preserved in PEF treated juice compared to heat pasteurized juice in this study and this has also been documented by other authors (Yeom et al. 2000b; Min and Zhang 2003) The conditions used for heat treatment were severe in terms of temperature and time (94.6 C for 30 s and 92 C for 90 s, respectively). T he current

PAGE 68

68 study shows improved color stability in PEF cider compared to thermal cider during storage even under equivalent process conditions. Conclusion Non thermal processing has been extensively researched over the past few years as an alternative to heat pasteurization. However, the non thermal process was often compared to thermal processes using conditions that did not achieve the same reduction in concerned microorganism(s), which made fair comparison of their effects on juice quality impossible. T his is the first report where conditions were carefully selected so that all three processes achieved a similar reduction (6 log) of E. coli The present study demonstrated that PEF was as efficient in microbial inactivation as heat pasteurization while ma intaining the color of the apple cider. Heat pasteurization negatively affected the color of apple cider. UV pasteurization as a non thermal technique was successful only for a shelf life of 2 weeks after the processing conditions used in this study. Compa ring the three techniques, PEF shows promise as a pasteurization technique for the preservation of apple cider.

PAGE 69

69 Figure 3 1. UV inactivation of inoculated E coli K 12 in apple cider at different treatment times Treatment conditions: wavelength 254 nm outlet temperature < 15 C Figure 3 2 PEF inactivation of inoculated E coli K 12 in apple cider at different electric field strengths. Treatment conditions: pulse duration of 2.5s, total treatment time of150 s, outlet temperature 49 51 C.

PAGE 70

70 F igure 3 3. T hermal inactivation of inoculated E coli K 12 in apple cider at different temperatures. Treatment condition: 1.3 s hold time, outlet temperature <15 C. Figure 3 4. Total aerobic plate count for control, thermal, UV and PEF treated apple c ider samples stored at 4 c for 4 weeks. Vertical bar indicates standard deviation (n=4)

PAGE 71

71 Figure 3 5. Yeast and mold count in control, thermal, UV and PEF treated apple cider samples stored at 4 c for 4 weeks. Vertical bar indicates standard deviation ( n=4) Figure 3 6. Change in total color difference (de) in thermal, UV and PEF treated apple cider during storage. Vertical bar indicates standard deviation (n=3)

PAGE 72

72 Table 3 1. C hange in pH and Brix values for fresh* thermal, UV and PEF treated apple ci der stored at 4 C for 4 weeks Weeks of storage pH Brix Fresh Thermal UV PEF Fresh Thermal UV PEF 0 2 4 3.76 a ** 3.76 a 3.76 a 3.770 a 3.750 a 3.750 a 3.76 a 3.75 a 3.72 a 3.76 a 3.75 a 3.75 a 13.13 a 13.13 a 13.13 a 13.00 a 13.00 a 12.88 a 13.0 0 a 12.90 a 12.10 b 13.10 a 13.00 a 12.90 a Fresh apple cider refers to untreated cider stored at 17 C through 4 weeks of storage. **Values with different initials within same row are significantly different from each other at p<0.05.

PAGE 73

73 Table 3 2. C hange in Hunter color values** for fresh* thermal, UV and PEF treated apple cider stored at 4 C for 4 weeks Weeks of storage L A b Fresh Thermal UV PEF Fresh Thermal UV PEF Fresh Thermal UV PEF 0 2 4 32.58 a 32.60 a 32.59 a 32.94 b 33.33 c 33.38 c 32.88 b 32.63 b 33.44 c 32.80 b 32.80 b 32.55 b 3.32 a 3.31 a 3.32 a 2.01 b 2.00 b 1.96 c 2.18 b 2.00 b 1.98 c 2.21 b 2.13 b 2.14 b 5.55 a 5.57 a 5.58 a 5.93 a 6.63 b 6.78 c 5.31 a 6.50 b 7.40 d 5.74 a 6.19 b 6.62 b Fresh apple cider refers to untreated cider stored at 17 C through 4 weeks of storage. **Values with different initials within same row for each hunter value are significantly different from each other at p<0.05.

PAGE 74

74 CHAPTER 4 IMPACT OF THERMAL AN D NON THERMAL PROCESSING T ECHNOLOGIES ON APPLE CIDER AROMA Introduction A la rge amount of microbial information concerning thermally and non thermally processed apple juice can be found in the literature but little information exists on the effects of these processes on the flavor of apple cider. A decrease in ester concentration s due to thermal pasteurization of apple juice has been reported (Kato et al. 2003; Su and Wiley 1998) and a single study compared 8 apple juice volatiles in PEF and thermally processed juice (Aguillar Rosas et al. 2007) Because a major motivation for non thermal processing technologies is a minimal change in org anoleptic and nutritional properties, an in depth analysis of the effect of the above processes on the flavor profile of apple cider and its relation to sensory quality is necessary. The present work focused on : 1) comparing the aroma volatiles from therma lly processed apple ciders with non thermal treatments where each treatment was optimized to achieve a n equivalent of 6 log reduction in microorganisms and 2) examining changes in aroma active and major volatiles in the treated apple ciders stored at 4C at week 0 and week 4. Materials and Methods The s tandard compounds ethyl acetate, ethyl propanoate, 2 methyl propyl acetate, methyl butyrate, ethyl butyrate, ethyl 2 methyl butyrate, hexanal, butyl 2 methyl acetate, butyl propanoate, 2 methyl 1 butanol, ( E) 2 hexenal, ethyl hexanoate, hexyl acetate, (E) 2 hexen 1 ol acetate, 6 methyl 5 hepten 2 one, hexyl propanoate, (Z) 2 hexen 1 ol, hexyl butyrate, hexyl 2 methyl butyrate, hexyl hexan farnesene, damascenone, hexane, methanol and 1

PAGE 75

75 butanol were purchased from Aldrich (St. Louis, MO). Butyl acetate, benzaldehyde, 1 octanol, butyl butyrate, propyl butyrate, 1 hexanol, p allylanisole, butyl 2 methylbutyrate, pentyl acetate, propyl hexanoate, dimethyl sulfide and an a lkane standard solution (C8 25) were purchased from Fluka (Steinheim, Switzerland). Apple C ider Two hundred and twenty seven liters of unpasteurized apple cider with no additive s was procured from Ziegler Juice Company (Lansdale, Pennsylvania). A var ietal blend of apples, including Ginger Gold, Golden Delicious, Red Delicious, Empire, Macintosh, Gala and Cortland were used for the juice in this study. Apples were inspected for q uality (no visible mold or decay) and were washed and graded to remove loose stems, leave s and other foreign materials. After further inspection and grading, apples were passed through a Westphalia decanter press to express juice and separate solids. No ad ditives were added to the ci der. The cider was packaged in high density polyethylene (HDPE) 3.78 L (1 US Gallon) containers. The containers were transported to the USDA facility within 2 hours of juicing and stored in a 17 C deep freezer. The juice rema ined for in frozen condition for 6 days before being thawed at 4 C overnight for processing. Apple Cider Processing Preliminary experiments were performed to determine equivalent processing conditions using heat, pulse electric field and ultraviolet radi ation for apple cider as described in chapter 3 Pasteurized apple cider (Ziegler Juice Co., Lansdale, PA) was inoculated with E. coli K12 (ATCC 23716) from a stationary phase culture to give approximately 7 log CFU/mL population. Microbial assays were con ducted for ciders heated from 60 76 C, with UV exposure times of 17 68 s and 5 23 kV/cm electric field

PAGE 76

76 strengths for PEF treatment. Final optimized processing conditions resulted in approximately 5 log reductions of E. coli K12 and were used for the remai nder of the study. Heat treatment Apple cider was heat pasteurized using a miniature scale HTST processing system (Armfield, Jackson, NJ, FT74 30 MkIII 33 34) as described in chapter 3. Apple cider was introduced into the system via the feed tank at a fl ow rate of 15 L/hr with hot water circulation set at 76 C such that the juice was held at 76 C for 1.3s and cooled rapidly. The inlet and outlet temperatures were continuously monitored and were in the range of 13 17 C and 24 30 C respectively during processing. Unpasteurized apple cider passed through the thermal processing system without heat at room temperature and was used as the control sample for microbial studies. Ultraviolet treatment A low pressure mercury lamp surrounded by a coil of UV t ransparent Chemflour tubing was used for UV processing of apple cider (Geveke 2005) as described in chapter 3. Cider was pumped through the tubing at flow rates of 25 L/hr which translates to a treatment time of 17 s per bulb. The energy used was 34 J/ml. Apple cider was exposed to a total treatment time of 51 s. The inlet and outlet temperatures recorded were between 10 15 C during proce ssing. Pulsed electric field treatment A bench scale continuous PEF system described in chapter 3 (OSU 4F, Ohio State University, Columbus, OH ) was used to treat the inoculated apple cider. The cider was pumped through the system at flow rate of 7.2 L/ hr. The square wave pulse duration was 2.5 s and the electric field strength was 23 kV/cm. The mean total

PAGE 77

77 treatment time was calculated as 150 s. Apple cider sequentially flowed through all the chambers via steel coils immersed in a water bath set at 48 C. The inlet and outlet cider temperatures were continuously monitored using thermocouples and were in a range of 30 34 C and 49 51 C, respectively. Packaging and S torage Processed apple cider was collected directly from the processing unit outlet int o sterile 1L media glass bottles (Corning Inc, Corning, NY) inside a sanitary laminar hood equipped with a HEPA air filter (Forma Scientific Inc., Marietta, OH). The hood was sanitized by UV lighting at 254nm for 30min before use and then wiped with 100% a lcohol. The packaged juice was stored at 4 C for storage studies. Storage studies were conducted for 4 weeks on the various processed apple ciders stored at 4 C. Microbial analyses were performed every week, whereas volatile and sensory analyses were don e in weeks 0 and 4. Fresh unpasteurized cider samples maintained at 0 C were used as control s for volatile and sensory analysis. Microbial S tability The microbial analysis was performed according to method by Fan and Geveke ( 2007) Microbial tests were conducted every week during the four weeks of storage. Total aerobic plate count and yeast and mold count were determined using plate count agar (PCA) and yeast and mold (YM) pertrifilms. The PCA plates were incubated at 37 C for 24 hours while YM plates were incubated at room temperature for 5 da ys before counting. All samples were analyzed in duplicate and two replicates of each dilution were prepared and plated.

PAGE 78

78 Volatile E xtraction Aliquots (27 ml) of apple cider were placed in 40 ml glass vials with screw cap containing Teflon coated septa simi lar to the procedure used by Dreher and others (2003) The cider was equilibrated for 10 min at 36 C with stirring. A 2 cm 50/30m, exposed in the equilibrated headspace for 45 min at 36 C. The fiber was desorbed for 5 min in the GC i njection port at 250 C. All samples were analyzed in quadruplicate. G as C hromatography M ass S pectrometry A nalysis A Gas chromatography/Mass spectrometry (6890N GC, 5973N MS, Agilent Technologies, Santa Clara, CA) was used for separation and analysis of v olatiles. The instrument was also equipped with a Pulsed Flame Photometric Detector, PFPD, (model 5380, OI Analytical, College station, Texas, USA). Samples were run separately on a polar DB Wax and non polar DB 5 column with identical dimensions (30 m x 0.32 mm x 0.5 m from J&W Scientific, Folsom, CA). The column oven temperature was programmed from 40 C to 110 C at 7 C/min then raised 15 C/min to 250 C with 3 min hold. Injector and detector temperature were 250 C. Mass spectrometry conditions wer e as follows: transfer line temperature at 275 C, mass range 30 to 300 amu, scan rate 5.10 scan/s and ionization energy 70 eV. Helium was used as the carrier gas at a flow rate of 2 mL/min. Mass spectral matches were made by comparison with NIST 2002 stan dard spectra. Authentic standards were used for confirmation. Alkane linear index values were determined on both columns (Girard and Tv 1996)

PAGE 79

79 Quantification of Apple Cider V olatiles Volatile free apple cider was prepared according to Fan and others (2001) by concentrating 500 mL of apple cider using a vacuum rotary evaporator (Brinkmann Instruments Inc., Westbury, N.Y., U.S.A.) from 11.0 to 28.0 Brix. Any residual volatiles were extracted wi th hexane and discarded. Any trace hexane residue in the concentrated juice was removed using a vacuum rotary evaporator. The concentrated juice was diluted back to initial Brix of 11.0 using distilled water and checked for residual volatiles. A mixture of 29 standards in methanol was serially diluted with deodorized apple cider and added in concentration of 0.5 to 3 times their estimated concentrations in cider. Volatiles were analyzed by SPME GC/MS using the same conditions described in GC/MS analysis s ection. A quantitation database for standards was created using MSD Chemstation software. Response factor curves were created by plotting target ion count (base peak) against standard concentrations in volatile less apple cider. Parameters used for compoun d identification were retention time, target ion and secondary ions. Compounds were quantified using target ion values and response factors generated from standard curves. Sensory E valuation A discriminative triangle test (Meilgaard and others 1999) was employed to orthonasally detect difference s in aroma between unpasteurized and pasteurized app le storage at 4 C. The statistical power for the triangle test was recorded as 0.9. The analysis was conducted in a sensory panel facility at Eastern Regional Research Center (Wyndmoor, PA) which has 6 booths with computers. T he sensory analysis was designed and conducted using Compusense five (Compusense Inc., Ontario, CA). Samples were prepared by pouring 40 mL apple cider into 100 mL

PAGE 80

80 glass bottles and closed with airtight caps. The bottles were stored in boxes at 4 C overni ght. On the day of testing, the bottles were taken out 1hr before testing. Cider temperature was 10 12 C during testing. Testing was performed under red light so that color and other visible differences were masked from panelist s Each panelist was given five sets of apple ci der samples (3 samples per set) one at a time. All samples were randomly assigned three digit codes. The order of presentation of sample sets among panelist was also randomized. In total, 50 untrained panelists from ERRC evaluated t he samples and each panelist performed five triangle tests which included control versus PEF, control versus UV, control versus thermal, thermal versus UV and thermal versus PEF treated apple cider samples. After each test, the panelist s w ere also asked w hich sample was preferred among the three test samples and to give a reason for their preference. Statistical Analysis All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc., Raleigh, N.C., USA). Data were from a single sample for each treatment. Each sample was analyzed in quadruplicate. The tests for statistical significance of difference between treatments for storage data on volatiles was performed by analysis of variance (ANOVA) at a of treatments on means of = 0.05). Microbial d ata was analyzed using MS Excel 2003 and standard deviations were presented.

PAGE 81

81 Results and Discussion M icrobial Stability Durin g S torage The total aerobic count for processed apple cider was maintained below 20.24 log CFU/ mL through 4 weeks of storage at 4 C. The yeast and mold count in initial cider at 0 day was 20.12 log CFU/ mL. PEF and thermal processed apple cider did not show any yeast and mold growth throughout storage. After 2 weeks storage, the yeast and mold count in UV treated samples increased to 3.8 0.52 log CFU/ mL. A visible mold growth was observed after 4 weeks storage. Deterioration in quality of UV treated apple juice due to yeast and mold growth after 2 weeks of storage has also been observed by others (Tandon et al. 2003; Donahue et al. 2004) Donahue and coworkers confirmed the presence of injured microbial cells in UV treated cider using selective enrichment media. They suggested that the decreas e in efficiency of cell inactivation by UV could possibly be due to high turbidity and inadequate mixing of apple cider as it flows through the tubes (Donahue et al. 2004) Volatile C omposition A total of 34 volatile compounds were identified using MS in the initial untreated ap ple cider after separation on a DB Wax column (Table 4 1). Apple juice volatiles have been previously quantified using both external and internal standard methods (Komthong and others 2006b; Mehinagic et al. 2006) The current study is the first report w h ere apple cider volatiles have been quant ified using external calibration from an odorless apple cider to compensate for matrix effects. Response factor plots were determined for 29 compounds in Appendix B 1. No response factors were determined for peaks 2, 3, 6, 14 and 30 because their signal/no ise ratio was less than 3. The

PAGE 82

82 standard addition plots for 29 compounds are given in Appendix B 1. The plots for most compounds had a linear correlation coefficient (R 2 ) above 0.95. Esters, aldehydes and alcohols comprised the major volatiles in apple cid er accounting for 40%, 43% and 16% of the total volatiles identified, respectively. However, it should be kept in mind that the volatile composition of apple juice depends on various factors like variety, maturity and storage conditions of fruit used for pressing (Dixon and Hewett 2000; Girard and Lau 1995) Certain apple cultivars like Jonathan and Cox Orange Pippin are reported to have 5 100 fold highe r aldehyde content compared to Golden D elicious cultivars (Dixon and Hewett 2000) A pple juice also has higher C6 aldehyde concentration compared to fruit due to oxidation of the fatty acids linoleic and linolenic acid produced by lipoxygenases soon after crushing (Paillard and Rouri 1984b) Hexanal and 2 (E) hexenal were the most abundant aldehyde s identified in apple cider in this study (see Table 4 1). Apple cider esters can be classified into acetic, butyr ic, propanoic and hexanoic groups. Acetate esters are reported to be the major volatiles in apple juice and high concentrations of hexyl acetate and butyl acetate are considered to be normal characteristic s of many apple cultivars (Dixon and Hewett 2000; Young and others 1996; Girard and Lau 1995) The most abundant acetate esters in this study were hexyl acetate, 2 methyl butyl acetate and butyl acetate. Odor Activity V alues To assess the contribution of each vol atile to apple cider aroma, OAV were calculated as the ratio of concentrations found in apple cider to its odor threshold value in water (Table 4 2) (Takeoka and others 1990; Flath and others 1967a; Buttery and other s 1988) Apple cider odor threshold values were not available. Aqueous threshold

PAGE 83

83 values should be similar to actual apple cider values since apple cider is approximately 90% water. The highest OAV values found in initial apple cider were for hexyl acetate (69), hexanal (41), 2 methyl butyl acetate (25), 2 methyl ethyl butyrate (23) and 2 (E) hexenal (14). These results are consistent with the work of Fuhrmann and others (2002) where 2 methyl ethyl butyrate, hexyl acetate and 2 methyl butyl acetate were hexanal and 2 (E) hexena l are responsible for the green or fresh aroma of apple cider and are essential to apple juice aroma due to their high correlation with apple aroma intensity (Durr and Schobinger 1981) Durr and others (1981) have shown that even though esters give the fruity aroma to cider, the concentration of aldehydes is essential for sensory impression of juice odor. Alcohols like 1 hexanol are low aroma impact components and have been identified as a negati ve contributor to apple aroma (Bult and others 2002) In this study, only UV treated cider possessed a hexanol OAV value which might suggest that it was aroma active. Effect of Treatment and Storage on Volatiles The con centration of volatiles was not significantly (p<0.05) affected by treatments immediately after processing (week 0 data not shown). However, pronounced volatile differences between treatments were observed after 4 weeks at 4 C (Table 4 1). Figure 4 1 comp ares the levels of volatiles with significant differences between treated and control apple ciders after 4 weeks of storage at 4 C. Hexyl acetate concentrations decreased during storage in all processed apple cider samples with a concomitant increase in 1 hexanol by the action of residual esterase present in apple cider pulp (Schreier and others 1978) Thermally treated ciders lost 30% of their original ester and

