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Novel Blue Mussel (Mytilus edulis) Extract Inhibits Polyphenol Oxidase in Fruit and Vegetable Tissue

Permanent Link: http://ufdc.ufl.edu/UFE0024150/00001

Material Information

Title: Novel Blue Mussel (Mytilus edulis) Extract Inhibits Polyphenol Oxidase in Fruit and Vegetable Tissue
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Bent, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: browning, enzymatic, hypotaurine, inhibition, melanin, mussel, polyphenol, polyphenoloxidase, ppo
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 in processed fruits and vegetables, mediated by the catalyst polyphenol oxidase (PPO; EC number 1.14.18.1), is a significant source of waste of both financial and other resources. Though there are a number of traditional browning inhibitors in commercial use today, no single inhibitor has yet been found that can be successfully used in every application. Our goal was to characterize the anti-browning and kinetic properties of a novel PPO inhibitor isolated from blue mussel (Mytilus edulis). To isolate the inhibitor, frozen mussels were thawed and the drip loss extracted. The mussel meats were also squeezed to extract the aqueous contents. The liquid from the mussels was filtered through a glass filter followed by a 0.45 micron filter, both under vacuum. Following filtration, the liquid was dialyzed using 500 dalton molecular weight cutoff membrane for 24 hours against distilled water, including three water changes. After dialysis, the extract was run through a Sephadex G-25 size exclusion chromatography column, which eluted two fractions. The fraction that eluted second was freeze-dried and the resulting powder reconstituted when needed for experimentation. Apple, banana, avocado, and potato PPOs were extracted from the crops into an acetone powder and were then reconstituted for use at a ratio of 1g powder to 50 mL of 0.1 M KH2PO4/Na2HPO4, pH 7.2 buffer. The mixture was stirred at 4 degrees Celsius for 30 minutes, centrifuged at 12000xg for 30 minutes, and the supernatant collected. Machine Vision was used to study the inhibitory effects of the new compound on purees of the four crops mentioned earlier. 3 grams of puree was mixed with 1.5 mL of mussel extract reconstituted to a volume of 5 mL with distilled water. Pictures were taken in a lightbox to control illumination at times 0, 1, 5, 10, 30, 60, and 120 minutes, as well as 24, 48, 72 hours and 7 and 14 days. Color analysis was performed using LensEye software to determine percentage surface area covered by each of 4096 color blocks. Average L-star values also were recorded and analyzed for changes over time. Kinetic analysis was conducted using Dixon and Cornish-Bowden plots in concert, to determine the types of inhibition, as well as Prism software to perform non-linear regression in order to ascertain the kinetic parameters Km, Vmax, and Ki. Analysis of the data from the Machine Vision studies showed that the inhibitor reduced browning in all four substrates, though not equally well in all. L-star values showed that browning was significantly reduced in all crops tested but avocado by one hour. Avocado showed a significant decrease in browning by 24 hours. Kinetic analysis showed that in the cases of apple and avocado PPO, the novel compound acts as a competitive inhibitor, while in avocado and potato, it is a mixed inhibitor. In addition, the Ki values calculated for the inhibitor in all crops tested compared favorably to other PPO inhibitors. Over the last couple of decades, much research has attempted to find suitable inhibitors for PPO in processed crop products. This novel inhibitor shows a number of qualities, including strong inhibition on a number of different forms of PPO as well as long lasting inhibition, that make it a good option for many food products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Robert Bent.
Thesis: Thesis (M.S.)--University of Florida, 2009.
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 2009
System ID: UFE0024150:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024150/00001

Material Information

Title: Novel Blue Mussel (Mytilus edulis) Extract Inhibits Polyphenol Oxidase in Fruit and Vegetable Tissue
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Bent, Robert
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: browning, enzymatic, hypotaurine, inhibition, melanin, mussel, polyphenol, polyphenoloxidase, ppo
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 in processed fruits and vegetables, mediated by the catalyst polyphenol oxidase (PPO; EC number 1.14.18.1), is a significant source of waste of both financial and other resources. Though there are a number of traditional browning inhibitors in commercial use today, no single inhibitor has yet been found that can be successfully used in every application. Our goal was to characterize the anti-browning and kinetic properties of a novel PPO inhibitor isolated from blue mussel (Mytilus edulis). To isolate the inhibitor, frozen mussels were thawed and the drip loss extracted. The mussel meats were also squeezed to extract the aqueous contents. The liquid from the mussels was filtered through a glass filter followed by a 0.45 micron filter, both under vacuum. Following filtration, the liquid was dialyzed using 500 dalton molecular weight cutoff membrane for 24 hours against distilled water, including three water changes. After dialysis, the extract was run through a Sephadex G-25 size exclusion chromatography column, which eluted two fractions. The fraction that eluted second was freeze-dried and the resulting powder reconstituted when needed for experimentation. Apple, banana, avocado, and potato PPOs were extracted from the crops into an acetone powder and were then reconstituted for use at a ratio of 1g powder to 50 mL of 0.1 M KH2PO4/Na2HPO4, pH 7.2 buffer. The mixture was stirred at 4 degrees Celsius for 30 minutes, centrifuged at 12000xg for 30 minutes, and the supernatant collected. Machine Vision was used to study the inhibitory effects of the new compound on purees of the four crops mentioned earlier. 3 grams of puree was mixed with 1.5 mL of mussel extract reconstituted to a volume of 5 mL with distilled water. Pictures were taken in a lightbox to control illumination at times 0, 1, 5, 10, 30, 60, and 120 minutes, as well as 24, 48, 72 hours and 7 and 14 days. Color analysis was performed using LensEye software to determine percentage surface area covered by each of 4096 color blocks. Average L-star values also were recorded and analyzed for changes over time. Kinetic analysis was conducted using Dixon and Cornish-Bowden plots in concert, to determine the types of inhibition, as well as Prism software to perform non-linear regression in order to ascertain the kinetic parameters Km, Vmax, and Ki. Analysis of the data from the Machine Vision studies showed that the inhibitor reduced browning in all four substrates, though not equally well in all. L-star values showed that browning was significantly reduced in all crops tested but avocado by one hour. Avocado showed a significant decrease in browning by 24 hours. Kinetic analysis showed that in the cases of apple and avocado PPO, the novel compound acts as a competitive inhibitor, while in avocado and potato, it is a mixed inhibitor. In addition, the Ki values calculated for the inhibitor in all crops tested compared favorably to other PPO inhibitors. Over the last couple of decades, much research has attempted to find suitable inhibitors for PPO in processed crop products. This novel inhibitor shows a number of qualities, including strong inhibition on a number of different forms of PPO as well as long lasting inhibition, that make it a good option for many food products.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Robert Bent.
Thesis: Thesis (M.S.)--University of Florida, 2009.
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 2009
System ID: UFE0024150:00001


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1 NOVEL BLUE MUSSEL ( Mytilus edulis ) EXTRACT INHIBITS POLYPHENOL OXIDASE IN FRUIT AND VEGETABLE TISSUE By ROBERT SCHWARTZ BENT 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 2009

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2 2009 Robert Schwartz Bent

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3 To my family.

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4 ACKNOWLEDGMENTS First and forem ost, I thank my family for their unconditional support in this journey through graduate school. It would have been impossible without their help. I thank Dr. Marshall and my committee for their time, ef fort, and personal atte ntion over the last th ree years. I thank Emilia for her patience and company through many la te nights in the lab. Finally, I thank Dr. Kurt Schulbach for his incredible dedication to this project and for the numerous lessons he taught me during my time working with him.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... .............11 CHAP TER 1 INTRODUCTION .................................................................................................................. 13 2 LITERATURE REVIEW .......................................................................................................17 Problems Surrounding PPO .................................................................................................... 17 Biochemistry of PPO Reactions ............................................................................................. 18 Plant PPO ................................................................................................................................20 Properties of Apple, Banana, Potato and Avocado PPOs ....................................................... 24 Arthropod PPO ................................................................................................................. ......26 Hypotaurine ................................................................................................................... .........29 Blue Mussel ............................................................................................................................30 Inhibition of Enzymatic Browning ......................................................................................... 30 Objectives and Hypothesis .....................................................................................................33 3 MATERIALS AND METHODS ...........................................................................................38 Machine Vision Analysis of Color Development ................................................................... 38 Preparation of Inhibito r from Blue Mussel ..................................................................... 38 Preparation of Puree for Testing Browni ng Inhibition of Blue Mussel Extract .............. 39 Machine Vision Analysis ........................................................................................................40 Enzyme Kinetics ............................................................................................................... ......40 Polyphenol Oxidase Preparation ..................................................................................... 40 Determination of IC50 ..................................................................................................... 42 Electrophoretic Separation of PPO Isoforms .................................................................. 43 4 RESULTS AND DISCUSSION ............................................................................................. 45 Machine Vision Analysis of Color Development ................................................................... 45 Enzyme Kinetics ............................................................................................................... ......48 Comparison of Novel Inhibitor to Other Browning Inhibitors ...............................................50 5 CONCLUSIONS ................................................................................................................... .75 APPENDIX ADDITIONAL DATA .......................................................................................... 77

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6 LIST OF REFERENCES ...............................................................................................................90 BIOGRAPHICAL SKETCH .......................................................................................................101

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7 LIST OF TABLES Table page 2-1 pH and temperature optima for apple, ba nana, potato, and avocado PPOs, as well as substrate specificities ordere d from highest to lowest ....................................................... 35 3-1 Camera settings for Machine Vision experim ents ............................................................. 44 4-1 Kinetic parameters for apple, avocado, banana, and potato .............................................. 53

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8 LIST OF FIGURES Figure page 2-1 Locations within a plant cell of pheno lic com pounds, as well as polyphenol oxidase and peroxidase enzymes (Toivonen and Brummell 2008). ...............................................35 2-2 Mechanism of action for PPO upon its substrate compounds (Toivonen and Brumm ell 2008). ............................................................................................................... .36 2-3 PPO-catalyzed oxidation of L-DOPA to m elanin .............................................................. 37 4-1 Untreated samples in the first apple trial; 0-5 m inutes a nd 10-120 minutes; color blocks and percentage surface areas ..................................................................................54 4-2 Untreated samples in the first apple trial; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................55 4-3 Treated samples in the first apple trial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..............................................................................................56 4-4 Treated samples in the first apple trial; 24-72 hours and 1-2 w eeks; color blocks and percentage surface areas ....................................................................................................57 4-5 Untreated samples in the second apple tr ial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................58 4-6 Untreated samples in the second apple tria l; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................59 4-7 Treated samples in the second apple tr ial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................60 4-8 Treated samples in the second apple tria l; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................61 4-10 Untreated samples in the first banana tr ial; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................63 4-12 Treated samples in the first banana tria l; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................65 4-9 Untreated samples in the first banana trial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................62 4-11 Treated samples in the first banana trial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................64

