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Polyphenol Oxidase Inhibitor(s) from German Cockroach (Blattella germanica) Extract

Permanent Link: http://ufdc.ufl.edu/UFE0021880/00001

Material Information

Title: Polyphenol Oxidase Inhibitor(s) from German Cockroach (Blattella germanica) Extract
Physical Description: 1 online resource (81 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apple, blattella, browning, cockroach, enzymatic, german, germanica, oxidase, polyphenol, potato, ppo, sulfites
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Enzymatic browning causes millions of dollars in losses yearly to the food industry by discoloration of fruits and vegetables. The Food and Drug Administration's (FDA) banning of sulfites in 1986 has created a large field of research in search of natural, effective and economic inhibitors of enzymatic browning. The objective of this research was to demonstrate inhibition of plant(s) polyphenol oxidase (PPO; EC 1.10.3.1) using inhibitor(s) from a whole body extract of German cockroach, Blattella germanica. German cockroach samples were homogenized and extracted in a 0.1 M Na2PO4, pH 6.5 buffer and centrifuged for 15 min at 10,450xg, 4 degrees C. The extract was then filtered and centrifuged again at 105,000xg for 1 hr, filtered, and the supernatants collected. Apple and potato PPO were extracted from an acetone powder. Reaction mixture contained 2.45 mL buffer, 0.3 mL 0.5 M catechol, 0.2 mL inhibitor or control buffer, and 0.05 mL PPO. Activity was determined spectrophotometrically at 420 nm. Crude inhibitor(s) was characterized based on temperature stability, pH extraction profile, incubation time, dialysis, ultrafiltration, treatment with proteases and kinetic analysis. Crude cockroach extract inhibited 60-70% of apple PPO activity and 15-25% of potato PPO, but it was not effective on banana and mushroom. Inhibition in the reaction mixture occurred fairly rapidly. The inhibitor(s) was stable when stored at refrigerated temperatures as well as freeze-thaw cycling, but upon boiling at 100 degrees C up to 60 min, inhibition was totally inactivated. Further characterization showed that the inhibitor(s) was unstable to changes in extraction pH, being most stable at pH 6.5. The percent inhibition dropped drastically when the pH moved in either direction. Incubation time was found to affect the inhibitor(s) when the inhibitor and PPO were allowed to sit up to 30 minutes, however when the inhibitor and substrate were incubated there was no effect on inhibition. Ultrafiltration and dialysis studies showed that the unknown inhibitor(s) has an apparent molecular weight (MW) larger than 100,000 nominal molecular weight limit (NMWL). Kinetic studies using Lineweaver-Burk showed the inhibitor(s) displayed non-competitive inhibition on apple PPO. Successful identification of inhibitor(s) of PPO from German cockroach would be useful to the fruit and vegetable segments of the food industry, due to the losses they incur from enzymatic browning. Further studies should be conducted to purify and identify the unknown inhibitor(s).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Marshall, Maurice R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021880:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021880/00001

Material Information

Title: Polyphenol Oxidase Inhibitor(s) from German Cockroach (Blattella germanica) Extract
Physical Description: 1 online resource (81 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: apple, blattella, browning, cockroach, enzymatic, german, germanica, oxidase, polyphenol, potato, ppo, sulfites
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Enzymatic browning causes millions of dollars in losses yearly to the food industry by discoloration of fruits and vegetables. The Food and Drug Administration's (FDA) banning of sulfites in 1986 has created a large field of research in search of natural, effective and economic inhibitors of enzymatic browning. The objective of this research was to demonstrate inhibition of plant(s) polyphenol oxidase (PPO; EC 1.10.3.1) using inhibitor(s) from a whole body extract of German cockroach, Blattella germanica. German cockroach samples were homogenized and extracted in a 0.1 M Na2PO4, pH 6.5 buffer and centrifuged for 15 min at 10,450xg, 4 degrees C. The extract was then filtered and centrifuged again at 105,000xg for 1 hr, filtered, and the supernatants collected. Apple and potato PPO were extracted from an acetone powder. Reaction mixture contained 2.45 mL buffer, 0.3 mL 0.5 M catechol, 0.2 mL inhibitor or control buffer, and 0.05 mL PPO. Activity was determined spectrophotometrically at 420 nm. Crude inhibitor(s) was characterized based on temperature stability, pH extraction profile, incubation time, dialysis, ultrafiltration, treatment with proteases and kinetic analysis. Crude cockroach extract inhibited 60-70% of apple PPO activity and 15-25% of potato PPO, but it was not effective on banana and mushroom. Inhibition in the reaction mixture occurred fairly rapidly. The inhibitor(s) was stable when stored at refrigerated temperatures as well as freeze-thaw cycling, but upon boiling at 100 degrees C up to 60 min, inhibition was totally inactivated. Further characterization showed that the inhibitor(s) was unstable to changes in extraction pH, being most stable at pH 6.5. The percent inhibition dropped drastically when the pH moved in either direction. Incubation time was found to affect the inhibitor(s) when the inhibitor and PPO were allowed to sit up to 30 minutes, however when the inhibitor and substrate were incubated there was no effect on inhibition. Ultrafiltration and dialysis studies showed that the unknown inhibitor(s) has an apparent molecular weight (MW) larger than 100,000 nominal molecular weight limit (NMWL). Kinetic studies using Lineweaver-Burk showed the inhibitor(s) displayed non-competitive inhibition on apple PPO. Successful identification of inhibitor(s) of PPO from German cockroach would be useful to the fruit and vegetable segments of the food industry, due to the losses they incur from enzymatic browning. Further studies should be conducted to purify and identify the unknown inhibitor(s).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Marshall, Maurice R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021880:00001


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1 POLYPHENOL OXIDASE INHIBITOR(S) FROM GERMAN COCKROACH (Blattella germanica) EXTRACT By PAUL JUSTIN KURT GROTHEER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Paul Justin Kurt Grotheer

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3 In loving memory of my grandfather Charlie M. Rowden (1917-2006) and my grandmother Elvira Meyn Gr otheer (1914-2006).

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4 ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Maurice Marshall, for his support and friendship throughout my research. Without his encouragement this study could not have been completed. I would also like to thank my committee members, Dr. Amy Simonne, Dr. Steven Valles, and Dr. Allen Wysocki, for all thei r help and guidance throughout my research. I would also like to thank my parents, Col (Ret) Kurt and Dr. Priscilla Grotheer, for their constant love and support. They have always be lieved in me, especially at times when I had trouble believing in myself. I also want to ac knowledge their constant financial support during both my undergraduate and graduate degrees. I would also like to thank my sister, Laura, for always being there for me when I need to tal k, and always being supporti ve of the decisions I make. Lastly I want to thank the friends I made at the University of Florida. In particular, I thank some of my roommates who ha ve become my closest friend s: Michael Robuck, Robby Etzkin, and Aaron Vieira. In addition I acknowledge good friends Timothy Darby and his wife Brittany, Charles Angeles, and Gary McClai n, and all the other friends I have made in Gainesville over the years.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAP TER 1 INTRODUCTION..................................................................................................................11 2 LITERATURE REVIEW.......................................................................................................14 Enzymatic Browning..............................................................................................................14 Biochemical Foundation of Enzym atic Browning................................................................. 15 Polyphenol Oxidase (PPO)..................................................................................................... 16 Mechanism of PPO.................................................................................................................16 Plant PPO.........................................................................................................................17 General Properties of Apple and Potato PPO..................................................................21 Substrate Specificity.......................................................................................................... .....21 Effects of pH and Temperature...............................................................................................23 Insect PPO..............................................................................................................................26 Inhibition of Enzymatic Browning......................................................................................... 29 Reducing Agents.............................................................................................................30 Sulfites......................................................................................................................30 Ascorbic acid............................................................................................................32 Chelating Agents............................................................................................................. 33 Acidulants........................................................................................................................34 Physical Treatments......................................................................................................... 34 Natural Inhibitors............................................................................................................. 35 German Cockroach, Blattella germanica ...............................................................................36 Objectives...............................................................................................................................37 3 MATERIALS AND METHODS........................................................................................... 38 German Cockroach Samples................................................................................................... 38 Preparation of Inhibitor Extract.............................................................................................. 38 Preparation of Plant PPO................................................................................................. 38 Assay of PPO Activity and Inhibition.............................................................................40 The pH Optimum of Plant PPO....................................................................................... 40 Effect of Timed Incubation on Inhibitor(s)..................................................................... 41 Effect of Heating and Freezing and Thawing on Inhibitor(s) ......................................... 41 The pH Extraction Profile............................................................................................... 41

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6 Dialysis of Inhibitor(s)....................................................................................................41 Ultrafiltration...................................................................................................................42 Treatment of Inhibitor(s) with Trypsin............................................................................ 42 Treatment of Inhibitor(s) with Papain.............................................................................43 Inhibition Kinetics........................................................................................................... 43 4 RESULTS AND DISCUSSION............................................................................................. 44 Optimization of pH Conditions fo r P PO Activity and Inhibition........................................... 44 Characterization of Crude Inhib itor(s) From German Cockroach.......................................... 51 Effect of Timed Incubation on the Inhibitor(s) ............................................................... 51 Temperature Stability ...................................................................................................... 52 The pH Extraction Profile............................................................................................... 53 Dialysis of Inhibitor(s)....................................................................................................54 Ultrafiltration...................................................................................................................55 Treatment of Inhibitor(s) with Trypsin............................................................................ 56 Treatment of Inhibitor(s) with Papain.............................................................................57 Inhibitor Kinetics.............................................................................................................58 5 CONCLUSIONS.................................................................................................................... 60 APPENDIX A RAW DATA...........................................................................................................................62 LIST OF REFERENCES...............................................................................................................71 BIOGRAPHICAL SKETCH.........................................................................................................81

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7 LIST OF TABLES Table page 2-1 Relative substrate specificity of PPOs from apple, potato, and banana........................... 22 2-2 The pH optimum of PPO for different plant sources....................................................... 25 2-3 Effect of temperature on PPO activity.............................................................................25 4-1 Ultrafiltration...................................................................................................................56 A-1 The pH optimum of potato PPO......................................................................................62 A-2 Optimum pH for potato PPO inhibition...........................................................................63 A-3 The pH optimum of apple PPO........................................................................................ 64 A-4 Inhibition of apple PPO activity by inhibitor(s) from German cockroach...................... 64 A-5 Optimum pH for apple PPO inhibition............................................................................ 65 A-6 The pH extraction profile of inhibitor..............................................................................66 A-7 Dialysis................................................................................................................. ...........67 A-8 Treatment of inhibitor( s) with trypsin and papain ........................................................... 68 A-9 Ultrafiltration...................................................................................................................69 A-10 Inhibitor kinetics..............................................................................................................70

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8 LIST OF FIGURES Figure page 2-1 Reaction for enzymatic browning.................................................................................... 16 2-2 Inhibition of enzymatic browning using a reducing agent............................................... 31 4-1 The pH optimum of mushroom PPO............................................................................... 44 4-2 The pH optimum of banana PPO..................................................................................... 45 4-3 The pH optimum of potato PPO...................................................................................... 46 4-4 Optimum pH for PPO inhibition by cockroach extract................................................... 47 4-5 The pH optimum of apple PPO........................................................................................ 48 4-6 Inhibition of apple PPO activity by inhibitor(s) from German cockroach...................... 49 4-7 Optimum pH for PPO inhibition by cockroach extract................................................... 50 4-8 Incubation time study.......................................................................................................52 4-9 The pH extraction profile of inhibitor(s)......................................................................... 54 4-10 Percent inhibition after dialys is with water or control buffer. ......................................... 55 4-11 Treatment of inhibitor(s) with trypsin.............................................................................. 57 4-12 Treatment of inhibitor(s) with papain..............................................................................58 4-13 Inhibitor kinetics:..............................................................................................................59

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science POLYPHENOL OXIDASE INHIBITOR(S) FROM GERMAN COCKROACH (Blattella germanica) EXTRACT By Paul Justin Kurt Grotheer May 2008 Chair: Maurice R Marshall Major: Food Science and Human Nutrition Enzymatic browning causes millions of dollars in losses yearly to the food industry by discoloration of fruits and vegetables. Th e Food and Drug Administrations (FDA) banning of sulfites in 1986 has created a large field of resear ch in search of natural, effective and economic inhibitors of enzymatic browning. The objective of this research was to demonstrate inhibition of plant(s) polyphenol oxidase (PPO; EC 1.10.3.1) using inhibitor(s) from a whole body extract of German cockroach, Blattella germanica German cockroach samples were homogenized and extracted in a 0.1 M Na2PO4, pH 6.5 buffer and centrifuged for 15 min at 10,450xg, 4C. The extract was then filtered and centrifuged again at 105,000xg for 1 hr, filtered, a nd the supernatants collected. Apple and potato PPO were extracted from an acetone powder. Reaction mixture contained 2.45 mL buffer, 0.3 mL 0.5 M catechol, 0.2 mL inhibitor or control buffer, and 0.05 mL PPO. Activity was determined spectrophotometrically at 420 nm. Crude inhibitor(s) was characterized based on temperature stability, pH extrac tion profile, incubation time, dial ysis, ultrafiltration, treatment with proteases and kinetic analysis. Crude cockroach extract inhibited 60-70% of apple PPO activity a nd 15-25% of potato PPO, but it was not effective on banana and mu shroom. Inhibition in the reaction mixture

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10 occurred fairly rapidly. The inhi bitor(s) was stable when stored at refrigerated temperatures as well as freeze-thaw cycling, but upon boiling at 100C up to 60 min, inhibition was totally inactivated. Further characterizat ion showed that the inhibitor(s ) was unstable to changes in extraction pH, being most stable at pH 6.5. The percent inhibition dropped drastically when the pH moved in either direction. Incubation time was found to aff ect the inhibitor(s) when the inhibitor and PPO were allowed to sit up to 30 minutes, however when the inhibitor and substrate were incubated there was no effect on inhibition. Ultrafiltrati on and dialysis studies showed that the unknown inhibitor(s) has an ap parent molecular weight (MW) larger than 100,000 nominal molecular weight limit (NMWL). Kinetic studies us ing Lineweaver-Burk showed the inhibitor(s) displayed non-competitive inhibition on apple PPO. Successful identification of inhi bitor(s) of PPO from German cockroach would be useful to the fruit and vegetable segments of the food industry, due to the losses they incur from enzymatic browning. Further studies should be conducted to purify and identify the unknown inhibitor(s).