PAGE 84

84 aldehyde content during storage with significant decreases (p<0.05) in butyl acetate, 2 methyl butyl acetate, hexanal and 2 (E) hexa nal concentrations (Table 4 1 and Figure 4 1). Although thermal treatment is known to inactivate most enzymes, the cider in this study was exposed to a temperature of 76 C for only 1.3 s. This may not have completely inactivated flavor altering enzymes ev en though it reduced microbial concentrations to the desired level. UV treated cider was characterized by a complete absence of hexanal and 2 (E) hexenal, a decrease in hexyl acetate and an increase in 1 hexanol compared to control after 4 weeks of storage The increase in 1 hexanol concentration is associated with decreases in precursors such as hexanal, 2 (E) hexenal and hexyl acetate (Schreier et al. 1978) PEF cider lost less than 2% of total ester and aldehyde volatiles during storage suggesting that it more effectively inactivated indige nous enzymes than thermal or UV treatments. A s ignificant decrease (p<0.05) in volatile concentrations were observed only in hexyl acetate and 2 (E) hexenal. 1, 3 Pentadiene was detected only in UV samples after 4 weeks of storage. It is an unsaturated hy drocarbon produced by molds such as Zygosaccharomyces and Penicillium in beverages. It possesses a petroleum odor that is often associated with microbial spoilage (Casas and others 1999) An interesti ng change observed in both thermal and PEF cider was an increase in benzaldehyde concentrations after 4 weeks storage. Sumitani and coworkers found a similar increase in benzaldehyde in high pressure treated peaches during storage due to release from its b glucosidases present in the peach (Sumitani and others 1994) Similar enzyme action is possible in

PAGE 85

85 thermal and PEF ciders since apple seeds are known to have amygadalin as a major constitue nt (Lu and Foo 1998) Effect of Treatment and Storage on Odor Activity Values of V olatiles Odor activ ity values were calculated for quantified volatiles in control and all treated samples to assess the impact of storage and treatment on apple cider aroma (Table4 2). Significant odor value losses (p<0.05) were observed for thermal and UV cider samples afte r 4 weeks storage. Hexanal and 2 (E) hexenal OAV decreased 35% and 43% respectively in thermally treated cider during storage. Decreases in aldehyde odor values in thermally treated cider would likely reduce aroma strength as well as perceived freshness. Significant decreases (p<0.05) in essential esters such as hexyl ace tate and 2 methyl butyl acetate could further deteriorate the aroma quality of thermal cider. In UV treated cider, 100% of the original hexanal and 2 (E) hexanal was lost during 4 weeks o f storage. During the same time the OAV for 1 hexanol increased from 0 to 2 in UV cider only. This volatile has a green, musty aroma and is characterized as a negat ive contributor to apple aroma (Bult et al. 2002) They demonstrated that an increase in 1 also developed a perceivable fermented odor after 4 weeks storage due to microbial spoilage. Due to its high odor threshold, benzaldehyde OAV was l ess than one for all treatments. Therefore, increases in benzaldehyde concentrations observed in PEF and thermal ciders probably did not impact the aroma of these appl e ciders. PEF volatile losses during storage were minor (less than 2% loss of total ester and aldehydes) except for hexyl acetate. This key odorant was greatly diminished during

PAGE 86

86 storage in all treated ciders. In the case of PEF treated cider, only 35% was retained after 4 weeks storage. However, losses of hexyl acetate were even greater in thermally and UV treated ciders. Aroma Sensory S tudies An aroma triangle test comparison between the control (unpasteurized cider samples maintained at 0 C for 4 weeks ) and all treated apple cider s at day 0 found no significant difference at p<0.05. However, after 4 weeks of storage at 4 C, 22 of the 50 panelists detected a difference between the aroma of the thermally treated sample and the untreated cider (control). The aroma of thermally processed cider was less preferred compared to fresh untreated (control) samples. Aroma of UV treated cider differed significantly (p<0.05) from the control. Thirty six of the 50 panelists correctly detected the difference. Of those 36, 35 panelists (97%) preferred control apple cider compared to UV cider. No significant difference (p<0.05) was detected by panelists between PEF treated and control apple cider. Triangle test results indicated that UV and PEF treated samples differed si gnificantly from heat treated cider (p<0.05). Ninety one percent of the triangle test panelists who correctly differentiated between PEF and thermal cider preferred the odor of PEF treated cider over that of thermally treated cider. The preference for PEF cider coincides with the greater retention of apple aroma volatiles in PEF cider compared to thermal cider. Preference results from this study do not necessarily reflect true consumer preference, and further sensory studies would be necessary to evaluate t he processing effects on preference in the general consumer population.

PAGE 87

87 Conclusion The key volatile components identif i ed in apple cider based on OAV values were hexyl acetate, hexnanal, 2 methyl butyl acetate, 2 methyl ethyl butyrate and 2 (E) hexenal. Th e pasteurization of apple cider by thermal and non thermal techniques at equivalent processing conditions resulted in a decrease in volatile components after 4 weeks of storage. PEF treated cider showed better volatile retention compared to both thermal an d UV cider. UV treated cider had a shelf life of 2 weeks. PEF cider aroma was undistinguishable from fresh untreated cider by triangle sensory analysis after 4 weeks of storage. PEF shows good promise as a viable pasteurization technique for apple cider.

PAGE 88

88 Figure 4 1. Effect of treatment and storage (after 4weeks) on major volatiles of apple cider 1 = butyl acetate, 2 = hexanal, 3 = 2 methyl butyl acetate, 4 = 2 (E) hexanal, 5 = hexyl acetate, 6 = benzaldehyde, 7 = 1 hexanol, a,b,c,d = different letters for each volatile indicate significant difference(p<0.05), control refer s to fresh unpasteurized cider maintained at 0 C for 4 weeks

PAGE 89

89 Table 4 1. Effect of thermal and non thermal treatments on apple cider volatiles compared to fresh untreated cider after 4 weeks of storage at 4 C Pk no. Compound name LRI Wax Mean concentration (g/L) (week 4) Control § Thermal PEF UV 1 1,3 pentadiene 714 nd a nd a nd a nq b* 2 dimethyl sulfide* 753 nq nq nq nq 3 ethyl acetate* 810 nq nq nq nq 4 ethyl propanoate 961 0.730.09 b 0.730.04 b 1.000.07 a 1.550.15 a 5 methyl butyrate ** 994 2.530.17 b 2.460.90 b 2.730.20 a 2.930.35 a,b 6 2 methyl propyl acetate* 1024 nq nq nq nq 7 ethyl butyrate ** 1050 3.000.52 a 2.860.20 b 3.650.55 a 3.751.35 a 8 ethyl 2 methyl but yrate ** 1067 2.360.27 a 2.300.43 a 2.600.13 a 2.000.12 a 9 butyl acetate ** 1088 1206.83 a 98.82.09 b 1319.38 a 89.41.95 b 10 hexanal ** 1098 2038.73 a 13313.2 b 21913.1 a nd c 11 butyl 2 methyl acetate ** 1135 1276.64 a 96.71.48 b 1285.02 a 1219.53 a 12 propyl butyrate ** 1137 6.500.34 b 5.700.12 b 10.00.25 a 5.550.76 b 13 butyl propionate ** 1153 4.340.36 a 4.230.37 a 5.130.10 a 4.560.26 a 14 1 butanol* 1158 nq nq nq nq 15 pentyl acetate ** 1183 1.300.31 a 0.760.07 b 1.460.23 a 0.360.10 b 16 2 methyl 1 butanol ** 1214 6.833.25 d 15.061.17 c 32.13.73 b 56.607.85 a 17 butyl butyrate ** 1225 7.660.41 b 6.800.07 b 8.710.83 a 4.560.20 b 18 2 (E) hexenal ** 1227 2312.62 a 1443.39 c 1867.12 b nd d 19 butyl 2 methyl butyrate ** 1236 1.500.09 b 1.430.12 b 1.530.1 0 a 1.500.08 b 20 ethyl hexanoate ** 1238 nd a nd a nd a nd a 21 hexyl acetate ** 1278 13710.74 a 43.22.07 b 48.68.92 b 11.78.54 c 22 propyl hexanaoate ** 1326 1.800.26 a nd b nd b nd b 23 (E) 2 hexen 1 ol acetate ** 1342 0.570.11 b 0.600.17 b 0.730.10 b 1.7 00.12 a 24 6 methyl 5 hepten 2 one ** 1348 0.100.03 a 0.100.26 a 0.200.05 a 0.100.03 a 25 hexyl propionate ** 1349 0.070.00 ab 0.000.00 b 0.130.01 a nd c 26 1 hexanol ** 1366 1645.91 c 2536.24 b 2415.76 b 80312.83 a 27 (Z) 2 hexen 1 ol ** 1423 0.170.23 a 0. 530.48 a 0.160.25 a nd c 28 hexyl butyrate ** 1432 5.340.23 b 4.260.29 b 6.930.22 a 1.550.12 c 29 hexyl 2 methyl butyrate ** 1443 2.900.22 a 3.060.20 a 3.730.18 a 3.400.13 a 30 2 methyl 6 hepten 1 1480 nq nq nq nq 31 benzaldehyde ** 1546 3.730.26 c 3 6.73.73 b 62.22.92 a 0.862.20 c

PAGE 90

90 Table 4 1 Continued Pk no. Compound name LRI Wax Mean concentration (g/L) (week 4) Control § Thermal PEF UV 32 1 octanol** 1568 nd a nd a nd a 10.45 0.13 b* 33 hexyl hexanaote ** 1620 0.430.00 a 0.40 0.03 a 0.46 0.00 a nd b 34 p allyl anisole ** 1683 0.270.07 a 0.20 0.10 a 0.73 0.15 b 0.13 0.05 a 35 farnesene ** 1747 bq bq bq bq Mean concentrations given as g/L standard deviations of quadruplicate analyses on single samples aMean concentration ug/L, (n = 4) ;nq = not quantitated; bq = below quantitation; nd = not detected d; different letters in the same row indicate significant differences (p<0.05). b* 1,3 pentadiene was detected in stored UV cider but it was not quantitated; ** represent 29 standards used for quantitation of volatiles; compounds standard available §control = fresh unpasteurized cider maintained at 0 C for 4 weeks

PAGE 91

91 Table 4 2. Effect of thermal and non thermal treatments on odor activity values (OAV) of apple cider volatiles after 4 weeks of storage at 4 C Compound b RI (Wax) OT a (g/L) OAV Control § Therm al PEF UV Esters ethyl propanoate 961 10 <1 a <1 a <1 a <1 a methyl butyrate 994 60 <1 a <1 a <1 a <1 a ethyl butyrate 1050 1 3 a 3 a 4 a 4 a ethyl 2 methyl butyrate 1067 0.1 23 a 23 a 24 a 20 b butyl acetate 1088 66 2 a 1 a 2 a 1 a butyl 2 methyl acetate 1135 5 25 a 19 b 26 a 24 a propyl butyrate 1137 18 <1 a <1 a <1 a <1 a butyl propionate 1153 25 <1 a <1 a <1 a <1 a pentyl acetate 1183 43 <1 a <1 a <1 a <1 a butyl butyrate 1225 100 <1 a <1 a <1 a <1 a but yl 2 methyl butyrate 1236 17 <1 a <1 a <1 a <1 a ethyl hexanoate 1238 1 ----hexyl acetate 1278 2 69 a 22 b 24 b 6 c hexyl propionate 1349 8 <1 a -b <1 a -b hexyl butyrate 1432 250 <1 a <1 a <1 a <1 a hexyl 2 methyl butyrate 1443 22 <1 a < 1 a <1 a <1 a Aldehydes hexanal 1098 5 41 a 27 b 44 a 0 c 2 (E) hexenal 1227 17 14 a 8 b 11 c 0 d benzaldehyde 1546 350 <1 a <1 a <1 a <1 a Alcohols 2 methyl 1 butanol 1214 300 <1 a <1 a <1 a <1 a 1 hexanol 1366 500 <1 a <1 a <1 a 2 b 1 octanol 1568 130 -a -a -a <1 b (Z) 2 hexen 1 ol 1423 70 <1 a <1 a <1 a <1 a Others 6 methyl 5 hepten 2 one 1348 50 <1 a <1 a <1 a <1 a a OT = odor threshold values in water from literature, b compounds identified based on RI on DB Wax and DB 5 column using standards, § control refer to fresh unpasteurized cider maintained at 0 C for 4 weeks

PAGE 92

92 CHAPTER 5 COMPARISON OF THERMA L AND NON THERMAL TECHNIQUES O N GRAPEFRUIT JUICE STORAGE QUALIT Y UNDER EQUIVALENT P ROCESS CONDITIONS Introduction Inc reased consumer demand for fresh like products with minimum flavor and nutritional losses has paved the way for alternatives to heat processing. PEF treatment as a non thermal technique for pasteurization of juices has been studied extensively in recent ye ars (Jin and Zhang 1999; Min et al. 2003b; Yeom et al. 2000b) PEF processed juice at a commercial scale had a microbial shelf life of 196 days at 4 C with a higher sensory preference compared to heat pasteurized j uice (Min et al. 2003a) Studies on UV pasteurization have shown extension of shelf life of apple juice and orange juice by 7 and 5 days re spectively, with minimal loss of sensory quality (Donahue et al. 2004; Tran and Farid 2004b) Radio frequency electric field is a recently developed non thermal technique that is not FDA approved. The effect of RFEF treatment on microbial quality of apple and orange juice has been reported in literature (Geveke et al. 2007; Geveke and Brunkhorst 2004) Geveke and others (2004) achieved a 3.3 log reduction in E. coli in RFEF treated orange juice relative to untreated juice. There are several studies in the literature comparing the effect of thermal and non thermal treatments on juice quality (Yeom et al. 2000b; Tandon et al. 2003; Sanchez Vega et al. 2009) However, most often the conditions used for thermal processing are severe in terms of temperature and time of exposure to heat. Thus, shelf life studies comparing the effect of non thermal to thermal techniques on the acceptability of juice using conditions that do not achieve the same reduction of the target microorganism would result in incorrect conclusions (Aguillar Rosas et al. 2007; Yeom et al. 2000b;

PAGE 93

93 Rivas et al. 2006) T herefore, for a fair comparison of the effects on quality of juice, both thermal and non thermal process es must achieve an equivalent reduction in microorganism level s The current work focuses on comparing the effect of thermal and non thermal processing techniques on grapefruit juice quality where each treatment is optimized to achieve approximately equivalent 5 log reduction s in E. coli K12 population Materials and Methods Determination of Equivalent Treatment Conditions Pasteurized grapefruit juice w as inoculated with E. coli K12 (ATCC 23716) obtained from the American Type Culture Collection (ATCC) (Manassas, VA). The bacterium was maintained on Tryptic Soy Agar (Remel, Lenexa, KS, USA) at 4 C. Prior to inoculation of product, the organism was cult ured in Tryptic Soy Broth (Remel) with shaking at 37 C for 16 18 h. Jui ce was inoculated from the stationary phase culture to give approximately 7 log CFU/mL population. Heat treatment Inoculated grapefruit juice was heat pasteurized using a miniature sca le HTST processing system (model FT74 30 MkIII 33 34, Armfield, Jackson, NJ, USA) described in chapter 3. Juice was pumped through the system at a flow rate of 15 L/hr which translated to a hold time of 1.3s. Microbial assays were performed at holding tub e outlet temperatures of 60, 62, 64, 66, 68 and 70 C. Ultraviolet treatment The UV apparatus used for pasteurization of juice is described in chapter 3. The experimental system included a feed tank, a peristaltic pump and four UV lamps of the same dimensi ons connected in a series. Juice was pumped through the tubing at a flow

PAGE 94

94 rate of 28.8 L/hr which translated to a treatment time of 14.75s per bulb. Microbial assays were conducted using 1, 2 3 and 4 UV lamps which corresponds to UV treatment times of 14 .75, 29.57, 44.25 and 59s. Pulsed electric field treatment A bench scale continuous PEF system (Valappil et al. 2009) at USDA, Wyndmoor, P A ( as described in chapter 3 ) was used to treat the inoculated grapefruit juice. The juice was pumped through the system at a flow rate of 7.2 L/hr. The square wave pulse duration was set at 2.5 tested were 5, 10, 13, 17, 21 and 23 kV/cm. Grapefruit juice sequentially flowed through all the treatment cha mbers via steel coils immersed in a water bath set at 30 C. R adio frequency electric field treatment The RFE F system used in the previous study has been previously described by Geveke and others (2007). The system was equipped with an 80 kW RF power supply (Ameritherm, Scottsville, NY model L 80) interfaced with a custom designed network that enabled RF energy t o be applied to a resistive load (Ameritherm) over a frequency range of 21.1 to 40.1 kHz. The RFEF power supply was connected to a series of treatment chambers. Grapefruit juice was supplied into the RFEF system from a feed tank using a progressing cavity pump. The processing parameters were set at a frequency of 20 kHz, a flow rate of 33 L/hr and field strength of 15kV/cm. The juice was quickly cooled to less than 25 C after exiting the treatment chambers using a stainless steel heat exchanger sample cool er (Madden manufacturing, Elkhart, IN, model SC0004).

