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Quality Assessment, Antioxidant and Antimicrobial Activities, and Acrylamide Inhibition in Muscadine Grapes

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Title:
Quality Assessment, Antioxidant and Antimicrobial Activities, and Acrylamide Inhibition in Muscadine Grapes
Creator:
Xu, Changmou
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (159 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science
Food Science and Human Nutrition
Committee Chair:
MARSHALL,MAURICE R,JR
Committee Co-Chair:
WYSOCKI,ALLEN F
Committee Members:
GU,LIWEI
SIMONNE,AMARAT
WYSOCKI,ALLEN F
LU,JIANG
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Antibacterials ( jstor )
Antioxidants ( jstor )
Biofilms ( jstor )
Enzymes ( jstor )
Ethanol ( jstor )
Grapes ( jstor )
Hydrolysis ( jstor )
Polyphenols ( jstor )
Skin ( jstor )
Solvents ( jstor )
Food Science and Human Nutrition -- Dissertations, Academic -- UF
acrylamide -- antimicrobial -- antioxidant -- enzymes -- muscadine -- polyphenols -- quality
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Food Science thesis, Ph.D.

Notes

Abstract:
The muscadine grape (Vitis rotundifolia Michx.) industry in the southeastern United States has expanded in recent years as more varieties become available. To provide quality and nutraceutical information for this industry, 58 muscadine varieties were evaluated for their fruit quality, phenolic profiles and antioxidant activities during two growing seasons. Then an enzymatic extraction method for polyphenols was proposed to improve the functionality of grape pomace. Lastly, the antimicrobial properties and acrylamide inhibiting properties of polyphenols from grape pomace were evaluated for the potential of utilizing winery wastes. In the first objective, data related to 58 muscadine cultivar's fruit quality (color, weight, size, pH, titratable acidity, soluble solids content, ratio), and nutraceutical (polyphenol contents and profiles) and antioxidant properties were developed for a database. In the second objective, enzyme hydrolysis shortened the polyphenols extraction time but didn't increased the total phenolic yield compared with solvent (50% ethanol) alone. Interestingly, enzyme hydrolysis was found to modify the galloylated form of polyphenols to low molecular weight phenolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity. In the third objective, antioxidant activity for different polyphenols varied greatly ranging from 5.0 to 11.1 mmol Trolox/g. Antioxidant and antibacterial activity for polyphenols showed a weak positive correlation (r = 0.53). Muscadine polyphenols exhibited a broad spectrum of antibacterial activity against tested foodborne pathogens. Muscadine polyphenols at 4 x MIC caused nearly a 5 log10 CFU/mL drop in cell viability for S. aureus in 6 h with lysis, while at 0.5 x MIC, they inhibited its biofilm formation, and at 16 x MIC, they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log10 CFU/mL drop in cell viability at sub- inhibitory concentration. In the fourth objective, muscadine grape polyphenols could significantly reduce acrylamide formation in both in vitro chemical and potato chip model systems. The reduced rates of grape polyphenols were higher than 90% at 100 u/mL. These results suggest that muscadine grape polyphenols may be used as a natural food additive to mitigate acrylamide formation in heat-treated commercial starchy foods. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: MARSHALL,MAURICE R,JR.
Local:
Co-adviser: WYSOCKI,ALLEN F.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-02-28
Statement of Responsibility:
by Changmou Xu.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Xu, Changmou. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
2/28/2015
Resource Identifier:
968131667 ( OCLC )
Classification:
LD1780 2014 ( lcc )

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1 QUALITY ASSESSMENT, ANTIOXIDANT AND ANTI MICROBIAL ACTIVITIES , AND ACRYLAMIDE INHIBITIO N IN MUSCADINE GRAPE S By CHANGMOU XU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2 © 2014 Changmou Xu

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3 To my family for the initial impetus that gave me the love to explore science