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11 CHAPTER 1 INTRODUCTION Discoloration of fruits and vegetables by enzymatic browning causes millions of dollars in losses yearly to the food indus try (Whitaker and Lee 1995). This browning is associated with the enzyme polyphenol oxidase (PPO; EC 1.10.3.1). It is a copper-cont aining enzyme, which catalyzes the hydroxylation of monophenols (monophenolase activ ity) and the oxidation of odiphenols into o-quinones (diphenolase activity) using oxygen as the primary oxidant (Lax and Vaughn 1991; Whitaker 1994; Lerch 1995; Yoruk a nd Marshall 2003). The resulting quinones undergo further nonenzymatic condensation reacti ons leading to the formation of undesirable dark brown melanins (Sapers 1993). The catalytic action of PPO has a large effect on the quality of several fruit and vegetable crops, which can resu lt in the alteration of color, flavor, texture, and nutritional value (Yoruk and Marshall 2003). Enzymatic browning commonly occurs in frui t and vegetable crops after harvest during handling, processing and storage (Jang and othe rs 2002). Intact plant cells do not undergo browning because phenolic compounds in vacuol es are separated from PPO in cytoplasm (Yemenicioglu and others 1997). Severe brow ning of plant products occurs when PPO and substrate come into contact due to stress condit ions from subcellular decompartmentalization and oxygen penetration (Vamos-Vigyazo 1981). Enzymatic browning is considered an undesirabl e reaction in raw fruits and vegetables due to the change in color and devel opment of off-flavors, as well as loss of nutritional quality from the breakdown of vitamins (Murata and others 1995; Weemaes and others 1997). Apples and potatoes are popular crops worldwide, with apple production exceeding 85 million tons/y (Becker and others 2000; Yoruk and others 2004). In the United States, apple production is valued at more than $1.6 billion dollars annua lly, while total potato production was estimated at

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12 424 million hundredweight (cwt) in 2004 (Miller 2005; USDA 2006). Historically, sulfites have been used to slow and/or prevent browning. FDAs banning of sulfites in 1986 has created a large field of research in search of natural, effective and economic inhibitors of enzymatic browning. PPO is found in insects as well as plants (S ugumaran 1998). Insect PPO is considered to play a key role in important physiological proc esses in insects such as cuticular tanning and sclerotization, as well as in wound healing and defense reac tions against foreign pathogens (Tsukamoto and others 1992; Sugumaran and Ne llaiappan 2000; Wang and others 2004). It seems possible that natural inhibitors within in sects regulate this development. Previous literature has demonstrated endogeno us inhibitors in insects such as the housefly (Tsukamoto and others 1992; Daquinag and others 1995, 1999). Sugumaran and Nellaiappan (2000) isolated a heat-labile, high molecular weight (380000) glyc oprotein inhibitor of PPO from the larval cuticle of tobacco horn worm, Manduca sexta. In addition, Yoruk and others (2003) discovered a new natural apple PPO inhibitor(s) from housefly, Musca domestica L. that was stable to heat, but lost all but 10% PPO inhibition after ultrafiltration with a 100,000 NWML membrane. Preliminary studies in our laboratory eval uating insects found a new natural plant polyphenol oxidase (PPO) inhibitor(s) from German cockroach ( Blattella germanica ). Crude inhibitor(s) isolated via buffer extraction inhibited the activites of apple and potato PPO at pH values of 5.5 and 6.0, respectively. Heat treatment showed th at inhibition was totally inactivated. Ultrafiltrat ion and dialysis studies showed the unknown inhibitor(s) was larger than 100 kDa. There is no previously known research examining the applicability of inhibitors from German cockroach to food systems. The objectives of this study were to provide evidence for the existence of plant PPO inhibitor(s) in Germ an cockroach and to demonstrate inhibition of

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13 plant PPO activity using these inhibitor(s). Ch aracterization of crude inhibitor(s) included temperature stability, pH extrac tion profile, incubation, dialysis, ultrafiltration, treatment with proteases and kinetics.

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14 CHAPTER 2 LITERATURE REVIEW Enzymatic Browning Polyphenol oxidase (PPO; 1,2-benzenedio l:oxygen oxidoreductase; EC 1.10.3.1) causes enzymatic browning in food products by catal yzing the conversion of natural phenolic compounds to quinones (Nicolas and others 1994; Yoruk and Marshall 2003; Yoruk and others 2004). PPO is a generic name for a group of en zymes frequently referred to as tyrosinase, polyphenol oxidase, phenolase, catec hol oxidase, cresolase, or catecholase. PPO is capable of catalyzing reactions for several phenols to produce o-quinones (Whitaker 1994). The catalytic action of PPO has a large effect on the quality of several fruit and vegetables, which can result in the alteration of color, flavor, texture, and nutritional value (Yoruk a nd Marshall 2003). Those are four attributes c onsidered by consumers when making food choices. Browning commonly occurs in fruit and vegetable crops after harvest during handling, processing and storage (Lee and others 2001; Jang 2002). PPO is most likely present in all plants, but is in especially high concentrations in mushrooms, potatoe tubers, apples, peaches, bana nas, and avocados (Sanchez-Ferrer and others 1993b; Whitaker 1994). These crops ar e especially susceptible to browning reactions. It is estimated that over 50 percent of fruit losses occur due to enzymatic browning (Whitaker and Lee 1995). On the contrary, some cases of enzymatic browning are considered desirable, for example during the harvest of date s and in flavoring of beverages such as tea, coffee, and cocoa (Le Bourvellec and others 2004). In addition to its general occurrence in plants, PPO also occu rs in aquatic foods, primarily in crustaceans such as lobster and shrimp (Sim pson and others 1988). Aquatic organisms rely on PPO to impart important physiological functions fo r their development, such as hardening of the

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15 shell (sclerotization) af ter molting, as in crustaceans such as shrimp and lobsters. PPO is also responsible for wound healing. The mechanism of wound healing in aquatic organisms is similar to that of plants in that the compounds produ ced as a result of the polymerization of quinones exhibit both antibacterial and an tifungal activities. However, PPO-catalyzed browning of the shell after harvest has an adverse effect on both the quality and consumer acceptability of these products (Marshall and others 2000). Although black spot fo rmation does not affect the nutritional quality of seafood, it is perceived as spoilage by consumers (Chen and others 1992). Biochemical Foundation of Enzymatic Browning Enzymatic browning is a two-step process. The first step involves the enzymatic oxidation of monophenols (monophenolase activity) or o -diphenols (diphenolase activity) to yield o -quinones. In the second step, the o-quinones undergo condensation to form dark brown melanins (Sapers 1993; Whitaker 1994, 1995; Sanc hez-Ferrer and others 1995). Figure 2-1 shows the reaction for enzymatic browning a nd subsequent nonenzymatic condensation The involvement of copper as a prosthetic group of PPO is essential for its activity. Monophenolase activity is always coupled to diph enolase activity; however diphenolase activity is not always preceded by monophenolase activity (Whitaker 1994). In the two general reactions, phenols and oxygen are the substrates and BH2 represents an o -diphenolic compound, which acts as an election donor. In the absence of an o-diphenol (BH2), there is a characteristic lag or induction period in the m onophenolase reaction prior to atta ining a steady state rate. The duration of the lag period depends on the substr ate concentration, the pH and the presence of catalytic amounts of o-diphenol (Sanchez-Ferre r and others 1993c).

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16 Figure 2-1. Reaction for enzymatic browning (A dapted from Walker, J.R.L. 1977. Enzymatic Browning in foods: Its chemistry and control. Food Technol. NZ, 12: 19-25) Polyphenol Oxidase (PPO) A large field of research shows that PP O is found throughout nature (Whitaker 1994). PPO is widely distributed and can be found in animals, plants, and microorganisms (Whitaker and Lee 1995). PPO is located in the plastids of all tissue types of sp ecies that contain the enzyme (Sherman and others 1995). Generally, PPO is associated with pigmentation of tissues, acting as protection in animals, while the role of the enzyme is not known with certainty in higher plants (Whitaker and Lee 1995). Mechanism of PPO The mechanism of the catalytic action of PPO was deduced based on the geometric and electronic structure of the copper active site (Wilcox and others 1985; Solomon and others 1992). The active site of the enzyme consists of a bi-nuclear copper clus ter in the ligand field with three conserved histid ine residues existing in three forms: met-PPO (Cu+2), deoxy-PPO (Cu+1), and oxy-PPO (Cu+2) (Gandia-Herrero and others 2005). The resting form of the enzyme is thought to be met-PPO, which is kinetically capable of oxidizing diphenol but not monophenol substrates (Solomon and others 1992; Solomon and Lowery 1993; Gandia-Herrero and others

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17 2005). Oxidation of one molecule of catechol to o -benzoquinone takes place to reduce met-PPO to deoxy-PPO. The deoxy-PPO then reacts with dioxygen to produce an oxy-PPO intermediate (Solomon and others 1992). Oxy-PPO hydroxylates monophenols to generate o-diphenols and the met-PPO site. PPO is converted into deoxy-PPO and ready for another round without cycling through the resting form. In the diphenol oxidation pathway, diphenolic substrates react with both oxy-PPO and met-PPO. Met-PPO is first reduced to its deoxy form leading to the formation of oxy-PPO, which then forms an o diphenol-PPO complex. Finally, af ter the oxidation of catechol to obenzoquinone, the enzyme is reduced to the re sting form of met-PPO (Yoruk and Marshall 2003). Kinetic data indicate that PPO first bi nds oxygen and later monophenol in an ordered sequential mechanism (Wilcox and others 1985). Th erefore, the mechanism is thought to be an Ordered Sequential Bi Bi Mechanism (Whitaker 1994). Plant PPO PPO is present in most plant tissues (Sherman and others 1995) and is in especially high concentrations in mushrooms, potato tubers, p eaches, apples, bananas, avocados, tea leaves, coffee beans, and tobacco leav es (Whitaker 1994). PPO has been extensively studied by scientists in food and plant science due to it s adverse browning effect on plant products. The chemistry of PPO-catalyzed browning reactions in higher plants beyond the o-quinone step has only been elucidated in a few cases, because of insolubility of the pr oducts (Whitaker 1995). A sequence of biochemical reactions leads to the formation of melanin from the oxidation of phenolic amino acid tyrosinase. The produc ts through indole-5,6-qui none are soluble in in vitro systems (Whitaker 1995).