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9 4-13 Apple and avocado: comparison between c ontrol and treated L* values at each tim e point ......................................................................................................................... ..........66 4-14 Banana and potato: comparison between contro l and treated L* values at each time point ......................................................................................................................... ..........67 4-15 Apple kinetics plots............................................................................................................68 4-16 Avocado kinetics plots .......................................................................................................69 4-17 Banana kinetics plots .........................................................................................................70 4-18 Potato kinetics plots ...........................................................................................................71 4-23 Results of native gel electrophoretic separation of crude PPO isolates. ............................ 74 4-19 Apple: plots of percent activity vs. in hibitor concentration to determ ine IC50 .................72 4-20 Avocado: plot of percent activity vs. inhibitor concentrati on to determ ine IC50 ............. 72 4-21 Banana: plot of percent activity vs. in hibitor concentration to determ ine IC50 ................73 4-22 Potato: plot of percent activity vs. in hibitor concentration to determ ine IC50 .................. 73 A-1 Untreated samples in the first avocado trial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................77 A-2 Untreated samples in the first avocado tr ial; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................78 A-3 Treated samples in the first avocado trial; 0-5 m inutes a nd 10-120 minutes; color blocks and percentage surface areas ..................................................................................79 A-4 Treated samples in the first avocado tr ial; 24-72 hours and one week; color blocks and percen tage surface areas ..............................................................................................80 A-5 Untreated samples in the first potato trial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................81 A-6 Untreated samples in the first potato trial; 24-72 hours and 1-2 weeks; color blocks and percen tage surface areas ..............................................................................................82 A-7 Treated samples in the first potato tr ial; 0-5 m inutes and 10-120 minutes; color blocks and percentage surface areas ..................................................................................83 A-8 Treated samples in the first potato trial; 24-72 hours and 1-2 week s; color blocks and percen tage surface areas ....................................................................................................84

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10 A-9 Apple: browning progression in treate d and untreated sam ples during Machine Vision experiment ............................................................................................................. .85 A-10 Avocado: browning progression in treated and untreated sam ples during Machine Vision experiment ............................................................................................................. .86 A-11 Banana: browning progression in treate d and untreated sam ples during Machine Vision experiment ............................................................................................................. .87 A-12 Potato: browning progression in treate d and untreated sam ples during Machine Vision experiment ............................................................................................................. .87 A-13 Time zero SAS output for apple machine vision L* data .................................................. 88 A-14 Time 24 hours SAS output for apple m achine vision L* data ........................................... 88 A-15 Color block reference legend. ............................................................................................ 89

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11 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 NOVEL BLUE MUSSEL ( Mytilus edulis ) EXTRACT INHIBITS POLYPHENOL OXIDASE IN FRUIT AND VEGETABLE TISSUE By Robert Schwartz Bent May 2009 Chair: Maurice R. Marshall Major: Food Science and Human Nutrition Enzymatic browning in processed fruits and vegetables, mediated by the catalyst polyphenol oxidase (PPO; EC number 1.14.18.1), is a significant source of waste of both financial and other resources. Though there are a number of traditional browning inhibitors in commercial use today, no single inhibitor has yet been found that can be successfully used in every application. Our goal was to characteri ze the anti-browning and kinetic properties of a novel PPO inhibitor isolated from blue mussel ( Mytilus edulis ). To isolate the inhibitor, frozen mussels were thawed and the drip loss extracted. The mussel meats were also squeezed to extract the aq ueous contents. The liquid from the mussels was filtered through a glass f ilter followed by a 0.45 micron filter, both under vacuum. Following filtration, the liquid was dialyzed usin g 500 dalton molecular weight cutoff membrane for 24 hours against distilled water, including thr ee water changes. Afte r dialysis, the extract was run through a Sephadex G-25 size exclusi on chromatography column, which eluted two fractions. The fraction that eluted sec ond was freeze-dried and the resulting powder reconstituted when needed for experimentation. Apple, banana, avocado, and potato PPOs were extracted from the crops into an acetone powder a nd were then reconstituted for use at a ratio of

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12 1g powder to 50 mL of 0.1 M KH2PO4/Na2HPO4, pH 7.2 buffer. The mixture was stirred at 4 C for 30 minutes, centrifuged at 12000xg for 30 minutes, and the supernatant collected. Machine Vision was used to study the inhi bitory effects of the new compound on purees of the four crops mentioned earlie r. 3 grams of puree was mixed with 1.5 mL of mussel extract reconstituted to a volume of 5 mL with distilled water. Pictur es were taken in a lightbox to control illumination at times 0, 1, 5, 10, 30, 60, and 120 minutes, as well as 24, 48, 72 hours and 7 and 14 days. Color analysis was performed us ing LensEye software to determine percentage surface area covered by each of 4096 color blocks. Average L* values also were recorded and analyzed for changes over time. Kinetic analysis was conducted using Dixon and CornishBowden plots in concert, to determine the type s of inhibition, as well as Prism software to perform non-linear regression in order to ascertain the kinetic parameters Km, Vmax, and Ki. Analysis of the data from the Machine Visi on studies showed that the inhibitor reduced browning in all four substrates, though not equally well in all. L* values showed that browning was significantly reduced in all crops tested but avocado by one hour. Avocado showed a significant decrease in browning by 24 hours. Kinetic analysis showed that in the cases of apple and avocado PPO, the novel compound acts as a co mpetitive inhibitor, while in avocado and potato, it is a mixed inhibitor. In addition, the Ki values calculate d for the inhibitor in all crops tested compared favorably to other PPO inhibitors. Over the last couple of decades, much research has attempted to find suitable inhibitors for PPO in processed crop products. This novel inhibitor shows a number of qualities, including strong inhibition on a number of different forms of PPO as well as long lasting inhibition, that make it a good option for many food products.

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13 CHAPTER 1 INTRODUCTION Polyphenol oxidase (EC numb er 1.14.18.1), also known as tyrosinase, plays a number of roles in various ecological systems. It has been widely studied as a browning agent in fruits and vegetables (Akissoe and others 2005, Ganda-Herre ro and others 2005, Spagna and others 2005). Despite its generally negative re putation regarding browning, it also plays a positiv e role in the production of tea, coffee, and cocoa, all of wh ich require polyphenol oxi dase (PPO)-mediated browning. It appears as well in crustacean and fungal systems (Jaenicke and Decker 2003). In these species, as well as in some insects, PPO catalyzes reactions n ecessary for the proper development of the organism. According to Lee and Whitaker (1995), over 50 % of fruit losses occu r due to enzymatic browning. The fruit and vegetable industry is a steadily growing sector that supplies a large amount of nutritious food eaten in the US. In 2002, fruit and berry production supplied $11.2 billion in farm cash receipts to growers. Vegetable and melon sales accounted for $12.8 billion (Kipe 2004). Supporting these large sales numbers 76% of families in the US buy fresh-cut produce at least once a month, a commodity highly susceptible to enzyma tic browning, (Govt of Ontario 2006). This trend of in creased sales and consumption is not confined to the US. In 2002, Canada produced $517 million in fruit crops. Canadians also showed a per capita consumption of 72 kg of fruit per year. This figu re is 19% higher than on e decade earlier (Govt of Saskatchewan 2005). The largest producer of apples in the world has, for the past few years, been China. In 2004, China produced over 23 million metric tons of apples. The US came in second with almost five million metric tons of apples produce d. The value of only the apples exported from the US in 2004 was over 383 million dollars (U SDA Economic Research Service 2007a).

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14 Banana production is an economically impor tant crop to many countries, including the US. The highest production of bananas comes from India, with almost 17 million metric tons of bananas grown in 2004. One of the largest exporte rs of bananas in the world is Ecuador which, in 2004, exported over a billion dollars worth of bananas (USDA Econo mic Research Service 2007b). Avocado production is a major economic factor for Mexico, which produced just under a million metric tons of avocados in 2004. Th e US produced over 162,000 metric tons during the same year. The value of avocados exporte d by Mexico in 2004 was over 211 million dollars (USDA Economic Research Service 2007c). In the case of potatoes, China is again the largest producer with over 70 million metric tons in 2004. The US comes in fifth with just over 20 million metric tons. Potato exports from the US accounted for 72 million dollars, though other countries totaled far more in potato sales. The Netherlands, which produced only about 7.5 m illion metric tons of potatoes, sold over 497 million dollars worth of potatoes in 2004 (US DA Economic Research Service 2007d). Damage to potato crops due to impact injuries and th e consequent PPO-mediat ed browning could cause over 20% product loss (Storey and Davies 1992). Clearly, given the large amounts and values of these four commodities produced every year throughout the world, the potential for economic loss due to enzymatic browning is significant. The large numbers of fruits and vegetables produced, as well as their value in the marketplace, demonstrate why enzymatic browning losses every year garner great interest in finding a widely applicable, safe, and economical means of browning inhibition. While there are a number of methods to help prevent enzymatic br owning, not all of these ar e useful in fresh-cut fruit and vegetables, either due to taste, safety, or regulatory concerns.

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15 In addition to fruit and vegetable losses, postharvest melanosis of crustaceans, specifically lobster and shrimp, also causes se vere monetary losses. Although melanosis does not actually degrade the taste or safety of the crustaceans, buyers will reject the products simply due to the brown or black discoloration (Marshall and others 2000). The prevalence of aquaculture as a method for producing seafood is growing throughout the world, especially in China, Chile, and Thailand. In addition, the US inte rest in aquaculture is significant. The value of US aquaculture products in 2001 was 935 million dollars (Harvey 2004). Prevention of post-mortem degradation of shellf ish species has historically been of great interest due to the high perish ability of the food products crea ted from marine animals. Commercial, temperature-dependent methods for preservation of aquatic species include storage in flake ice, refrigerated seawater, brine so lutions, and more recently, modified atmospheres (Aubourg and others 2007). As well, sulphites are a very common and powerful chemical PPO inhibitor used in seafood preservation. However, sulphites have been implicated in allergic reactions in asthmatics (Lpez-Caballero and othe rs 2007). A chemical PPO inhibitor that does not cause adverse r eactions in consumers could be beneficial to many food industries, including seafood products. In addition, the combination of a powerful anti-browning compound in conjunction with current commercial preservation methods may yield even better results than present commercial practices. As mentioned previously, a number of methods for inhibition of PPO-mediated browning exist. Yet none is universally applicable, and most show clear limitations in their usefulness as commercial browning inhibitors. Sulphites are a very effectiv e browning inhibitor, but are banned in fresh fruit and vegetable products. As corbic acid is another very useful browning

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16 inhibitor that is widely used commercially, es pecially in conjunction wi th citric acid. The ascorbic acid works as a pH modifier, bringi ng the food product out of the pH range that is optimal for the function of PPO. It also acts as a reducing agent. When PPO oxidizes its odiphenol substrates to o-quinones, ascorbic acid reduces the latter b ack to the former, slowing the browning process. Unfortunately, the ascorbic acid oxidizes to dehydroascorbic acid, which has no anti-browning property. Citric acid acts as a chelator, binding the copper atoms essential to the proper function of PPO (Jiang a nd others 1999). The anti-browni ng effect of ascorbic acid is limited; however, to the time that it maintains it s reducing ability. Thus, foods preserved with ascorbic acid eventually brown after all the ascorbic acid is oxidized to dehydroascorbic acid. The search for a more effective, safe, and economical PPO inhibitor is of great interest to fruit, vegetable, and seafood industries. The potential benefit of economic savings by way of a reduction in product loss due to enzymatic browning is large. An inhibitor was found in blue mussel and is presently being identified. It shows a number of properties, which may make it useful in the prevention of enzymatic browni ng. Research was performed to examine the effectiveness of the blue mussel inhibitor on a number of differen t fruits and vegetables (apple, banana, avocado and potato) known to be prone to enzymatic browning. Since sulphites are still used postharvest in the seafood industry to prevent melanosis in many aquatic animals, it was necessary to determin e that no sulphites were applied to the frozen mussels used in this project prior to their freezi ng and packaging. To this end, live blue mussels were acquired from Canada and tested for inhibi tion of apple PPO. The inhibitory strengths of the extracts from the live and frozen mussels we re the same, indicating that no sulphites were added to the frozen mussels during processing.