PAGE 95

95 Shelf Life Study 150 L of unpasteurized red grapefruit juice with no additives frozen in 5 gallon buckets and packed in styrofoam boxes with dry ice layers were shipped from a commercial juice facility in Florida. The juice was received after 2 days of shipping and stored immediately in a 17 C freezer. Juice was filtered through 2 layers of cheesecloth under hygienic conditions due to high pulp content Th e filtered juice was pasteurized by UV, PEF, RF EF and thermal techniques as described below. Grapefruit juice pasteurization All processing equipment w as sanitized by pumping 5% bleach solution through the system followed by a distilled water rinse. Grapefruit juice was heat pasteurized at 70 C for 1 .3s. Prior to UV pasteurization juice was preheated to 50 C and then exposed to UV lamps for 44.25s. PEF processing was performed at an electric field processing was per formed at 20 kHz frequency, 15kV/cm field strength, an outlet temperature 55 C and a treatment time of Packaging and storage Processed grapefruit juice was collected directly into sterile 1L media glass bottles (Corning Inc, Corning, NY) inside a sanitary laminar hood equipped with a HEPA air filter (Forma Scientific Inc., Marietta, OH). The hood was sanitized by UV lighting at 254nm for 30 min before use and then wiped with 100% alcohol. The quality of grapefruit juice stored at 4 C was evaluat ed every week for 4 weeks (0, 1, 2, 3 and 4) on the basis of microbiology, pectin methyl esterase activity (PME), non enzymatic browning (NEB), ascorbic acid content (AA) and physical properties (pH, Brix and

PAGE 96

96 color). All assa ys were performed in triplicate Microbial assays were performed in duplicate. Microbial assay Microbial tests were conducted as described in chapter 3. One mL aliquots of juice were pour plated with Tryptic Soy Agar (Remel, Lenexa, KS, USA) and incubated at 37 C for 24 h. The E. coli K12 population was expressed as CFU/mL of grapefruit juice. Data for each replicate were normalized against the control and plotted as the log reduction versus temperature, time, treatment cycle or electric field strength. For the storage study, the tota l aerobic plate count (TPC) and yeast and mold count (YMC) were determined using plate count agar (PCA) and yeast and mold (YM) pertrifilms. After appropriate dilutions (if needed), samples (1 mL) were then pour plated with PCA or pertrifilm. The PCA plate s were incubated at 37 C for 24 hrs while YM plates were incubated at room temperature for 5 days before counting. All samples were analyzed in duplicate and two replicates of each dilution were prepared and plated. Pectin methyl esterase activity assay PME activity was determined according to a modified method described by Sampedro and others ( 2009) .Two milliliters of juice sample was added to a 30mL mixtur e of 0.35% citrus pectin solution and 125mM NaCl solution. The pH was first adjusted to 7.5 using 0.1M NaOH (pre titration). The hydrolysis at 22 C was maintained at pH 7.5 by adding 0.1M NaOH solution using automated pH stat titrator (Mettler Toledo T 70 Titrator, Schwerzenbach, Switzerland). After the initial 30s, the consumption of NaOH was recorded every 1 s for a 5 20 min reaction period. The slope

PAGE 97

97 (dV/dt) from linear plot between volume of NaOH consumed (dV) vs. time (dt) is used to calculate PME act ivity using following formula (Yeom and others 2000a) : (5 1) Percentage of residual PME activity was ca lculated in relation to the activity of the untreated sample. Ascorbic acid assay The ascorbic acid (Vitamin C) content in grapefruit juice was assayed by the HPLC method by Geveke and others (2007) Grapefruit juice was centrifuged at 10,000g for 10 min at 5 C in a Sorvall RC 2 refrigerated centrifuge (Kendro Laboratory Products, Newtown, CT). An aliquot of 0.5ml of supernatant was mixed with 5% meta phosphoric acid. The solution was filtered through a (Gelman Sciences, Ann Arbor, MI). The filtered material was analyzed using a HP Ti series 1050 HPLC s ystem (Agilent Technologies, Palo Alto, CA) with a photodiode array detector. An Aminex HPX 87H organic acids column (300 7.8 mm) fitted with a microguard cation H+ was used for separation. The mobile phase of 5mM sulfuric acid was passed through the col umn at a flow rate of 0.5 ml/min. Column temperature was maintained at 30 C using a column heater (Bio Rad Laboratories, Hercules, CA). Ascorbic acid was monitored at 245nm. A calibration curve was prepared by external standard method using standard ascor bic acid solutions in a range from 100 1000 g/mL in water. Non enzymatic browning Browning in grapefruit juice was assayed using the method by Fan and Thayer ( 2002) Juice was centrifuged at 10,000g for 10min at 5 C. An aliquot of 2ml of

PAGE 98

98 supernatant was mixed with 2ml of 100% ethanol. The s olution was vortexed and left at room temperature for 1 hr. The mixture was centrifuged again and the absorbance of supernatant was measured at 420nm using a spectrophotometer (Shimadzu UV 1601, Shimadzu Scientific Instruments, Columbia, MD). pH The pH meter used for all analysi s was an Orion 420A+ (Thermo Electron Corp., Beverly, MA). The pH meter was calibrated at pH 4.0 and pH 7.0 using standard solutions on each day of analysis. Brix A Bausch & Lomb Abbe refractometer (B&L Corp., Rochester, NY) was used. To measure Brix of j uice sample, a drop of sample was placed on the refractometer and the corresponding refractive index was recorded which gave the measure of soluble solids in juice. Color Color was measured in CIE L* a* b* 3 dimesional color space using ColorQuest XE spe ctrophotometer (Hunter Associates Lab, Reston, VA) as described in chapter 3. The lamp was allowed to warm up for 30 min prior to measurement. The system was calibrated using black and white standard tiles on each day of analysis. Forty mL of juice sample in a 20 mm path length glass cell was used for measuring color. The effect of processing methods on color of juice was represented using total color difference (dE) calculated for all the samples using the following equation 2: ( 5 2)

PAGE 99

99 w here L = lightness of treated sample at time t; L0 = lightness of untreated sample at day 0; a = redness of treated sample at time t; a0 = redness of untreated sample at day 0; b = yellowness of t reated sample at time t; and b0 = yellowness of untrea ted sample at day 0. Statistical Analysis All data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc., Raleigh, N.C., USA). Statistical significance of the differences among the treatments was tested using the analysis of variance (ANOVA) The effect of storage and processing te chniques were compared using Results and Discussion Effect of Treatments on Microbial Inactivation Pasteurized grapefruit juice was inoculated with E. coli K12 to give approximately 7 log CFU/ml population. The initial microbial count in pasteurized juice was below 40 CFU/mL indicating a very low microbial load before inoculation. The juice was treated by thermal, UV, PEF and RFEF te chniques to achieve an approximately equivalent 5 log reduction in inoculated microbial levels. The inlet and outlet temperatures recorded during thermal treatment were between 16 18 C due to a built in cooling section. The inoculated E. coli K12 populat ion decreased from 5.6 logs to 2.09 logs as temperature increase d from 60 to 70 C (Fig 5 1). Treatment of juice with temperatures above 62 C resulted in more than 4 log reduction in population. Thermal treatment at 70 C for 1.3 s was selected for microb ial stability studies as it resulted in a population reduction by 4.91 logs which was close to the target 5 log reduction.

PAGE 100

100 For UV treatment inoculated juice was passed through 1, 2, 3 and 4 UV bulbs connected in series at a flow rate of 28.8L/hr. Based on flow rate, the time of exposure to UV light translates to 14.75s, 29.57s, 44.25s and 59s. The inlet temperature was recorded at 50 C (pre treatment temp) and outlet temperatures recorded were from 48 50 C. The microbial population reduced with increas ing number of UV bulbs (Fig 5 2). UV treatment using three bulbs (44.25 s) reduced the microbial population by 5.05 logs. These conditions were used for microbial stability studies. The e ffect of PEF processing on E. coli K12 inactivation at an electric f ield strength from 15 to 22 kV/cm with a pulse duration of 2.5 s and a mean total treatment time of 150 s is shown in Figure 5 3. The inlet and outlet temperatures of the filtered juices were between 20 21 C and 34.6 50 C respectively, during the len gth of treatment. An e lectric field strength at 20kV/m gave a 5.23 log reduction in E. coli population. Yeom and others (2000b) found a 7 log reduction in the inoculated microbial population of unfiltered orange juice treated with PEF at 35kV/cm for 59 s showed that higher electric field strength and longer treatment times decreased microbial levels. RFEF is a relatively new non thermal technique that is not yet FDA approved for commercial use. It is similar to PEF except that the high vol tage is applied continuously using an AC generator. Previous work by Geveke and others (2007) ha s shown a 2.1 log reduction in inoculated E. coli K12 population in orange juice at conditions of 21.1 kHz frequency, 15kV/cm electric field strength, an outlet temperature of 60 C and processed at 20 kHz frequency, 15 kV/cm field strength, an outlet temperature of

PAGE 101

101 reduction in E. coli K12 population (Fig 5 4). After 2 treatment cycle s 5.26 log reduction was achieved. Effect of Treatments on Microbial Stability During Storage The effect of treatments on total aerobic count and yeast and mold count in grapefruit juice during 4 weeks of storage at 4 C are shown in Fig 5 5 and Table 5 1 The initial TAC in untreated grapefruit juice (control) was 1.57 log cfu/mL. After 4 weeks of storage the microbial population reached 3.52 log cfu/mL. The intial TAC for all treated samples reduced to < 5 cfu/mL at week 0. Thermal and RFEF treated juic e maintained the population below 1 log cfu/mL for 3 weeks of storage. However, in RFEF treated juice the microbial population increased to 3.34 log cfu/mL after 4 weeks of storage. In UV and PEF juice, the microbial population increased after 2 weeks of storage and reached 3.33 and 3.51 log cfu/mL respectively after 4 weeks of storage. The increase in microbial count during storage could be due to two reasons : 1) ineffective inactivation of microbial spores and their subsequent growth during storage or 2) incomplete inactivation or injury to microbial cell s and later recovery during storage. An increase in microbial count has been previously reported in PEF treated tomato juice due to incomplete inactivation of microbial spores (Min et al. 2003b) Because RFEF works on a similar principle of microbial in activation as PEF, it may also be ineffective in inactivation of spores. UV treatment has been shown to cause only injury to cells in cider due to lower penetration power. Tran and others (2004b) also found an increas e in aerobic count from 2.47 to 4.33 log cfu/mL after 12 days of storage in UV treated orange juice. The initial YMC in control juice was 2.46 log cfu/mL and it increased to 3.90 log cfu/mL by the end of 2 weeks storage. After 2 weeks, the mold growth was uncountable

PAGE 102

102 due to high growth and the colonies appeared too diffused on the petriflim. In thermal and RFEF no Y&M was detected at week 0 indicating effective inactivation of Y&M. In thermal samples the Y&M count reached 3.33 log cfu/mL after 4 weeks o f storage. In RFEF, PEF and UV, the mold growth was too high to be counted after 2 weeks of storage. Similar to total aerobic count, the reason for the increased Y&M growth during storage could be due either to sublethal injury to cells or resistance of sp ores to different treatments. Yeast and mold growth are the most common spoilage agents in refrigerated citrus juices (Wyatt and Parish 1995) They are also more tolerant to high temperatures compared to bacteria and are therefore difficult to inactivate. Mold ascospores like bacterial spores are known to be more resistant to PEF treatment (Grahl and Markl 1996) The results from microbial stability studies show that thermally treated juice had good microbial quality for 4 weeks of storage. Thermal treatment is mor e efficient in limiting bacterial and yeast and mold growth compared to non thermal treatment. RFEF treated juice had a shelf life of 3 weeks whereas PEF and UV treated juice spoiled after 1 week of storage. It could be concluded that heat treatment is mo re effective at microbial inactivation than non thermal techniques at equivalent process conditions. Effect of Treatments on P ectin M ethyl E sterase Activity The effect of heat, PEF, RFEF and UV treatment on PME activity is shown in Figure 5 6. PME is prima rily responsible for the clarification of citrus juice cloud. PME causes demethylation of carboxyl groups of pectin. These unprotected COOH groups react with calcium ions in juice and form pectin aggregates which precipitate out of juice (Wolfgang, 1990). Previous studies on heat pasteurization of orange juice have shown 90 100% inactivation in PME activity at temperatures higher than 90 C for 20 60 s

PAGE 103

103 (Elez Martinez et al. 2006; Rivas et al. 2006) Thermal treatment at 70 C for 1.3 s decreased the PME activity in gr apefruit juice by 80.47 1 .99%. Tran and others (2004b) reported a 70% PME inactivation in orange juice treated at 70 C for 2s. RFEF, PEF and UV treatments decreased the PME activity by 81.66 3.10%, 59.17 2.64% and 48 .52 3.62% respectively. Studies on the effect of PEF on PME activity by Yeom and others ( 2000a) s howed 88% PME inactivation in PEF treated orange juice at 35kV/cm and a 60 C outlet temperature. Rivas and others (2006) found 75.6% PME inactivation it is not surprising to find a lower inactivation of PME at the milder treatment conditions used in this 48.52 3.62% in grapefruit juice. Tran and others (2004b) found only 5% reduction in PME activity in orange juice at high UV dose at ambient temperatures. The pretreatment of juice to 50 C could possibly have an additive effect on the inactivation of PME in grapefruit juice. RFEF treatment gave the highest PME inactivation compared to other treatments. It is possible that the electric strength in combination with an output temperature of 55 C resulted in increased inactivation. H owever, there are no previous data on RFEF effect on citrus PME for comparison. During storage RFEF and thermal ly treated juices maintained residual PME activity during 4 weeks of storage indicating irreversible inactivation of PME. PME exist as isozymes in citrus juices with different degrees of thermostability. The thermolabile enzyme is quickly inactivated whereas the thermostable enzyme is gradually inactivated by exposure to high temperature s for extended periods of time. The residual

PAGE 104

104 PME activity o bserved in juices is mostly due to the presence of a thermostable enzyme. A decrease in activity during storage was observed in PEF and UV treated juice (Figure 5 6). In the control juice, the PME activity reduced to 65% after 4 week of storage. Since a lkaline is optimum pH for PME, a loss in activity during storage under acidic conditions is expected ( McFeeters RF 1983) Because grapefruit juice is acidic in nature, a loss in PME activity during storage is expected. An exponential decay in PME activity in untreated orange juice during storage has been reported by Elez and others (200 6) Effect of Treatments on Non enzymatic Browning The effect of thermal and non thermal treatments on the brown color of grapefruit juice during 4 weeks of storage at 4 C is given in Figure 5 7. All treated juice showed a significant increase (p<0.05) in the browning index compared to the control value of 0.069 at week 0. The browning index values at week 0 for thermal, RFEF, UV and PEF were 0.0835, 0.080, 0.073 and 0.072 respectively. Browning in citrus juice is mainly due to Maillard reactions betwee n sugars amino acids and ascorbic acid degradation products (Clegg 1964) Browning is commonly observed in heat treated citrus juice and is affected by storage temperature and time (Nagy et al. 1990) The Maillard reaction between reducing sugars and amino acids results in formation of melanoids which give the brown color to juice. However, in citrus juices the importance of Maillard type browning is minor due the acidity of these juices which is not favorable for Maillard type reactions. A s ignificant increase (p<0.05) in brown color was noted in all treated juice samples during 4 weeks of storage compared to initial week 0 values. The browning index after 4 weeks for thermal, RFEF, UV, PEF and control were 0.097, 0.094, 0.086,

PAGE 105

105 0.083 and 0.083 respectively. The browning index for thermal and R FEF treated juices are significantly higher (p<0.05) compared to the control, PEF and UV treated juices. Effect of Treatments on Ascorbic Acid Degradation The initial concentration of ascorbic acid in grapefruit juice was measured at 69.86 1.92 mg/100mL of juice. After pasteurization, a significant loss (p<0.05) in ascorbic content was noted in all treated samples ( Figure 5 8 ) Thermal processing reduced the ascorbic acid content by 29.19% in grapefruit juice. RFEF, PEF and UV treatment showed 26.33%, 16. 77% and 19.72% loss respectively, in ascorbic acid content at week 0. Ascorbic acid is a sensitive biomolecule and therefore treatment temperature and oxygen availability affects its rate of degradation (Sadler and others 1992; Lee and Coates 1999) Since all treated jui ce samples were exposed to temperatures above ambient, a decrease in ascorbic acid content is expected. During storage a further decrease wa s noted in all treated samples. After 4 weeks of storage, thermally treated samples had the highest ascorbic acid l oss (p<0.05) compared to non thermally treated juice samples. Ascorbic acid undergoes oxidative degradation to form dehydroascorbic acid which further reacts with amino acids to form brown color compounds. L ee and Nagy (1988) reported a high correlation between the percentage loss of ascorbic acid and an increase i n browning in grapefruit juices. To confirm whether similar correlation exists in present study, a linear regression plot of ascorbic acid content versus browning index is given in Figure 5 9. A negative correlation of 0.97 and 0.92 respectively were foun d for RFEF and thermally treated juices. The high correlation coefficients indicate that degradation of ascorbic acid is a probable explanation for the increase in brown color in treated samples during storage. Untreated grapefruit juice had the highest as corbic acid content compared to treated juice samples

PAGE 106

106 after 4 weeks of storage Sadler and others (1992) also found greater ascorbic acid retention in untreated orange juice compared to thermally treated juice during a 50 day storage period. It was propose d that increased microbial population in untreated juice may have lowered the dissolved oxygen content and prevented AA oxidation. Effect of Treatments on Color The effect of different treatments on color of grapefruit change was determined using Hunter L*, a* and b* values during 4 weeks of storage at 4 C. The change during storage on L* and b* values are shown in Fig. 5 10 and 5 11 respectively. The L* values increased significantly (p<0.05) for thermal and RFEF pasteurized juices compared to control juice at week 0. This is contradictory to the browning index value where an increase in browning was noted for thermal and RFEF samples. Therefore these results need to be considered with caution as they could be due to analytical error or to bottle to bottle sampling variation. No significant change (p<0.05) in L* was noted in PEF and UV treated juice compared to control at week 0. A significant increase (p<0.05) in b* values for all treated samples were noted at week 0 indicating a yellowing of sample s. However, during 4 weeks of storage, the b* values significantly decreased for thermal and RFEF juice. The decrease in b* values could possibly be due to increased browning in grapefruit juice. The linear regression plot between b* versus the browning in dex gave a correlation coefficient of 0.94 and 0.91 for thermal and RFEF treated juices, respectively (Figure 5 12). These value s indicate a strong relationship between browning and decrease in b* values in stored RFEF and thermal samples. Cortes and other s (2008) report ed a decrease in b value during storage in grapefruit juice. No significant (p<0.05) effect of treatment was observed for a* values during storage ( data not shown).

PAGE 107

107 Total color difference values (dE) were calculated to determine the magnitude of color diff erence between treated and control grapefruit juice ( Fig ure 5 13 ) A delta value of two or higher must be present in order for noticeable visual difference to be observed (Lee and Coates 1999) In the present study, calculated delta values were less than 2 for all treated juices throughout the storage period. Even though L* and b* value changed during storage, visually no noticeable differences were found in trea ted grapefruit juices compared to control juice. Effect of Treatments on pH, Brix and Total Acidity Measured pH values in control, thermal, UV, RFEF and PEF treated grapefruit juice are given in Table 5 2. The pH values were not significantly (p<0.05) affe cted by either processing or storage for treated juice compared to control juice Brix value for control grapefruit juice was measured at 10.42. No significant differences (p<0.05) in Brix values were noted at week 0 in treated samples. During storage, a significant decrease (p<0.05) was noted in PEF and UV after 4 weeks of storage. This decrease could be attributed to the utilization of sugars during fermentation by microorganism. A s imilar change in Brix due to microbial growth has been reported by many authors (Yeom et al. 2000b; Min et al. 2003a) Total titratable acidity for control and treated juice was measured and expressed as gm citric acid equivalents/100mL of grapefruit juice. Titratable acidity values we re not significantly (p<0.05) affected by either processing or storage for treated juice compared to control juice Conclusion The effect of thermal and non thermal pasteurization techniques on the quality of red grapefruit juice were compared to untreated juice for 4 weeks of storage at 4 C.