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18 The oxidation of phenols and formation of melanins are normal physiological processes of PPO, although the significance of the enzyme ac tivity in living intact plant tissues has not been unequivocally determined (Vaughn and others 1988; Sherman and others 1995; Yoruk and Marshall 2003; Maki and Morohash i 2006). This is surprising given a century of intensive research as a result of the economic importance of the enzyme (Sommer and others 1994). It has been frequently suggested that PPOs have a defensive role against pathogens and pests (Vaughn and others 1988; Maki and Morohashi 2006). Location of PPO has always been of special inte rest to researchers, because one must take into account its subcellular lo cation in order to assign a physio logical function to the enzyme (Mayer and Harel 1979; Vaughn a nd Duke 1984b). PPO is located in several subcellular fractions, including peroxisomes, mitochondria and microsomes (Mayer and Harel 1979; Martinez-Cayuela and others 1989; Yoruk and Ma rshall 2003). Research on the inheritance of isozyme patterns in species and interspecific hybrids (Lax and ot hers 1984; Kowalski and others 1990), suggests that PPO is a nucle ar-coded enzyme. The widely distributed enzyme is found in the plastids of all tissue type s in species possessing PPO activ ity (Vaughn and others 1988; Sherman and others 1995; Murata and others 1997) It is implausible th at the presence of a different form of the enzyme in aplastidic tissu es exists in tentoxin-tr eated plants lacking PPO activity. The toxin has been show n to inhibit the import of precurs or PPO protein into plastids (Sommer and others 1994). PPO has been described as tightly bound to thylakoids, but others have suggested it is located in plastid envelopes or the thylakoid lumen (Vaughn and others 1988; Sherman and others 1991; Sommer and others 1994). More recent research s uggests that PPO is synthesized on cytoplasmic ribosomes and is practically in active until the time it is integrated into the

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19 plasmid (Yoruk and Marshall 2003). Molecular chap erones, a class of cell ular proteins that facilitate correct folding and assembly of prot eins, stabilize a partia lly unfolded protein and prevent its refolding. This allows the protein to maintain its competence for insertion into thylakoid membranes (Yalovsky and others 1992; Marques and others 1995; Yoruk and Marshall 2003). The size of the native enzyme has also b een in question. Higher molecular weight nuclear-coded precursor proteins that possess a transit peptide sequence at the amino-terminal end are directed to the chloroplast. These si gnal sequences are later removed by specific stromal peptidases during incorporation, producing a lo wer molecular weight mature protein (Sommer and others 1994; Koussevitzky and others 1998). Molecular weights of precursor PPO proteins are approximately 68 kDa, while the mature pr oteins are approximately 60 kDa (Yoruk and Marshall 2003). Intense photochemical activity occurs in th e thylakoid membranes of chloroplasts. The location of PPO in the thylakoid has led researchers to believe that PPO may play a role in photosynthesis of functional chloroplast (Vaughn and Duke 1984a; Vaughn and others 1988; Thygesen and others 1995). The association of PPO with photosystem complexes suggested a possible role in photosynthetic electron transport (Lax and Vaughn 1991; Sheptovitsky and Brudvig 1996). Tolbert (1973) suggested PPO plays a role in pseudocyclic photophosphorylation, which is ATP production with oxygen as a terminal electron acceptor (Vaughn and Duke 1984a; Sherman and others 1995) However, it appears the activity of photosystem II membrane-associated PPO is pH dependent, with an optima around pH 8.0. During light-induced O2 evolution, the pH of the lumen is rath er acidic. Therefore, it appears

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20 PPO is important mostly for dark processes in the thylakoid lumen, when the pH is close to optimum for PPO (Sheptovitsky and Brudvig 1996). A number of PPO genes from different pl ant species have now been isolated and characterized. Tomato PPO is routed to the thylakoid lumen in a two step process (Sommer and others 1994). First a 67 kDa precursor is importe d into stroma and then processed to 62 kDa by stromal peptidases. Finally, the resulting inte rmediate is translocated into the lumen and processed to a 59 kDa mature protein. The ra tio between the intermediate and mature forms depends on the plant species, age, and growth conditions (Sommer and others 1994). In some species, PPO genes are present as multigene families while in others only a single PPO gene has been identified. However, all the PPO genes char acterized so far are nuc lear encoded (Cary and others 1992; Robinson and Dry 1992; Shahar and others 1992; Hunt and others 1993; Thygesen and others 1995). Involvement of PPO in oxidation of phenolic compounds in various plants is its most common role (Yoruk and Marshall 2003). Severe oxidative browning does not occur in healthy intact plant tissues perhaps because PPO activity in vivo is limited by lack of phenolic substrates physically separated from the enzyme inside the vacuoles (Vaughn and Duke 1984a; Macheiz and others 1990). It is generally believed that senescence or inju ry results in destruction of the biological barriers between PPO and polyphenols and the enzyme is active only when it unites with its phenolic substrat es (Vaughn and Duke 1984a; Vaughn and others 1988; Eskin 1990; Murata and others 1997). However, it is unkn own whether other factors, in addition to disruption of membrane integr ity, are involved in formation of such active enzymes for synthesizing or oxidizing polyphenols, or that a ffect the amount of active enzyme form and the level of enzyme activity during senescen ce, developmental stages or injury.

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21 General Properties of Apple and Potato PPO Apples are popular throughout the world, with world production estimated to be 42 million metric tons in the marketing year (M Y) 2004/2005 (USDA 2005). In the United States, apple production is valued at more than $1.6 billion dollars annually (Miller 2005) with production estimated at 4.6 million metric t ons in MY 2004/2005 (USDA 2005). According to Miller (2005), apples are regard ed as the third most valuable fruit crop in the United States behind grapes and oranges. During the 2004/200 5 marketing year (MY), apple production is expected to increase to about 4.3 million tons (USDA 2005), while apple consumption in the United States is expected to increase 10 percent. Total potato production in the United Stat es in 2005 was estimated at 424 million hundredweight (cwt), up 1 percent from the esti mate made in the January Annual Crop Summary (USDA 2006). Cwt is the commonly accepted meas urement for potatoes and equivalent to 100 lbs. According to USDA (2006), the value of U.S. all potatoes sold in 2005 is estimated at $2.76 billion, up 18 percent from the previous year. Enzymatic browning has a large impact on th e quality of both apples and potatoes, and their products, as it results in alteration of color, flavor, text ure, and nutritional value (Sapers 1993; Nicolas and others 1994). Therefore, research that discovers ways to maintain the original color of these fresh plant tissues is of great importance to the food industry. Substrate Specificity The primary substrates of apple and potat o PPOs are phenolic compounds (Table 2-1). The type of natural phenols and their relative concentrations vary widely from plant to plant source (Yoruk and Marshall 2003). Catechin, epicatechin and caffeic acid derivatives have been found to be common natural substrates of fru it PPOs (Macheix and others 1990). In apples, chlorogenic acid is the major phenolic compound in mature fruit while catechin is the major

PAGE 22

22 phenolic compound in immature fruit (Murata a nd others 1995). Activity of apple PPO on tyrosine was shown to be much lower than on o-diphenol (Zhou and others 1993). In addition, catechin was reported to be a major cause of browning in ap ples (Murata and others 1995). Table 2-1 shows the difference in activity of apple, potato, and banana PPOs for different types of substrates. PPO is most active with substrates that have a high specificity for the enzyme. Factors such as the nature of th e side chain, number of hydroxyl groups and their position on the benzene ring have a large effect on the catalytic activity of the enzyme (Macheix and others 1990). It appears that substrate spec ificity of PPO is also dependent on species and cultivars. In addition, PPO isoforms in a tissu e of interest may also demonstrate differential substrate specificities and va riations in their relative activities towards monophenols and odiphenols (Park and Luh 1985; Mach eix and others 1990). Four PPO isoforms differing in substrate specificities were discovered in apple tissues (Har el and others 1965). Table 2-1. Relative substrate specificity of PPOs from apple, potato, and banana Percent Activity Substrates Applea Potatob Bananac 4-Methylcatechol 181 93 Catechol 100 100 34 Chlorogenic acid 102 5 d-Catechin 54 12 Caffeic acid 0.7 1 Pyrogallol 38 27 1 Dopamine 37 100 DL-DOPA 12 0.6 8 Tyrosine 3 DOPA: Dihydroxyphenylalanine; References: aZhou and others (1993) bCho and Ahn (1999) cYang and others (2000)

PAGE 23

23 Effects of pH and Temperature The catalytic activity of PPO is greatly influenced by changes in pH. This is because changes in ionization of prototr opic groups in the activ e site of an enzyme at lower acid and higher alkali pH values may prevent proper confor mation of the enzymes active site. Changes in the active sites conformation alter the binding of substrates, and/or ca talysis of the reaction (Tipton and Dixon 1983; Whitaker 1994; Yoruk and Marshall 2003). Factor s that influence the stability of an enzyme do so by affecting the s econdary, tertiary, and/or quaternary structure of proteins. Therefore, irre versible denaturation of the protein a nd/or reduction of the stability of the substrate as a function of pH could also in fluence the catalytic activity of enzymes (Valero and Garcia-Carmona 1998; Yoruk and others 2003). The optimum pH of PPO varies widely depend ing on the plant source, but is generally in the range of 4.0-8.0, (Table 2-2) (Fraignier and others 1995; Marques and others 1995; Sheptovitsky and Brudvig 1996; Yoruk and Marsha ll 2003). Apple PPO has been found to have an acidic pH. While a single pH optimum of 4.55.0 has been reported in some cases (JanovitzKlapp and others 1989; Zhou and others 1993), ot hers have reported two pH optima at around 5.0 and another around 7.0 (Stelzig and others 1972) The optimum pH value for PPO is affected by several experimental factors such as ex traction methods, temperature, nature and concentration of the buffer, natu re and concentration of the subs trate, and purity of the source preparation (Stelzig and othe rs 1972; Janovitz-Klapp and others 1989; Zhou and others 1993; Whitaker 1994). The modulator sodium dodecyl sulfate (SDS) can also affect the pH optimum for PPO. The general behavior of apple PPO with changing pH was altered in the presence of SDS, with the activity inhibited at acidic pH and activated at pH above 5.0 in the presence of 3.5 mM SDS

PAGE 24

24 regardless of substrate (Marques an d others 1995). SDS also causes a shift in the pH optimum of PPO from low to a higher pH value with di fferent values for different substrates. The catalytic activity of PPO is also greatly influenced by temperature. On one hand at low temperature the kinetic energy of the reactant molecu les decreases and thus corresponds to a slower rate of reaction (Lehni nger and others 1993; Yoruk a nd Marshall 2003). On the other hand, high temperature caused disrup tion of the integrity of the th ree-dimensional structure of the enzyme molecule as well as alteration of the solubility of oxygen. Oxygen is a required substrate for the catalytic ac tivity of PPO (Whitaker 1994). Optimum temperature of PPO varies dependi ng on the plant source (Table 2-3). Apple PPO activity has an optimum temperature around 30 C, while potato PPO has an optimum around 40C (Zhou and others 1993; Cho and Ahn 1999). Optimum temperature of the activity is also affected by the substrate used in the assay. A study on Monroe apple PPO showed when the temperature was increased from 20 to 40 C, the relative activity of PPO decreased from 100 to 96%. When the temperature was increased from 50 to 70 C, the relative activity decreased from 87 to 6%, indicating rapid denaturation of the enzyme at higher temperatures (Zhou and others 1993). Thermal inactivation of PPOs from several sources was shown to follow first-order kinetics (Lee and others 1983; Yemenicioglu and others 1997 ). A study was done on several cultivars of apple (Yemenicioglu and others 1997) examining heat inactivation kinetics at temperatures of 68, 73, and 78 C. Results showed that after an initial increase in PPO activity, which can be attributed to the activation of a latent form, activity decreased following a first order kinetic model. In addition, a variance in the thermal stability between the different cultivars of apple was seen.

PAGE 25

25 Table 2-2. The pH optimum of PP O for different plant sources Source of Enzyme Substrate pH Optimum Applea Catechol 5.5 Apricotb 4-Methylcatechol 5.0-5.5 Cherryb 4-Methylcatechol 4.5 Grapec 4-Methylcatechol 3.5-4.5 Peachb 4-Methylcatechol 5.0 Plumb 4-Methylcatechol 4.0-5-5 Potatod,e Chlorogenic acid 4.5-5.0 and 6.0-6.5 Catechol 6.6 Strawberryf Catechol 5.5 Sweet Potatog Catechol 6.5 References: aYoruk and others (2003) bFraignier and others (1995) cValero and others (1998) dSanchez-Ferrer and others (1993a) eCho and Ahn (1999) fWesche-Ebeling and Montgomery (1990) gLourenco and others (1992) Table 2-3. Effect of temperature on PPO activity Source of Enzyme Substrate Temperature Opt. (C) Applea Catechol 30 Bananab Dopamine 30 Grapec 4-Methylcatechol 25-45 Potatod Catechol 40 Strawberrye Gallic acid 50 References: aZhou and others (1993) bYang and others (2000) cValero and others (1998) dCho and Ahn (1999) eSerradell and others (2000)

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26 Insect PPO PPO is found in insects as well as plants (Sugumaran 1998; Marsha ll and others 2000). Insect PPO is considered to play a key role in important physiological pr ocesses in insects such as cuticular tanning and sclerotization, as well as in wound healing and defense reactions against foreign pathogens (Tsukamoto and others 1992 ; Sugumaran and Nellaiappan 2000; Feng and Fu 2004; Wang and Jiang 2004; Zufelato and others 2004). The general properties of PPO appear to be simlar for plant and animals; the enzyme acts upon a number of phenolic substrates and oxidizes them into quinone compounds (Sugumaran 1998; Yoruk and Marshall 2003). In many ways, the molecular biology of PPOs fr om plants and insects is similar. Recent studies concerning the molecular cloning of PPOs from different species of insects have shown that there are 2 conserved c opper-binding sites showing high se quence similarity to arthropod hemocyanins (Sugumaran 1998). On the other hand, insect PPOs differ from plant PPOs in that they lack signal peptide sequences, directi ng nuclear-coded precursor of plant PPOs to the chloroplast envelope for subsequent processing, as discussed above (Yoruk and others 2003). PPO is present as a soluble enzyme in the blood of insects, which is stored in an inactive proform, and activated via a number of mech anisms during a time of need (Sugumaran and Nellaiappan 2000). The blood of insects is rich in potenti al phenoloxidase activity, exhibiting both monophenolase and diphenolase activities. Howeve r, the cuticle only contains diphenolase activity (Brunet 1980). All known PPOs, whether plant or insect, are capable of oxidizing odiphenols, making diphenolase activity the most common function of the enzyme (Yoruk and others 2003). There is a large fluctuation of PPO ac tivity during insect metamorphosis (Hara and others 1993).