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17 CHAPTER 2 LITERATURE REVIEW Problems Surrounding PPO Polyphenol oxidase is one name for a class of enzymes that catalyze the oxidation of omonophenols to o-diphenols and odiphenols to o-quinones. The oxidation reaction also uses molecular oxygen as substrate. There is large variation in PP O structure and function between different species of plants and animals. It is found in almost every class of plant and animal, as well as fungi and bacteria. PPO types are ge nerally classified as either having mainly monophenolase (a.k.a. tyrosinase) or diphenolase (a.k.a. catechol oxidase) activity, though many PPOs exhibit both types of activity. PPO is located in numerous organelles within th e cells of plants and animals. It has been found in chloroplasts of photosynthe tic plants, aerial roots of orch ids, the leaves and seeds of coffee, in the mycelium and extra cellular matrix of fungi, and in the skin under the exoskeleton of crustaceans (Mayer 2006, Opoku-Gyamfua and ot hers 1992). Figure 2-1 shows some of the locations in which phenolic subs trates of PPO and the enzymes themselves have been found in plants. Generally, PPO is separated, by way of cellular compartmentalization, from its phenolic substrates. Still, physic al injury to a plant can cause the breakdown of these intracellular barriers, allowing for the contact of PPO and its substrates. Physical injury can include puncture wounds, impact injuries, abrasions, and commercial processing steps, including cutting, grinding, and pureeing. As PPO is present in almost all crops, it is a problem for numerous food commodity industries. Some crop industrie s that battle losses due to en zymatic browning include apple, avocado, banana, cucumber, grape, pineapple, ma ngo, peach, apricot, eggplant, cabbage, lettuce,

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18 potato, water chestnut, carambola, and stra wberry (Yoruk and Marshall 2003, Yueming and others 2004, Teixeira and others 2008, Chisar i and others 2007). Large volumes of crop products are wasted every year due to quality loss caused by PPO-mediated browning. The search for safe, cheap, and effective inhibitors for PPO draws great interest because of the widespread economic damage done by this single class of enzymes. In addition to plants, PPO also causes quality degradation in postharvest seafood products. Some marine species that undergo enzymatic browning af ter harvest include lobster, prawns, and shrimp (Slatery and others 1995, Aubourg and others 2007, Simpson and others 1997). PPO is activated and deactivated throughout the life-cycle of many marine species as a shell-hardening agent and possi bly as a disease-resistance mechanism (Yoruk and Marshall 2003). However, after harvest polyphenol oxida se causes the common problem known as black spot in many marine species. Although not a food safety or taste concern, the visual quality of the products and therefore marketab ility are significantly reduced by the black spots that appear on the flesh of the animal. Biochemistry of PPO Reactions Polyphenol oxidase is a copper-dependant oxidase enzyme that acts upon monophenol and o-diphenol compounds, oxidizing them into o-quinones. For the reactions to take place, PPO must come into physical contact with both molecular oxygen as well as substrate molecules. These reactants are normally separated from each other by way of intracellular compartmentalization. Under conditions of inju ry, whether in nature or during commercial processing, the breakdown of intracellular barrie rs can allow the three reactants to interact (Toivonen 2004). Depending on which substrate the enzyme is acting on, there will be either one or two main steps catalyzed by PPO to generate the fina l o-quinone structure. If the substrate is a

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19 monophenolic compound, such as tyrosine, the fi rst reaction is the oxidation of the monophenol to an o-diphenol. Once the o-diphenol has been formed, or if the substrate begins as an odiphenol, such as catechol, the fi nal step catalyzed by PPO is the oxidation of the o-diphenol into an o-quinone. Following the formation of the o-quinone product, numerous non-catalyzed oxidative condensation reactions take place duri ng which the o-quinone polymerizes with amino acids, phenolic compounds, proteins, and other o-qui nones. As the polymerization continues, the large, final products are the brownish melanins commonly seen on untreated, fresh-cut fruits and vegetables (Martinez and Whitaker 1995). Figure 2-2 diagrams a simp lified version of the reactions catalyzed by PPO, leading from th e monophenol substrate to the oxidized o-quinone product. Copper plays an instrumental role in the f unction of PPO. The act ive site of all PPO compounds contains two copper ions, which in thei r normal states are in the form of met-PPO (Lerch 1983). Each of the two copper atoms is bound by three conserved histidine residues. Both molecular oxygen and phenolic substrates bi nd to the copper-contain ing active site (Van Gelder and others 1997). When acting as a monophenol oxidase, PPO is reduced to deoxy-PPO by oxidizing one molecule of diphenol substrate to the o-quinone structure. After this reduction, the active site binds with a molecule of oxygen, giving the third form of PPO, oxy-PPO. Monophenolic compounds can then bind to the oxy-PPO active site, forming a PPOmonophenol-oxygen complex. After the monophenol is converted to an o-quinone, it is released from the active site and the cycle begins again. When acting as a diphenol oxidase, the first step is again the reduction of met-PPO to deoxy-PPO by the oxidation of one molecule of odiphenol substrate. Following this reduction,

PAGE 20

20 the PPO is then available to bind to a mol ecule of oxygen, forming oxy-PPO. Oxy-PPO then binds to a second molecule of o-diphenol, whic h is oxidized to the fi nal o-quinone product. The products and intermediate compounds of PPO-mediated oxidation of phenolic substrates can result in nutritive degradation of food products. The oxidized products of the PPO reactions can covalently bind to food proteins and amino acids, possibly changing their tertiary structure. This change in three dimensional conformation may degrade the ability of enzymes and other bioactive food proteins to perform th eir expected functions in the human body, since form and function are integrally tie d in the case of proteins. In addition, tyrosine free radicals produced during the PPO-catalyzed browning processes can also interact with food proteins, negatively affecting their func tional and nutritive characterist ics (Matheis and Whitaker 1984, Prigent and others 2007). Plant PPO Polyphenol oxidase is essent ially ubiquitous throughout the plant kingdom (Yoruk and Marshall 2003). It is located in a nu mber of different locales w ithin the cell, and is generally separated from its phenolic substrates. Howeve r, when the crop sustains injury, there is commonly a breakdown of intracellular compartm entalization, leading to enzyme-substrate contact and the browning reactions normally seen in fresh-cut fruits and vegetables (Toivonen and Brummell 2008). PPO catalyzes the first two major reactions in the transformation of mono and diphenols to o-quinones, which go on to form brown mela nin pigments by non-enzymatic condensation. The reactions that occur following the two PPO-catal yzed steps have historically been hard to document because they are generally insolubl e (Lee and Whitaker 1995). Figure 2-4 shows the steps leading from the diphenolic PPO substrate L-DOPA to its final form in a melanin-protein complex.

PAGE 21

21 Describing the possible functions of PPO in a living plant is problematic. The enzyme has been implicated in defense against pat hogens and insect infestation, as well as wound healing. The intermediate hydroxyphenolic compounds and fina l o-quinone products of PPOmediated oxidation reactions exhib it virucidal and bacteriocidal prope rties. In addition, the large polymerized phenolic complexes resulting from the non-catalyzed condensation of o-quinones and other compounds show f ungicidal action (Mayer and Harel 1979, Vaughn and others 1988, Macheix and others 1990, Scalbe rt 1991, Shaw and others 1991, Zawistowski and others 1991). The insoluble products of o-quinone condensation following enzymatic oxidation also may serve as wound caps, much like scars in humans (Vamos-Vigyazo 1981, Vaughn and others 1988, Zawistowski and others 1991). An insoluble blocking agent within an injury could also potentially decrease water loss following injur y. Water loss is a major influence on the postharvest quality of fruits and vegetable products. Dehydration signi ficantly decreases the quality and salability of many commodities (Smith and others 2006, Thomas and others 2006, Porata and others 2005). Finally, o-quinone products of enzymatic br owning reactions, as well as intermediate phenolic radical compounds may modi fy the structure of amino acids and proteins. This scheme of modification is generally termed an antinutriti ve defense mechanism th at is thought to deter insects from attacking a plant (Duffey and St out 1996). Quinone products from PPO-catalyzed oxidation of phenolic compounds ar e highly reactive compounds. Th ey are able to alkylate the amino groups of lysine and tryptophan residues, as well as other thiol groups within a proteins structure, crosslink proteins into large, insoluble complexes, and destroy many amino acids outright (Duffey and Felton 1991, Felton and others 1989, Felton and others 1992, Pierpoint 1969, Rawel and others 2001, Rawel a nd others 2002). Alkylation of lysine residues within

PAGE 22

22 plant proteins decreases the number of sites at wh ich an insects protease trypsin can attack the protein. Trypsin specifically cl eaves peptide chains at the carboxyl end of lysine and arginine residues. The crosslinking of pr otein molecules occurs either directly across an o-quinone, or by way of a phenolic or protein fr ee radical. The large size of the resulting protein complex causes its precipitation (Du ffey and Stout 1996). PPO has been shown to be encoded by nuclear genes, synthesized on cytoplasmic ribosomes, and remains membrane-bound and in a latent state until activated (Yoruk and Marshall 2003, Tolbert 1973, Lax and others 1984) Activation mechanisms shown to be effective in vitro include exposure to white and red light, treatment with trypsin or acids, aging, and the addition of the anionic detergent SD S (Tolbert 1973, SanchezFerrer and others 1993, Lerner and others 1972). In vivo activity of PPO may be influenced by a number of different sources, including developmental age, transcripti on-level upregulation of PPO expression due to injury, or exposure to certain fatty acids or e ndogenous proteases (Van Gelder and others 1997). The size of activated and latent PPOs varies wide ly between species. PPOs have been isolated weighing between 32 and 200 kDa, though most exist between 35 and 70 kDa (Flurkey 1986, Fraignier and others 1995, Sherman and others 1991, Steffens and others 1994, Van Gelder and others 1997, Yang and others 2000). Some in vitro studies have attributed the activation of PPO to cleavage of a peptide cap covering the activ e site of the enzyme. In one study using broad bean, latent PPO weighing 60 kDa was observed usi ng protease inhibitors. PPO from this crop is generally considered to be a 45 kDa protein. Following in vitro proteolytic cleavage of the latent enzyme using both trypsin and thermolysin, active proteins weighing 42 kDa, as well as smaller inactive fractions were observed (Robinson a nd Dry 1992). These data suggest that exogenous,