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4 ACKNOWLEDGMENTS I want to express my sincere appreciation to my major advisor, Dr. Maurice R. Marshall , for his patience, advice and mentorship. Without his guidance and support, this research could not be accomplished. I am grateful for my committee members, Dr. Amarat Simonne , Dr. Liwei Gu , Dr. Jiang Lu and Dr. Allen F. Wysocki , for their valuable time and suggestions. I cherished the friendship with my la b members: Wei Yea Hsu, Lu Zhao, David L. Guderian , Adriana Carolina Matheus, Evan James , Senem Guner , Dr. Wlodzimierz Borejsza Wysocki , Amanda Hogle and all other IR 4 staff . They were always willing to offer me helping hands and emotional support Last and most important, I express my deepest gratitude to my parents for their constant love and commitment to my education. They were my first teachers and gave me many good lessons. They always encouraged me to pursue my dream. Because of the ir love, pa tience, and unconditional support, I gained the strength to complete this program.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURE S ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 A BSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 AN O VERVIEW OF THE MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX.): FRUIT QUALITY, POLYPHENOLS, AND POTENTIAL HEALTH BENEFITS ................................ ................................ ................................ .............. 15 Muscadine Grapes ................................ ................................ ................................ .. 15 Fruit Quality ................................ ................................ ................................ ............ 15 Polyphenols ................................ ................................ ................................ ............ 16 Extraction of Po lyphenols ................................ ................................ ....................... 16 Potential Health Benefits ................................ ................................ ......................... 18 Antimicrobial and Antibiofilm Activity of Polyphenols ................................ ........ 18 Acrylamide Inhibition Activity of Polyphenols ................................ .................... 19 Research Question and Objectives ................................ ................................ ........ 20 2 FRUIT QUALITY AND NUTRACEUTICAL PROPERTIES OF MUSCADINE GRAPE ( VITI S ROTUNDIFOLIA MICHX.) ................................ .............................. 22 Background ................................ ................................ ................................ ............. 22 Materials and Methods ................................ ................................ ............................ 23 Grape Materials ................................ ................................ ................................ 23 Chemicals ................................ ................................ ................................ ......... 23 Sample Preparation ................................ ................................ .......................... 24 Phenolic Compounds Extraction ................................ ................................ ...... 24 Color Analysis ................................ ................................ ................................ .. 25 Physicochemical Analysis ................................ ................................ ................ 25 Phenolic Content and Antioxidant Activity Analysis ................................ .......... 26 Statistical Analyses ................................ ................................ .......................... 29 Results and Discussion ................................ ................................ ........................... 29 Color Analysis ................................ ................................ ................................ .. 29 Physicochemical Analysis ................................ ................................ ................ 29 Phen olic Compounds and Antioxidant Activity Analysis ................................ ... 31 Principal Component Analysis ................................ ................................ .......... 32 Summary ................................ ................................ ................................ ................ 33