PAGE 27

27 The discovery of a quinone tanning process invo lving PPO goes back to early research by Pryor (1940) on the sclerotization of cockroach oothecae and insect cuticle (Hopkins and Kramer 1992; Sugumaran 1998; Suderman and others 2006). The cuticle, or exoskeleton, of insects is responsible for body architecture, allowing loco motion, and acting to protect them against pathogen attack and environmental stress (Ande rsen and others 1996; Moussian and others 2005). Insect cuticle is composed of structural proteins, chitin, enzymes, catecholamines, lipids, and minerals. The relative ratio of these compon ents varies from insect to insect, cuticle to cuticle, and even region to region according to varying demands of function and growth (Andersen and others 1996; Sugumaran 1998). In the case of cockroaches, the egg case (ootheca) is totally devoid of chitin with 8.5% proteins, 5% diphenol and 1% lipids, while the adult cuticle of cockroaches has about 40% chitin, 50% protein, 5-10% catechol, and 1% lipid (Sugumaran 1998). The main roles of PPOs in insects include sclerotization (hardeni ng) and tanning of the insect cuticle (Hopkins and Kramer 1992; Anderson and others 1996; Sugumaran 1998). Sclerotization of the cuticle is a vital process fo r the survival of most insects, which protects susceptible soft parts of their bodies (Sugumara n and Ricketts 1995). Sc lerotins are chemically modified protein-containing components of in soluble and stiff skeletal structures. In cockroaches, sclerotins occur in the ootheca and exoskeleton (Peter 2006). Cuticular sclerotization involves the introduction of covalent crosslinks between proteins as well as chitin, which results in the formation of protein-protein and protein-chitin crosslinks (Sugumaran and others 1992). A co mbination of studies over the years indicates that quinones, quinone methides and semiquinones all act as sclerotizing agents (Sugumaran 1998). According to a unified mechanism, catecholamine sclerotizi ng agents precursors are converted by cuticular

PAGE 28

28 phenoloxidases to quinones that ei ther participate in the quinone tanning process or serve as substrates for quinone isomerase that converts 4-alkylquinones to hydroxylp-quinone methides (Sugumaran 1998; Sugumaran and Nelson 1998). Inte raction of the sclero tizing agents with certain side groups on cuticular co mponents, such as protein and ch itin, results in the hardening of the cuticle (Sugumaran 1998). Cuticles can vary in hardness, color, and flexibility depending on the sclerotization mechanisms and precurs ors used (Sugumaran and Ricketts 1995). Insect PPO is also suspected to play critic al roles in defense (c olor and camouflage), wound healing, and disease resistance (Sugumar an and Nellaiappan 2000; Chase and others 2000; Yoruk and others 2003). The onset of an infection stimulates a multifaceted defensive response in insects (Gillespie and others 1997). The response triggers an activation of the PPO zymogen present in the hemolymph. Secondary r eaction products of PPOs appear to fulfill some functional roles in insects, just like they do in plants (Gillespie and others 1997; Sugumaran and Nellaiappan 2000; Yoruk and Marshall 2003). The oxidative polymerization of quinones forms insoluble melanin over wounds to prevent loss of blood from infected ti ssues (Gillespie and others 1997; Sugumaran and Nellaiappan 2000). Another important defensive role of melanization in insects is its action in pathoge n sequestration (Gillespie and others 1997). Parasites and pathogens which ar e too large to be pha gocytosed are encapsulated in a melanin coat in the insects blood. This acts to not onl y limit the growth and development of the foreign invader, but also acts to create a physical barrier to prevent damage to the host (Chase and others 2000; Sugumaran and Nellaiappan 2000). PPO is present throughout the life cycle of insects in order to perform these multiple functions. PPO in the open circulatory system of insects occurs in the inactive proenzyme form, and is then activated when needed (Hara a nd others 1993; Chase and others 2000; Sugumaran

PAGE 29

29 and Nellaiappan 2000). The regul atory biochemical mechanisms of endogenous PPO activity include inactivation, polymerization, complexation with other proteins, and inhibition (Sugumaran and Nellaiappan 2000). It is suggested that PPO activity might also be regulated by intrinsic inhibitors throughout th e course of development (Tsuka moto and others 1992; Daquinag and others 1995, 1999; Sugumaran and Nellaiapp an 2000; Yoruk and others 2003). Tsukamoto and others (1992) isolated inhi bitors from the pupal extracts of house fly and demonstrated inhibition on house fly PPO. The potent house fly inhibitors were found to be heat-stable lowmolecular weight (approximately 3000 to 4200) peptid es with an inhibition constant in the nM range (Tsukamoto and others 1992). Further, Sugumaran and Nellaiappan (2000) have isolated a high molecular weight (380000) glycoprotein inhibitor of PPO from larval cuticle of the tobacco horn worm, Manducta sexta, and proposed that the presence of endogenous PPO inhibitor(s) would ensure direct control of PPO activity in insects. The specific e ndogenous PPO inhibitor(s) of Manducta sexta inhibited both insect and plant PPOs. Additionally, Yoruk and others (2003) isolated inhibitor(s) from the common house fly ( Musca domestica). The isolated inhibitor(s) were found to be fairly heat-stable and lo w-molecular weight. The inhibitor(s) from Musca domestica inhibited apple PPO. Inhibition of Enzymatic Browning Enzymatic browning causes millions of dollars in crop losses every year (Whitaker 1996; Kim and others 2000). The economic impact of these losses in fruits, vegetables, and seafood has spurred a large amount of research set to control the reaction in order to extend product shelf-life (Yoruk and Marshall 2003). Inhibitors of enzymatic browning caused by PPO fall into six groups based on their mode of action according to McEvily and others (1992): Reducing agents (ascorbic aci d and analogues, sulfites)

PAGE 30

30 Chelating agents (ethylenediaminetetraacet ate [EDTA], sodium diethyldithiocarbamate [DIECA], sodium azide) Complexing agents (cyclodextrins, chitosan) Acidulants (ascorbic acid, citric acid, malic acid, phosphoric acid) Enzyme inhibitors (substrate analogs, halides) Enzyme treatments (proteases, o-methyltransferase) Reducing Agents Reducing agents are widely used in the f ood industry to inhibit enzymatic browning. They act to prevent formation of me lanin by preventing the accumulation of o-quinones, or by forming stable colorless produc ts (Eskin and others 1971; Ni colas and others 1994; Kim and others 2000). The action of a reducing agent on the enzymatic browning can be seen in Figure 22. Sulfites Sulfites are inorganic salts that have antio xidant and preservativ e properties. Many compounds capable of producing sulfite called sulfiti ng agents, have been used as food additives since antiquity to help prevent enzymatic and nonenzymatic browning; control growth of microorganisms; act as bleaching agents, antioxida nts, or reducing agents; and carry out various other technical functions (Taylor and others 1986; Sapers 1993; Girelli and others 2004). Examples of sulfiting agents in clude sulfur dioxide, sodium su lfate, sodium and potassium bisulfites and metabisulfites. Specifically, sulf ites are used on fruits and vegetable to prevent unpleasant browning, applied to shrimp and lobster to prevent melanosis or black spot, used in wines to discourage bacterial growth, as a conditioner in dough and to bleach certain food starches and cherries.

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31 In fresh fruits and vegetables, sulfites prevent PPO from working properly by preventing brown pigment formation (Sayavedra-Soto a nd Montgomery 1986; Sapers 1993). Sulfites are thought to inhibit browning by acting as a redu cing agent that combines with the ortho -quinones and converts them back to colorless diphenols. This prevents the nonenzymatic condensation of o-quinones to complex brown polymer s (Figure 2-2) (Weaver 1974). Figure 2-2. Inhibition of enzymatic browning us ing a reducing agent. (Adapted from Walker, J.R.L. 1977. Enzymatic Browning in foods: Its chemistry and control. Food Technol. NZ, 12: 19-25) A small percentage of the U.S. population is suspected to be sensitive to sulfites. Sensitivity to sulfites can develop at any tim e during a human lifespan, with some initial reactions not showing up until a person has reached their forties or fifties. The manifestations of sulfite sensitivity include a la rge array of dermatological, pu lmonary, gastrointestinal, and cardiovascular symptoms. Adverse reactions to su lfites in nonasthmatics are extremely rare, but asthmatics that are steroid-dependent or have a great degree of airway hyperreactivity may be at an increased risk of having a r eaction to a sulfite containing f ood (Lester 1995). Varying degrees

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32 of bronchospasm, angiodema, urticaria, naus ea, abdominal cramping, and diarrhea are commonly reported (Knodel 1997). Sulfites have such a long history of use th at when the Federal Food, Drug, and Cosmetic Act was amended in 1958 to regulate preservativ es and other food additives, FDA classified sulfites as generally recognized as safe (GRAS) (Papazian 1996). However, in response to a 1982 FDA proposal to affirm the GRAS status of sulfating agents, FDA began to receive reports of adverse health reac tions to sulfites (Pap azian 1996; Sapers 1993). FDA contracted the Federation of American Societies for Experi mental Biology (FASEB) to examine the link between sulfites and repo rted health claims. The FASEB submitted its final report to FDA in 1985, concluding that sulfites are safe for most people, but could pose a hazard of unpredic table severity to asthmatics and others who are sensitive to them. Based on this report, F DA took the following regulatory actions in 1986: sulfites were prohibited to maintain color and cris pness of fresh fruits and vegetables, such as in salad bars or fresh produce. Companies were re quired to list sulfites or chemical components that give rise to sulfites with at least 10 part per million (ppm) or higher, and any sulfiting agents that had a technical or functi onal effect in the food regardle ss of the amount present (FDA 1988a; Papazian 1996). Currently, sulfiting agents are no t considered GRAS for use in meats, foods recognized as a major source of vitamin B-1, or fruits or vegetabl es intended to be serv ed raw to consumers or to be presented to consumers as fresh (F DA 1988b). Sulfites have been found to destroy thiamin and have therefore been banned for use in foods that are a majo r source of vitamin B-1. Ascorbic acid Ascorbic acid (vitamin C), which is acidic in nature, is known as the best alternative reducing agent to sulfites. It forms neutral salts w ith bases, and is highly water soluble (Marshall

PAGE 33

33 and others 2000). Ascorbic acid is commonl y used as an antibr owning agent in the manufacturing of fruit juices, purees, frozen sl iced fruits, and canned fruits and vegetables (Yoruk and Marshall 2003). Asco rbic acid retards the formation of melanins by directly reducing o -quinones back to corresponding reactants, o-diphenols, before the quinones can undergo additional non-enzymatic polymerization to form dark brown melanins, (Figure 2-2). Unfortunately, ascorbic acid is irreversibly oxi dized to dehydroascorbic acid during the reduction process, which allows browning to occur upon its depletion, limiting its usefulness as an inhibitor (Sapers 1993). There is an interest in investigation of us ing ascorbic acid in combination with other inhibitors for better control of enzymatic browni ng. Eskin and others (1 971) and Sapers (1993) found ascorbic acid in combination with citric acid to be a more effective inhibitor than ascorbic acid alone. This increased inhibition is likely due to the increased stability of ascorbic acid in an acidic environment as well as inhibition of the acid ic environment on the catalytic activity of the enzyme (Yoruk and Marshall 2003). Citric acid can also function as a PPO inhibitor through its chelating action (Eskin and others 1971); and ascorbic acid thr ough the site-directed specificity toward histidine residues on the PPO protein (Osuga and others 1994). Chelating Agents Chelating agents are another group of enzymatic browning inhibitors. Copper is critical for PPO activity because the enzyme is a coppe r metalloprotein (Lerch 1995; Van Gelder and others 1997). The active site of PPO is co mprised of two copper atoms through which the enzyme interacts with its phenolic substrates and oxygen (Lerch 1995; Van Gelder and others 1997). Chelating agents have th e ability to react with metals through chelation, making copper at the active site unavai lable, thereby inhibiting PPO (McEvi ly and others 1992). Examples of agents serving as chelators for copper include EDTA, phosphates, citric acid,