PAGE 23

23 as well as some possibly endogenous, proteases ma y cleave the larger, latent form of PPO to form the smaller, active form. In many organisms, PPO activity changes throughout the lifecycle. Generally, PPO activity has been shown to be higher in younger plants and decreas es as ripening and senescence takes place (Cipollini and Redman 1999, Murata and others 1995, Serradell and others 2000, Shahar and others 1992). There are a number of factor s that may contribute to this change in activity, including changes in PPO concentratio n, changes in concentrations of phenolic substrates for PPO, conformational changes and/ or denaturation of PP O, or a decrease in concentration of PPO activators (Murata and othe rs 1995, Laveda and others 2000). It is worth noting that the changes in PPO concentrations at different points during development may occur due to transcription-level decreases in expression of PPO genes and not simply because of denaturation or other means of protein degr adation (Chevalier a nd others 1999, Gooding and others 2001). Another means of increasing PPO activity in crops is through mechanical injury or infection. This increase in activ ity is believed to contribute no t only to wound healing, but also to defense against insect and microbiological infection (Duffey and St out 1996, Mayer and Harel 1979, Vamos-Vigyazo 1981). Commercial harv esting and processing methods pose serious threats to food crops. There is often physical damage, by way of cutting, grinding, peeling, and transport, incurred by a fruit or vegetable during these processes that eventually leads to a decline in quality such that it is no longer sala ble. Susceptibility to mechanical damage is influenced by a number of physical properties, including cultivar, dry ma tter content, mineral content, turgidity, temperature, shape, and size of the crop. In addition, vulnerability to bruising due to mechanical injury also depends on chemical factors, including PPO content and

PAGE 24

24 distribution, as well as concentr ation and location of phenolic s ubstrates for PPO (Partington and others 1999). It is not clear whether this in crease in PPO activity is a loca lized response to the wound or infection, or whether it is a systemic action under taken throughout the plant. There is evidence that, in a number of crops, inju ry induces an increase in PPO activity systemically by way of increased levels of available PPO mRNA. Whether this change is due to increased transcription of PPO mRNA or a physical modification of the mRNA is unknown (Thipyapong and others 1995). On the other hand, there is evidence that the increase in PPO activity may be localized. Both PPO activity and the concentration of phenolic substances increased around the wounded tissue of potato following injury. In this study, PPO was considered to be one of a number of factors highly associated w ith resistance to infection (Ray and Hammerschmidt 1998). Properties of Apple, Banana, Potato and Avocado PPOs The enzym es that comprise the class comm only referred to as PPOs are varied by many physicochemical properties. As with any enzyme the structure of the protein, including its tertiary characteristics, influences the ability of the PPO to interact with a specific substrate. Hence, each PPO has a different range and order of phenolic substrate specificities with which it best reacts. In addition, the fam ily of PPOs is also widely vari ed in other properties, including the temperature and pH at which each specific PPO best operates. Temperature and pH optima in enzymatic systems are determined by a number of different factors. In the case of both extreme temperature and pH protein quaternar y and tertiary structures are changed. Quaterna ry structure is affected by a di sassociation of protein subunits from each other, or a change in the spatial arrangement of subunits to each other. Both of these modifications may change the biochemical functionality of the protein.

PAGE 25

25 Tertiary structure is also affected by extr eme temperature and pH. Low pH will tend to cause H+ ions to protonate carboxyl groups on some amino acids as well as bind to the unoccupied pair of electrons on the nitrogen atoms of amino groups within amino acids. These changes will disrupt the normal elect rostatic interactions between amino acids that help form the tertiary structure of the protein, causing the protei n to unfold. In addition, as heat is added to a molecule, the strength of hydrogen bonds decrease. As the bonds become weaker, the stabilizing force usually given to the prot eins structure by these already weak bonds is lost. Acids and bases can also disrupt salt bri dges usually held together by io nic charges. Finally, reducing agents have the ability to destroy the disulf ide bonds formed by the oxidation of sulfhydryl groups on cysteine molecules (Ophardt 2003). The substrate specificity of different forms of PPO is not surprising. Different crops contain various possible phenolic substrates for PPO and these di fferent substrates contain a wide variety of structures. It is logical to assu me that, over time, plants evolved such that their specific type of PPO best fit the polyphenols that exist in the highest quantities within the plant. This tight fit between enzyme and substrate woul d assure the highest le vel of enzyme activity and the greatest affinity for substrate. It has been shown that even within a single plant; a number of different PPOs may exist within different tissues. The properties of thes e PPOs may vary widely. In red clover, three different PPOs exist. They differ in a number of qua lities. In their latent states, the pH optima of the three PPOs range from 5.1 to 6.9. After activ ation by room temperature incubation for six to eight days, the activities of th e three PPOs increased by 10-40 fold. Finally, the three PPOs showed different orders of substr ate specificity when assayed with caffeic and chlorogenic acids,

PAGE 26

26 catechol, dopamine, and other common substrates, though all showed the highest reactivity with caffeic acid (Schmitz and others 2008). It has been shown that, within one plant, the activity of PPO can be widely variable. One study looked at the distribution of PPO activity throughout the fruits of six different varieties of Japanese apples. PPO activity was determined to be 32-78 units (U)/mL pr eparation in the peel, 126-430 U/mL in the apple flesh, and 383-800 U/mL in the core (Wakayama 1995). Whether these differences were due to variations in PPO structures or distribution is unclear, but both are possibilities. These studies used four commodities grown throughout the world. Table 2-1 shows the differences in pH and temperature optima for PPO activity between the four crops. In addition, it also shows the preferred substrates for each specifi c PPO ordered from highest affinity to lowest. Arthropod PPO PPO is found throughout nature, including within the phylum arthropoda (OpokuGyamfua and others 1992, Chen and others 1991a). The PPOs found in arthropods, like those in plants, oxidize phenolic substrates to form o-quinone products that then polymerize into melanin compounds. However, arthropod PPOs are generally assayed for activity us ing Lor DL-DOPA, a substrate not usually used in plant PPO studies due to its low specificity to many plant PPOs (Opoku-Gyamfua and others 1992, Chen and ot hers 1991b, Yoruk and Marshall 2003). Though the mechanism of action and even the structure of arthropod PPO are rath er similar to those found in plants, the ways insects use the pro-ox idative properties of PPO is grossly different from plants (Opoku-Gyamfua and others 1992). As an arthropod grows, its hard exoskeleton becomes too small for its body, and it must molt its shell. This effect is commonly seen in softshell crabs, a culinary delicacy. After molting the hard shell, PPO begins to solidify the soft outside of the animal and form the new

PAGE 27

27 exoskeleton. As o-quinones are produced by th e reactions catalyzed by PPO, they form crosslinks between adjacent shell proteins, forming a stiff matrix of hardened proteins (Stevenson 1985). This hardening process is called sclerotization. Not surp risingly, PPO in arthropods has been shown to lie in high concentrations within the cuticles of the animals (Bartolo and Brik 1998). PPO has been shown to exist in arthropods in both latent and activated forms. The proenzyme of PPO found in Norw ay lobster can be activated exogenously using trypsin (Yan and Taylor 1991). Endogenous activ ation is thought to be the re sult of a number of natural proteases, as well as one or more unknown factors that have yet to be identified (Wang and others 1994, Zotos and Taylor 1996). PPO activity in lobsters and shrimp has b een shown to be sex-dependent (Ogawa and others 1984). In addition, PPO activity also changes throughout the lif ecycle of an arthropod. One study found a relationship between PPO activ ity and molting stage. Higher PPO activity was observed in spiny lobsters th at were close to molting (Ferre r and others 1989). In addition, there are yearly peak periods for molting in the Norway lobster, though molting occurs at some level throughout the year (Farmer 1975). Danish l obster processors have also noted an increase in blackspot-related problems around September of each year (Bartolo and Brik 1998). Blackspot is a quality defect caused by PPO activity in arthropods including shrimp, lobster and crabs. It can be brought on by inju ry to the animal while it is alive or by de gradation or storage processes after death (Lopez-Caballero and others 2007, Ogawa 1987). House fly ( Musca domestica ), another arthropod, has been in vestigated in the past as a source of new inhibitors for PPO due to the variable effects of PPO over the course of the flys lifetime. (Yoruk and others 2003). In that study, extracts from house flies at different stages of

PAGE 28

28 their lifecycle were tested for inhibitory prope rties against apple PPO. The most potent PPO inhibitor was isolated from thirdinstar larvae and 3-5 day old pupae. This compound inhibited apple PPO up to 90%. At the outset of this investigation, a crude, aqueous extract of blue mussel was simply added to a cuvette containing 0.1M pH 5.5 phosphate buffer, apple PPO, and 0.5M catechol. Though a reduction in browning was seen immediat ely, the identity of the compound within the blue mussel extract that effect ed the inhibition of PPO was u nknown for over a year (Schulbach 2008). The elucidation of the inhibitors identit y began following the successful purification of the compound through the use of high performan ce liquid chromatography (HPLC). With the inhibitor dissolved in methanol, HPLC-mass sp ectroscopy (HPLC-MS) analysis was attempted at the University of Floridas chemistry de partment. The methanol/inhibitor solution was assayed before HPLC-MS analysis and exhibited clear inhibition of apple PPO. However, when examined at the chemistry department, the result s indicated that the so lution was comprised of only pure methanol. It was hypot hesized that the mass sprectro scopy (MS) analysis of the inhibitor had failed due to its very low molecular we ight, which was known at that time from preliminary ultrafiltration st udies to be below 1000 daltons. In a search to remedy the problem of low molecular weight, another characteristic of the inhibitor was exploited. In addi tion to its ability to inhibit the catalytic action of PPO, the compound also exhibited a bleaching effect on the o-quinone products of PPO and its polyphenolic substrates. The mechanism of acti on of the bleaching effect was hypothesized to be through the binding of the inhibitor to th e colored enzyme-substrate products, creating a larger, colorless complex. Through the use of HPLC, this final colorless product was purified and sent to the chemistry department for MS analysis.