PAGE 108

108 The treatment conditions were optimized to achieve equivalent 5 log reductions in the inoculated E. coli population. Thermal treatment was more effective compared to non thermal treatment in terms of microbial stabili ty at after 4 weeks storage. However, RFEF treatment ensured microbial safety of grapefruit juice for 3 weeks of storage with significantly (p<0.05) higher Vitamin C content and equivalent PME inactivation compared to thermally treated juice. PEF and UV tr eatments maintained good microbial quality for 1 week with significantly (p<0.05) higher vitamin C content as well as less browning compared to thermal treatment The results from the study showed that at equivalent processing conditions, non thermally t reated juice had a shorter shelf life but higher vitamin C content and better physical qualities compared to thermally treated juice.

PAGE 109

109 Figure 5 1. Thermal inactivation of inoculate E. coli K12 in grapefruit juice Figure 5 2. UV Inactivation of inocu lated E. coli K12 in grapefruit juice

PAGE 110

110 Figure 5 3. PEF inactivation of E. coli K12 in grapefruit juice Figure 5 4. RFEF inactivation of inoculated E. coli K12 in grapefruit juice

PAGE 111

111 Figure 5 5 Total aerobic plate count for control, thermal, UV, PE F and RFEF treated grapefruit juice stored at 4 C for 4 weeks Table 5 1. Yeast & mold count in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks Week Control Thermal PEF RFEF UV 0 2.46 0.26 0 1.36 0.18 0 1.76 0.52 1 3.90 0.18 0 2.52 0.42 1.27 0.38 3.05 0.27 2 mold 0 mold mold M old 3 mold 2.24 0.36 mold mold M old 4 mold 3.33 0.24 mold mold mold

PAGE 112

112 Figure 5 6. Change in PME activity in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks Figure 5 7. Change in browning index of control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks

PAGE 113

113 Figure 5 8. Change in ascorbic acid content of control, thermal, UV, PEF and RFEF treated grapefruit ju ice stored at 4 C for 4 weeks Figure 5 9. Linear regression plot of ascorbic acid vs. browning index for thermal and RFEF treated grapefruit juice

PAGE 114

114 Figure 5 10 Change in color L* value of control, thermal, UV, PEF and RFEF treated grapefruit juice s tored at 4 C for 4 weeks

PAGE 115

115 Figure 5 11 Change in color b* values of control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks Figure 5 12. Linear regression plot of brown color vs. color b* values for thermal and RFEF t reated grapefruit juice

PAGE 116

116 Figure 5 13 Change in total color difference (dE) in control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks

PAGE 117

117 Table 5 2. pH, Brix and total acidity (TA) values for control, thermal, UV, PEF and RFEF treated grapefruit juice stored at 4 C for 4 weeks Weeks Control Thermal PEF RFEF UV pH 0 3.53 a 3.52 a 3.51 a 3.51 a 3.51 a 1 3.51 a 3.50 a 3.50 a 3.52 a 3.51 a 2 3.53 a 3.52 a 3.50 a 3.52 a 3.50 a 3 3.52 a 3.51 a 3.51 a 3.53 a 3.52 a 4 3 .53 a 3.50 a 3.52 a 3.53 a 3.51 a Brix 0 10.42 a 10.45 a 10.45 a 10.40 a 10.40 a 1 10.41 a 10.40 a 10.43 a 10.42 a 10.42 a 2 10.40 a 10.48 a 10.10 b 10.41 a 10.30 a 3 9.91 b 10.50 a 10.00 b 10.40 a 10.00 b 4 9.75 c 10.40 a 10.05 a 10.41 a 9.90 b TA 0 1.20 a 1.31 a 1.10 a 1.13 a 1.17 a 1 1.22 a 1.26 a 1.2 a 1.25 a 1.23 a 2 1.20 a 1.30 a 1.15 a 1.15 a 1.18 a 3 1.28 a 1.28 a 1.22 a 1.20 a 1.20 a 4 1.20 a 1.25 a 1.11 a 1.16 a 1.19 a

PAGE 118

118 CHAPTER 6 IMPACT OF THERMAL AN D NON THERMAL PROCESSIN G TECHNOLOGIES ON AROMA OF RED GRAPEFR UIT JUICE: GC OLFATOMETRIC COMPARS ION Introduction Non thermal techniques like pulsed electric field (PEF), ultraviolet (UV) and high pressure (HPP) have been examined in the past for their efficiency in extending shel f life of juice while minimizing nutritional and quality loss. Comparative studies on the effect of PEF and thermal processing on orange juice has shown reduced loss of flavor, color, and vitamin C in PEF juice compared to thermal processing (Ayhan et al. 2001) However, there is only a single literature reference on PEF processed grapefruit juice. The authors (Cserhalmi et al. 2006) investigated the effect of PEF treatment on physical proper ties like pH, Brix, color, non enzymatic browning and flavor components No was observed T he favor analysis was limited to 8 volatiles with no supporting sensory data. RFEF a recently developed non thermal technique (Geveke 2005) has not been FDA approved yet. Few studies have been conducted in citrus juice on microbial inactivation kinetics or on ascorbic acid cont ent and color of juice for non thermal treatments (Geveke et al. 2007) UV is commercially used for water di sinfection and apple cider pasteurization. Studies on UV treated orange juice is limited due to low transmittance of UV light through the high pulp juice (Tran and Farid 2004b) UV irradiation of unclarified orange juice extended th e shelf life by 5 days with 17% loss in ascorbic acid at 4C. No data on UV or RFEF treated grapefruit juice has been found in the literature. Even though the major motivation for non thermal processing technologies is minimal

PAGE 119

119 change to organoleptic proper ties, it is surprising to note that little research has been conducted in this area. Quantitative as well as qualitative studies on volatile constituents of grapefruit juice and essence have been extensively studied by many groups (Lin and Rouseff 2001; Coleman et al. 1972; Buettner and Schieberle 1999) Many odor active volatiles important to grapefruit aroma are present in low levels and are not usually detected by mass spectrometry or FID. GCO is a bioassay tha t measures human response to odorants separated by GC and is used to detect aroma active compounds. The present study focuses on comparing the impact of thermal and non thermal pasteurization techniques on aroma active volatiles of fresh unpasteurized grap efruit juice during 4 weeks of storage at 4 C using GCO. As discussed in chapter 5 the non thermal and thermal treatments were optimized to achieve equivalent 5 log reductions in microorganism s for fair comparison. Materials and Methods Standard compou nds ethyl propionate, ethyl 2 methyl propionate, methyl butyrate, ethyl butyrate, ethyl 2 methyl butyrate, hexanal, b pinene, b myrcene, limonene, 2 methyl butanol, ethyl hexanoate, p cymene, octanal, 2 methyl 3 furanthiol, nonanal, ethyl octanoate, methio nal, decanal, linalool, geranial, geraniol, a ionone, g decalactone, b damascenone and 2 methoxy 4 vinyl phenol were purchased from Aldrich (St. Louis, MO). Natural 4 mercapto 4 methylpentan 2 one (1% in PG) was obtained from Oxford Chemicals (Hartlepool, UK). Grapefruit Juice One hundred and fifty litres of unpasteurized red grapefruit juice with no additives was procured from a commercial Florida processor. Shipping, storage and initial

PAGE 120

120 treatment conditions have been described in chapter 5 Juice was fi ltered through 2 layers of cheesecloth before processing by PEF, RFEF, UV and thermal treatments Grapefruit Juice Processing Preliminary experiments were performed to determine equivalent processing conditions us ing heat, pulse electric field ( PE F) radiof requency electric field (RFEF) and ultraviolet radiation (UV) for grapefruit juice. Juice was inoculated with E. coli K12 (ATCC 23716) from a stationary phase culture to give an approximately 7 log CFU/mL population. Microbial assays were conducted for jui ce heated from 60 72 C with UV exposure times of 6 25s at 50 C, 15 22 kV/cm electric field strengths for PEF treatment and 0 2cycles at 20kHz frequency and 15kV/cm for RFEF treatment. Final optimized processing conditions resulted in approximately 5 log reductions of E. coli K12 and were used for the remainder of the study. Heat treatment Grapefruit juice was heat pasteurized using a miniature scale HTST processing system (Armfield, Jackson, NJ, FT74 30 MkIII 33 34) explained previously in chapter 5. Non t hermal juice treatments The UV, PEF and RFEF juice treatment conditions have been described in c hapter 5. Packaging and storage Processed grapefruit juice was collected directly from the processing unit outlet into sterile 1L media glass bottles (Corn ing Inc, Corning, NY) inside a sanitary laminar hood equipped with a HEPA air filter(Forma Scientific Inc., Marietta, OH). The hood was sanitized by UV lighting at 254nm for 30min before use and then wiped with 100% alcohol. The bottled juice was stored at 4 C for 4 weeks. Volatile analysis of fresh

PAGE 121

121 unpasteurized juice was performed at week 0. For all pasteurized juice samples, volatiles analysis and sensory analysis were conducted at week 0 and week 4 ( the end of storage study). The reference sample used for week 4 sensory evaluation was untreated grapefruit stored at 17 C and thawed overnight at 4 C one day before analysis. Extraction of Grapefruit Juice Volatiles Aliquots (25 ml) of grapefruit juice were placed in 40 ml glass vials with screw cap s co ntaining Teflon coated septa. Tetradecane (1 L of 1000ppm) was added as internal standard into the juice before equilibration. The juice was equilibrated for 15 min at 40 C with stirring. A 2 cm (Supelco,Bellefont e, PA) SPME fiber was exposed in the equilibrated headspace for 30 min at 40 C. The fiber was desorbed for 5 min in the GC injection port at 250 C. All samples were analyzed in quadruplicate. Volatile Analysis of Grapefruit Juice Volatiles A GC/MS/O inst rument (6890N GC, 5973N MS, Agilent Technologies, Santa Carla, CA) and olfactory detector (Gerstel, Baltimore, MD) were used for separation and analysis of volatiles. The GC effluent was split between MS/O in a ratio of 1:3 Samples were run separately o n a polar DB Wax and non polar DB 5 column with identical dimensions (30 m x 0.32 mm x 0.5 m from J&W Scientific, Folsom, CA). The column oven temperature was programmed between 40 C to 250 C at 7 C/min with a 5 min hold at 250 C. The i njector and det ector temperature s w ere set at 250 C. Mass spectrometry conditions were as follows: transfer line temperature at 275 C, mass range 30 to 300 amu, scan rate 5.10 scan/s and ionization energy 70 eV. Helium was used as the carrier gas at a flow rate of 2 mL/min. Mass spectral matches were

PAGE 122

122 compared with NIST 2002 standard spectra. Authentic standards were used for confirmation. Alkane linear index values were determined on both columns. GCO analysis was performed using two trained sniffers, both non smoker s between ages of 30 35 yrs. The panelists were trained in a similar manner as reported by Bazemore and others ( 1999) u sing standard solution s of varying concentration of 10 compounds usually found in citrus juices: ethyl butyrate, octanal, hexanal, linal ool, a pinene, limonene, geranial, neral, citronellal and a terpineol. The panelists were trained on intensity rating optimum positioning and breathing techniques. The p anelist seated at the sniffing port rated the intensity of volatil ity on a 4 point int ensity scale using an olfactory pad (Gerstel, Baltimore, MD) and the aroma description was audio recorded. The aroma intensity scale ranged from 0= no aroma perceived to 4 = strong aroma perceived. The ODP output was integrated into the MSD Chemstation sof tware (Agilent MSD Productivity Chemstation version D.02) to achieve TIC chromatogram and aromagram simultaneously. Samples were sniffed two times by each assessor resulting in four aromagrams. The results from 4 aromagrams were averaged for each sample. Odor active compounds producing intensity response s at the same retention time at least 50% times were selected. The odor active compounds were identified based on sensory descriptor, retention index calculated, comparing RI and descriptor from two columns and authentic standards. Sensory Evaluation Screening and training of panelists The s creening for panelist s was performed using two different commercially pasteurized grapefruit juice s and fresh squeezed grapefruit juice. A total of 40 panelists were scr eened for their ability to differentiate between the samples in two sessions.

PAGE 123

123 Panelist s who were consistently correct in differentiating juice samples were selected for further training. Twenty five panelists were selected after the initial screening. The panel cons isted of 12 males and 13 females in an age group between 25 45 yrs. Selected panelist s were further trained for 1 2 hours using the same juice samples They were trained to use verbal terminologies or descriptors to differentiate between sample s and to use scaling procedures to determine the degree of difference between the samples. Test method A difference from control type of sensory evaluation (Meilgaard and others 1999) was employed to orthonasally detect differences in aroma between unpaste urized and pasteurized grapefruit juice. All tests were conducted once in a sensory panel facility at Eastern Regional Research Center (Wyndmoor, PA) with 6 booths equipped with computers. The sensory analysis was designed and conducted using Compusense fi ve (Compusense Inc., Ontario, CA). A sample sensory ballot is shown in Appendix D. Samples were prepared by pouring 40 mL of juice into 100 mL glass bottles with airtight plastic caps and stored in boxes at 4 C overnight. On the day of testing, the bottl es were taken out 1hr before the testing. Juice temperature was measured at 10 12 C during testing. Testing was performed under red light so that color and other visible differences were masked from panelist s The control sample given to panelist s was fr esh untreated grapefruit juice and labeled as R. The experimental samples consisted of the 4 treated juice samples (thermal, UV, PEF & RFEF). Each panelist received 1 R sample and 5 coded samples corresponding to the 4 experimental samples and 1 blind co ntrol (unpasteurized). Panelist s were required to specify the degree of difference on a

PAGE 124

124 scale of 0 10, 0 = no difference and 10 = extremely large difference. Panelists were also asked to give verbal description s of the difference between samples. Statistic al Analysis Sensory data were subjected to statistical analysis using SAS 9.1 (SAS Inst. Inc., Raleigh, N.C., USA). The tests for statistical significance were performed by analysis of variance (ANOVA) at a significantly different from one another Results and Discussion Effect of Treatment and Storage on Grapefruit Juice V olatiles The volatiles in grapefruit jui ce were quanti fied using an internal standard (tetradecane) by GC/MS. A total ion chromatogram (TIC) of volatiles from untreated grapefruit juice is shown in Appendix Figure C 1. The volatile concentration s for untreated and treated grapefruit at week 0 an d week 4 are given in Appendix Table s C 1 and C 2 respectively. The percentage loss in volatile s was calculated for thermally and non thermally treated grapefruit juice compared to untreated juice during storage (Table 6 1). Concentrations of all volatile s except terpineol and linalool were diminished in treated samples at week 0 P cymenene lost more than 50% and nootkatone lost more than 40% during the same time period After 4 weeks storage, hexanal and ethyl hexanoate were completely lost and decana l and octanal lost more than 80% in all treated samples. The loss in aldehydes during storage occurs due to their conversion into corresponding alcohols (Petersen and others 1998) O ctanol levels increased (2 14%) in all treated juice samples. Alpa terpineol increased by more than 80% in all treated samples. Alpha terpineol is formed in stored orange jui ce by acid catalyzed hydration of limonene an d linalool (Petersen and others 1998). In the present

PAGE 125

125 study, a 15 30% loss in limonene and linalool content was noted in treated samples. The result s from week 0 and week 4 analysis suggest that the volatiles of grapefruit juice are more strongly affected by storage rather than treatment type. G as C hromatography O lfactometry Profile of Grapefruit Juice A total of 38 aroma active components were detected in the headspace of fresh untreated and treated grapefruit j uice as listed in Table 6 2. Odorants were identified based on retention indices on two dissimilar columns, odor description and GCO data from standard compounds ( except for compounds 24, 26 and 32 ) These compounds were identified based on retention inde x and odor descriptors. Nine odorants (1, 2, 19, 21, 28, 33, 34, 36 and 38) remain unknown. The odorants in GFJ could be classified into 5 groups based on similar aroma notes comparable to those of Lin and others (2002b) : fruity/sweet, green, citrus/grapefruit, floral/musky/fragrant, terpeny/ piney and cooked/ roasted/catty. Sweet/fruity The sweet/fruity category had the highest number of odorants (11 odorants). The majority were identified as esters: ethyl propionate, methyl butyrate, ethyl butyrate, ethyl 2 methyl butyrate, ethyl 3 methyl p ropionate, ethyl hexanoate and ethyl octanoate. Ethyl octanoate is one of the odorants with the highest aroma value in the present study. Other compounds belonging to this group were b damascenone (30), g decalactone (35) and two unknowns (33, 34). Fresh/ citrus Six odorants belonged to the fresh/citrus group. They include aldehydes (15, 17, 23, 27), alcohol (30) and terpene hydrocarbon (11). Aldehydes such as octanal, nonanal, decanal and geranial are common to citrus fruits like orange, grapefruit, and l emon (Allegr one and others 2006; Lin et al. 2002b) Limonene with

PAGE 126

126 citrus aroma has been shown to be an important ingredient in orange juice by omission studies in model flavor mixtures (Buettner and Schieberle 2001) Cooked/roasted/catty The group includes the 4 sulfur compounds: 2 methyl 3 furnathiol (MFT), methi onal, bis (2 methyl 3 furfuryl) disulfide (MFT MFT), 4 mercaptone 4 methyl 2 pentanone (4 MMP) and two unknowns (19 3 8). The meaty odorant MFT is a product of thiamine degradation under acidic conditions. It has low odor threshold at 0.007ppm in water. It can also be produced by Maillard reaction between cysteine and pentose sugar and is often found in heat treated citrus juices (Perez Cacho and Rouseff 2008) It read il y forms a dimer, MFT MFT, which also has a meaty odor Methional has a cooked potato odor and is reported in many fresh fruits like orange, tomato and grapefruit (Perez Cacho and others 2008). Its biosynthetic pathway in plants is unknown but it can be formed in processed juices by Strecker degradation from the amino acid methionine during thermal treatments and subsequent storage. Me thional and 2 methyl 3 furanthiol are reported as possible off flavors in orange juice (Bezman and others 2001) 4 MMP is significant for characteristic grapefruit aroma. It has very low odor threshold of 0.1 ng/L in water (Buettner and Schieberle 2001) It i s reported to possess a grapefruit aroma at very low concentrations and a catty sulfurous odor at higher concentrations. Green/fresh The green/ fresh odor of grapefruit juice is imparted by odorants hexanal, p cymene, decanal and 4, 5 epoxy (E) 2 decenal and one unknown (22). Odorant 4, 5 epoxy (E) 2 decenal has a green metallic odor and is a key ingredient in fresh grapefruit juice (Buettner and Schieberle 2001)