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34 diethyldithiocarbamate (DIECA), and sodium azide (Eskin and ot hers 1971; McEvily and others 1992; Yoruk and Marshall 2003). Acidulants Acidulants are another group of inhibitors us ed by the food industry to prevent enzymatic browning because they are often part of the natural food composition. Acidulants, such as ascorbic, citric, malic, and phosphoric acids, are applied to maintain the pH well below that required for optimum catalytic ac tivity an enzyme. These acids can shift the food pH to 3 or lower, and slow the action of PPO (Richards on and Hyslop 1985; Eskin 1990; Osuga and others 1994). PPO is typically more ac tive in a pH range from 4.0-8.0, a nd the activity drops steadily in acidic environments (Yoruk and Marshall 2003). C itric acid is one of the most widely used acidulants in the food industry. Under conditions of extreme acidic pH, resulting inhibition can be attributed to protonation of catalytic groups essential for catalysis, conf ormational changes in the active site of the enzyme, irreversible denaturation of the protein, and/or reduction in the stability of the substrate as a function of pH (Whitaker 1994). In additi on, acidity also diminish es the strong binding of the enzyme to its active site copp er, which increases the ability of citric acid to react with copper through chelation (Osuga and others 1994). Physical Treatments Physical treatments are another option in controlling enzymatic browning of food products. These include heating, freezing, refr igeration, dehydration, irradiation, and high pressure treatments (Ashie and others 1996; Kim and others 2000; Yoruk and Marshall 2003). Although effective, these physical treatments can also produce undesirable side effects, such as subcellular decompartmentalization, which leads to enzyme-substrate contact and deterioration in texture (Macheix and others 1990). Irradiation can result in tissue dark ening and decreased

PAGE 35

35 vitamin content, while freezing can lead also lead to changes in texture and freshness. A proper combination of pressure and temperature can be used to enhance enzyme activation in certain foods (Marshall and others 2000). Additional methods for preventing enzymatic browning that are being investigated include controlled atmosphere packaging, and edible films and coatings that act to prevent oxygen pe netration (Ahvenainen 1996). Natural Inhibitors The use of browning inhibitors by the food i ndustry is limited due to factors such as offflavors/odors, food safety, economic feasibility, and effectiveness of inhibition (Eskin 1990; McEvily and others 1992; Sapers 1993; Park 1999; Chen and others 2000; Yoruk and Marshall 2003). With sulfites banned by the FDA in 1996, ther e is an even greater research interest in finding new effective, natural, and safe antibrown ing agent(s) to prevent browning of fruits and vegetables. Substantial investigations are pe rformed on natural antibro wning agents, such as amino acids (Kahn 1985). Research has been pub lished involving natural inhibitors such as glucose (Tan and Harris 1995) honey (Osmianski and Lee 1990; Chen and others 2000), and pineapple juice (Lozano-de-Gonzal ex and others 1993). These na tural PPO inhibitors are not being commercially utilized on food at this time. There is research being c onducted on inhibition of PPO by naturally occurring oxalic acid. Oxalic acid in both sol uble and insoluble forms is a natural component of many higher plants. Oxalic acid was suggested to have some selective, fuctional si gnificance or adaptation during normal plant development (Ilarslan and ot hers 1997). Despite not being an approved food additive, oxalic acid is a common component of a large variety of plant foods such as asparagus, broccoli, Brussel sprouts, carro t, garlic, lettuce, onion, parsle y, pea, potato, purslane, spinach, tomato, and turnip (USDA 1984). Research by Sato (1980) demonstrated oxalic acid was a naturally occurring inhibitor of PPO in spinach, while Ferrar and Walker (1996) reported that

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36 oxalic acid produced by the phytopathogen Sclerotinia sclerotiorum inhibited PPO activity in infected apples and bean pods. German Cockroach, Blattella germanica The German cockroach ( Blattella germanica ) is about 5/8-inch in length and brown in color with two dark longitudinal streaks that run the length of the pronotum. The male is light brown and somewhat boat-shaped and slender, while the female is slightly darker in color with a broader and rounded posterior (S uiter and Koehler 2000). The Ge rman cockroach has three life stages typical of insects with incomplete metamorphosis: the egg, nymph, and adult (Valles 1996). The German cockroach produces more eggs per capsule than other pest cockroach species, and the life cycle can be completed in about 3 months (Koehler and Castner 1997; Suiter and Koehler 2000). They tend to breed continuo usly, with many overlapping generations present at any one time (Valles 2000). Although the German cockroach is a primitive insect, it is well adapted to the human environment (Lin and others 2002). It is found worldwide in associati on with humans (Valles 1996). In the United States, it is the principle domestic house hold cockroach species (Bhat and others 2003). It is prevalent in and around homes, apartments, supermarkets, food processing plants, and restaurants (Suiter and Koehler 2000). The major limiting factor for the survival of German cockroaches appears to be cold temperat ures, which prevent them from being able to colonize. Other limiting factors include availabi lity of food, water, and harborage (Valles 1996). The German cockroach is considered an aesth etic pest, therefore, the action threshold for it is dependent on the tolerance of the people living in the infest ed dwelling (Valles 1996). The most successful means of control is use of baits (stations or gel form). When the level of infestation is high, use of an insecticide mixed with an insect growth regu lator to act as a cleanout may be necessary (Suiter and Koehler 2000).

PAGE 37

37 Objectives Since insects have a natural PPO mechanism, which appears to be regulated during their growth and development, we hypothe size that this regulation is due to inhibitors in the insect. Thus, the objective of this research was to pr ovide evidence for the existence of plant PPO inhibitor(s) from German cockroach, Blattella germanica Past research supports findings of an inhibitor(s) in insects that infl uences the enzyme activity of po lyphenol oxidase. If such an inhibitor(s) is present in German cockroach, it may also be effec tive in inhibiting the browning in fruits and vegetables caused by PPO. Specific obj ectives included testing different plant PPOs to determine effectiveness with the extracts, and characterizing the extract as an inhibitor to determine the effect of temperature, pH, and incubation time on it. In addition, the approximate size of the inhibito r(s) was determined.

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38 CHAPTER 3 MATERIALS AND METHODS German Cockroach Samples Samples of Blattella germanica (L.) were provided by the U.S. Dept. of Agriculture, Center for Medical, Agricultu ral, and Veterinary Entomology (Gainesville, FL). The cockroaches were reared at 26 C, 55% relative humidity, and with a 12:12 light:dark photoperiod as described by Koehler and Patters on (1986). Newly molted adult males were collected using featherweight forceps. Preparation of Inhibitor Extract PPO inhibitor was extracted from German co ckroach following a procedure adapted from Valles and Yu (1995). Cockroaches were anaesth etizied with carbon dioxide and male samples were separated and put on ice. The cockroaches were decapitated, and the bodies were put in a 10 mL tube with 0.1 M sodium phosphate buffe r, pH 6.5. A Delta Model 11-990 12 inch drill press (Pittsburgh, PA) equipped with a Thomas Scientific Potter-Elv ehjem Teflon pestle (Swedesboro, NJ) was used to homogenize the co ckroaches. Next, the homogenate was filtered through a double layer of cheese cloth and centrifuged in a Beckman Model L8-M Ultracentrifuge (Fullerton, CA) at 10,000 g for 15 minutes at 4 C. The supernatant was filtered through glass wool and then centrifuged again for 1 hour at 105,000 g. The supernatant was filtered through glass wool once again and stored at -20 C in microcentrifuge tubes until used. The supernatant is the solu ble fraction and contains the cytosome. The resulting pellet containing the microsomes was discarded. Preparation of Plant PPO In preliminary studies, mushroom and banana were tested for PPO activity and inhibition. Fresh Crimini mushrooms and bananas were acqui red from a local grocery store and prepared

PAGE 39

39 into an acetone powder. Washed mushrooms and peeled bananas were cut up to small pieces (200g of each), which were homogenized in a pr e-chilled blender (Waring Products Inc., Torrington, CT) with acetone (-20 C) for 1 min, and then filtered through Whatman No. 1 filter paper. The residue was re-extracted three times with 200 ml of cold acetone (-20 C). The resulting white acetone powder was vacuum-dried in a FoodSaver Vac 350 (Tilia Inc., San Francisco, CA) at room temperature (25 C) and stored in commercial vacuum bags (Tilia Inc., San Franciso, CA) at -20 C until needed for PPO extraction. Russet potatoes were selected as a source of potato PPO because this variety has shown high enzyme activity in past research confirmed by visual tests conducted in our lab. Previous research indicated higher PPO activity in the Ru sset variety potato (Hsu and others 1988). A potato acetone powder was prepared from Russet potatoes acquired from a local grocery store. The potatoes were washed and sliced into small pieces and then prepared into an acetone powder as described above. Red Delicious variety of apple was selected as a source of apple PPO because past research indicated it to have the highest enzyme activity among the apple cultivars (Janovitz-Klapp and others 1989). Apple PPO was extracted from an apple acetone powder. Red Delicious apples, which were acquired from a local grocery store, were washed, peel ed, and cut into small pieces and then prepared into an acetone powder as described above. PPO extraction was accomplished by adding 1 gram of acetone pow der to 50 mL of 0.1 M KH2PO4/Na2HPO4, pH 7.2 buffer containing 1% Trit on X-100 (Bio-Rad Laboratories, Hercules, CA) and incubated for 20 min while stirring with a magnetic stirrer at 4 C. The suspension was centrifuged in a Beckman Model DU 640 Ultraviolet-Visibl e spectrophotometer (Beckman Instruments Inc., Fullterton, CA) for 30 min at 12,000 g, and the resulting

PAGE 40

40 supernatant was filtered through gl ass wool. Finally, the filtered supernatant was stored in microcentrifuge tubes at -20 C. Assay of PPO Activi ty and Inhibition PPO inhibition was quantified by measuring th e PPO activity of apple with and without the added inhibitor extract. A standard assay consisted of 2.45 mL of 0.1 M sodium acetateacetic acid, pH 5.5 buffer, 0.3 mL of 0.5M catecho l substrate (Sigma Chemical Co., St. Louis, MO), 0.2 mL of test extract or control buffer and 0.05 mL of the enzyme extract. PPO activity was measured by spectrophotometric determinati on of initial reaction rates at 420 nm and 25 C with a Beckman Model DU 640 Ultraviolet-Visible spectrophotometer (Beckman Instruments Inc., Fullerton, CA). The pH of the reaction mixture was checked after each assay to confirm pH values were maintained for the control and test systems. PPO activity of apple was also assayed by mixing it with an in hibitor extract in a standard reaction mixture where the pH of the main buffer (0.1 M sodium acetate-acetic acid) varied from pH 4.0 to 5.7. One unit of enzyme activity was defined as an increase in absorbance of 0.001 per min at 25C. The degree of inhibition was expressed as percent inhibition (I), calculated using the formula [100(A-B)/A], where A represents enzyme activity of the control system and B represents enzyme activity of the test system. The pH Optimum of Plant PPO The pH-activity profile for the oxidation of catechol by PPO was determined using 0.1 M sodium acetate, pH 4.0 to 5.5, and 0.1 M sodium phosphate, pH 6.0 to 7.0. The reaction mixture contained 0.05 mL of the enzyme solution, 0.3 mL of 0.5 M catechol and 2.65 mL of various buffer solutions. The reaction was initiated wi th the addition of the enzyme solution. PPO activity was determined as described above.