PAGE 29

29 The components of the known PPO-catechol prod uct, an o-quinone to which the inhibitor was bound, were removed from the results obtained through MS analysis of the entire colorless complex, leaving only the components of the inhib itor. The inhibitor wa s then identified as hypotaurine. Hypotaurine Hypotaurine is a sulfinic aci d that, in m ammals, is a meta bolic precursor to taurine formed from L-cysteine (Fontana and others 2004 ). The metabolic pathway from cysteine to hypotaurine consists of two steps. Cysteine is first oxidized to cy steinesulfinic acid by cysteine dioxygenase. Then, cysteinesulfinic acid is likely decarboxylated to hypotaurine by cysteinesulfinic acid decarboxylas e. Finally, hypotaurine is oxid ized to taurine; the mechanism of that oxidation is unknow n (Font and others 2001). Hypotaurine is a small (109.15 daltons), wa ter-soluble compound known to act as an antioxidant, binding hydroxyl radicals (Green and others 1991). It functions as an inhibitor for the sodium-dependant transport of gamma-aminobutyric acid (GABA) and -alanine in the cerebellar granule cells of rats (Saransaari and Oja 1993). Hypot aurine also acts as a GABA neuromodulator and inhibits N-methyl-D-aspar tic acid (NMDA), kainate, and quisqualate receptors (Quinn and Harris 1995, Dahchour and De Witte 2000, Kurachi and others 1983). In preliminary experiments using fresh, unpro cessed apple juice, no taste or odor changes could be detected due to the a ddition of hypotaurine as a browning inhibitor. Concentrations of hypotaurine in these trials were not measured, but enough hypotauri ne was added to maintain the original juice color, according to the naked eye, for at least f our weeks. In another set of preliminary experiments using fresh, unprocessed Thompson seedless grape ( Vitis vinifera ) juice, no odor or taste changes could be detected when hypotaurine was added to the juice at concentrations up to 200ppm (Sims 2008).

PAGE 30

30 Blue Mussel In this study, blue m ussel was investigated as another potential sour ce of PPO inhibitors. Blue mussel is a bivalve mollusk that is harvested from waters all over the world for food, and has been used for food by humans since at least 6000 BC. They grow in both marine and brackish waters with salinities as low as 4%, though their growth rate begins to drop at 18% salinity (FAO 2008). They also are the most e fficient feeders of all shellfish, filtering 10-15 gallons of water per day. They consume almost all the nutritious material from this filtered water and satisfy all of their en ergy and nutrition needs (Batten 2008). Mussels grown in aquaculture usually reach market size by two years. A number of aquaculture methods have been developed over time including long line or rope culture, raft culture, and on-bottom culture. However, the original form, known as intertidal wooden pole culture or bouchots, has been in use since at least the 13th century, when it was first used in France. Bouchots are still used for mussel aquaculture today (FAO 2008). Blue mussels begin their development as spherical eggs measuring less than a millimeter in diameter. After 15-35 days in this larval st age, they turn into a juvenile stage called a plantigrade. After reaching a size of 1 to 1.5mm in length, the young mussels use water currents to transport themselves to established blue mussel beds, to which they affix themselves and reach sexual maturity within one to two years (Newell 1989). Inhibition of Enzymatic Browning Enzym atic browning has long been a problem for the fruit and vegetable industry (Lee and Whitaker 1995, Brandelli and Lopes 2005). Browning in fruits and vegetables causes losses in post-harvest product year after year, putting a heavy ec onomic burden on the industry. Methods for browning inhibition have been of hi gh interest because the problem stems from one class of enzymes, known collectiv ely as polyphenol oxidases. However, despite the fact that a

PAGE 31

31 single type of protein is responsible for enzyma tic browning in countless varieties of fruits and vegetables, there is not a univers ally useful inhibition method. So me inhibitors do not work well against specific PPOs, as all PPOs are slightly different in struct ure. In addition, some powerful PPO inhibitors are not allowed in food products. There are currently six popular types of browning inhibito rs or inhibition methods, including reducing agents, chelators, complexi ng agents, acidulants, enzyme inhibitors, and enzyme treatments (McEvily and others 1992). All six of these inhib itors reduce a critical element in the browning process, which include the PPO enzyme, its phenolic substrates, the essential copper bound to the enzyme active site, and intermediate compounds during the course of the reactions. Some of the most popular PPO inhibitors used in the food industry today are reducing agents. Reducing agents prevent the formati on of polymerized melanin compounds by slowing down the buildup of o-quinone products of PPOcatalyzed oxidation reactions by reducing the oxidized o-quinones back to colorless o-diphenols Sulfiting agents, such as sulfur dioxide, sodium sulfite, and sodium bisulfite, are very powerful re ducing agents (Sapers 1993). It is also possible that they directly inhibit PPO (Lee and Whitaker 1995). However, they are banned from use in fresh fruit and vegetable products du e to concerns over adverse, breathing-related problems seen in some consumers (Gomez-Lop ez 2002b). Sulfites are still in use in seafood products, including shrimp, lobster, and crab, to prevent the formation of blackspot (Kim and others 2000). A popular alternative to sulfites is ascorbic acid. Also known as vitamin C, ascorbic acid works by reducing o-quinones back to o-dipheno ls, preventing the polymerization of the oquinones with other compounds to form brown me lanins. Unfortunately for the fruit and

PAGE 32

32 vegetable industry, while cheap and safe, ascorb ic acid is generally a short-lived inhibitor (Sapers 1993). The oxidized product of ascorb ic acid, dehydroascorbi c acid, does not inhibit browning and is not reduced back to ascorbic acid to act again upon the o-quinones (Eskin and Robinson 2000). Ascorbic acid is commonly paired with citric acid in order to boost its antibrowning efficiency. Ascorbic acid is more stable in an acidic environment, and the low pH can bring the enzyme out of its pH optimum. In addition, citric acid may act as a chelator (Eskin and others 1971). Another method to stop PPO-mediated browning in fruits and vegetables is the use of chelating agents. Chelators bind metal ions removing them from the PPO molecule and preventing the enzyme from binding its substrates PPO is a copper metalloprotein and the two copper ions associated with the active site of the enzyme are im perative to its function (Shahar and others 1992). A common commercial chelator is ethylenediaminetet raacetic acid (EDTA). It has a very strong affinity for a number of metals, including copper. One study found that EDTA prevented browning in avocado puree for up to three months (Soliva-Fortuny and others 2002). Other chelators used in the food industry include citric acid, diet hyldithiocarbamic acid (DIECA), oxalic acid, and phosphate s (McEvily and others 1992). Acidification also offers a method by wh ich enzymatic browning can be slowed. Acidifiers in foods include ci tric, phosphoric, lactic, malic, a nd ascorbic acid, among others. A low pH environment exerts a number of effects upon PPO. First, as the pH is lowered below about 4.5, PPO is generally no longer within its range of pH optima, reducing enzyme activity (Eidhin and others 2006, Unal 2007, Duangmal a nd Apenten 1999). This pH-mediated change in activity may be due to prot onation of groups essential to cat alytic action, denaturation or conformational changes in the tertiary structur e of the protein, lessening its ability to bind

PAGE 33

33 substrates, or a reduction in the stability of th e phenolic substrates th emselves (Tipton and Dixon 1983, Whitaker 1994). Besides chemical treatments, physical me thods, including freezing, heating, dehydration, modified atmosphere packaging, irradiation, pulsed el ectric fields, and high pressure can be used to help prevent enzymatic browning (Castro and others 2008, Kim and others 2000, Noci and others 2008, Teixeira and others 2007, Vijayanand and others 1995). These methods are not perfect since they may cause problems in product quality following treatmen t, such as a loss of firm texture and subcellular decompartmenta lization (Diaz-Tenorio and others 2007, Macheix and others 1990). Because no PPO inhibitor yet discovered is usef ul in all circumstances, interest in finding effective, safe, and economical inhibitors continues. One new facet in this vein of research is the use of natural PPO inhibitors, such as amino aci ds, Maillard reaction products, and cyclodextrins (Cheriot and others 2006, Kahn 1985, Sojo and ot hers 1999). In addition, a new and extremely innovative idea in PPO inhibition re search is the use of inhibito rs extracted from animals in which PPO plays a role in their life cycle. On the forefront of this area of investigation was a successful study into the use of proteinous extracts from the common housefly (Yoruk and others 2003). Objectives and Hypothesis Another PPO inhibito r, extracted from houseflie s, showed significant inhibition of apple PPO (Yoruk and others 2003). The biological functions of PPO within blue mussel and house fly have similarities, as they are both members of the phylum arthropoda (Chase and others 2000). Due to the relationship between the two organisms, the hypothesis for this project was that the inhibitor would decrease browning when used with all of the types of PPO tested.

PAGE 34

34 However, it was predicted that the inhibitor would not work equally well against all PPOs, due to differences in the three dimensional structures of the PPOs. From a review of the literature, it appeared that a large number of PPO inhibitors showed competitive-type inhibition (Richard-Forget and others 1992, Oktay and Dogan 2005, Dogan and others 2007). Therefore, it was e xpected that the inhibi tor investigated in this study would also show competitive inhibition. This project has a number of obj ectives, all of which relate to elucidating the biochemical and physical properties of a novel in hibitor of polyphenol oxidase, isolated from blue mussel. 1) To examine how effective the inhibitor is ag ainst browning in samples designed to mimic commercial processing on fresh fru its and vegetables. 2) To determine the type of inhibition taking place using enzyme kinetics plots, includi ng Dixon and Cornish-Bowden linear plots, as well as plots fit to the Michael is-Menten equation using non-linear regression. 3) To further elucidate the biochemical propertie s of the PPO inhibitor, the IC50 and Ki of the inhibitor were determined when used with each of the four different types of PPO used in the project.

PAGE 35

35 Table 2-1. pH and temperature optima for apple, banana, potato, and avocado PPOs, as well as substrate specificities ordere d from highest to lowest. Commodity pH Optimum Temp Optimum (C) Substrate Specificity Apple 5.0-7.5 (1) 18-30 (1) 4-methylcatechol > catechol > pyrogallol > (-)-epicatechin > caffeic acid > DL-dopa (1) Banana 6.5-7.0 (2,5) 30 (2,5) Dopamine > gallic acid (7); D-dopa > L-dopa (8) Potato 6.8 (4) 22,25 (4,6) 4-methylcatechol > caffeic acid > pyrogallol > catechol > chlorogenic acid > DL-dopa > dopamine (4) Avocado 4.5,7.5 (3) ? 4-methyl catechol > chlorogenic acid > pyrogallol > catechol > caffeic acid > DLDOPA (3) Note: 1. (Eidhin and others 2006); 2. (Una l 2007); 3. (Gomez-Lopez 2002); 4. (Duangmal 1999); 5. (Yang and others 2000); 6. (Vamos-Vigyazo 1981); 7. (Yang and others 2004); 8. (Galeazzi and Sgarbieri 1981). Figure 2-1. Locations within a plant cell of phenolic compounds, as well as polyphenol oxidase and peroxidase enzymes [Reprinted from Postharvest Biology and Technology, 48/1, Toivonen PMA and Brummell DA, Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables /Appearance, page 2, 2008, with permission from Elsevier].

PAGE 36

36 Figure 2-2. Mechanism of action for PPO upon its substrate compounds. Vmax values were observed through mushroom PPO acting upon L-tyrosine as the monophenol and LDOPA, the resulting o-diphenol [Repri nted from Postharvest Biology and Technology, 48/1, Toivonen PMA and Br ummell DA, Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables/Appearance, page 3, 2008, with permission from Elsevier].