PAGE 127

127 Floral/fragrant Linalool, 4 vinyl guaiacol, a ionone and 2 unknowns (1, 28) have a floral fragrant aroma. Linalool with its intense floral aroma is another component that is commonly found in citrus fruits. Para viny guaiacol is formed from ferulic acid present in citrus peels (Perez Cacho and Rouseff 2008) The presence of this compound is usually an indicator of thermally abused or aged juice. Alpha ionone is formed from their carot enoid precursors in orange juice (Mahattanatawee and others 2005) Terpeny Only two odorants, b pinene and b myrcene are responsible for the terpeny odor in grapefruit juice. In fresh untreated GFJ 24 odor active compounds were perce ived. GCO analysis revealed ethyl octanoate and octanal as having the highest aroma intensities followed by ethyl butyrate, ethyl hexanoate, methional and decanal. The results are different from Buettner and others (2001) who reported high FD values for ethyl butyrate, (Z) 3 hexenal, 1 he pten 3 one, 4 mercapto 4 methyl 2 pentanone, 1 p menthene 8 thiol, 4,5 epoxy (E) 2 decenal and wine lactone in fresh white Marsh grapefruit juice by aroma extract dilution analysis Except for ethyl butyrate and 4, 5 epoxy (E) 2 decenal other volatiles were not detected in fresh juice in our study. Lin and others (2001) a lso found qualitative as well as odor intensity differences for the majority of odor active compounds when compared to results from Bue tt ner and others (2001). Possible reason s for the differences could be due to v ariety fruit quality, juice extraction methods, storage time and temperature volatile extraction method and GCO analysis methods. The grapefruit variety used by Buettner and others (2001) was white Marsh whereas in the present study red grapefruit was used. Secondly, the extraction method used by Buettner and others (2001) was liquid extraction compared to static headspace

PAGE 128

128 SPME used in this work. The quantitative and qualitative volatile profile depends on the extr action method used. Rouseff and others ( 2001) showed that the terpene s limonene, a pinene and myrcene comprised 86% of the total FID area by SPME compared to 24% from liquid extract ion of the same orange juice. Low vapor pressure compounds are not readily extracted by SPME. This could be the reason for absence of wine lacto ne in our study. Reconstitution of odorants in a model grapefruit juice flavor mixture by Buettner and others (2001) showed that omission of 4 MMP resulted in a more orange like odor rather than grapefruit odor. The authors concluded that 4 MMP is importan t for typical grapefruit juice odor. Surprisingly in our studies the volatile was found only in UV and thermally treated juice (but not in fresh juice ) The possible reasons for the absence of 4 MMP in our studies could be the different extraction methods used. The concentration of 4 MMP in juice is very low ranging from 0.8 1ng/L (Buettner and others, 2001). Possibly the HS SPME used in the present study may not be as efficient as solvent extraction of 4 MMP at such low levels. Sulfur compound, 1 p ment ha 8 thiol with a typical grapefruit like odor is reported as a character impact odorant in grapefruit juice. However, the significance of this compound to grapefruit juice aroma is debatable since Lin and others (Lin et al. 2 002b) reported the presence of this thiol only in concentrated juice and not in the original juice They proposed that the compound was possibly a reaction product of limonene or a pinene with hydrogen sulfide in thermally abused juices and therefore may not be present in fresh untreated juice. Gas Chromatography Olfactometry Profile Comparison of Fresh Untreated and Treated Juice at Week 0 The total number of odorants perceived in fresh, UV, thermal, PEF and RFEF treated GFJ were 24, 27, 23, 18 and 20, respectively (Table 6 2). A comparative profile

PAGE 129

129 based on the six odor categories for treated and untreated juice is plotted as a spider web diagram in Figure 6 1. T he panel average aroma intensities for odorants belonging to same odor category are added to get the total odor intensi ty for that particular group (Table 6 3). It is evident from Figure 6 1 that the aroma profile of fresh juice was distinctly different from all treated juices. In general a decrease in fruity/sweet and citrus/fresh notes is obse rved for treated juices compar ed to fresh. Cooked/meaty/catty odor increased in thermal and UV juice considerably. Terpeny odor was the least affected by pasteurization techniques. The impact of treatments on odorants belonging to the six aroma categories are discussed in detail below. Thermal treatment The total odor intensity for fruity/sweet, citrus/fresh and green/metallic decreased in thermally treated juice by 4.7, 3.4 and 2.2 points respectively, compared to fresh juice. In the fruity/sweet category esters like ethyl butyrate, methyl butyrate and ethyl 2 mehtyl butyrate had lower a intensity whereas ethyl propionate was completely absent. N onanal, geraniol and geranial belonging to the fresh/citrus group were absent in thermal samples The green me tallic odorant 4, 5 epoxy (E) 2 decenal important for grapefruit odor was also missing in thermally processed juice. The results indicate the instability or degradation of the above compounds in the presence of heat. The sweet odorant b damascenone was d etected in thermal juice but not in untreated juice. The compound has been reported in reconstituted grapefruit juice which is thermally processed (Lin and others 2002 b ). They proposed that there is a release of gylcosydically bound b damascenone by acid hydrolysis during heating. A c ooked/meaty/catty odor was increased in thermal juice by 4.8 points compared to fresh juice due to the formation of MFT, MFT MFT, 4 MMP and an unknown (19). The m eaty

PAGE 130

130 odorant MFT could be formed either by Maillard reaction bet ween cysteine and pentose or from thiamin degradation. The level of cysteine present in red grapefruit juice is 15.3 mol/L and pentoses are present in ample amounts in grapefruit juice (Kusmierek and Bald 2008) Rouseff and others, 2001). F ormation of M FT via the Maillard reaction during heat treatment in grapefruit juice is possible. MFT dimerizes in juice to form the meaty odorant bis (2 methyl 3 furfurl) disulfide. 4 MMP is present in grapes as a cysteine conjugate that is released during fermentation by yeast enzymes. Thermal hydrolysis could result in the release of 4 MMP from such a precursor in heat treated juice. Ultraviolet treatment No major change in sweet/fruity intensity was noted in UV treated juice. A decrease in citrus/fresh and green/me tallic odor by 3 and 1.2 points respectively was noted due to the absence of odorants (Z) 2 nonenal, geranial, geraniol and 4,5 epoxy (E) 2 decenal. B eta damascenone was also detected in UV treated juice. Thermal degradation of neoxanthin in a model syst em containing peroxy acetic acid produced damascenone. However, the reaction temperatures used for this synthesis were high (60 90 C) and lasted for extended periods of time (Bezman et al. 2001) The mechanism of formation and release of damascenone in UV treated samples is not known. An increase in cooked/catty odor by 4.2 points was observed in UV treated juice. The catty odor of 4 MMP was more intense in UV treated juice than in thermal ly treated juice It is surprising to find the meaty odorant MFT MFT in UV juice because of the absence of its monomer MFT. The odor of MFT MFT is 8.9x10 11 mM in water whereas the odor threshold for MFT is 6.14x10 8 mM in water (Dreher and others, 2003).

PAGE 131

131 Because the odor threshold of the dimer is lo wer t han the monomer it is possible that the concentration of 2 methyl 3 furnathiol may be present below odor detection limit s in UV juice. Pulsed electric field treatment Similar to thermal treatment, a decrease in fruity/sweet as well as citrus/fresh od or was noted in PEF juice. The compound 4, 5 epoxy (E) 2 decenal was also missing in PEF treated juice. The increase in cooked/catty odor was less compared to that of thermal and UV treated juice. Radio frequency electric field treatment RFEF treated grap efruit had an odor profile similar to PEF juice. This is expected because both pasteurization techniques use electric field strength for inactivation of microorganism s The aroma profile had lower sweet/fruity and citrus character s compared to fresh juice and juice with a slightly higher cooked/meaty odor. Gas Chromatography Olfactometry Comparison of Fresh Untreated and Treated Juice at Week 4 After 4 weeks of storage, the total number of odorants decreased to 14, 13, 12 and 15 respectively for thermal, P EF, RFEF and UV treated juice (Table 6 4). 14 total odorants were missing in week 4 treated samples compared to untreated juice analyzed at week 0. Guaiacol with a medicinal odor was detected only in UV treated samples due to microbial contamination. Th is compound is formed by spoilage microorganism s like Alicyclobacillus sp. in stored juice (Gocmen D 2005) However, it was not detected in PEF and RFEF samples even though both these samples had microbial contamination. A possible reason for this discrepancy could be bottle to bottle variation in microbial levels during sampling

PAGE 132

132 Sensory Evaluation A difference from control type of sensory analysis was performed on grapefruit juice aroma s at week 0 and week 4 (Appendix D 1 and D 2). This type of sensory analysis tells us whether a difference exits between the samples and tells us the magnitude of difference between two samples. The data recorded by 25 panelists for difference s from the control test between pasteurized and unpasteurized grapefruit juice at week 0 is given in Appendix D 1. Statistical significance between samples was determined using an ANOVA test at 95% confidence levels through an F test. Table 6 5 shows that the Fcal value is greater than the Fcrit value This indicates a statistical difference between sample means at week 0. Tukey s test is a single step multiple comparison procedure It is performed in conjunction with ANOVA to find if there is significant differen ce between treatment means The HSD (hon estly significant difference) value calculated was 1.58 for week 0 mean values An HSD value higher than the difference between two sample means indicates a significant differen ce between two treatment means It is evident from Table 6 6 that the differenc e in mean values between treatments is lower than the HSD value. The mean values for treated juice are significantly (p<0.05) different from each other because the calculated HSD is greater than the treatment differences The difference in means is highest between UV and fresh untreated juice. The general comments from panelist s for UV treated juice were that the juice had a rotten or spoiled odor. Because no microbial spoilage was noted in UV samples at week 0, the sensory comments are possibly a reflectio n of increased 4 MMP levels in UV samples. Thermally treated juice was described as having a cooked odor. The presence of 2 methyl 3 furnathiol and bis (2 methyl 3 furfuryl) disulfide would give a cooked note to the juice. According to

PAGE 133

133 panelist s comments, both PEF and RFEF juice had weaker/less fresh aroma s compared to untreated juice The ANOVA results for week 4 sensory analyses ( Table 6 7 ) indicate a significant difference ( = 0.05) between sample means. The HSD value calculated was 1.85. Table 6 8 shows that the difference in mean s between PEF and thermal samples are higher than the HSD value indicating a significant difference (p<0.05) between the two samples. No signifi cant difference (p<0.05) in sample means wa s noted between RFEF, UV and thermal ly treated juices Panelists commented that all treated juice samples except thermal had a fermented rotten odor. This is due to microbial spoilage detected in PEF, RFEF and UV samples after 4 weeks of storage (chapter 5). Thermal samples had a cooked odor. Conclusion The results from th is study indicate that thermal as well as non thermal pasteurization techniques affected the aroma profile of fresh grapefruit juice. The effec t s of both thermal and non thermal treatments on juice aroma w ere characterized by loss es in desirable fruity and citrus odorants along with an increase in undesirable cooked/catty odorants. Aroma of treated juice s differed significantly (p<0.05) from fr esh juice by sensory evaluation further confirming GCO results. Further sensory studies such as preference evaluation need to be conducted to understand consumer preference s between thermally or non thermally treated grapefruit juice.

PAGE 134

134 Figure 6 1. Co mparative aroma profile for unpasteurized and pasteurized grapefruit juice at week 0

PAGE 135

135 Table 6 1.Change in GC MS grapefruit juice volatiles during 4 weeks of storage at 4 C RI Compound Identified %loss week0 PEF RFEF UV Thermal %loss week4 PEF RFEF UV T hermal 888 ethyl acetate 9.88 13.64 16.54 10.50 41.59 29.87 26.68 37.17 1037 a pinene 0.84 3.94 3.72 1.43 15.28 38.81 20.24 32.90 1049 ethyl butyrate 19.69 33.23 28.59 30.62 45.90 53.40 88.66 53.75 1098 H exanal 1.70 9.91 4.58 2.69 100.00 100.00 100.00 100.00 1174 b myrcene 3 .47 6.39 7.40 7.55 8.55 15.92 20.42 31.76 1219 L imonene 4.59 8.20 13.34 12.01 14.38 26.99 25.09 24.77 1243 ethyl hexanaote 21.05 20.53 29.09 18 .55 100.00 100.00 100.00 100.00 1260 3 carene 18.02 26.74 27.10 14.34 82.91 3 1.74 57.6 4 58.31 1285 p cymene 35.71 29.30 25.45 25.98 44.67 40.00 53.48 55.83 1299 O ctanal 6.55 13 .58 17.16 5.20 94.72 95.54 95.38 94.39 1400 N onanal 6.01 23.40 14.78 23.17 40.85 69.26 55.48 71.35 1436 ethyl octanoate 15.49 32.89 4.61 13.11 19 .81 29.21 24.15 2 2 .71 1447 p cymenene 54.37 57.67 67.79 51.16 75.91 72.78 76.10 78.63 1501 D ecanal 4.74 2.18 8.75 3.35 80.84 56.23 83.77 87.71 1506 a copaene 1.74 29.81 16.12 14.29 29.32 41.45 46.41 57.69 1537 L inalool 1 4.33 1 0.66 1 3.74 1 3.52 14.86 30 .30 27.01 2 3.88 1 549 1 octanol 0.57 1.98 4.42 5.53 2.14 6.00 6.11 1 3.78 1608 b caryophyllene 0.14 1 .31 1.48 0.91 5.16 2 .17 4.39 1 0.15 1670 a caryophyllene 2.16 2.00 1.73 2.53 19.37 10.88 3.84 11.95 1709 V alencene 11.35 32.55 1 9.00 1 2.55 65.39 67.03 62.01 63.28 1 721 a selinene 0.38 3.52 3.16 2 .02 4.46 2.11 1 1.12 1 1.44 1728 a terpineol 13.97 3.35 4 .91 8.43 80.87 99.71 98.34 85.67 1735 d cadinene 1 3.71 27.72 10.97 23.95 31.93 34.74 31.93 34.74 1747 a panasinsen 7.81 9.19 5.21 11.94 21.89 28.72 25.21 31.97 2677 Nootkatone 39.35 46.81 47.98 47.65 48.10 53.52 56.80 55.97

PAGE 136

136 Table 6 2 GCO analysis results of pasteurized and unpasteurized grapefruit juice No DB 5 DB Wax descriptor C ompound untre ated UV T her mal PEF RFEF 1 811 floral lemony U nknown 1.3 2 823 bad off U nknown 0.5 3 932 ethanol ethyl propanoate 1.5 1.0 4 912 1018 fruity ethanol ethyl 2 methyl propionate 2.3 2.0 1.3 1.8 1.5 5 782 1033 S weet methyl butyrate 2.5 2.5 1.5 1.0 1.5 6 803 1046 fruity sweet ethyl butyrate 2.5 2.5 2.5 2.3 1.5 7 1058 fruity strawberry ethyl 2 methyl butyrate 2.0 1.0 1.3 1.0 0.8 8 1091 green grass H exanal 1.0 1.0 0.8 1.0 1.0 9 1117 P iney b pinene 1.0 1.0 0.5 0.8 1.0 10 974 1172 musty terpene b myrcene 1.3 1.0 1.8 1.5 1.3 11 1022 1217 fresh citrusy minty L imonene 1.3 1.0 1.3 1.3 1.0 12 1030 1227 sweet fruity 2 methyl butanol 0.8 13 979 1241 sweet fruity ethyl hexanoate 2.5 2.3 2.0 2.5 2.0 14 1285 green apple skin p cymene 0.8 15 985 1296 fresh citrus O ctanal 3.0 2.5 2.3 1.8 2.0 16 856 1326 meaty roasted 2 methyl 3 furanthiol 1.5 17 1372 green citrus N onanal 1.5 1.0 0.5 1.0 0.8 18 961 1400 sulfur catty 4 mercapto 4 methylpentan 2 one 2.5 1.0 19 1428 cooked unpleasant U nknown 0.8 20 1177 1434 F r uity ethyl octanoate 3.0 1.5 1.0 1.8 2.0 21 1448 grass musky U nknown 0.8 22 889 1460 cooked potato M ethional 2.3 2.0 2.5 2.3 2.0 23 1190 1504 citrus green D ecanal 2.0 1.5 1.5 1.5 1.8 24 1517 fatty green (Z) 2 nonenal* 1.0 25 26 1081 1535 floral fresh L inalool 1.8 1.5 2.0 1.0 1.5 1661 cooked meaty bis (2 methyl 3 furfuryl) disulfide* 1.5 1.3 1.5 1.3

PAGE 137

137 Table 6 2 Continued No. DB 5 DB Wax descriptor C ompound untrea ted UV therm al PEF RFEF 27 1749 sweet citrus gerania l 0.8 28 1789 floral unknown 1.5 29 1822 sweet b damascenone 0.5 0.5 30 1840 citrusy floral geraniol 0.5 31 1873 floral sweet a ionone 1.5 32 1985 green metallic 4,5 epoxy (E) 2 decenal* 1.0 33 20 13 sweet fruity unknown 0.5 34 2055 fresh fruity unknown 0.8 35 1489 2069 buttery fragrant peachy g decalactone 1.5 1.8 1.8 1.0 1.5 36 2163 sweet ethanolic unknown 1.8 37 2196 2187 musky cologne 4 vinyl guaiacol 1.8 2.0 1.5 1. 8 2.0 38 2240 bad solvent unknown 1.0 Odorants identified based on odor descriptor and retention indices with reported data due to unavailability of data

PAGE 138

138 T able 6 3 Calculated total aroma intensity values for pasteurized and unpasteurized g rapefruit juice belonging to five odor categories Aroma intensity Odor category Compounds fresh UV ther mal PEF RFE F fruity sweet ethyl propanoate 1.5 1.0 ethyl 2 methyl propionate 2.3 2.0 1.3 1.8 1.5 methyl butyrate 2.5 2.5 1.5 1.0 1.5 et hyl butyrate 2.5 2.5 2.5 2.3 1.5 ethyl 2 methyl butyrate 2.0 1.0 1.3 1.0 0.8 2 methyl butanol 0.8 ethyl hexanoate 2.5 2.3 2.0 2.5 2.0 b damascenone 0.5 0.5 unknown 0.5 unknown 0.8 g decalactone 1.5 1.8 1.8 1.0 1.5 unknown 1.8 total intensity 15.5 16.6 10.8 9.6 8.8 fresh/citrus limonene 1.3 1.0 1.3 1.3 1.0 octanal 3.0 2.5 2.3 1.8 2.0 nonanal 1.5 1.0 0.5 1.0 0.8 geranial 0.8 decanal 2.0 1.5 1.5 1.5 1. 8 geraniol 0.5 total intensity 9.0 6.0 5.6 5.5 5.6 green/metallic hexanal 1.0 1.0 0.8 1.0 1.0 p cymene 0.8 unknown 0.8 (Z) 2 nonenal 1.0 4,5 epoxy (E) 2 decenal 1.0 total intensity 3.0 1.8 0.8 1.0 1.8 floral/fragrant linalool 1.8 1.5 2.0 1.0 1.5 unknown 1.5 a ionone 1.5 -4 vinyl guaiacol 1.8 2.0 1.5 1.8 2.0 total intensity 5.0 3.5 3.5 2.8 5.0 T erpeny b pinene 1.0 1.0 0.5 0.8 1.0 b myrcene 1.3 1.0 1.8 1.5 1.3 total intensity 2.3 2.0 2.3 2.3 2.3