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6 3 ENZYME RELEASE OF PHENOLICS FROM MUSCADINE GRAPE ( VITI S ROTUNDIFOLIA MICHX.) SKINS AND SEEDS ................................ ..................... 68 Background ................................ ................................ ................................ ............. 68 Materials and methods ................................ ................................ ............................ 70 Grape Materials ................................ ................................ ................................ 70 Sample Preparation ................................ ................................ .......................... 71 Chemicals ................................ ................................ ................................ ......... 71 Enzymes ................................ ................................ ................................ ........... 72 Extraction of Phenolic Compounds ................................ ................................ .. 72 Phenolic Content and Antioxidant Activity Analysis ................................ .......... 74 Ratio of Antioxidant Activity to Total Phenolics ................................ ................. 76 Statistical Analyses ................................ ................................ .......................... 77 Results and Discussion ................................ ................................ ........................... 78 Different Combinations of Solvent or Enzyme on Release of Phenolics from Grape Skin and Seeds ................................ ................................ .................. 78 Enzyme Type and Incubation Time on Release of Phenolics from Grape Skin and Seeds ................................ ................................ ............................. 80 Enzyme Type and Hydrolysis Time on Antioxidant Activities from Grape Skin and Seeds ................................ ................................ ............................. 85 Antioxidant Activity after Enzyme Hydrolysis ................................ .................... 85 Summary ................................ ................................ ................................ ................ 87 4 ANTIOXIDANT, ANTIBACTERIAL AND ANTIBIOFILM PROPERTIES OF POLYPHENOLS FROM MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX.) POMACE AGAINST SELECTED FOODBORNE PATHOGENS .............. 95 Background ................................ ................................ ................................ ............. 95 Materials and Methods ................................ ................................ ............................ 96 Bacter ial Strains ................................ ................................ ............................... 96 Antibiotics and Chemicals ................................ ................................ ................ 97 Grape Materials, and Extraction, Separation, and Identification of Grape Phenolic Compounds ................................ ................................ .................... 97 Phenolic Content and Antioxidant Activity Assay ................................ ........... 100 Time kill and Synergy Assay ................................ ................................ ......... 102 Antibiofilm Activity Assay ................................ ................................ ................ 103 Statistical Analysis ................................ ................................ .......................... 104 Results ................................ ................................ ................................ .................. 104 Muscadine Grape Phen olic Compounds and Antioxidant Activity .................. 104 Antibacterial Activity of Phenolic Compounds ................................ ................ 105 Evaluation of Bacterial Killing and Synergistic Effect by Phenolic Compounds ................................ ................................ ................................ . 107 Antibiofilm Activity of Phenolic Compounds ................................ .................... 108 Discussion ................................ ................................ ................................ ............ 109 Summary ................................ ................................ ................................ .............. 113

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7 5 MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX.) POLYPHENOLS REDUCE ACRYLAMIDE FORMATION IN BOTH ASPARAGINE/GLUCOSE AND P OTATO CHIP MODEL SYSTEM S ................................ ............................. 123 Background ................................ ................................ ................................ ........... 123 Materials and Me thod ................................ ................................ ........................... 124 Chemicals ................................ ................................ ................................ ....... 124 Grape Materials ................................ ................................ .............................. 124 Sample Prepa ration ................................ ................................ ........................ 124 Phenolic Compounds Extraction ................................ ................................ .... 125 Phenolic Compounds Analysis ................................ ................................ ....... 125 Antioxidant Activities Analysis ................................ ................................ ........ 126 Chemical Model Maillard Reactions ................................ ............................... 126 Fried Potato Chip Model Maillard Reactions ................................ .................. 127 Simulated Physiological Conditions Trapping Reactions ................................ 127 Acrylamide Bromination ................................ ................................ .................. 128 GC ECD Analysis ................................ ................................ ........................... 129 HPLC ESI MS n Analysis ................................ ................................ ................. 129 Color Analysis ................................ ................................ ................................ 130 Statistical Analyses ................................ ................................ ........................ 131 Results and Discussion ................................ ................................ ......................... 131 Effect of Phenolic Compounds Concentration on the Formation of Acrylamide ................................ ................................ ................................ .. 131 Effect of Muscadine Grape Phenolic Compounds on the Formation of Acrylamide ................................ ................................ ................................ .. 132 Correlation between An tibrowning Activity and Inhibiting Acrylamide Formation of Phenolic Compounds ................................ ............................. 132 Correlation between Antioxidant Activity and Inhibiting Acrylamide Formation of Phenolic Compounds ................................ ............................. 134 Inhibitory Mechanism of Phenolic Compounds and Proposed Pathway ......... 135 Muscadine Grape Phenolic Compounds Reducing Acrylamide in Potato Chip ................................ ................................ ................................ ............. 136 Acrylamide Trapping Capability of Phenolic Com pounds in Simulated Physiological Conditions ................................ ................................ ............. 136 Summary ................................ ................................ ................................ .............. 137 6 CONCLUSIONS ................................ ................................ ................................ .... 148 LIST OF RE FERENCES ................................ ................................ ............................. 150 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 159