PAGE 37

37 Figure 2-3. PPO-catalyzed oxidation of L-DOPA to melanin [Adapted from Double KL, Riederer P, and Gerlach M. 1999. Significance of Neuromelanin for Neurodegeneration in Parkinsons Disease. Drug News and Perspectives 12(6):333].

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38 CHAPTER 3 MATERIALS AND METHODS Machine Vision Analysis of Color Development Preparation of Inhibitor from Blue Mussel Crude inhibitor used in the color developm ent studies was isolated from blue mussels ( Mytilus edulis ). Two pound bags of frozen mussels imported from China were bought from a local seafood company and thawed in warm water. The drip loss from the mussels was collected. In addition, the mussels were opene d by hand and the mussel meats were wrapped in cheesecloth and squeezed to extract the liquid from the mussels. This liquid was added to the drip loss. The liquid mussel extract was then filtered through a Whatman (Whatman Plc, Maidstone, Kent, UK) glass filter under vacuum, using celite if necessary to aid in the filtering process. It was again filtered th rough a 0.45um Whatman filter under vacuum. Following filtration, the extract was poured in to approximately six inch sections of 500Da molecular weight cut off (MWCO) dialysis membrane (Spectrum Laboratory Products, Inc., New Brunswick, NJ) and dialyzed against distilled water for 24 hour s with three water changes. The dialysis was performed at 4C and th e water was stirred throughout the process. After dialysis, the mussel extract was r un through a Sephadex G-25 size exclusion chromatography column (Sigma-Aldrich, Co., St. Louis, MO). Two peaks were observed, and the second peak was collected and divided into smaller containers for freeze drying. The containers were put into the freezer overnight until frozen solid and then freeze dried using a Labconco freeze dryer (Labconco Co., Kansas Cit y, MO). Following freeze drying, each vial of extract, which contained approximately 35 mL of liquid extract, was reconstituted using 5 mL distilled water.

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39 Preparation of Puree for Testing Browning Inhibition of Blue Mussel Extract The fruits and vegetables used in this experiment, Hass avocado ( Persea Americana), banana ( Musa acuminate), red delicious apple ( Malus domestica ), and russet potato (Solanum tuberosum ) were bought from a local supermarket. A ll crops were purchased at a ripeness level at which they would be eaten or prepared im mediately without need for storage or further ripening. At the time of each e xperiment, the crop was diced into small pieces and three grams of material was transferred into a mortar alre ady containing 1.5 mL of either pH 5.5 phosphate buffer (control samples) or reconstituted mussel extract (test samples). The mixture was ground into a homogenous puree using a pestle and then tr ansferred into a small, round, white bottle cap. The same type of cap was used for stor age and imaging throughout the Machine Vision experiments. The cap was chosen because it allowed a relatively flat, uniform surface for photographs taken from above. For each of two experiments per fruit or vegetable, three samples each of control and test samples were prepared and photographed. Pictures of the puree were taken using a M achine Vision system comprised of two main components. The first is a light box designed to help ensure greater uniformity between pictures taken throughout the experiments. Its constr uction and design are described by Luzuriaga (1997). A Nikon D200 digital camera with a VR 18-200mm F/3.5-5.6 G lens at 50mm was used to photograph the samples within the sealed light box. The settings for the camera are shown in table 3-1. Pictures were taken immediat ely after grinding (0 minutes), at one, five, 10, 60, and 120 minutes, as well as at one, two, and three days, a nd finally at one and two weeks. Samples were stored in sealed plastic contai ners at 4C with a water bath inside the container to decrease drying of samples during storage. During pi cture taking, samples were removed from the container and the container was resealed. The ambient temperature in the room during picture

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40 taking was 20-25C. Following each picture, th e samples were returned immediately to their storage container. Machine Vision Analysis Two m ethods of analysis were performed on the data from the photographs. The first was performed using LensEye software (Engineering and CyberSolutions, Gainesville, FL). The photographs were analyzed per pixel to determin e both average L*, a*, and b* values as well as its color block identity. With in the software, the visible spect rum was divided into 4096 parts. Each pixels R,G,B value was used to determine in to which color block the pixel best fit. The percentage of sample surface area taken up by eac h color block was then calculated. Through the specific definition and tabula tion of the colors within each picture, the color development was compared over time. As various color blocks emerge and others are replaced, the shift in color and in the general intensity of the sample over time can be observed. The second method of analysis used the aver age L* value from the samples at each time point to determine the general darkening of the crops over time. L* was used because it gives the most direct indication of browning. For eac h time point, the average L* value for the three control samples was compared to the average L* value of the three test samples at the same time point. One-way analysis of variance was calcula ted for each time point using SAS. Means were separated using the least significan t difference test at a probability level of 0.05, also calculated using SAS. Enzyme Kinetics Polyphenol oxidase preparation Enzyme kinetic experiments were performe d on PPO extracted from the four crops in order to determine kinetic parameters, as well as the type of inhibition in each case. Unlike the Machine Vision experiments, the kinetics tria ls used purified hypotaurine (Sogo Pharmaceutical

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41 Co., Tokyo, Japan) as the inhibitor instead of the crude mussel extrac t. During the course of this study, a compound, hypotaurine, was purified and identified as the browning inhibitor from blue mussels. Also note that the kine tic experiments using the hypotau rine were further complicated by the presence of a number of isoforms of PPO in each crop PPO extract because the preparation was only partially purified; however, the data c ould be analyzed successfully. PPO extraction began with the whole, raw fr uit or vegetable. The crop was washed, cut into large chunks and placed into a pre-chilled blender containing 400 mL of acetone at -20C. The mixture was blended for one minute and then filtered through a Whatman #1 filter under vacuum until all of the visible wetness had disappeared from the top of the solids. The solids were then placed back into the blender with 200 mL of chilled acetone and blended again for one minute. The mixture was filtered again, and this re-extraction process was repeated another two times for a total of one primary extraction and th ree secondary extractions. At the end of the third re-extraction, the solids were left under vacuum overnight to dry. The resulting powder was packed under vacuum into storage bags and placed at -20C until needed. To extract the PPO powder for use in the e xperiments, one gram of powder was mixed into 50 mL of 0.1 M KH2PO4/Na2HPO4, pH 7.2 buffer and stirred in 4C for at least 30 minutes. The slurry was the centrifuged at 12000xg for 30 minutes, and the supernatant was removed and filtered through glass wool. The filtrate wa s crude PPO extract for the enzyme kinetic experiments. The PPO extract was frozen until needed. Using a Beckman Model DU 640 UltravioletVisible spectrophotometer, a plot of substrate concentration vs. reacti on velocity was determined, in order to find the correct substrate concentrations to use in the kinetics experiment for that crop. After the substrate concentrations were chosen, the lowest concentration was used to determine the amount of inhibitor needed to

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42 provide a wide range of activity. A range of approximately 20% to 80% inhibition was satisfactory, though in a number of cases a wider range was used. To determine the activity at each point, a 3 mL assay was used, containing 0.05 or 0.1 mL of PPO extract, depending on how active the particular extract was, inhibitor at the specified amount, catechol (Sigma Chemical Co., St. Louis, MO) as substrate at the specified amount for that series, and the balance in buffer at eith er pH 5.5 or 6.0, depending upon the pH optimum for the crop PPO being examined. In the case of av ocado, 100 uL of 1% trypsin solution was also used in each cuvette in order to activate the PPO The enzyme was added last, and the contents were covered in parafilm and shaken three times to mix the reagents. The spectrophotometer measured absorbance at 420nm for 90 seconds and th e greatest initial rate was determined using the spectrophotometers kinetic software. Each point was assayed in duplicate and each experiment was replicated. To determine the type of inhibition taki ng place in the case of each crop, a Dixon plot was constructed for each trial. In addition, a Co rnish-Bowden plot of substrate concentration over reaction velocity vs. inhibi tor concentration was constructed in order to specifically ascertain the inhibition t ype (Cornish-Bowden 1974). Prism software (Graphpad Software, Inc., La Jolla, CA) was used to determine Km and Vmax by nonlinear regression. Ki was determined from the results of th e IC50 test along with the Km using the Cheng-Prusoff equation, whic h reads, Ki = IC50 / (1+[S]/Km) (Cheng 1973). Determination of IC50 To find the level of inhibitor that would give a 50% reduction in reaction rate, it was necessary to determine the reac tion rates of assays containing one substrate concentration and one enzyme concentration while varying the amount of inhibitor. In these experiments, the substrate level used for each crops trial was the same as the highest substrate concentration used

PAGE 43

43 in the enzyme kinetics studies for that crop. All of the crop s used a range of 0.038174mM to 0.61078mM of inhibitor to ensure a wide range of inhibitory strengths. Each point was assayed in duplicate and each experiment was replicated. The curve was fit using a second order polynomial equation and the IC50 was interpolated. Electrophoretic Separation of PPO Isoforms To determine if the PPO extract contai ned one or more PPO isoforms, native gel electrophoresis was conducted. Native gel electrophor esis was used to allow the staining of only the PPO proteins within the mixture by way of their natural enzymatic action. Instead of staining the gel using a protein stain, as in the case of de natured protein samples, the intact enzymes were allowed to react with their natura l substrates to elucidate their lo cations within the gel as they formed melanin. The experiment was performed using a Bio-Rad Mini-Protean III system (BioRad Laboratories, Inc., Hercules, CA). 7.5% acrylamide gels were purchased from Bi o-Rad, as was the running buffer and sample buffer. The PPO extracts were diluted in a 2:1 ratio of sample buffer to sample. Eight wells were filled, two for each crop. One well for each crop was loaded with 25uL of sample and the other was loaded with 45uL. The gels were run using a Bio-Rad Powerpac 3000 voltage supply unit (Bio-Rad Laboratories, Inc., Hercules, CA) at 200 volts until the blue dye was about 2-3mm from the end of the gel. After washing the gels, they were placed in a bath of 10mM L-DOPA (Sigma-Aldrich, Co., St. Louis, MO) on a shaker plate for 10 mi nutes. After 10 minutes, solid catechol was added to the bath in excess to comple te the staining process. The gels remained in the substrate bath until no further staining could be observed.