PAGE 41

41 Effect of Timed Incubation on Inhibitor(s) Initiation of the assay described above was done in two different ways to compare the differences between incubating the inhibitor(s) with the PPO or s ubstrate for varying periods of time. The main reaction buffer (sodium acetate-a cetic acid, pH 5.5) and inhibitor were placed in the cuvette with either the PPO or the catechol substrate and incubated at room temperature (25 C) for 0, 1, 5, 15, and 30 min. Reactions were initiated with the component not used for incubation and initial reaction rate s were calculated at 420nm and 25 C. Effect of Heating and Freezing and Thawing on Inhibitor(s) Aliquots of 2 mL of the i nhibitor preparation were incubated in boiling water, 100 C, for 5, 15, 30, and 60 min. The precipitate was sepa rated by centrifugation for 5 min at 10000 g at room temperature. The clear supernatants were collected in clean micr ocentrifuge tubes. PPO inhibition was determined at 25 C as described above. Aliquots of 2 mL for the inhibitor prepara tion were exposed to repeated freezing and thawing and storage (3X). In the last step, the samples were thawed after storage at -20 C. Aliquots were also stored at -20 C for up to 6 months The pH Extraction Profile The inhibitor extract was extracted according to the methods described above but at pH values ranging from 4.5-7.5 with 0.1 M sodium phosphate or sodium acetate buffer. The samples were stored at -20 C in microcentrifuge tubes until they were used to compare PPO inhibition as described above. Dialysis of Inhibitor(s) Dialysis of inhibitor extracts was pe rformed using 500, 2000, and 25000-Da molecularweight cut off (MWCO) dialysis membrane s (Spectrum Laboratory Products, Inc., New

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42 Brunswick, NJ) with constant stirring for 24 hours against 2 and 3 changes of 0.1 M sodium phosphate buffer, pH 6.5, and distilled wate r. The dialysates were kept at 4 C until they were tested for PPO inhibition. Ultrafiltration Ultrafree-4 centrifugal filter units (Millipore Cor poration, Bedford, MA) with the nominal molecular weight limit (NMWL) membranes of 10000, 50000, and 100000 were used. Filter units were rinsed with di stilled water to remove a trace amount of glycerin. Aliquots of 1.5 mL of inhibitor sample were placed onto the filter units and concentrated by centrifugation at 4C and 7,500 g for 35 min. Filtrates were collected and kept at 4C. The sample was reconstituted back to 2 mL by adding 0.1 M s odium phosphate buffer, pH 6.5, and centrifuged again. Concentrated inhibitor sample on the membrane surface, rententate, was reconstituted back to 2 mL by adding distilled water and assa yed for PPO inhibitor activity as described above. Control inhibitor samples in centrifuge tubes also were subjected to centrifugation with test samples. Treatment of Inhibitor(s) with Trypsin A trypsin solution was created by dissolving 0.01g of trypsin (Sigma Chemical Co., St. Louis, MO) in 10 mL of a pH 7.2 sodium phosphat e buffer. Next, 0.3 mL of the trypsin solution was added to 5.7 mL of crude co ckroach inhibitor extract. A c ontrol solution was also made up by adding 0.3 mL of control bu ffer to 5.7 mL of cockroach in hibitor solution. 0.2 M TRIS was added to the control and test solu tions to reach the optimum pH for trypsin of 7.6 as stated by the manufacturer. The solutions were incubated in a water bath at room temperature (25 C) for 1 hour. Finally the solutions were titrated back to pH 5.5 using 0.2 M acetic acid and assayed for inhibitor activity.

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43 Treatment of Inhibitor(s) with Papain A papain solution was made by dissolving 0.15 g of papain (Sigma Chemical Co., St. Louis, MO) in 10 mL of pH 6.5 sodium phosphate Then, 0.15 mL of the papain solution was added to 2.85 mL of inhibitor extract. A cont rol solution was also made by adding 0.15 mL of control buffer to 2.85 mL of inhib itor extract. Since papain has an optimum pH range of 6-7, no further pH adjustment was necessary. The soluti ons were incubated in a water bath at room temperature (25 C) for 1 hour. Finally, the solutions were assayed for inhibitor activity. Inhibition Kinetics Plots based on experimental data are often complicated by substrate inhibition or activation; therefore it is be st to use methods of plotting v0 versus (A0) data to display them in linear form (Whitaker 1994). The Lineweaver-Burk method, first described in 1934, is the method most frequently used. A plot of substrate-velocity data was ma de according to the Lineweaver-Burk method. Inhibition by cockroach extract on apple PPO was determined in the presence of three different concentrations of inhibitor solution (1x, X, and X) for five different fixed concentrations of catechol (1, 0.5, 0.25, 0.125, and 0.0625M) at pH 5.5.

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44 CHAPTER 4 RESULTS AND DISCUSSION Optimization of pH Conditions fo r PPO Activity and Inhibition The pH activity profile for oxidation of cat echol by mushroom and banana PPO were both determined in preliminary studies (Figures 4-1 and 4-2 respectively). Maximum activity for mushroom PPO for the range of pH 4.5-6.5 was found at around pH 6.5, although activity at pH 6.0 was similar to 6.5, especially given the st andard deviation. At pH 5.5, only about 72% activity was found relative to that at pH 6.5. Mi nimal activity was detected at pH 4.5, only 37% relative to that at pH 6.5. In comparison, Gouzi and Benmansour (2007) found two pH optima for mushroom PPO, at 5.3 and 7.0. Meanwhile Co lak and others (2007) found a pH optimum of 7.0 for three different wild mushroom species. 0 20 40 60 80 100 120 4 555 566 5pH% Relative activity Figure 4-1. The pH optimum of mu shroom PPO. Activities are expressed as % relative activity to the maximal activity determined at pH 6.5. Each data point represents mean standard deviation of 2 experi ments each with 3 replicates.

PAGE 45

45 Maximum activity for banana PPO for the ra nge of pH 4.0-6.5 was also found at pH 6.5. Activity between pH 4-5 ranged from a minimum of 38% at pH 4 up to around 50% at pH 5. However, at pH 5.5 the activity jumped to around 73%. At pH 6, about 80% activity was observed relative to that at pH 6.5. Yang and ot hers (2000) also found a pH optimim of 6.5 for banana pulp, while Palmer (1963) found a pH op timum of 7.0 for banana when catalyzing the oxidation of dopamine. Although some inhibition st udies were carried out using these plants, there was not enough evidence of significant inhib ition to continue more comprehensive studies. 0 20 40 60 80 100 120 44 555 566 5pH% Relative activity Figure 4-2. The pH optimum of banana PPO. Activiti es are expressed as % relative activity to the maximal activity determined at pH 6.5. Each data point represents mean standard deviation of 2 experi ments each with 3 replicates. The pH activity profile for oxidation of cat echol by potato PPO for the pH range of 4.06.5 was determined (Figure 4-3). Maximum activity for potato PPO was found around pH 6.0, while the minimum activity was found at pH 4.0. A steady increasing trend occurred from pH

PAGE 46

46 4.0 up to the maximum at 6.0. At pH 6.5, activity dr opped down to about 82% relative to that at 6.0. Potato PPO has been reported to have tw o pH optima, the first between 4.5-5.0 and the second between 6.0-6.5 (Sanchez-Ferrer and others 1993a). 0 20 40 60 80 100 120 3.544.555.566.577.5pH% Relative Activity Figure 4-3. The pH optimum of pot ato PPO. Activities are expressed as % relative activity to the maximal activity determined at pH 6.0. Each data point represents mean standard deviation of 2 experiment s each with 3 replicates. The ability of the cockroach extract to inhibit potato PPO was demonstrated spectrophotometrically by mixing the inhibitor extract with an activ e extract of potato PPO at the determined pH optimum of 6.0. The crude inhibitor preparation reduced potato PPO activity by 20%. The inhibition was fairly rapid, and occu rred during the assay anal ysis. The amount of inhibition by the cockroach extract on potato PPO varied with changi ng pH (Figure 4-4). Inhibition showed an increasing tr end from pH 4.0, although the inhi bition at pH 5.0 was slightly

PAGE 47

47 higher than that seen at pH 5.5. At a pH of 6.5, only 13% inhibition was seen, compared to the 20% seen at the pH optimum of 6.0. -5 0 5 10 15 20 25 44 555 566 5pH% Inhibition0 20 40 60 80 100 120% Relative Activity Figure 4-4. Optimum pH for PPO inhibition by cockroach extract. This was determined by measuring the PPO activity of potato mixe d with the inhibitor(s) in a standard reaction mixture where the pH of the main buffer (0.1 M acetate-acetic acid) varied from 4.0 to 6.5. Bars indicate %inhibition wh ile the dashed like indicated %relative PPO activity without inhibitor(s). Each da ta point represents mean standard deviation of 2 experiment s each with 3 replicates. The pH activity profile for oxidation of cat echol by apple PPO was also determined (Figure 4-5). Maximum activity for apple PPO for the range of pH 4.0-7.0 was found around pH 5.5, with a steady decreasing trend above th at. At pH 5.0, around 71% activity was found relative to that at pH 5.5. Minimal activity was detected at pH 6.0 or higher. The pH optimum of apple PPO is commonly found to be acidic, with other researchers finding the pH optimum in the range from 4.5-5.5 (Janovitz-Klapp and ot hers 1989; Zhou and others 1993; Yoruk and

PAGE 48

48 Marshall 2003). Stelzig and othe rs (1972) reported the PPO of apple peel to have two pH optima, 4.2 and 7.0. For all plant PPOs, several factors can affect the optimum pH value, such as the method of extraction, temperature, nature of the phenolic substrate, and type of buffer system used in determination of maximum activity (Ste lzig and others 1972; Ja novitz-Klapp and others 1989; Zhou and others 1993; Whitake r 1994; Yoruk and Marshall 2003). 0 20 40 60 80 100 120 3.544.555.566.57 pH% Relative activity Figure 4-5. The pH optimum of apple PPO. Activities are expressed as % relative activity to the maximal activity determined at pH 5.5. Each data point represents mean standard deviation of 2 experiment s each with 3 replicates. The ability of the cockroach extract to inhibit apple PPO was demonstrated spectrophotometrically by mixing the inhibitor extract with an activ e extract of apple PPO at the determined pH optimum of 5.5. A comparison of absorbance at 420 nm be tween the control and test assays for 120 seconds was plotted (Figure 4-6). The crude inhibitor preparation reduced apple PPO activity by 60%. As w ith potato PPO, the inhibition was fairly rapid, occurring in the

PAGE 49

49 assay mixture, and did not require a long incubati on. In depth studies of PPO inhibitor(s) assay conditions demonstrated the extreme importance of reaction pH on inhibition activity by the cockroach extract (Figure 4-7). The amount of inhibition by the cockroach extract on apple PPO varied with changing pH. Inhi bition showed an increasing tr end from pH 4.0 and higher with greatest inhibition of 72% seen at pH 5.7. At the pH optimum of 5.5, 60% inhibition was observed. However, below pH 5.0 very low (levels < 5%) inhibition was observed. It should also be noted that at pH levels above 5.5 the increased amount of inhi bition may in fact be attributed to the decline in PPO activity on th e activity profile as a result of changing pH. 0 0.1 0.2 0.3 0.4 0.5 0.6 020406080100120Time (s)Absorbance at 420 nm PPO alone PPO plus inhibitors Figure 4-6. Inhibition of apple PPO activity by inhibitor(s) from German cockroach. Connected line indicates results of PPO alone, while the dashed line indicated PPO with inhibitor(s). Inhibition activit y was observed in the 3 mL standard reaction mixture.

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50 0 10 20 30 40 50 60 70 80 4 4.5 5 5.3 5.5 5.7 p H% Inhibition0 20 40 60 80 100 120% Relative PPO activity Figure 4-7. Optimum pH for apple PPO inhibiti on by cockroach extract. This was determined by measuring the PPO activity of apple mixed with the inhibitor(s) in a standard reaction mixture where the pH of the main buffer ( 0.1 M acetate-acetic acid) varied from 4.0 to 5.7. Bars indicate %inhibition while the dashed like indicated %relative PPO activity without inhibitor(s). E ach data point represents m ean standard deviation of 2 experiments each with 3 replicates. Inhibition of PPO by insect tissues has been reported previously. Tsukamoto and others (1992) isolated inhibitors from the pupal extrac ts of houseflies and demonstrated inhibition on house fly PPO. Sugumaran and Nellaiappan (2000) isolated an endogenous inhibitor in the larval cuticle of the tobacco horn worm ( Manduca sexta). This PPO inhibitor from Manduca sexta inhibited the activity of both insect and pl ant PPOs and lacase. Yoruk and others (2003) isolated an inhibitor(s) from the common house fly ( Musca domestica). The isolated inhibitor(s) was found to be fairly heat-stable and low-molecular weight. The inhibitor(s) from Musca domestica inhibited the activity of ap ple PPO up to 90% at pH values above 5.0. PPO is thought to play a role in several regulatory bioche mical mechanisms of endogenous PPO activity in

PAGE 51

51 insects (Sugumaran and Nellaiappan 2000). Inse ct PPO activity may also be regulated by intrinsic inhibitors during development (Tsukamoto and others 1992; Sugumaran and Nellaiappan 2000). Characterization of Crude Inhibi tor(s) From German Cockroach Effect of Timed Incubati on on the Inhibitor(s) Incubation had a large effect on the unknown inhibitor(s) when the inhibitor and PPO were left to sit up to 30 min (Figure 4-8). When the time of incubation increased, rate of reaction at 420 nm also increased, which corresponds to a decrease in inhibiti on. However, incubation had minimal effect when inhibitor and substrat e were incubated over a length of 30 min. The rate at 420 nm remained fairly c onstant at each time after sligh tly decreasing from 0 to 1 min. Therefore, PPO inhibition studies were carried out initiating the reaction with the PPO as opposed to the substrate.