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8 LIST OF TABLES Table page 2 1 Muscadine grape evaluation: fruit quality and nutraceutical properties in juice ... 34 2 2 Muscadine grape evaluati on: grape seed polyphenols and antioxidant ac tivity 41 2 3 Muscadine grape evaluation : grape skin polyphenols a nd antioxidant activity .... 48 2 4 Correlation matrix of grape fruit quality ................................ ............................... 55 2 5 Correlation matrix of grape seed polyphenols and antioxidant activities ............. 56 2 6 Correlation matrix of grape skin polyphenols and antioxidant activities. ............. 57 3 1 Treatment conditions for 10 combinations of solvent and enzyme . 89 3 2 Ethanol, enzyme type and incubation time on extraction of total phenolics and anthocyanins from grape skins and seeds ................................ ......................... 90 3 3 Effect of ethanol, enzyme type and incubation time on grape skins and seeds antioxidant activities (DPPH values) ................................ ................................ ... 91 4 1 Phenolic content and antioxidant activity of experimental compounds . 115 4 2 Antibacterial activity of experimental compounds against selected foodborne pathogens using the disc diffusion method ................................ ....................... 116 4 3 Minimum inhibitory concentration (MIC) of experimental compounds against human pathogenic bacteria ................................ ................................ .............. 117 4 4 Minimum inhibitory concentration (MIC) of Nalidixic acid against pathogenic bacteria and Minimum Biofilm Inhibitory Concentration (MBIC) against their preformed biofi lm reduction ................................ ................................ .............. 118 4 5 Minimum inhibitory concentration (MIC) of experimental compounds against S. aureus ATCC 35548, and Minimum Biofilm Inhibitory Concentration (MBIC) against its biofilm formation and preformed biofilm reduction ............... 119 5 1 Correlation between color development and inhibi tion of acrylamide formation by phenolic compounds 138 5 2 Aqueous chemical model reactions for mechanism study ................................ .. 139

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9 LIST OF FIGURES Figure page 2 1 Distribution of individual polyphenols from Muscadine grape seed .................... 58 2 2 Distribution of individual polyphenols from Muscadine grape skin ..................... 59 2 3 Distribution of total polyphenols from Muscadine ................................ ............... 60 2 4 Distribution of total antioxidant activity from Muscadine ................................ ..... 6 1 2 5 Component plot of pr incipal component analysis of muscadine grape fruit quality based on principal components 1, 2 and 3. ................................ ............. 62 2 6 Component plot o f principal component analysis of muscadine grape seed polyphenols based on principal components 1, 2 and 3 ................................ ..... 63 2 7 Component plot of principal component analysis of muscadine grape skin polyphenols based on principal components 1, 2 and 3. ................................ .... 64 2 8 Score plots of principal component analysis of grape fruit quality based on principal components 1, 2 and 3. ................................ ................................ ........ 65 2 9 Sc ore plots of principal component analysis of grape seed polyphenols based on principal components 1 and 2 ................................ ................................ ........ 66 2 10 Score plots o f principal component analysis of grape skin polyphenols based on principal components 1, 2 and 3 ................................ ................................ .... 67 3 1 Total phenolics and anthocyanins of grape skin and seed extracted by 10 com 92 3 2 HPLC chromatogram (280 nm) of the predominant phenolic compounds present in Noble seeds by different treatments. ................................ ................. 93 3 3 The ratio of antioxidant activities (DPPH values) to total phenols for different fractions of Noble seeds by various treatments. ................................ ................. 94 4 1 Phenolic compounds and their structures found i n muscadine grape skin and . . 120 4 2 Evaluation of killing (a, b, c) and lytic (d, e, f) action of experimental compounds and their synergism with antibiotics against S. aureus ATCC 35548 in MHB. ................................ ................................ ................................ .. 121 4 3 Percent reduction of Alamar blue (bars) and Log 10 CFU/mL ( ) for phenolic compounds on S. aureus ATCC 35548. ................................ ............. 122