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44 Table 3-1. Camera settings for Machine Vision experiments Parameter Value ISO 100 Exposure Manual Shutter speed 1/3s F/11 Exposure compensation -1.0 EV Focus Manual Sharpening Normal Tone compensation Normal Color mode Mode 1 Saturation Normal Hue adjustment 0 White balance Direct Sunlight Zoom Manual

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45 CHAPTER 4 RESULTS AND DISCUSSION Machine Vision Analysis of Color Development Figures 4-1 through 4-4 show the graphical representations of color blocks emerging over time during the first apple trial fo r the untreated (contro l) and treated (expos ed to hypotaurine) samples. Figures 4-5 through 4-8 display the same data from the replication of the first experiment. As there was little variation between replications for all cr ops, only representative data from the first experiment for each crop is shown for banana, avocado, and potato. Two types of analysis were used to exam ine the data provided by the Machine Vision studies. The first was to graph the color blocks at each time point versus the percentage surface area of the sample that they covered. By ove rlaying these graphs by time, it is possible to observe a shift in color blocks towards the lo wer numbers, which indicate darker colors. A smaller shift in color blocks indicates a lower ra te of browning during that time period. In the case of the first apple trial, fi gures 4-1 and 4-3 show a particul arly large decrease in browning was seen in the samples treated with hypotaurine versus the browning s een in the untreated control samples between time points 10 minutes a nd 120 minutes. This inhibition of browning is indicated by the smaller range of color block values in the treated samples. Figure 4-1B shows a range of over 1400 color blocks between 10 and 120 minutes in the untreated samples. On the other hand, figure 4-3B shows that the treated sa mples experienced a range of only just over 300 color blocks over the same time period. Avoca do and potato show the same sort of browning inhibition during the same time period. In the case of banana, the large differences in browning show up in the time period from 24 hours to two w eeks. Over that period, though the range of blocks was larger for the untreated sample, th e values of the blocks show the browning

PAGE 46

46 inhibition. The untreated samples from 24 hours to two weeks showed values from about 2700 to 1600, as seen in figures 4-10A and 4-10B. Over the same time period, the treated samples showed values from about 4070 to 2150, as seen in figures 4-12A and 4-12B. Hence, the treated samples were darker, though the amount of browning displayed by the treated samples from 24 hours to two weeks was greater. The difference in browning is probably due to the fact that the untreated samples had already browned significantly by 24 hours, while the browning in the treated samples had been relatively inhibited until that point. Machine Vision data for untreated and trea ted banana samples during the time points 0120 minutes are shown in figures 4-9 and 4-11. In contrast to Machine Vision data obtained using apple, as shown in figures 4-1 and 4-3, the shift in color primitives is similar between treated and untreated banana samples. The sim ilarity between the ranges of color primitives observed during the time period 0-12 0 minutes indicates that there was not an effective reduction in browning taking place over those observation times. The second analysis method used on the M achine Vision data was to compare the L* values for the treated and control samples at each time point. L* was analyzed because it is a measure of the lightness or darkness of a sample, which is the aspect of the samples color most directly changed by PPO-mediated br owning. L* values were averag ed using both tr ials of each crop, providing six L* values per point for bot h treated and control samples. A one-way ANOVA was performed at each tim e point and the means were compared by way of the LSD test. Thus, it was determined whether or not the control and tr eated samples L* values were significantly different at each time point.

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47 As shown in figures 4-13A and 4-14B, respect ively, in the case of apple and potato, the control samples were statistically different from each other at time zero, though the difference was very small. For all four crops, however, the difference between control and treated samples grew with increased storage leng th. Figures 4-13 and 4-14 show that the treated samples for all of the crops except avocado exhibited a signi ficant decrease in browning beginning after one hour. The treated samples of avocado showed a significant difference st arting at 24 hours. These results indicate that in all of the tria ls, the inhibitor was su ccessful in slowing the browning process. The differences in inhibition between the crops are likely due to varying tertiary structures among the various PPOs. In the same way that a particular PPO has varying affinities to specific substrates, the affinity of the inhi bitor to the binding site on the surface of the PPO molecule is likely determined by its unique thr ee-dimensional shape. Both the size and shape of a binding site can affect the affi nity of one molecule to anothe r (Bahadur and Zacharias 2007). In the case of PPO, various sizes of the enzyme have been observed, indicating possible variations in the size and/or shape of the inhibitor binding s ite (Flurkey 1986, Fraignier and others 1995, Sherman and others 1991, Steffens and others 1994, Van Gelder and others 1997, Yang and others 2000). In other Machine Vision studies of PPO-me diated browning, similar results have been shown. In a study investigating the inhibitory effects of oxalic acid on browning in banana and apple slices, Machine Vision was also used to measure changes in L* values. In that study, decreases in L* value were observed over time as the fruits browned following slicing (Yoruk and others 2003). The same decrease in L* value was seen in this study following pureeing of the fruit. In the case of banana, oxalic acid treatment up to 20 mM led to approximately the

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48 same change in L* value over the first two hours following slicing as the banana puree did in this study following grinding and treatment with hypotaur ine. However, in the case of apple, even treatment with just 3 mM of oxalic acid inhibite d browning slightly better during the first two hours than the hypotaurine treatment did over the same time period. In another study evaluating melanosis in shrimp, Machine Vision readings we re compared with grades of melanosis given by a trained shrimp evaluator. In that study, 64 color blocks were used and 12 were designated as colors associated with melanosis. Over the fi rst 15 days, melanosis increased linearly in the shrimp, and there was an accompanying increase in the levels of melanotic colors over the same time period (Luzuriaga and others 1997). The parallel increases in both the prominence of melanotic colors and scores of melanotic grad e indicate that Machine Vision analysis is an accurate tool that can be used to quantify changes in browning-related color over time. Enzyme Kinetics To determ ine the type of inhibition in each crop, Dixon plots were constructed along with Cornish-Bowden plots. Both types of plots al one are limited. The Dixon plot cannot establish the difference between mixed and competitive inhibition, while the Cornish-Bowden plot cannot differentiate between uncompetitive and mixed inhi bition. Therefore, when used in concert the two plots can unambiguously determine the type of inhibition (Cornish-Bowden 1974). Figure 4-15 shows both the Dixon and Cornis h-Bowden plots generated from the apple PPO experiments. The pattern of intersecting lines in the first quadrant in the Dixon plot and the parallel lines shown in the Cornish-Bowden plot indicate competitive inhibition. The same pattern is seen in Figure 4-16, wh ich shows the same type of pl ot generated using the avocado data. On the other hand, the results are not as clea r when examining the plots of the banana and potato data. Figures 4-17 and 4-18 show the plot s for banana and potato, respectively. In each

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49 case, the Dixon plot indicates either mixed or competitive inhibition. However, in both cases, the Cornish-Bowden plot shows a pattern not ex pected with this analysis. These unexpected results may be due to the presence of more than one PPO isozyme within each of the PPO extracts. Figure 4-23 shows the results of native gel el ectrophoresis on the four PPO isolates. Though not a high resolution separation, in all four cases it appears that there is more than one PPO isozyme in the extract. It is possible that, in the cases of potato and banana, these multiple PPO isozymes exhibit different kinetic inhibition, leading to the unexpected results seen in the Cornish-Bowden plots. A number of sulfur-containing compounds have been evaluated as PPO inhibitors and it has been shown that they vary in their respective types of inhibition. In a study investigating the use of L-cysteine as an inhibitor for apple PPO L-cysteine was found to act as a non-competitive inhibitor (Gacche and others 2006). Glutathione, another sulfur-containing compound that is known to inhibit PPO, was evaluated in another study using PPO isolated from artichoke. When glutathione was used with 4-met hylcatechol acting as substrate for the PPO, glutathione showed mixed-type inhibition. Yet, when pyrogallol wa s used as the substrat e instead, glutathione showed noncompetitive inhibition (Dogan and others 2005). Finally, a third study found that p-aminobenzenesulfonamide behaved as a competitive inhibitor of wild pear PPO (Yerliturk 2008). Thes e results are not surprising, given the fact that sulfur-containing PPO inhibitors vary in the types of inhibiti on effected, with differences observed even as a function of the PPO substrate us ed in the system. When used with apple and avocado PPOs, for example, hypotaurine exhibits competitive inhibition, like p-

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50 aminobenzenesulfonamide. When used with ba nana and potato PPOs, however, hypotaurine acts as a mixed-type inhibitor. To establish an IC50 for each crop, the maxi mum substrate concentration used in the kinetics experiments for each fruit or vegetable was assayed in the spectrophotometer using the standard 3 mL mixture in the presence of a wide range of inhibitor concen trations. A quadratic curve was fit to the resulting data, allowing the interpolation of the IC50 value, which is the concentration of inhibito r required to effect a 50% reduction in reaction velocity. Figures 4-19 through 4-22 show the data from each crops trial. Figure 4-19 shows both the initial apple trial as well as the replication. 7 To establish Km and Vmax parameters for each crop, non-linear regression was performed on the untransformed data. Prism soft ware from GraphPad Software was used to fit the data to the Michaelis-Menten model. Nonlinear regression was chosen in favor of more traditional methods of Km and Vmax determina tion, such as using a Lineweaver-Burk (double reciprocal) plot because non-lin ear regression fits the data without modification, while the double reciprocal plot requires edit ing of both substrate concentration and reaction velocity data in order to construct the plot (Lineweaver and Burk 1934). The i ndirect analysis of the data results in a distorted distribution of error. Th erefore, estimates of kinetic parameters given by fitting the data using non-linear regression are more statistica lly valid (Leatherbarrow 1990). Table 4-1 shows Km and Vmax, as well as IC50 a nd Ki values, for each crop. Ki was calculated using the Cheng-Prusoff equation, which takes in to account Km, substrate concentration, and the IC50 value (Cheng and Prusoff 1973). Comparison of Novel Inhibitor to Other Browning Inhibitors Compared to popular browning inhibitors th at are used commercially now, hypotaurine has a number of distinct advantages. First, it is able to decrease the maximal amount of

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51 browning in a product. In the case of ascorbic acid, another common browning inhibitor used in fresh cut fruits, only the lag phase of the browning process is affected. Because ascorbic acid slows browning, at least when inhibiting some type of PPO, solely by acting upon the oxidized reaction products. Ascorbic acid is able to re duce the products back to their colorless forms. The resulting oxidized form of ascorbic acid, de hydroascorbate, does not re generate back to its reduced form, however, and it is no longer able to prevent browning. In time, therefore, the same level of browning will be achieved as a sample without ascorbic acid (Arias and others 2007). By contrast, the new inhi bitor not only in creases the period before browning is observed but also decreases the maximum browning observed, due to the fact that it inhibits the PPO molecule directly. Another advantage of the new inhibitor over some older types of br owning inhibitors is that it requires very low concentrations to ach ieve a high level of inhibition. In one study, a crude extract of burdock root PPO was tested against bisulfite, ascorbic acid, 4-hexylresorcinol and citric acid as browning inhibi tors. At a concentration of 0.5% w/v, citric and ascorbic acids provided approximately 10% i nhibition of browning activity, 4-hexylresorcinol showed approximately 85% inhibition, and bisulfite provided 100% inhibition (Lee-Kim and others 1997). It was also noted that ci tric acid is generally applie d to foods commercially at a concentration of 0.5% to 2% w/v, which supports the argument that citric acid requires high concentrations in order to aff ect significant browning inhibition (Marshall and others 2000). In comparison, the inhibitor examined in this study provides over 80% inhibition to most of the PPOs tested at a concentration of less than 1 mM (0.1095% w/v). Finally, unlike other browning inhibitors, such as ascorbic acid and EDTA, the novel browning inhibitor examined in this study can be used alone in food pr oducts instead of in