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52 Figure 4-8. Incubation time study. Ra te of reaction at 420 nm was determined after incubation of inhibitor/PPO compared to incubation of inhibitor/substrate over a time of 30 min. Each data point represents mean standard deviation of 3 replicates. Temperature Stability Temperature stability was st udied in order to measure th e effect of temperature on inactivation of inhibito r(s). The unknown inhibitor(s) from German cockroach extract used in this study was not very heat stable. It was completely inactivated by heating at 100 C for as little as 5 min. This is similar to results found by Sugumaran and Nellaiappan (2000) with tobacco horn worm, which was a heat-labile glycoprotein, inactivated by heating at 100 C for 10 min (Sugumaran and Nellaiappan 2000). Howeve r, Tsukamoto and others (1992) found their inhibitor(s) from housefly to be quite stable to heating, retaining 60% of their activity when heated to 80 C for 1 hour. Similarly, Yoruk and othe rs (2003) found their house fly inhibitor(s) to also be relatively heat stable, losing onl y 26% of its inhibitory activity on apple PPO upon 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0151 53 0 Time (min)Rate at 420nm Inhibitor/PPO Incubation Inhibitor/Substrate Incubation

PAGE 53

53 boiling at 100 C up to an hour. This is a crude way to characterize the inhibitor(s) since a number of enzymes may act on a comm on complex substrate (Whitaker 1994). The inhibitor(s) from German cockroach wa s found to be stable to repeated freezing and thawing cycles, losing only about 9% of its inhib itor activity. This is similar to results found by Yoruk and others (2003) with hous e fly, which lost only about 8% of its inhibito r activity to repeated freezing and thawing. Inhibitor activity of cockro ach extract stored at -20 C was found to remain stable up to 6 months. The pH Extraction Profile Inhibitor(s) extracted at a range of pH values from 4.5-7.5 was compared for its inhibitory activity on apple PPO to determine the pH extraction profile (Fi gure 4-9). The pH had a dramatic effect on the inhibitor(s ). It was most stable at a pH of 6.5. At a pH of 5.5 only about 40% relative inhibition was observed compared to pH 6.5. At pH levels of 4.5 and 7.5 only about 20% relative inhibition was observed when compared to those at pH 6.5. Similarly, pH was also reported to have a dramatic effect on the inhibitor from the tobacco horn worm (Sugumaran and Nellaiappan 2000). This inhibitor from the tobacco horn worm was also most stable around a neutral pH, losing its total activity upon exposure to pH 10 for 10 min. Yoruk and others ( 2003) also reported the inhibito r from common house fly to be unstable to changes in pH. In contrast to the German cockroach and t obacco horn worm, the housefly inhibitor(s) was most stable at acidic pH values and least stable at alkaline values. However, Tsukamoto and others (1992) observed the inhibitor(s) in house fl y pupae was quite stable over a wide pH range

PAGE 54

54 from 4.0 to 10.0. The extent of stability obt ained in these studies was not presented. 0 20 40 60 80 100 120 44 555 566 577 58pH% Relative inhibition Figure 4-9. The pH extraction prof ile of inhibitor(s). I nhibitor was extracted at pH values of 4.5, 5.5, 6.5, and 7.5 with 0.1 M sodium phosphate or sodium acetate buffer. Each data point represents mean standard deviati on of 2 experiments each with 2 replicates. Dialysis of Inhibitor(s) Dialysis of inhibitor extracts was pe rformed using 500, 2000, and 25000-Da molecularweight cut off (MWCO) dialysis membranes with water and buffer (Figure 4-10). Due to the changes pH have on the inhibitor(s) from coack roach extract, the extracts that went through dialysis with distilled water lost more than 40% inhibition compared to that of the control. When dialysis was performed using the control buffe r, a much smaller loss in %inhibition was observed. The 500-Da molecular weight cut off me mbrane lost only 8% inhibition compared to that of control. There was a slight decreasi ng trend as the molecular weight membrane got

PAGE 55

55 larger, with the 25000-Da membrane losing about 17% compared to that of the control. Since it appeared the unknown inhibitor(s) may in fact be larger than 25000-Da, ultrafiltration studies were performed to better approximate the molecular weight. 0 10 20 30 40 50 60 70 Control 500 2K 25KType of dialysis tubing% Inhibition Water Buffer Figure 4-10. Percent inhibition afte r dialysis with water or control buffer. Ea ch buffer data point represents mean standard deviation of 2 experiments ea ch with 3 replicates. Each water data point represents mean standard deviation of 3 replicates. Ultrafiltration Ultrafiltration is a type of membrane filtration in which hydr ostatic pressure forces liquid against a semipermeable membrane to separate so lids and solutes of high molecular weight from water and low molecular weight solutes which pass through the membrane (Matella and others, 2006). Inhibitor preparation was also characte rized by ultrafiltration st udies (Table 4-1). Ultrafiltration studies were conducted to determine the appr oximate molecular weight of the unknown inhibitor(s) required for futu re purification and to compar e with sizes from previous

PAGE 56

56 studies. The results indicated that the unknown i nhibitor(s) from German cockroach is larger than 100000 NMWL as more than 98% inhibitor ac tivity remained in the retentate at all 3 size ultrafiltration units. Ultr afiltration units with 10000, 50000, and 100000 NMWL retained 98.0, 98.7, and 99.5% respectively. These results may be similar to those f ound by Sugumaran and Nellaiappan (2000), from the inhibitor in the tobacco horn worm. They reported their endogenous glycoprotein to have a molecular weight of 380000 on SDS-PAGE gels. The size results are much larger than those found by Tsukamoto and others (1992) based on Sephadex G-25 gel filtration information, which showed the inhibitor to range in size from 3000 to 3500. Yor uk and others (2003) reported ultrafiltration results on the inhibitor(s) from the common house fly to retain 83, 67, and 51% of the inhibitor activity on 10000, 30000, and 50000 NMWL membranes. This would indicate a smaller inhibitor than the resu lts from this study, although th e crude mixture used in the cockroach and common house fly for ultrafiltration gives only an estimate of inhibitor size. Table 4-1. Ultrafiltration Relative % Inhibitor Activity NMWL Filtrate Retentate Control 0 100 10000 4.6 1.0 98.0 1.5 50000 5.4 1.8 98.7 0.8 100000 4.0 2.4 99.5 2.4 Ultrafiltration was performed using centrifugal filter units with specified nominal molecular weights limit (NMWL) membranes. Each data poin t represents mean standard deviation of an experiment with 3 replicates. Treatment of Inhibitor(s) with Trypsin Trypsin is a pancreatic seri ne protease. Trypsin has a pH optimum of 8. Treatment of inhibitor(s) with trypsin was ine ffective due to the instability of the inhibitor to changes in pH. By raising the pH of the inhibitor above 6.5 and back down again, the inhibition was lost in both control and test trypsin samples (Figure 4-11). Th erefore, its inconclusive whether there was an

PAGE 57

57 effect on the inhibitor(s) by tryps in, or if it was just the pH ch ange that caused the loss of inhibition being shown in both. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Control Cont. Trypsin Trypsin InhibitorRate at 420 nm Figure 4-11. Treatment of inhibito r(s) with trypsin. Each data point represents mean standard deviation of 2 experiment s each with 3 replicates. Treatment of Inhibitor(s) with Papain Papain is a sulfhydryl protease from Carica papaya latex. Treatment of inhibitor(s) with papain proved effective in decreas ing the inhibition in the test sa mple but not the control papain sample. The German cockroach inhibitor was too sensitive to pH changes required for treatment with trypsin to determine its eff ect on it. However, papain has an optimum pH of 6.0-7.0, which is similar to the German cockroach inhibitor, and the re sults indicated that cysteine protease may be effective in disrupting the inhibition (Figur e 4-12). The control had an average rate of 0.3284 compared to an average of 0.3058 fo r the test sample with papai n. The control papain sample

PAGE 58

58 had an average rate of 0.1598 compared to an average rate of 0.1467 for regular inhibitor, showing only a small decrease on the level of inhibition. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Control Control Papain Papain InhibitorRate at 420nm Figure 4-12. Treatment of inhibito r(s) with papain. Each data point represents mean standard deviation of 2 experiment s each with 3 replicates. Inhibitor Kinetics Inhibition by German cockroach extract on apple PPO was characterized using the Lineweaver-Burk method. Results indicated no n-competitive inhibition, as both the vertical intercept and slope terms of the equation are affected. (Figur e 4-13). The lines intercept at a point left of the y-axis and below the x-axis. Non-competitive inhibition is when a reversible inhibitor can bind to the enzyme at a site that is distinct from the active site. Inhibitor kinetics results from different i nhibitor sources vary. Oszmianski and Lee (1990) found the inhibitory effect of honey on apple PPO to also be non-competitive using a

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59 Lineweaver-Burk plot. In contrast, Son and ot hers (2000) found the inhibitory mode of oxalic acid on mushroom PPO to be of a competitive type. Kineticsy = 4.0964x 2.0171 R2 = 0.9982 y = 9.6659x 0.6628 R2 = 0.9993 y = 8.2383x 3.202 R2 = 0.9998 y = 5.8209x 2.4512 R2 = 0.9983 -25 -5 15 35 55 75 95 115 135 155 -2024681012141618 1/Ao1/Vo Control Inhib 1X Inhib 1/2X Inhib 1/4X Linear (Control) Linear (Inhib 1X) Linear (Inhib 1/2X) Linear (Inhib 1/4X) Figure 4-13. Inhibitor kinetics: Plot of the substrate-velocity data according to Lineweaver Burk method. Inhibition by cockroach extract on apple PPO was determined in the presence of three different concentrations of inhibitor solution (1X, X, X) for five different fixed concentrations of catechol (1, 0.5, 0.25, 0.125, and 0.0625M).

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60 CHAPTER 5 CONCLUSIONS A new type of inhibitor(s) was di scovered from German cockroach ( Blattella germanica ). Although minimal inhibition was found on mush room and banana, the crude inhibitor preparation reduced apple PPO activity up to 70 % and potato PPO up to 25% at a reaction pH optimal for the enzyme activity. The potential PPO inhibitor(s) was characterized using temperature stability, extraction pH adjustment, incubation time, treatment with protease, dialysis, ultrafiltration studies, and enzyme inhibitor kinetics. The inhibitor(s) from German cockroach was found to be heat labile, becoming inactivated when heated to 100 C for 5 min. However, the inhib itor was stable to freeze-thaw cycles and refrigerated storage. It was also discovered that the inhibitor(s) activity was influenced by changes in extraction pH, being most stable at pH 6.5 and dropping in % inhibition drastically when the pH moved in either direction. Incubation time was found to affect the inhibitor(s) when the inhibitor and PPO were allowed to incubate together for 30 min, however when the inhibitor and substrate were inc ubated, there was no effect on inhibition. Treatment with trypsin was inconclusive because of the sensitivity of the inhibitor(s) to changes in pH, which conflicts with the pH activit y range of trypsin. Therefore, treatment of the inhibitor(s) with papain was attempted since it has a pH optimum that is similar to the unknown inhibitor(s). Results showed a loss of inhibition, which may indi cate the presence of a protein. Sensitivity to changes in pH also proved to be a problem when performing dialysis with distilled water, but the inhibitor retained most of its inhibition when a sodium phosphate control buffer was used in place of water. Ultrafiltration studi es were performed to better estimate the molecular weight of the unknown i nhibitor(s). Results indicated that the i nhibitor(s) may be larger than 100000 NMWL, as more than 90% inhibi tion was retained compar ed to the control.

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61 Kinetic studies were performed using a Line weaver-Burk plot, and results indicated a noncompetitive inhibition of the cockroach inhibitor(s) on apple PPO. In order to study the amino acid composition, molecular weight, tertiary structure, substrate specificity, or kinetic parameters of the inhibitor(s), it must be completely separated from all other substances. These results are the first attempt at identifying an inhibitor(s) from the German cockroach on PPO from higher plants ; therefore further res earch is needed to completely purify the unknown inhibitor(s). Furt her studies may help clarify the mechanism by which this inhibitor(s) works on other plant PPOs, especially those from apple and potato.