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10 5 1 Effect of different concentrations of phenolic compounds on the formation of acrylamide in chemical models containing 0.8 mM each of asparagine and glucose in 2.5 mL of water and heated at 180 ± 5 °C for 60 min. ..................... 140 5 2 Effect of different varietal muscadine seed and skin phenolic compounds (100 µg/mL) on the formation of acrylamide in chemical models containing 0.8 mM each of asparagine and glucose in 2.5 mL of water and heated at 180 ± 5 °C for 60 mi n. ................................ ................................ ................................ ......... 141 5 3 Concentration study on the effect of phenolic compounds on color development in acrylamide producing chemical models. ................................ . 142 5 4 Correlation analysis between antioxidant activity (DPPH method) and inhibition of acrylamide by phenolic compo unds at 50 µg/mL. .......................... 143 5 5 HPLC chromatograms (280 nm) of chemical model reaction G (0.8 mM asparagine + 0.8 mM glucose + 0.35 mM epicatechin) before heating and heated at 180 ± 5 °C for 60 min. ................................ ................................ ....... 144 5 6 MS/MS spectrum demonstrating the possible existence of epicatechin acrylamide adduct (m/z 432) and proposed pathway for the scavenging of amide source to inhibit acrylamide formation by epicatechin. ........................... 145 5 7 Relationship between acrylamide levels and different immersion solutions of muscadine (Carlos skin) polyphenols for chips before frying (immersion time: 60 min, n = 6).. ................................ ................................ ................................ . 146 5 8 Activity of epicatechin in direct trapping of acrylamide in simulated physiological conditions. ................................ ................................ ................... 147

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11 LIST OF ABBREVIATIONS ANOVA Analysis of variance DAD Diode array detector DPPH 2, 2 diphenyl 1 picrylhydrazyl g Gram g Relative centrifugal force h Hour (s) HPLC High performance liquid chromatography Kg Kilogram L Liter m/z Mass to charge ratio MS Mass spectrometer Microgram Microliter Micromole MBIC Minimum Biofilm Inhibitory Concentration MIC Minimum inhibitory concentration mL Milliliter mm Millimeter min Minute (s) m mol Milli mole MW Molecular weight psi Pounds per square inch

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12 PCA Principal component analysis PC1 Principal component 1 PC2 Principal component 2 PC3 Principal component 3 rpm Revolutions per minute s Second (s) Trolox 6 Hydroxy 2,5,7,8 tetramethylchroman 2 carboxylic acid SS C S oluble solids content UV Ultraviolet Vis Visible V Volume W Weight

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy QUALITY ASSESSMENT, ANTIOXIDANT AND ANTI MICROBIAL ACTIVITIES , AND ACRYLAMIDE INHIBITIO N IN MUSCADINE GRAPE S By Changmou Xu August 2014 Chair: Maurice R. Marshall Major: Food Science The muscadine grape ( Vitis rotundifolia Michx.) industry in the southeastern United S t ates has expanded in recent years as more varieties become available. To provide quality and nutraceutical information for this industry, 58 muscadine varieties were evaluated for their f ruit quality, phenolic profiles and antioxidant activities during two growing seasons . Then an enzymatic extraction method for polyphenols was proposed to improve the functiona lity of grape pomace. Lastly , the antimicrobial properties and acrylamide inhibiting properties of polyphenols from grape pomace were evaluated for the potential of utilizing winery wastes. In t h e first objective, data related to 58 muscadine grape cultiv (color, weight , size, pH, titratable acidity , soluble solids content, °Brix/acid ratio ) , and nutraceutical (polyphenol contents and profiles) and antioxidant properties were developed for a database . In the second objective, enzyme hydrolysis shorten ed the polyphenols extraction time total phenolic yield compared with solvent (50% ethanol)