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52 concert with other browning inhi bitors. A typical commercial an ti-browning cocktail for fruits and vegetables could consist of three or more aci difiers, chelators, and chemical reducing agents (Marshall and others 2000). A comparable and successful reduction in browning can be achieved by using relatively low concentr ations of the novel inhibitor alone. The novel compound also compares favorably to other PPO inhibito rs in regards to kinetic parameters. Ki is the dissociation constant of the enzyme-inhibitor complex. A lower Ki value equates to a lower IC50 value, and the lower the IC50 value, the more strongly a compound inhibits the PPO being tested. In one study, novel PPO inhibito rs were synthesized using raw materials that did not show a high level of PPO inhi bition. The best PPO inhibitor synthesized during this study showed a Ki of 40 uM (Tricand de la Goutte 2001). In comparison, the Ki values found fo r the novel inhibitor in this st udy ranged from approximately 3-14 uM, depending on the crop from which th e PPO was isolated. In another study, diethyldithiocarbamic acid was investigated as an i nhibitor for PPO from three different sources. Again, the novel inhibitor from this study show ed lower Ki values than any shown using diethyldithiocarbamic acid. The lowest Ki value calculated for diethyldithiocarbamic acid was when using it as an inhibitor for mushroom PPO with 4-methylcatechol as a substrate for the enzyme. The Ki value was 0.015 mM, which is greater than the maximum Ki calculated in this study using the novel inhibitor, whic h was 0.0138 mM (Dogan and others 2008). Another study investigated a number of subs trate/inhibitor pairings, using yacon PPO. When chlorogenic acid was the substrate for PPO, p-coumaric and cinnamic acids had Ki values of 0.017 and 0.011 mM, respectively (N eves and Silva 2007). In that enzyme-substrate-inhibitor system, the cinnamic acid achieved up to 95% inhibi tion, an inhibitory abili ty comparable to the value documented in this study for the novel inhibitor and apple PPO.

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53 Table 4-1. Kinetic parameters for ap ple, avocado, banana, and potato. Parameter Apple Avocado Banana Potato Km (Mm) 40.47 10.26 25.52 8.322 Vmax (Absorbance) 0.5247 0.3997 0.5376 0.4049 IC50 (Mm) 0.2177 +/0.0009 0.2253 +/0.0224 0.2853 +/0.03552 0.1962 +/0.0008 Ki (Mm) 0.0138 +/7.774x10-5 0.01004 +/0.0018 0.0038 +/5.324x10-4 0.0027 +/1.552x10-5 Note: Km and Vmax determined by non-linear regr ession fit to Michaelis-Menten model. Ki calculated from Km and average IC50 using th e Cheng-Prusoff equati on (Cheng and Prusoff 1973). Error is given as one standard deviation.

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54 A B Figure 4-1. Untreated samples in the first ap ple trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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55 A B Figure 4-2. Untreated samples in the first apple trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time points 1-2 weeks.

PAGE 56

56 A B Figure 4-3. Treated samples in the first apple tr ial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

PAGE 57

57 A B Figure 4-4. Treated samples in the first apple tr ial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time points 1-2 weeks.

PAGE 58

58 A B Figure 4-5. Untreated samples in the second appl e trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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59 A B Figure 4-6. Untreated samples in the second apple trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time points 1-2 weeks.

PAGE 60

60 A B Figure 4-7. Treated samples in the second apple trial; 0-5 minut es and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

PAGE 61

61 A B Figure 4-8. Treated samples in th e second apple trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time points 1-2 weeks.

PAGE 62

62 A B Figure 4-9. Untreated samples in the first banana trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

PAGE 63

63 A B Figure 4-10. Untreated samples in the first banana trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time point 1-2 weeks.

PAGE 64

64 A B Figure 4-11. Treated samples in the first bana na trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

PAGE 65

65 A B Figure 4-12. Treated samples in the first banana trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time point 1-2 weeks.

PAGE 66

66 A B Figure 4-13. Apple and avocado: comparison between control and treated L* values at each time point. Time point labels followed by an asterisk indicate a si gnificant difference between control and treated samples at that time point (p<0.05). Error bars show one standard deviation below and above average. A) Values for apple. B) Values for avocado.

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67 A B Figure 4-14. Banana and potato: comparison between control and treated L* values at each time point. Time point labels followed by an asterisk indicate a si gnificant difference between control and treated samples at that time point (p<0.05). Error bars show one standard deviation below and above average. A) Values for banana. B) Values for potato.

PAGE 68

68 A B Figure 4-15. Apple kinetics plots. These two plots, in conjunc tion, show that the inhibitor is competitive with regard to apple polyphenol oxidase. A) Dixon and B) CornishBowden plots for apple kineti c data (Cornish-Bowden 1974).

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69 A B Figure 4-16. Avocado kinetics plots. These two plots, in conjuncti on, show that the inhibitor is competitive with regard to avocado pol yphenol oxidase. A) Dixon and B) CornishBowden plots for avocado kine tic data (Cornish-Bowden 1974).

PAGE 70

70 A B Figure 4-17. Banana kinetics plots. These tw o plots, in conjunction, are inconclusive but indicate either competitive or mixed i nhibition. A) Dixon and B) Cornish-Bowden plots for banana kinetic data (Cornish-Bowden 1974).

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71 A B Figure 4-18. Potato kinetics plots. These two pl ots, in conjunction, are inconclusive but indicate either competitive or mixed inhibition. A) Dixon and B) Cornish-Bowden plots for potato kinetic data (Cornish-Bowden 1974).

PAGE 72

72 A B Figure 4-19. Apple: plots of per cent activity vs. inhib itor concentra tion to determine IC50. The replicate experiment (B) is shown for this crop but not for the others, as variance between trials was small. IC50 for trials one and two are 0.21832 mM and 0.217066 mM, respectively. IC50s are indi cated by points marked in pink. Figure 4-20. Avocado: plot of percent activity vs. inhibitor co ncentration to determine IC50. IC50 for trials one and two are 0.209467 mM and 0.241135 mM, respectively. Average IC50 is indicated by point marked in pink.

PAGE 73

73 Figure 4-21. Banana: plot of percent activity vs. inhibitor concentration to determine IC50. IC50 for trials one and two are 0.310414 mM and 0.260183 mM, respectively. Average IC50 is indicated by point marked in pink. Figure 4-22. Potato: plot of percent activity vs. inhibitor concentration to determine IC50. IC50 for trials one and two are 0.195615 mM and 0.196749 mM, respectively. Average IC50 is indicated by point marked in pink.

PAGE 74

74 Figure 4-23. Results of native gel electrophoretic se paration of crude PPO isolates. From left to right, the lanes were loaded as follo ws 25ug banana PPO, 25ug potato PPO, 45ug potato PPO, 45ug banana PPO, 25ug avocado PPO, 45ug avocado PPO, 25ug apple PPO, and 45ug apple PPO. All volumes include 1:2 dilution of PPO extract with running buffer.

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75 CHAPTER 5 CONCLUSIONS A novel polyphenol oxidase inhibitor was extracted from blue mussel ( Mytilus edulis ), identified as hypotaurine, and characterized with regards to its inhibitory properties in avocado, potato, banana, and apple and the modes of inhibiti on displayed in these four species of crops. Over a storage period of two weeks, all sample s showed significant decreases in browning when exposed to the inhibitor compound. Significant differences in browning appearance between control and experimental samples were observed to begin from one minute after the start of the experiment to one day after the start of the experiment, dependi ng on the species of the specific sample. The inhibitor was also shown to exhibit both competitive and mixed-type inhibition, depending upon the species of crop on which it wa s tested. Problems arose when attempting to graphically determine the types of inhibition in the cases of banana and potato. It is believed that these results are due to the presence of more th an one PPO isozyme in the enzyme extract from the fruits that each exhibit different types of inhibition. In order to further elucidate the benefits of this novel enzyme inhibitor, the Machine Vision experiments should be cond ucted again using a purified form of the inhibitor. Closely controlling the concentration of th e inhibitor in the samples would greatly benefit the ability to compare the effect of the inhibitor between various crop species. There are clearly differences in how well the compound is able to inhibit the action of PPO in different crops, but without being able to exactly control the inhi bitor concentration it is hard to compare the samples accurately side by side. In addition, it w ould be beneficial to be able to perform the enzyme kinetics experiments again using purified PPO extracts, as opposed to the crude ones used in this study.

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76 By utilizing only one PPO isozyme at a time and th e purified hypotaurine as the inhibitor, clearer results may be obtained. Finally, further work should be done in order to allow the purificati on of hypotaurine at a lower cost. One major hurdle for this inhibitor with regard to the commercial market is its excessive price. The inhibitor has many benefits over conventional PPO inhib itors. It is widely found in nature, colorless, odorless, tasteless, an d is able to provide very high levels of PPO inhibition at relatively low concentr ations. However, it is most likely cost-prohibi tive at this time for commercial use in most industries. By making the isolation process more efficient, costs to supply the inhibitor c ould be reduced and would make th e prospect of commercial use of the inhibitor more likely.

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77 APPENDIX ADDITIONAL DATA A B Figure A-1. Untreated samples in the first avoc ado trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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78 A B Figure A-2. Untreated samples in the first avocad o trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time point one week.

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79 A B Figure A-3. Treated samples in the first avoc ado trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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80 A B Figure A-4. Treated samples in the first avoca do trial; 24-72 hours and one week; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time point one week.

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81 A B Figure A-5. Untreated samples in the first potato trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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82 A B Figure A-6. Untreated samples in the first potato trial; 24-72 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time points 24-72 hours. B) Time point one week.

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83 A B Figure A-7. Treated samples in the first potat o trial; 0-5 minutes and 10-120 minutes; color blocks and percentage surface areas. A) Time points 0-5 minutes. B) Time points 10-120 minutes.

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84 A B Figure A-8. Treated samples in the first potato trial; 2472 hours and 1-2 weeks; color blocks and percentage surface areas. A) Time poin ts 24-72 hours. B) Time point one week.

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85 Figure A-9. Apple: browning pr ogression in treated and untreated samples during Machine Vision experiment.

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86 Figure A-10. Avocado: browning progression in treated and untre ated samples during Machine Vision experiment.

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87 Figure A-11. Banana: browning pr ogression in treated and untreated samples during Machine Vision experiment. Figure A-12. Potato: browning pr ogression in treated and untr eated samples during Machine Vision experiment.

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88 Figure A-13. Time zero SAS output fo r apple machine vision L* data. Figure A-14. Time 24 hours SAS output for apple machine vision L* data.

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89 Figure A-15. Color block reference legend.

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101 BIOGRAPHICAL SKETCH Robert Schwartz Bent was born in Takom a Park, MD to a biot echnology patent lawyer and a zoological neuroscientist. He has one brother three years older than himself. He attended Montgomery Blair high school in Si lver Spring, MD and participated in the math and science magnet program there. After high school, Rob m oved to Florida to atte nd the University of Florida. After graduating with a bachelors degree in food science, he began pursuing a masters degree in food science with Dr. Marshall at the Un iversity of Florida. Outside of school, Rob enjoys powerlifting, bu ilding cars, and traini ng in mixed martial arts. After graduation, Rob hopes to pursu e a career in food product development.