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62 APPENDIX A RAW DATA Table A-1. The pH optimum of potato PPO pH 1 2 3 4 5 6 Average St Dev % Rel. Act. 4 0.02931 0.03086 0.029360.029010.028920.02774 0.0292 0.00100419.8121 4.5 0.02947 0.03067 0.032970.037110.038750.03463 0.033933 0.00361323.0236 5 0.05075 0.05215 0.050930.055670.053610.05277 0.052647 0.00183735.7205 5.5 0.09095 0.09355 0.088530.097650.095350.09648 0.093752 0.00347463.6100 6 0.15604 0.15607 0.170720.127620.140980.13288 0.147385 0.016352100.0000 6.5 0.11579 0.13151 0.129910.119790.119090.1125 0.121432 0.00765882.3908

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63 Table A-2. Optimum pH for potato PPO inhibition pH c I % inhibition Average St Dev 4 0.02901 0.02957 -1.93037 0.02874 0.02871 0.104384 0.02783 0.02782 0.035932 0.02856 0.02874 -0.63025 0.02892 0.02981 -3.07746 0.02774 0.02768 0.216294 -0.880241.341958 4.5 0.03875 0.0386 0.387097 0.03723 0.03616 2.874026 0.03476 0.03351 3.596087 0.03746 0.03627 3.176722 0.03711 0.0342 7.841552 0.03463 0.03331 3.811724 3.6145352.410945 5 0.05567 0.04855 12.78965 0.05503 0.04866 11.5755 0.05434 0.04758 12.44019 0.05385 0.048094 10.68895 0.05361 0.04662 13.03861 0.05277 0.04787 9.285579 11.636421.440139 5.5 0.09765 0.08936 8.489503 0.09638 0.08495 11.85931 0.09726 0.08832 9.191857 0.09673 0.08714 9.914194 0.09535 0.08396 11.94546 0.09648 0.08723 9.587479 10.164631.427687 6 0.15604 0.11852 24.04512 0.15315 0.12147 20.6856 0.14976 0.12032 19.65812 0.15589 0.12676 18.68625 0.14098 0.11956 15.19364 0.15607 0.12086 22.56039 20.138193.105412 6.5 0.11979 0.10619 11.3532 0.11934 0.10345 13.3149 0.11385 0.10057 11.66447 0.11857 0.10511 11.35194 0.11909 0.09897 16.89479 0.1125 0.10027 10.87111 12.575072.277233

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64 Table A-3. The pH optimum of apple PPO pH 4 4.5 5 5.3 5.5 5.7 6 6.5 7 0.22845 0.30943 0.4230.538970.5815 90.210570.13228 0.061630.04653 0.22633 0.30893 0.411070.538260. 594140.218420.13394 0.062860.0401 0.22462 0.30631 0.418960.542430. 592510.217650.12858 0.060050.04166 0.21856 0.30539 0.422920.526140. 586250.215340.12987 0.061290.04456 0.21965 0.30169 0.415330.542620. 575250.214980.12398 0.060670.04243 0.21853 0.30455 0.411550.539260. 583060.215490.13845 0.061380.04521 Average 0.22269 0.30605 0.4171383330.537947 0.5854670.2154080.131183 0.0613130.043415 St Dev 0.00433 0.00288 0.0053372180.0060 70.007080.0027460.00494 0.0009490.00242 % R.A. 38.03632 52.27454 71.2488613191. 883410036.7925922.40663 10.472567.415452 % Rel St Dev 0.739627 0.491972 0.911617752 1.03673300.4690080.843697 0.1621710.413301 Table A-4. Inhibition of apple PPO activity by inhibitor(s) from German cockroach Control Inhibitor 0 0.0078 0.0085 10 0.0252 0.0159 20 0.0554 0.0296 30 0.1024 0.0466 40 0.1427 0.0687 50 0.1851 0.0891 60 0.2338 0.1071 70 0.2937 0.1293 80 0.3432 0.1506 90 0.3899 0.1702 100 0.4303 0.1905 110 0.4806 0.208 120 0.5179 0.2305

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65 Table A-5. Optimum pH for apple PPO inhibition % inhibition at varying pH pH c I % inhibition Average St Dev 4 0.13576 0.13657-0.59664113 0.13478 0.132981.33550972 0.13268 0.130471.665661743 0.12945 0.127741.320973349 0.13239 0.13081.200997054 0.12826 0.126481.387806019 1.0523840.82247 4.5 0.19705 0.184236.505962954 0.19688 0.184216.435392117 0.19345 0.184564.595502714 0.18078 0.173673.932957186 0.1809 0.173733.963515755 0.18011 0.173413.71994892 4.858881.282483 5 0.32254 0.2864811.18000868 0.32304 0.2869911.15960872 0.32277 0.2854611.55931468 0.32234 0.2891310.30278588 0.32166 0.292179.168065659 0.32249 0.284311.84222767 10.868670.981699 5.3 0.36795 0.242834.01277347 0.34745 0.2626624.4035113 0.34731 0.2377231.55394316 0.35744 0.281821.16159355 0.34983 0.2632924.73772975 0.34586 0.262424.13115133 26.666784.968462 5.5 0.33058 0.1145565.34878093 0.32784 0.1250661.85334309 0.32213 0.1313659.22143234 0.32957 0.1288860.89449889 0.28904 0.123657.23775256 0.28611 0.1236656.77886128 60.222443.198734 5.7 0.08827 0.023873.03727201 0.08832 0.0239272.91666667 0.08735 0.0267869.34172868 0.08769 0.021575.48181092 0.08752 0.0254670.9095064 0.08947 0.0247972.29238851 72.32992.084577

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66 Table A-6. The pH extrac tion profile of inhibitor 4.5 C I % inhibition % relative 0.33963 0.2967512.62550423 0.33755 0.2818616.49829655 0.32779 0.293110.58299521 0.38387 0.3439610.3967489 0.38385 0.347739.409925752 0.37544 0.355985.183251651 Average 0.358022 0.31989710.78278705 18.45855 St Dev 3.728032792 5.5 C I % inhibition % relative 0.32017 0.2425124.25586407 0.32803 0.2519223.20214615 0.33137 0.2407527.34707427 0.3877 0.268430.77121486 0.38776 0.2958923.6924902 0.38447 0.3106719.19525581 Average 0.356583 0.26835724.74400756 42.35812 St Dev 3.939876636 6.5 C I % inhibition % relative 0.31495 0.1302158.65692967 0.31595 0.1361956.89507834 0.30746 0.1445852.97599688 0.32784 0.1250661.85334309 0.32213 0.1313659.22143234 0.32957 0.1288860.89449889 Average 0.31965 0.13271358.4162132 100 St Dev 3.180310962 7.5 C I % inhibition % relative 0.07942 0.070611.10551498 0.07611 0.0662113.00748916 0.0767 0.069439.478487614 0.07192 0.069143.865406007 0.06458 0.059477.91266646 0.07055 0.0611813.28136074 Average 0.073213 0.0660059.77515416 16.73363 St Dev 3.549459924

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67 Table A-7. Dialysis Dialysis C I % Inhibition Average St Dev Control 0.22959 0.0930859.45816455 0.22526 0.0851162.2169937 0.23955 0.0842664.8257148862.166962.684125 500 0.32677 0.2684217.85659638 0.32031 0.2698115.76597671 0.32747 0.2710217.2382202916.95361.073979 2K 0.32947 0.2795815.14250159 0.34214 0.265422.42941486 0.32647 0.2660118.5193126518.697083.646708 25K 0.25895 0.241136.881637382 0.24773 0.226698.493117507 0.24659 0.234195.0285899676.8011151.733667 500 buffer 0.33536 0.1475156.01443225 0.32008 0.1498853.17420645 0.31817 0.1538351.65163277 0.32716 0.1440655.96649957 0.31128 0.1492252.06245181 0.31804 0.1494353.0153439853.647431.90261 2K buffer 0.26329 0.1345548.89665388 0.26252 0.1368447.87444766 0.26281 0.1421445.91530003 0.26879 0.1405947.69522676 0.26523 0.1399547.23447574 0.26169 0.136547.839046247.575860.978894 25K buffer 0.28562 0.1623143.17274701 0.29943 0.1667144.32421601 0.28842 0.1637743.21822342 0.2984 0.1577247.14477212 0.298 0.1588646.69127517 0.29031 0.1580545.5581964145.018241.716992

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68Table A-8. Treatment of inhib itor(s) with tryps in and papain Trypsin Papain Control Cont. Trypsin Trypsin Inhibitor Control Control Papain Papain Inhibitor 0.33737 0.265890.281430.15159 0.326450.152350.301930.14985 0.33511 0.279240.279720.1555 0.327070.171340.291450.15139 0.3316 0.274950.281480.13843 0.331430.157820.314560.14523 0.32706 0.275940.280320.14863 0.328430.162380.305430.14679 0.33338 0.266730.281560.14513 0.335670.153560.314020.14673 0.31583 0.273910.281330.1464 0.321450.161430.307540.14045 Average 0.330058333 0.27277670.280973330.147613333 Average0.3284166670.1598133330.305821670.14674 St Dev 0.007795197 0.0053250.000766070.005848414St Dev 0.0048147050.0069403790.008583320.003825

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69Table A-9. Ultrafiltration Ultrafiltration % Inhib Avg Control 0.29496 0.11734 60.2183361.35971 0.29018 0.11344 60.90702 0.29393 0.10889 62.95376 Control Filtrate Retentate % Inhib F % Inhib R Avg. F Avg R. Std Dev F Std Dev R 10,000 0.25625 0.25128 0.106041.93951258.618 542.82144360.15248 1.0479091.511957 0.25423 0.24776 0.101192.5449460.19746 0.24649 0.23668 0.094553.97987761.64145 50,000 0.29496 0.28594 0.115963.05804260.686 193.34106560.58759 1.827710.800619 0.29018 0.28533 0.116821.67137659.74223 0.29393 0.27837 0.113655.29377761.33433 100,000 0.25625 0.24633 0.095233.8712262.837 072.42949461.07837 2.3549192.378966 0.25423 0.24481 0.096543.70530662.02651 0.24649 0.2472 0.10261-0.2880458.37154 NMWL Filtrate St Dev +/Retentate Std Dev +/Control 0 100 10000 4.6 1.0 98.01.5 50000 5.4 1.8 98.70.8 100000 4.0 2.4 99.52.4

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70Table A-10. Inhibitor kinetics Ao Vo rep 1 Vo rep 2 1/Ao 1/Vo rep 1 1/Vo rep 2 1/Vo Avg Inhib Conc Slope Control 1 0.31212 0.3089713.20389593.23656023.220228 19.6659 0.5 0.15186 0.1559126.5850 1256.41395686.499485 0.58.2383 0.25 0.07609 0.07308413.142 33113.68363413.41298 0.255.8209 0.125 0.03464 0.03323828.8683630.09328929.48082 04.0964 0.0625 0.01558 0.015531664.18485264.391564.28818 Inhib 1X 1 0.13398 0.1313217.46380067.61498637.539393 0.5 0.05341 0.05292218.72308618.89644718.80977 0.25 0.02535 0.02725439.44773236.69724838.07249 0.125 0.01216 0.01318882.23684275.87253479.05469 0.0625 0.006656 0.00643216150.23136155.47264152.852 Inhib 1/2X 1 0.20159 0.1575614.96056356.34678855.653676 0.5 0.08604 0.06521211.62250115.33507113.47879 0.25 0.03976 0.02936425.15090534.05994629.60543 0.125 0.01984 0.01381850.40322672.41129661.40726 0.0625 0.008594 0.00703716116.35754142.10399129.2308 Inhib 1/4X 1 0.27142 0.2042713.68432694.89548154.289904 0.5 0.1325 0.0833127.547169812.0033619.775265 0.25 0.06678 0.03793414.97454326.36435520.66945 0.125 0.03355 0.01872829.80625953.41880341.61253 0.0625 0.01539 0.0084241664.977258118.7140991.84567

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81 BIOGRAPHICAL SKETCH Paul Justin Kurt Grotheer wa s born in Springfield, Virginia outside Washington D.C. while his dad was working at the Penta gon. His dads career in the Air Force led his family to move frequently while he was growing up, including stay s in Tampa, Florida; Stuttgart, Germany; and Vicenza, Italy. When his dad finally retired fr om the military, Paul finished up high school in Tampa, Florida, before coming to the University of Florida. Paul earne d a Bachelors degree in Food Science and Human Nutrition specializing in Dietetics and also kept busy as a member of the Florida Drumline playing at home and away football games. Paul then decided to pursue a Masters of Food Science, working w ith Dr. Marshall in the Food and Environmental Toxicology Lab. He is also completing a minor in Food and Resource Economics. Paul enjoys playing and watching sp orts, traveling, kayaking, and brewing beer in his spare time. He plans to pursue a career in food safety in the food industry upon completion of his degree.