PAGE 139

139 Table 6 3 Continued Aroma intensity Odor category Compounds fresh UV ther mal PEF RFE F cooked/meaty/cat ty 2 methyl 3 furanthiol 1.5 unknown 0.5 4 mercapto 4 methyl pentan 2 one 2.5 1.0 methional 2.3 2.0 2.5 2.3 2.0 bis (2 methyl 3 furfuryl) disulfide 1.5 1.3 1.5 1.3 unknown 0.8 total intensity 2.3 6.5 7.1 3.8 3.3

PAGE 140

140 Table 6 4 GCO comparison of pasteurized gr apefruit juice at week 4 to unpasteurized grapefruit juice at week 0 No DB 5 DB Wax descriptor compound untre ated UV therm al PEF RFEF 1 932 E thanol ethyl propanoate 1.5 2 912 1018 fruity ethanol ethyl 2 methyl propionate 2.3 3 782 1033 S weet methyl butyrate 2.5 1 .5 1.0 1.0 1.0 4 803 1046 fruity sweet ethyl butyrate 2.5 1.3 1.0 1.5 1.0 5 1058 fruity strawberry ethyl 2 methyl butyrate 2.0 1.0 1.0 1.0 0.5 6 1091 green grass Hexanal 1.0 7 1117 Piney b pinene 1.0 8 97 4 1172 musty terpene b myrcene 1.3 9 1022 1217 fresh citrusy minty Limonene 1.3 10 1030 1227 sweet fruity 2 methyl butanol 0.8 11 979 1241 sweet fruity ethyl hexanoate 2.5 12 985 1296 fresh citrus octanal 3.0 1.0 1.5 1.0 1.0 13 856 1326 meaty roasted 2 methyl 3 furanthiol 1.0 14 1372 green citrus Nonanal 1.5 15 961 1400 sulfur catty 4 mercapto 4 methylpentan 2 one 1.0 0.5 16 1177 1434 F ruity ethyl octanoate 3.0 1.0 1.0 1. 0 1 .0 17 889 1460 cooke d potato methional 2.3 2.0 2.5 2.3 2.0 18 1190 1504 citrus green decanal 2.0 1.5 1.5 1.5 1.8 19 1517 fatty green (Z) 2 nonenal* 1.0 20 21 22 1081 1535 floral fresh linalool 1.8 1.5 2.0 1.0 1.5 1664 stinky, rotten unknown 1.0 0.5 1661 cooked meaty bis (2 methyl 3 furfuryl) disulfide* 1.5 1.3 1.5 1.3 23 17 20 grassy floral unknown 1.3 24 1749 sweet citrus geranial 0.8 25 1822 sweet b damascenone 0.5 26 27 1445 1840 1875 citrusy floral medicinal geraniol guaiacol 0.5 0.5

PAGE 141

141 Table 6 4. Continued No. DB 5 DB Wax descriptor compound untreated UV thermal PEF RFEF 28 1873 floral sweet a ionone 1.5 29 1985 green metallic 4,5 epoxy (E) 2 decenal* 1.0 30 2 055 fresh fruity unknown 0.8 31 1489 2069 buttery peachy g decalactone 1.5 1.0 1.5 1.0 1.5 32 2196 2187 musky cologne 4 vinyl guaiacol 1.8 2.5 2 .5 2 3 2.0 33 1358 2647 dirty musty unknown 0.8 Odorants identified based on odor descri ptor and retention indices with reported data due to unavailability of standards

PAGE 142

142 Table 6 5 Results of ANOVA for mean values from difference from control test ANOVA Source of Variation SS df MS F P value F crit Between Treatments 249.57 4 62.39 23.3 3E 14 2.447 Within panelists 321.76 120 2.681 Total 571.33 124 Table 6 6 Difference in means between pasteurized and unpasteurized grapefruit juice Mean values control PEF RF thermal UV Mean values 1.32 3.24 3.36 3.64 4.60 C ontrol 1.32 1.92 2.04 2.32 3.28 PEF 3.24 0.12 0.40 1.36 RF 3.36 0.28 1.24 T hermal 3.64 0.96 UV 4.60 Table 6 7. Results of ANOVA of mean values from difference control test at week 4 Source of Variation SS df MS F P value F crit B etween tre atments 414.38 4 103.59 18.6 1 1.05E 11 2.45 W ithin panelists 612.34 110 5.56 Total 1026.73 114 Table 6 8. Difference in means between pasteurized and unpasteurized grapefruit juice at week 4 control PEF RF thermal UV mean value s 1.96 7.13 6.43 5.08 6.82 Control 1.96 5.17 4.47 3.12 4.86 PEF 7.13 0.70 2.05 0.31 RF 6.43 1.35 0.39 Thermal 5.08 1.74 UV 6.82

PAGE 143

143 CHAPTER 7 CONCLUSION Non thermal processing has been extensively researched over the past few years as a n alternative to heat pasteurization. However, non thermal processes are often compared to thermal processes using conditions that did not achieve the same reduction in concerned microorganism(s) This made a fair comparison of their effects on juice quali ty impossible. Therefore the objective of this study was to compare the quality of apple cider and grapefruit juice treated by thermal and non thermal techniques where treatment conditions were carefully selected to achieve a similar reduction in E. coli k12 population. The non thermal techniques selected for the study were PEF, RFEF and UV. The quality attributes studied were aroma, sensory, microbial, physical, enzymatic and nutritional. In apple cider microbial inactivation by PEF technique was compar able to heat pasteurization UV treated cider had a shelf life of only 2 weeks. Other attributes like aroma volatile s color and sensory quality was better preserved in PEF treated cider compared to thermal ly and UV treated cider Comparing the three techn iques, PEF shows the best promise as a future pasteurization technique for the preservation of apple cider. In grapefruit juice the thermal treatment was more effective when compared to non thermal treatment s in terms of microbial stability after 4 weeks storage. The sensory quality of juice was affected by all treatments. UV treated grapefruit juice differed the most compared to the control juice followed by thermal, RFEF and PEF respectively The impact of treatments on odorants was highlighted by a lo ss of desirable fruity and citrus odorants along with an increase in undesirable cooked or

PAGE 144

144 catty odorants. RFEF treatment ensured grapefruit juice microbial safety for 3 weeks of storage with higher Vitamin C content and equivalent PME inactivation compar ed to thermally treated juice. PEF and UV treatments maintained good microbial quality for only 1 week. Non thermally treated juice had a shorter shelf life but better nutritional and physical qualities compared to thermally treated juice. T he current stud y has shown that the effects of thermal and non thermal treatments on juice quality are not the same at equivalent process conditions The t hermal technique consistently showed good microbial quality during storage compared to non thermal techniques. Howev er, organoleptic and nutritional qualities of juice were better preserved in non thermally treated juice than in thermal ly treated juice The efficacy of non thermal treatment in extending shelf life also depends on the type of food product. Though it is d ifficult for any non thermal technique to completely replace thermal process some of them might find niche applications for certain products that are sensitive to heat. For commercial success, non thermal technologies should show cost effectiveness and p athogen control comparable to thermal treatment. To achieve this goal, combination s of these techniques with traditional or emerging food preservations techniques where a synergistic effect in terms of improving food quality and safety is achieved needs to be explored. This combination of new and traditional techniques is commonly called hurdle technology. Moreover, it is necessary to find the niche products for applications of these technologies by conducting more detailed research on nutritional and org anoleptic properties of the product. In the future consumer demands

PAGE 145

145 for fresh and minimally processed food would definitely help in the acceptance and commercialization of non thermal processing techniques by the food industry.

PAGE 146

146 APPENDIX A CHANGE IN APPL E CIDER APPEARANCE D URING STORAGE Figure A 1. Change in appearance in treated and untreated apple cider during 4 weeks of storage at 4 C Control UV PEF Thermal Week 0 Week 2 Week 4

PAGE 147

147 APPENDIX B CALIBRATION TABLE FOR APPLE CIDER VOLA TILES Table B 1. Regression equation for standard compounds C ompound R egression equation C orrelation B ase ion hexyl hexanoate y = 139805x 26161 0.9769 117 p allyl anisole y = 57923x + 46022 0.9825 148 butyl propionate y = 105603x + 65342 0.9703 57 2 methyl hexyl butyrate y = 70481x 4 0381 0.9604 103 1 octanol y = 10696x 25739 0.9949 56 6 methyl hepten 5 en 2 one y = 759936x + 10268 0.9851 43 pentyl acetate y = 184623x + 56243 0.8015 70 2 hexen 1 ol, acetate y = 54292x + 11218 0.9784 67 2 hexen 1ol (z) y = 83730x + 51963 0.9921 5 7 ethyl propionate y = 316024x 592703 0.8861 57 ethyl butyrate y = 23796x + 144461 0.9619 71 ethyl hexanoate y = 121026x 32819 0.998 88 F arnesene y = 5E+08x + 24019 0.9057 93 butyl 2 methyl butyrate y = 106763x + 15192 0.9679 103 hexyl acetate y = 43260x + 544732 0.9839 56 2methyl butyl acetate y = 29856x + 250422 0.97 70 2methyl butanol y = 1820.2x + 28272 0.9183 57 1 hexanol y = 12069x + 111609 0.9967 56 butyl acetate y = 16391x + 184478 0.9514 56 H exanal y = 12165x + 140960 0.9609 56 2 me thyl ethyl butyrate y = 79357x 147057 0.9332 102 2 e hexenal y = 11392x 226626 0.9395 69 propyl butyrate y = 107921x 408031 0.9754 71 B enzaldehyde y = 51164x + 361796 0.9422 106 butyl butyrate y = 92039x 52362 0.9639 71 hexyl propionate y = 86 064x 117617 0.9467 57 propyl hexanoate y = 125122x 319336 0.8724 99 hexyl butyrate y = 71844x 38727 0.9595 71 methyl butyrate y = 21678x 51380 0.8882 74

PAGE 148

148 APPENDIX C GRAPEFRUIT JUICE VOL ATILE CONCENTRATION

PAGE 149

149 Table C 1.Volatile concentration ( g/L) in untreated (control) and treated grapefruit juice at week0 RT LRI compound identified control PEF RFEF UV Thermal 4.45 888 ethyl acetate 6.490.73 5.850.29 5.61 1.25 5.42 2.03 6.40 2.47 6.50 1037 a pinene 1.360.09 1.350.12 1.30 0.03 1.17 0.2 1 1.34 1.04 6.70 1049 ethyl butyrate 9.470.34 7.601.32 6.32 2.17 6.76 2.74 6.57 2.46 7.56 1098 H exanal 2.980.06 2.930.05 2.09 0.08 2.84 1.02 2.90 2.12 9.06 1174 b myrcene 129.014.73 124.403.68 120.76 4.23 119.46 4.29 119.27 5.79 9.97 1219 L imonen e 2309.5021.47 2203.5835.92 2120.1123.28 2001.39 42.83 2032.12 28.71 10.46 1243 ethyl hexanaote 32.565.03 25.713.18 25.88 3.73 23.09 3.25 26.48 2.08 10.82 1260 3 carene 15.542.83 12.742.25 11.38 2.04 11.33 5.27 13.31 2.56 11.33 1285 p cymene 15.6 30.92 10.052.63 11.05 1.27 11.66 2.99 11.57 2.18 11.61 1299 O ctanal 78.872.84 73.703.78 68.22 2.69 65.34 5.24 74.77 4.43 13.61 1400 N onanal 8.871.47 8.332.16 6.79 1.30 7.56 1.47 6.81 3.23 14.29 1436 ethyl octanoate 1.350.06 1.140.02 0.91 0.03 1. 29 0.93 1.18 0.07 14.51 1447 dimethyl styrene 5.150.46 2.350.27 2.18 1.02 1.66 1.11 2.51 1.16 15.52 1501 D ecanal 16.322.82 15.552.32 15.97 3.26 14.89 2.46 15.78 3.52 15.63 1506 a copaene 6.031.10 5.92 2.17 4.23 2.38 5.05 2.25 5.17 2.76 16.20 1537 L inalool 1.790.22 1.57 0.14 1.81 0.44 1.89 0.29 1.86 0.23 16.44 1549 1 octanol 9.892.91 9.83 2.34 9.69 2.82 10.32 2.47 10.43 3.73 17.44 1602 terpinen 4 ol 2.920.61 3.33 1.80 3.02 1.99 2.97 1.72 3.17 2.01 17.55 1608 b caryophyllene 135.994.83 135.79 5.20 86.61 4.27 133.97 4.88 134.75 6.23 18.77 1670 a caryophyllene 14.942.58 14.62 1.79 15.24 3.23 14.69 4.16 14.57 4.29 19.54 1709 V alencene 3.701.19 3.28 1.03 2.49 1.17 4.03 1.81 3.79 2.08 19.77 1721 a selinene 4.071.38 4.08 1.22 3.11 1.83 4.60 2.3 8 4.88 2.74 20.05 1735 d cadinene 4.632.37 4.46 2.18 3.34 2.08 4.12 1.32 3.52 1.72 20.31 1747 a panasinsen 1.450.03 1.34 0.06 1.32 0.70 1.38 1.28 1.28 0.84 30.56 2677 N ootkatone 6.132.82 3.72 1.38 3.26 2.68 3.19 2.01 3.21 2.22

PAGE 150

150 Table C 2. Volatile c oncentration ( g/L) in untreated (control week 0) and treated grapefruit juice at week 4 RT LRI compound identified C ontrol PEF RFEF UV thermal 4.45 888 ethyl acetate 6.490.73 3.79 1.28 4.55 2.25 4.76 1.29 4.08 0.84 6.50 1037 a pinene 1.360.09 1.15 0.03 0. 83 0.04 1.08 0.88 0.91 0.07 6.70 1049 ethyl butyrate 9.470.34 1 5.12 2.37 4.41 1.48 1.07 0.27 4.38 1.81 7.56 1098 H exanal 2.980.06 Nd Nd Nd Nd 9.06 1174 b myrcene 129.014.73 117.98 5.23 108.47 3.49 102.67 6.38 88.04 3.67 9.97 1219 limonene 2309.5021 .47 1977.48 53.38 1686.27 30.28 1730.11 29.47 1737.39 51.84 10.46 1243 ethyl hexanaote 32.565.03 Nd Nd Nd nd 10.82 1260 3 carene 15.542.83 2.66 2.12 10.71 2.43 6.58 2.71 6.48 2.48 11.33 1285 p cymene 15.630.92 8.65 3.42 9.38 2.83 7.27 4.29 6.91 3.89 11.61 1299 octanal 78.872.84 4.16 2.78 3.52 1.67 3.64 1.93 4.43 2.50 13.61 1400 nonanal 8.871.47 5.25 3.15 2.73 1.38 3.95 2.10 2.54 1.37 14.29 1436 ethyl octanoate 1.350.06 1.09 0.29 0.96 1.31 1.03 0.03 1.05 0.79 14.51 1447 dimethyl styrene 5.150.4 6 1.24 0.28 1.40 0.72 1.23 1.12 1.10 0.09 15.52 1501 decanal 16.322.82 3.13 3.02 7.14 2.89 2.65 1.37 2.01 1.18 15.63 1506 a copaene 6.031.10 4.26 2.44 3.53 1.21 3.23 2.63 2.55 2.45 16.20 1537 linalool 1.790.22 1.53 0.09 1.14 0.70 1.31 0.37 1.73 1.08 16.44 1549 1 octanol 9.892.91 9.67 3.10 10.48 2.56 10.49 2.89 15.20 5.27 17.44 1602 terpinen 4 ol 2.920.61 5.28 2.43 5.83 3.83 5.79 2.71 5.42 2.09 17.55 1608 b caryophyllene 135.994.83 143.00 4.82 134.39 6.88 130.02 12.32 108.58 4.76 18.77 1670 a ca ryophyllene 14.942.58 12.05 3.84 16.57 3.74 14.37 4.20 13.16 2.94 19.54 1709 valencene 3.701.19 1.28 0.89 1.22 1.04 1.40 1.41 1.36 0.65 19.77 1721 a selinene 4.071.38 3.48 2.46 3.98 2.79 3.21 2.63 2.79 2.36 20.05 1735 d cadinene 4.632.37 3.15 1.82 3 .02 3.38 3.15 2.85 3.02 2.89 20.31 1747 a panasinsen 1.450.03 1.14 1.03 7.69 2.45 1.09 0.67 0.99 1.01 30.56 2677 nootkatone 6.132.82 3.18 3.20 2.85 3.52 2.65 3.49 2.70 2.73

PAGE 151

151 Figure C 1. TIC of grapefruit vola tiles extracted by HS SPME A. untreated juice (week 0) B. UV treated juice (week 4). Separation on DB Wax column. Highest change in peak nos. 4 = hexanal, 7 = ethyl hexanoate, 10 = octanal, 14 = decanal. A B

PAGE 152

152 APPENDIX D GRAPEFRUIT JUICE SEN SORY RESULTS Table D 1. Difference from control data between hidden control (unpasteurized juice) and pasteurized juices (thermal, PEF, RFEF and UV) at week 0 Panelist thermal PEF RF UV control 1 9 2 2 6 1 2 5 4 2 7 2 3 6 3 3 8 3 4 4 2 2 6 0 5 5 4 4 8 2 6 2 4 2 4 0 7 6 2 2 9 0 8 4 3 5 7 3 9 3 3 2 5 1 10 4 2 5 5 0 11 6 2 3 6 0 12 3 2 4 3 1 13 5 4 6 9 2 14 3 3 4 8 0 15 2 2 3 3 2 16 3 4 2 5 2 17 4 2 3 5 1 18 6 2 3 3 0 19 4 3 3 7 3 20 4 3 4 9 2 21 2 4 4 4 2 22 5 6 6 6 2 23 3 3 3 7 1 24 4 4 2 4 2 25 5 6 3 2 1 Sum 107 79 82 146 33 Average 3.64 3.24 3.36 4.6 1.32 Variance 4.407 4.523 2.323 8.083 1.060

PAGE 153

153 Table D 2. Difference from control data between hidden control (unpasteurized fresh uice ) and pasteurized juices (thermal, PEF, RFEF and UV) at week 4 Panelist PEF RF thermal UV control 1 7 5 0 8 1 2 4 7 7 5 1 3 8 3 4 7 1 4 10 10 10 10 4 5 10 9 2 9 0 6 8 10 5 5 1 7 8 7 6 8 2 8 8 9 7 4 4 9 9 6 9 9 3 10 9 9 7 9 1 11 10 10 8 10 6 12 6 4 1 5 1 13 4 6 6 4 3 14 10 10 9 5 0 15 6 3 3 9 2 16 7 1 8 8 3 17 7 7 6 4 3 18 4 4 2 4 2 19 3 6 5 3 3 20 9 4 4 8 2 21 5 7 5 8 2 22 8 5 3 5 0 23 4 6 0 10 0 24 8 6 5 6 3 25 6 7 5 8 1 sum 178.00 161.00 127.00 171.00 49.00 average 7.12 6.44 5.08 6.84 1.96 variance 5.02 6.71 8.35 5.43 2.31

PAGE 154

154 APPENDIX E SA MPLE SENSORY BALLOT FOR DIFFERENCE FROM CONTROL TEST Difference from control test Name ____________ Date: ____________ Type of sample ___________ Test# ___________ INSTRUCTIONS Please DO NOT taste the samples. Evaluate the sam the vial and sniff headspace of the vial and remember the odor of the sample. Wait 10 seconds and then sniff the sample marked with three digit code. Assess the overall sensory difference between the two samples using scal e below. Mark the scale to indicate the size of the overall difference. Scale Mark to indicate difference 0 No Difference ___________ 1 ___________ 2 ___________ 3 ___________ 4 ___________ 5 ___________ 6 __________ 7 ___________ 8 ___________ 9 ___________ 10 Extremely different ___________ Comments: please give verbal descriptors for the odor ______________________ ___________

PAGE 155

155 LIST OF REFERENCES Aaby K, Haffner K & Skrede G. 2002. Aroma quality of Gravenstein apples influenced by regular and controlled atmosphere storage. Lebens Wiss Technol 35(3):254 259. Adler E, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJP & Zuker CS. 2000. A novel family of mammalian taste receptors. Cell 100(6):693 702. A FDO. 2001. AFDO requirements and recommendations for apple cider processing operations. J. Assoc. Food Drug Office 65(1):40 58. Aguillar Rosas SF, Ballinas Casarrubias ML, Nevarez Moorillon GV, Martin Belloso O & Ortega Rivas E. 2007. Thermal and pulsed e lectric fields pasteurization of apple juice: Effects on physicochemical properties and flavour compounds. J. Food Eng. 83(1):41 46. Allegrone G, Belliardo F & Cabella P. 2006. Comparison of volatile concentrations in hand squeezed juices of four differen t lemon varieties. J. Agric. Food Chem. 54(5):1844 1848. AMS. 1983. http://www.ams.usda.gov/standards Accessed 16th Nov. 2008 Ananthakumar A, Variyar PS & Sharma A. 2006. Estimation of aroma glycosides of nutmeg and their changes during radiation processing. J. Chromatogr. 1108(2):252 257. Araneda RC, Kini AD & Firestein S. 2000. The molecular receptive range of an odorant receptor. Nat. Neurosci. 3(12):1248 1255. Ayhan Z, Yeom HW, Zhang QH & Min DB. 2 001. Flavor, color, and vitamin C retention of pulsed electric field processed orange juice in different packaging materials. J. Agric. Food Chem. 49(2):669 674. Ayhan Z, Zhang QH & Min DB. 2002. Effects of pulsed electric field processing and storage on the quality and stability of single strength orange juice. J. Food Prot. 65(10):1623 1627. Bazemore R, Goodner K & Rouseff R. 1999. Volatiles from unpasteurized and excessively heated orange juice analyzed with solid phase microextraction and GC olfactome try. J. Food Sci. 64(5):800 803. Berry RE, Wagner CJ & Moshonas MG. 1967. Flavor studies of nootkatone in grapefruit juice J. Food Sci. 32(1):75 &. Besser RE, Lett SM, Weber JT, Doyle MP, Barrett TJ, Wells JG & Griffin PM. 1993a. An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157 H7 in fresh pressed apple cider. J. Am. Med. Assoc. 269(17):2217 2220.

PAGE 156

156 Bezman Y, Rouseff RL & Naim M. 2001. 2 methyl 3 furanthiol and methional are possible off flavors in stored orange juice: Aroma similarity, NIF/SNIF GC O, and GC analyses. J. Agric. Food Chem. 49(11):5425 5432. Biasioli F, Gasperi F, Aprea E, Colato L, Boscaini E & Mark TD. 2003. Fingerprinting mass spectrometry by PTR MS: heat treatment vs. pressure treatment of red orange juice a case study. Int J Mass Spect 223(1 3):343 353. Boff JM, Truong TT, Min DB & Shellhammer TH. 2003. Effect of thermal processing and carbon dioxide assisted high pressure processing on pectin methylesterase and chemical changes in orange juice. J. Food Sci. 68(4):1179 1184. Braddock RJ. 1999. Single strength orange juices and concentrate. New York: Wiley Interscience, p 53 83 Buck L & Axel R. 1991. A novel multigene family may encode odorant receptors a molecular basis for odor recognition. Cell 65( 1):175 187. Buettner A & Schieberle P. 1999. Characterization of the most odor active volatiles in fresh, hand squeezed juice of grapefruit (Citrus paradisi Macfayden). J. Agric. Food Chem. 47(12):5189 5193. Buettner A & Schieberle P. 2001. Evaluation of key aroma compounds in hand squeezed grapefruit juice (Citrus paradisi Macfayden) by quantitation and flavor reconstitution experiments. J. Agric. Food Chem. 49(3):1358 1363. Bult JHF, Schifferstein HNJ, Roozen JP, Boronat ED, Voragen AGJ & Kroeze JHA. 2 002. Sensory evaluation of character impact components in an apple model mixture. Chem. Senses 27(6):485 494. Buttery RG, Turnbaugh JG & Ling LC. 1988. Contributions of volatiles to rice aroma. J. Agric. Food Chem. 36(5):1006 1009. Cadwallader KR & Xu Y. 1994. Analysis of volatile components in fresh grapefruit juice by purge and trap gas chromatography. J. Agric. Food Chem. 42(3):782 784. Casas E, Valderrama MJ & Peinado JM. 1999. Sorbate detoxification by spoilage yeasts isolated from marzipan products Food Technol. Biotechnol. 37(2):87 91. Castro AJ SB, Barbosa Canovas, Zhang QH. 2001. Pulsed electric field modification of milk alkaline phosphatase activity. Lancaster, PA: Technomic Publishing Company, Inc. CFSAN. 2004. Juice HACCP Hazards and Contr ols Guidance. p. http://www.cfsan.fda.gov/~dms/juicgu10.html Accessed 17th Nov. 2008

PAGE 157

157 Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng LX, Guo W, Zuker CS & Ryba NJP. 2000. T2Rs function as b itter taste receptors. Cell 100(6):703 711. Chang JCH, Ossoff SF, Lobe DC, Dorfman MH, Dumais CM, Qualls RG & Johnson JD. 1985. UV inactivation of pathogenic and indicator microorganisms. Appl. Environ. Microbiol. 49(6):1361 1365. Charles Rodriguez AV, N evarez Moorillon GV, Zhang QH & Ortega Rivas E. 2007. Comparison of thermal processing and pulsed electric fields treatment in pasteurization of apple juice. Food Bioprod. Process. 85(C2):93 97. Choi LH & Nielsen SS. 2005. The effects of thermal and nonth ermal processing methods on apple cider quality and consumer acceptability. J. Food Qual. 28(1):13 29. Clegg KM. 1964. Non enzymatic browning of lemon juice. J. Sci. Food Agric. 15(12):878 &. Coleman RL, Shaw PE & Lund ED. 1972. Analysis of grapefruit es sence and aroma oils J. Agric. Food Chem. 20(1):100. Cortes C, Esteve MJ & Frigola A. 2008. Color of orange juice treated by High Intensity Pulsed Electric Fields during refrigerated storage and comparison with pasteurized juice. Food Control 19(2):151 15 8. Crupi F & Rispoli G. 2002. Citrus Juice Technology. NY, USA: Taylor & Francis. Cserhalmi Z, Sass Kiss A, Toth Markus M & Lechner N. 2006. Study of pulsed electric field treated citrus juices. Innov. Food Sci. Emerg. Technol. 7(1 2):49 54. Cunningham DG, Acree TE, Barnard J, Butts RM & Braell PA. 1986. Charm analysis of apple volatiles. Food Chem. 19(2):137 147. da Silva MAAP & Lundahl DS. 1994. The capabilities and psychophysics of Osme: a new GC Olfactometry Technique. Elsevier: New York.p 191 209 David JRD, Graves RH & Carlson VR. 1996. Aseptic processing and packaging of food: a food industry perspective. Boca Raton, Florida: CRC Press LLC.p 14 Deak T & Beuchat LR. 1993. Yeasts associated with fruit juice concentrates. J. Food Prot. 56(9):777 782 Demole E & Enggist P. 1983. Further investigation of grapefruit juice flavor components (citrus paradisi macfayden) valencane type and eudesmane type sesquiterpene ketones. Helv. Chim. Acta 66(5):1381 1391.

PAGE 158

158 Demole E, Enggist P & Ohloff G. 1982. 1 Par a menthene 8 thiol a powerful flavor impact constituent of grapefruit juice (citrus paradisi macfayden). Helv. Chim. Acta 65(6):1785 1794. Dever MC, Cliff M & Lau OL. 1992. Maturity and ripening effects on chemical and sensory properties of apple juice. J. Sci. Food Agric. 60(3):355 360. Dimick PS & Hoskin JC. 1983. Review of apple flavor state of the art. Crit. Rev. Food Sci. 18(4):387 409. Dixon J & Hewett EW. 2000. Factors affecting apple aroma/flavour volatile concentration: a review. New. Zeal. J. Crop Hort. Sci. 28(3):155 173. Donahue DW, Canitez N & Bushway AA. 2004. Uv inactivation of E coli O157 : H7 in apple cider: Quality, sensory and shelf life analysis. J. Food Process. Pres.. 28(5):368 387. Dreher JG, Rouseff RL & Naim M. 2003. GC olf actometric characterization of aroma volatiles from the thermal degradation of thiamin in model orange juice. J. Agric. Food Chem. 51(10):3097 3102. Duffy S, Churey J, Worobo RW & Schaffner DW. 2000. Analysis and modeling of the variability associated wit h UV inactivation of Escherichia coli in apple cider. J. Food Prot. 63(11):1587 1590. Durr P & Schobinger U. 1981. The contribution of some volatiles to the sensory quality of apple and orange juice odour. In: walter de Gruyter & Co, B. N. Y., editor). Fl avour '81 Berlin: Walter de Grutyer & Co. p 179 86 Elez Martinez P, Soliva Fortuny RC & Martin Belloso O. 2006. Comparative study on shelf life of orange juice processed by high intensity pulsed electric fields or heat treatment. Eur. Food Res. Technol. 2 22(3 4):321 329. Evrendilek GA, Jin ZT, Ruhlman KT, Qiu X, Zhang QH & Ritcher ER. 2000a. Microbial safety and shelf life of apple juice and cider processed by bench and pilot scale PEF systems. Innov. Food Sci. Emerg. Technol. 1(1):77 86. Evrendilek GA, Jin ZT, Ruhlman KT, Qiu X, Zhang QH & Ritcher ER. 2000b. Microbial safety and shelf life of apple juice and cider processed by bench and pilot scale PEF systems.Innov. Food Sci. Emerg. 1: 77 86 Evrendilek GA, Dantzer WR, Streaker CB, Ratanatriwong P & Zha ng QH. 2001. Shelf life evaluations of liquid foods treated by pilot plant pulsed electric field system. J Food Process Pres 25(4):283 297.

PAGE 159

159 Fan XT & Gates RA. 2001. Degradation of monoterpenes in orange juice by gamma radiation. J. Agric. Food Chem. 49(5) :2422 2426. Fan XT & Geveke DJ. 2007. Furan formation in sugar solution and apple cider upon ultraviolet treatment. J. Agric. Food Chem. 55(19):7816 7821. Fan XT & Thayer DW. 2002. Nutritional quality of irradiated orange juice. J. Food Process. Pres. 26 (3):195 211. FDA. 2001. FDA Publishes Final Rule to Increase Safety of Fruit and Vegetable Juices. http://www.cfsan.fda.gov/~lrd/hhsjuic4.html FDA/CFSAN Web site. Accessed 4th October, 2009. Fl ath RA, Black DR, Guadagni DG, McFadden WH & Schultz TH. 1967a. Identification and organoleptic evaluation of compounds in delicious apple essence J. Agric. Food Chem. 15(1):29 &. Fuhrmann E & Grosch W. 2002. Character impact odorants of the apple cultiva rs Elstar and Cox Orange. Nahrung 46(3):187 193. Garcia D, Hassani M, Manas P, Condon S & Pagan R. 2005. Inactivation of Escherichia coli O157 : H7 during the storage under refrigeration of apple juice treated by pulsed electric fields. J. Food Saf. 25(1) :30 42. Garruti DS, Franco MRB, da Silva M, Janzantti NS & Alves GL. 2003. Evaluation of volatile flavour compounds from cashew apple (Anacardium occidentale L) juice by the Osme gas chromatography/olfactometry technique. J. Sci. Food Agric. 83(14):1455 1 462. Genovese DB, Elustondo MP & Lozano JE. 1997. Color and cloud stabilization in cloudy apple juice by steam heating during crushing. J. Food Sci. 62(6):1171 1175. Geveke DJ. 2005. UV inactivation of bacteria in apple cider. J. Food Prot. 68(8):1739 17 42. Geveke DJ. 2008. UV Inactivation of E coli in Liquid Egg White. Food Bioproc. Technol. 1(2):201 206. Geveke DJ & Brunkhorst C. 2004. Inactivation of Escherichia coli in apple juice by radio frequency electric fields. J. Food Sci. 69(3):E134 E138. Ge veke DJ & Brunkhorst C. 2008. Radio frequency electric fields inactivation of Escherichia coli in apple cider. J. Food Eng. 85(2):215 221.

PAGE 160

160 Geveke DJ, Brunkhorst C & Fan XT. 2007. Radio frequency electric fields processing of orange juice. Inn. Food Sci. E merg.Technol. 8(4):549 554. Girard B & Lau OL. 1995. Effect of maturity and storage on quality and volatile production of 'Jonagold' apples. Food Res. Int. 28(5):465 471. Girard B & Tv. 1996. Retention index calculation using Kovats constant model for li near temperature programmed gas chromatography. J. Chromatogr. 721(2):279 288. Gocmen D EA, Williams T, Parish M, Rouseff RL. 2005. Identification of medicinal off flavours generated by Alicyclobacillus species in orange juice using GC olfactometry and GC MS. Lett. Appl. Microbiol. 40(3):172 177. Grahl T & Markl H. 1996. Killing of microorganisms by pulsed electric fields. Appl. Microbiol. Biotechnol. 45(1 2):148 157. Guerrero Beltran JA, Welti Chanes J & Barbosa Canovas GV. 2009. Ultraviolet c light pro cessing of grape, cranberry and grapefruit juices to inactivate saccharomyces cerevisiae. J. Food Process Eng. 32(6):916 932. Guiavarc'h Y, Segovia O, Hendrickx M & Van Loey A. 2005. Purification, characterization, thermal and high pressure inactivation o f a pectin methylesterase from white grapefruit (Citrus paradisi). Innov Food Sci Emerg Technol 6(4):363 371. Hatt H. 2000. Follow your nose: Mechanisms of signal transduction. Zool. Anal. Complex Syst. 102(2 3):120 126. Heinz V, Toepfl S & Knorr D. 2003 Impact of temperature on lethality and energy efficiency of apple juice pasteurization by pulsed electric fields treatment. Inn.Food Sci.Emerg. Technol. 4(2):167 175. Hoyer O. 1988. Testing performance and monitoring of UV systems for drinking water dis infection. Water Supply Pap. U.S. Geol. Surv. 16(1 2):424 429. Hulsheger H, Potel J & Niemann EG. 1983. Electric field effects on bacteria and yeast cells. Radiat. Environ. Biophys. 22(2):149 162. Jella P & Rouseff R. 1997. Comparison of extraction metho ds of volatile aroma compounds in processed grapefruit juice. Abstracts of Papers of the American Chemical Society 214:78 AGFD. Jella P, Rouseff R, Goodner K & Widmer W. 1998. Determination of key flavor components in methylene chloride extracts from proc essed grapefruit juice. J Agric. Food Chem. 46 (1)p. 242 247.

PAGE 161

161 Jeyamkondan S, Jayas DS & Holley RA. 1999. Pulsed electric field processing of foods: A review. J. Food Prot. 62(9):1088 1096. Jia MY, Zhang QH & Min DB. 1999. Pulsed electric field processing effects on flavor compounds and microorganisms of orange juice. Food Chem. 65(4):445 451. Jin ZT & Zhang QH. 1999. Pulsed electric field inactivation of microorganisms and preservation of quality of cranberry juice. J. Food Process. Pres. 23(6):481 497. Kale PN & Adsule PG. 1995. Citrus. Handbook of fruit science and technology:production, composition, storage and processing New York: Marcel Dekker, Inc. p. 56. Kataoka H, Lord HL & Pawliszyn J. 2000. Applications of solid phase microextraction in food analysis. J. Chromatogr. 880(1 2):35 62. Kato T, Shimoda M, Suzuki J, Kawaraya A, Igura N & Hayakawa I. 2003. Changes in the odors of squeezed apple juice during thermal processing. Food Res. Int. 36(8):777 785. Keyser M, Muller IA, Cilliers FP, Nel W & Gouws PA. 2008. Ultraviolet radiation as a non thermal treatment for the inactivation of microorganisms in fruit juice. Innov Food Sci Emerg Technol 9(3):348 354. Komthong P, Katoh T, Igura N & Shimoda M. 2006. Changes in the odours of apple Juice during enzymatic browning. Food Qual. Pref. 17(6):497 504. Koutchma T. 2008. UV light for processing foods. Ozone Sci Eng 30(1):93 98. Koutchma T. 2009. Advances in Ultraviolet Light Technology for Non thermal Processing of Liquid Foods. Food Bioprocess Technol 2(2):138 -155. Koutchma T, Paris B & Patazca E. 2007. Validation of UV coiled tube reactor for fresh juices. J. Environ. Eng. Sci. 6(3):319 328. Kozempel M, McAloon A & Yee W. 1998. The cost of pasteurizing apple cider. Food Technol. 52(1):50 52. Krau twurst D, Yau KW & Reed RR. 1998. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95(7):917 926. Kusmiere K K & Bald E. 2008. Reduced and total glutathione and cysteine profiles of citrus fruit juices using liquid chromatography. Food Chem. 106(1):340 344. Laing DG & Jinks A. 1996. Flavour perception mechanisms. Trends Food Sci. Technol. 7(12):387 389.

PAGE 162

162 Lancet D, Benarie N, Cohen S, Gat U, Grossisseroff R, Hornsaban S, Khen M, Lehrach H, Natochin M, No rth M, Seidemann E & Walker N. 1993. Olfactory receptors transduction, diversity, human psychophysics and genome analysis CIBA Found. Symp. 179:131 146. Lee HS & Coates GA. 1999. Thermal pasteurization effects on color of red grapefruit juices. J. Food Sci. 64(4):663 666. Lee HS & Nagy S. 1988. Quality changes and nonenzymatic browning intermediates in grapefruit juice during storage. J. Food Sci. 53(1):168 &. Liang ZW, Cheng Z & Mittal GS. 2006. Inactivation of spoilage microorganisms in apple cider u sing a continuous flow pulsed electric field system. Food Sci Technol 39(4):351 357. Liang ZW, Mittal GS & Griffiths MW. 2002. Inactivation of Salmonella typhimurium in orange juice containing antimicrobial agents by pulsed electric field. J. Food Prot. 6 5(7):1081 1087. Lin J, Jella P & Rouseff RL. 2002a. Gas chromatography olfactometry and chemiluminescence characterization of grapefruit juice volatile sulfur compounds. Heteroatomic Aroma Compounds ACS Sym. Sr.p. 110 120. Lin JM & Rouseff RL. 2001a. Ch aracterization of aroma impact compounds in cold pressed grapefruit oil using time intensity GC olfactometry and GC MS. Flavour Frag. J. 16(6):457 463. Lin JM, Rouseff RL, Barros S & Haim M. 2002b. Aroma composition changes in early season grapefruit juic e produced from thermal concentration. J. Agric. Food Chem. 50(4):813 819. Lin JM, Rouseff RL & Barros SM. 2001b. Aroma impact and volatile component changes in early season grapefruit juice produced from thermal concentration. Abs. Papers Am. Chem. Soc. 222:U31 U31. Lindemann B. 1996. Taste reception. Physiol Rev 76(3):718 766. Linforth R, Martin F, Carey M, Davidson J & AJ T. 2002. Retronasal transport of aroma compounds. J. Agric. Food Chem. 50(5):1111 1117. Lu YR & Foo Y. 1998. Constitution of some chemical components of apple seed. Food Chem. 61(1 2):29 33. Macleod WD & Buigues NM. 1964. Sesquiterpenes .1. nootkatone new grapefruit flavor constituent J. Food Sci. 29(5):565 &.

PAGE 163

163 Mahattanatawee K, Rouseff R, Valim MF & Naim M. 2005. Identification an d aroma impact of norisoprenoids in orange juice. J. Agric. Food Chem. 53(2):393 397. Malnic B, Hirono J, Sato T & Buck LB. 1999. Combinatorial receptor codes for odors. Cell 96(5):713 723. Marcotte M, Stewart B & Fustier P. 1998. Abused thermal treatment impact on degradation products of chilled pasteurized orange juice. J. Agric. Food Chem. 46(5):1991 1996. Matich AJ, Rowan DD & Banks NH. 1996. Solid phase microextraction for quantitative headspace sampling of apple volatiles. Anal Chem 68(23):4114 4118 McDonald CJ, Lloyd SW, Vitale MA, Petersson K & Innings F. 2000. Effects of pulsed electric fields on microorganisms in orange juice using electric field strengths of 30 and 50 kV/cm. J. Food Sci. 65(6):984 989. McFeeters RF FHaTR. 1983. Effect of heat ing and acid storage on pectinesterase activity in cucumber slices. Abs. Papers Am. Chem. Soc. 186:131. McLellan MR & Padilla Zakour OI. 2004. Juice Processing. Boca Raton, Fl: CRC Press LLC. p 77 Mehinagic E, Royer G, Symoneaux R, Jourjon F & Prost C. 2 006. Characterization of odor active volatiles in apples: Influence of cultivars and maturity stage. J. Agric. Food Chem. 54(7):2678 2687. Meilgaard M, Civille GV & Carr BT. 1999. Sensory Evaluation Techniques 2 ed. Boca Raton, Fl: CRC Press. Meyerhof W, Batram C, Kuhn C, Brockhoff A, Chudoba E, Bufe B, Appendino G & Behrens M. 2010. The Molecular Receptive Ranges of Human TAS2R Bitter Taste Receptors. Chem. Senses 35(2):157 170. Min S, Jin ZT, Min SK, Yeom H & Zhang QH. 2003a. Commercial scale pulsed el ectric field processing of orange juice. J. Food Sci. 68(4):1265 1271. Min S, Jin ZT & Zhang QH. 2003b. Commercial scale pulsed electric field processing of tomato juice. J. Agric. Food Chem. 51(11):3338 3344. Min S & Zhang QH. 2003. Effects of commercia l scale pulsed electric field processing on flavor and color of tomato juice. J. Food Sci. 68(5):1600 1606. Moshonas MG. 1971. Analysis of carbonyl flavor constituents from grapefruit oil. J. Agric. Food Chem. 19(4):769 &.

PAGE 164

164 Moshonas MG & Shaw PE. 2000. Ch anges in volatile flavor constituents in pasteurized orange juice during storage. J. Food Qual. 23(1):61 71. Mueller KL, Hoon MA, Erlenbach I, Chandrashekar J, Zuker CS & Ryba NJP. 2005. The receptors and coding logic for bitter taste. Nature 434(7030):22 5 229. Murakami EG, Jackson L, Madsen K & Schickedanz B. 2006. Factors affecting the ultraviolet inactivation of Escherichia coli K12 in apple juice and a model system. J. Food Process Eng. 29(1):53 71. Nagy S, Lee H, Rouseff RL & Lin JCC. 1990. Nonenzym ic browning of commercially canned and bottled grapefruit juice. J. Agric. Food Chem. 38(2):343 346. Nelson G, Chandrashekar J, Hoon MA, Feng LX, Zhao G, Ryba NJP & Zuker CS. 2002. An amino acid taste receptor. Nature 416(6877):199 202. Nelson G, Hoon MA Chandrashekar J, Zhang YF, Ryba NJP & Zuker CS. 2001. Mammalian sweet taste receptors. Cell 106(3):381 390. Nijssen LM, Visscher CA, Maarse H & Willemsen L. 1996. Volatile Compounds in Foods, Qualitative and Quantitative Data 7th ed.: Zeist, The Netherl ands, TNO Nutrition and Food Research. Nunez AJ, Bemelmans JMH & Maarse H. 1984. Isolation methods for the volatile components of grapefruit juice distillation and solvent extraction methods. Chromatographia 18(3):153 158. Nunez AJ, Maarse H & Bemelma ns JMH. 1985. Volatile flavor components of grapefruit juice ( citrus paradisi macfadyen ). J. Sci. Food Agric. 36(8):757 763. Paillard NMM & Rouri O. 1984. Hexanal and 2 hexenal productions by mashed apple tissues. Lebensm. Wiss. Technol. 17(6):345 350. P arish ME. 1997. Public health and nonpasteurized fruit juices. Crit. Rev. Microbiol. 23(2):109 119. Peleg M. 1995. A model of microbial survival after exposure to pulsed electric fields. J. Sci. Food Agric. 67(1):93 99. Perez Cacho PR & Rouseff R. 2008. Processing and Storage Effects on Orange Juice Aroma: A Review. J. Agric. Food Chem. 56(21):9785 9796. Petersen MA, Tonder D & Poll L. 1998. Comparison of normal and accelerated storage of commercial orange juice Changes in flavour and content of volat ile compounds. Food Qual. Pref. 9(1 2):43 51.

PAGE 165

165 Pino J, Torricella R & Orsi F. 1986. Correlation between sensory and gas chromatographic measurements on grapefruit juice volatiles. Acta Aliment. 15(3):237 246. Plotto A, McDaniel MR & Mattheis JP. 1999. Cha racterization of 'Gala' apple aroma and flavor: Differences between controlled atmosphere and air storage. J. Am. Soc. Hort. Sci. 124(4):416 423. Plotto A, McDaniel MR & Mattheis JP. 2000. Characterization of changes in 'Gala' apple aroma during storage u sing osme analysis, a gas chromatography olfactometry technique. J. Am. Soc. Hort. Sci. 125(6):714 722. Pothakamury UR, Vega H, Zhang QH, BarbosaCanovas GV & Swanson BG. 1996. Effect of growth stage and processing temperature on the inactivation of E coli by pulsed electric fields. J. Food Prot. 59(11):1167 1171. Prescott J, Allen S & Stephens L. 1993. Interactions between oral chemical irritation, taste and temperature. Chem. Senses 18(4):389 404. Prescott J & Stevenson RJ. 1995. Pungency in food percep tion and preference. Food Rev. Int. 11(4):665 698. Qin BL, Barbosa Canovas GV, Swanson BG, Pedrow PD & Olsen RG. 1998. Inactivating microorganisms using a pulsed electric field continuous treatment system. IEEE T Ind. Appl. 34(1) p. 43 50. Qin BL, Chang FJ, BarbosaCanovas GV & Swanson BG. 1995. Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Food Sci.Technol. 28(6):564 568. Qin BL, Pothakamury UR, BarbosaCanovas GV & Swanson BG. 1996. Nonthermal pasteuriza tion of liquid foods using high intensity pulsed electric fields. Crit. Rev. Food Sci. Nutr. 36(6):603 627. Qiu X, Sharma S, Tuhela L, Jia M & Zhang QH. 1998. An integrated PEF pilot plant for continuous nonthermal pasteurization of fresh orange juice. Tr ans. ASAE 41(4):1069 1074. Raso J, Palop A, Pagan R & Condon S. 1998. Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. J. Appl. Microbiol. 85(5):849 854. Rivas A, Rodrigo D, Martinez A, Barbos a Canovas GV & Rodrigo M. 2006. Effect of PEF and heat pasteurization on the physical chemical characteristics of blended orange and carrot juice. Lwt Food Sci. Technol. 39(10):1163 1170.

PAGE 166

166 Rouseff R, Bazemore R, Goodner K & Naim M. 2001. GC olfactometry wi th solid phase microextraction of aroma volatiles from heated and unheated orange juice. Headspace Analysis of Foods and Flavors p. 101 112. Rouseff R, Lin J & Valim F. 2003. Combined GC O/GC PFPD detection of sulfur aroma impact compounds in citrus juic e. Flavour research at dawn of twenty first century; proceeding of the 10th Weurman flavour research symposium:476 481. Sadler GD, parish ME & Wicker L. 1992. Microbial, enzymatic and chemical changes during storage of fresh and processed orange juice. J Food Sci. 57 (5): 1187 91. Saguy I, Kopelmani J & Mizrahi S. 1978. Simulation of ascorbic acid stability during heat processing and concentration of grapefruit juice J. Food Process Eng. 2(3):213 225. Sale AJH & Hamilton WA. 1967. Effects of high ele ctric fields on microorganisms .I. Killing of bacteria and yeasts. Biochim. Et Biophys. Acta 148(3):781 &. Sampedro F, Geveke DJ, Fan X, Rodrigo D & Zhang QH. 2009. Shelf Life Study of an Orange Juice Milk Based Beverage after PEF and Thermal Processing. J. Food Sci. 74(2):S107 S112. Sanchez Vega R, Mujica Paz H, Marquez Melendez R, Ngadi MO & Ortega Rivas E. 2009. Enzyme inactivation on apple juice treated by ultrapasteurization and pulsed electric fields technology. J. Food Process. Preserv. 33(4):486 4 99. Schamp N, De Pooter H & Dirinck P. 1988. Aroma development in ripening fruits. In: Flavor chemistry: Trends and developments. Abs. Pap. Chem. Cong. North Amer. 3(1):AGFD 6: 23 34 Schreier P, Drawert F, Steiger G & Mick W. 1978. Effect of enzyme trea tment of apple pulp with a commercial pectinase and cellulase on volatiles of juice. J. Food Sci. 43(6):1797 1800. Sentandreu E, Carobonell L, Rodrigo D & Carbonell JV. 2006. Pulsed electric field versus thermal treatment: Equivalent processes to obtain e qually acceptable citrus juices. J. Food. Prot. 60(8): 2106 8. Shaw PE, Ammons JM & Braman RS. 1980. Volatile sulfur compounds in fresh orange and grapefruit juices identification, quantitation, and possible importance to juice flavor J. Agric. Food Che m. 28(4):778 781. Shaw PE, Moshonas MG, Hearn CJ & Goodner KL. 2000. Volatile constituents in fresh and processed juices from grapefruit and new grapefruit hybrids. J. Agric. Food Chem. 48(6):2425 2429.

PAGE 167

167 Shaw PE & Wilson CW. 1981. Importance of nootkatone to the aroma of grapefruit oil and the flavor of grapefruit juice. J. Agric. Food Chem. 29(3):677 679. Shaw PE & Wilson CW. 1982. Volatile sulfides in headspace gases of fresh and processed citrus juices. J. Agric. Food Chem. 30(4):685 688. Shewfelt RL. 1986. Flavor and color of fruits as affected by processing. In: Luh, J. G. W. a. B. S., editor). Commercial Fruit Processing Westport, CT: Avi Publishing Co. p. 481 529. Sommer R, Haider T, Cabaj A, Pribil W & Lhotsky M. 1998. Time dose reciprocity in U V disinfection of water. Water Sci Technol 38(12):145 150. Stevens SS. 1964. The psychophysics of sensory function. Sensory Comm.:1 33. Streif J & Bangerth F. 1988. Production of volatile aroma substances by golden delicious apple fruits after storage fo r various times in different co2 and o 2 concentrations. J. Hortic. Sci. 63(2):193 199. Su MS & Chien PJ. 2010. Aroma impact components of rabbiteye blueberry (Vaccinium ashei) vinegars. Food Chem. 119(3):923 928. Su SK & Wiley RC. 1998. Changes in apple juice flavor compounds poring processing. J. Food Sci. 63(4):688 691. Sumitani H, Suekane S, Nakatani A & Tatsuka K. 1994. Changes in composition of volatile compounds in high pressure treated peach. J. Agric. Food Chem. 42(3):785 790. Takeoka GR, Flath RA, Mon TR, Teranishi R & Guentert M. 1990. Volatile constituents of apricot (prunus armeniaca). J. Agric. Food Chem. 38(2):471 477. Tandon K, Worobo RW, Churey JJ & Padilla Zakour OI. 2003. Storage quality of pasteurized and UV treated apple cider. J. F ood Process. Preserv. 27(1):21 35. Tatum JH, Nagy S & Berry RE. 1975. Degradation products formed in canned single strength orange juice during storage. J. Food Sci. 40(4):707 709. Tominaga T, des Gachons CP & Dubourdieu D. 1998. A new type of flavor pre cursors in Vitis vinifera L cv Sauvignon blanc: S cysteine conjugates. J. Agric. Food Chem. 46(12):5215 5219. Tran MTT & Farid M. 2004. Ultraviolet treatment of orange juice. Innov.Food Sci. Emerg. Technol. 5(4):495 502.

PAGE 168

168 Ukuku DO, Geveke DJ, Cooke P & Z hang HQ. 2008. Membrane damage and viability loss of Escherichia coli K 12 in apple juice treated with radio frequency electric field. J. Food Prot. 71(4):684 690. Uljas HE, Schaffner DW, Duffy S, Zhao LH & Ingham SC. 2001. Modeling of combined processing steps for reducing Escherichia coli O157 : H7 populations in apple cider. Appl. Environ. Microbiol. 67(1):133 141. Valappil ZA, Fan XT, Zhang HQ & Rouseff RL. 2009. Impact of Thermal and Nonthermal Processing Technologies on Unfermented Apple Cider Aroma Volatiles. J. Agric. Food Chem. 57(3):924 929. VegaMercado H, Pothakamury UR, Chang FJ, BarbosaCanovas GV & Swanson BG. 1996. Inactivation of Escherichia coli by combining pH, ionic strength and pulsed electric fields hurdles. Food Res. Int. 29(2):117 12 1. Vikram VB, Ramesh MN & Prapulla SG. 2005. Thermal degradation kinetics of nutrients in orange juice heated by electromagnetic and conventional methods. J. Food Eng. 69(1):31 40. Wicker L. 2004. Frozen citrus juices. in Hui YH, editor. Handbook of Froz en Foods. New York, Marcel Dekker Inc; p483 498. Wolfgan A.1990. Enzymes in industry: production and application. Germany Wiley Weiheim VCH. P 161 165. Wouters PC, Alvarez I & Raso J. 2001. Critical factors determining inactivation kinetics by pulsed ele ctric field food processing. Trends Food Sci. Technol.12(3 4):112 121. Wyatt MK & Parish ME. 1995. Spore germination of citrus juice related fungi at low temperatures. Food Microbiol. 12(3):237 243. Yeom HW, Evrendilek GA, Jin ZT & Zhang QH. 2004. Proces sing of yogurt based products with pulsed electric fields: Microbial, sensory and physical evaluations. J. Food Process. Preserv. 28(3):161 178. Yeom HW, Streaker CB, Zhang QH & Min DB. 2000a. Effects of pulsed electric fields on the activities of microor ganisms and pectin methyl esterase in orange juice. J. Food Sci. 65(8):1359 1363. Yeom HW, Streaker CB, Zhang QH & Min DB. 2000b. Effects of pulsed electric fields on the quality of orange juice and comparison with heat pasteurization. J. Agric. Food Chem 48(10):4597 4605.

PAGE 169

169 Yoo ZW, Kim NS & Lee DS. 2004. Comparative analyses of the flavors from Hallabong (Citrus sphaerocarpa) with lemon, orange and grapefruit by SPTE and HS SPME combined with GC MS. B. Korean Chem. Soc. 25(2):271 279. Young H, Gilbert JM Murray SH & Ball RD. 1996. Causal effects of aroma compounds on Royal Gala apple flavours. J. Sci. Food Agric. 71(3):329 336. Young JC, Chu CLG, Lu XW & Zhu HH. 2004. Ester variability in apple varieties as determined by solid phase microextraction and gas chromatography mass spectrometry. J. Agric. Food Chem. 52(26):8086 8093. Zhang Q, Monsalve Gonzalez A, Qin B, Barbosa Canovas GV & Swanson BG. 1994. Inactivation of Saccharomyces cerevisiae by square wave and exponential decay pulsed. J. Food Proc. En g. 17:469 478. Zhang YF, Hoon MA, Chandrashekar J, Mueller KL, Cook B, Wu DQ, Zuker CS & Ryba NJP. 2003. Coding of sweet, bitter, and umami tastes: Different receptor cells sharing similar signaling pathways. Cell 112(3):293 301. Zozulya S, Echeverri F & Nguyen T. 2001. The human olfactory receptor repertoire. Genome Biol 2(6):Research0018.

PAGE 170

170 BIOGRAPHICAL SKETCH Zareena Azhu Valappil grew up in Delhi, India. She attended Delhi University and graduated in 1996 with an undergraduate degree in biological s cie nces. She r eceived iochemistry from Hamdard University in 1998 After earning her master s degree she joined the Bhabha Atomic Research Center as a Scientific Officer and worked there for a period of 6 years. She was awarded the pr estigious Graduate Alumni Fellowship to pursue a doctoral degree in food science at the University of Florida in 2005 In 2006 s he moved from Gainesville, F L to the United States Department of Agriculture in Wyndmoor Pennsylvania to conduct her doctoral research. In 2008 she accepted a position at Takasag o International Corporation in Rockleigh, N ew J ersey. She completed her PhD in 2010