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14 alone. Interestingly , enzyme hydrolysis was found to modify the galloylated form of polyphenols to low molecular weight ph enolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity. In t he third objective , antioxidant activity for different polyphenols varied greatly ranging from 5 .0 11.1 mmol Trolox/g. Antioxidant and antibacterial activi ty for polyphenols showed a weak positive correlation ( r = 0.53). Muscadine polyphenols exhibited a broad spectrum of antibacterial activity aga inst tested foodborne pathogens . Muscadine polyphenols at 4 × MIC caused nearly a 5 log 10 CFU/mL drop in cell vi ability for S. aureus in 6 h with lysis, while at 0.5 x MIC, they inhibited its biofilm formation, and at 16 × MIC, they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log 10 CFU/mL drop in cell viabili ty at sub inhibitory concentration. In t he fourth objective , muscadine grape polyphenols could significantly reduce acrylamide formation in both in vitro chemical and potato chip model system s . The reduced rates of grape polyphenols were higher than 90% at 100 /mL. These results suggest that muscadine grape polyphenols may be used as a natural food additive to mitigate acrylamide formation in heat treated commercial starchy foods.

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15 CHAPTER 1 AN OVERVIEW OF THE MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX.): FRUIT QUALITY, POLYPHENOLS, AND POTENTIAL HEALTH BENEFITS Muscadine Grapes The muscadine grape ( Vitis rotundifolia Michx.) is native to the southeastern United States and was the first native grape species to be cultivated in No rth America. The natural range of muscadine grapes extends from Delaware to central Florida and occurs in all states along the Gulf Coast to east Texas. It also extends northward along the Mississippi River to Missouri ( 1 ) . Most scientists divide the Vitis genus into two subgenera: Euvitis (the European, Vitis vinifera L. grapes and the American bunch grapes, Vitis labrusca L.) and the Muscadania grapes (muscadine grapes). There are three species within the Muscadania subgenera ( Vitis munsoniana, Vitis popenoei and Vitis rotundifolia ). Euvitis and Muscadania have somatic chromosome numbers of 38 and 40, respectively ( 1 ) . There are over 100 impr oved cultivars of muscadine grapes . Muscadine grapes are tolerant of insect and disease pests, and homeowners can successfully grow muscadine grapes without spraying any pesticides. Muscadine grapes are consumed as fresh fruit or processed into wine, juice , jam or jelly. Fruit Quality Muscadine grapes have taste of some fruit juices ( 2 ) . Muscadine gra pes vary in size from 1/4 to 1 1/2 inches in diameter and 4 to 15 grams in weight. Skin c olor ranges from light bronze, pink, and purple to black, while f lesh is clear and translucent for all muscadine grapes ( 1 ) . Muscadine grapes have very

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16 thick and tough skin, which account s for about 40% of their fresh fruit weight. Muscadine b erries usually contain soluble solids around 14° Brix, which is lower than the Vitis vinifera L. grapes . Polyphenols Muscadine grapes have several uni que and distinguishin g chemical compositions , particularly the presence of ellagic acid. Ellagic acid is commonly present in other fruits, such as raspberry, strawberry, and blackberry, but is absent in all other Vitis spec ies. Ellagic acid in muscadine grapes is expressed as free ellagic acid, ellagic aci d glycosides, and ellagitannins ( 3 ) . Also uni que to muscadine grapes is the presence of myricetin in the bronze grapes, as this flavonol is not present in white V. vinifera grapes ( 4 ) . Another unique attribute of muscadin e fruit chemistry is the presence of anthocyan ins as 3,5 diglucosides of del phinidin, cyanidin, petunidin, peonidin, and malvidin in non acyla ted forms ( 4 ) . About 80% and 18% of polyphenols are located in the muscadine seeds and skin, respectively. The pulp contains very low amount of polyphenol s ( 5 ) . Extraction of Polyphenols In grape, phenolics in general can be classified as 1) cell wall phenolics, which are bound to polysaccharides by hydrophobic interactions, hydrogen bonds and covalent bonds, and 2) non cell wall phenolics, encompassing phenolics confined in the vacuoles of plant cells and phenolics associated with the cell ( 6, 7 ) . The cell wall of grape is a complex network composed of about 30% neutral polysaccharides (cellulose, xyloglucan, arabinan, galactan, xylan and mannan), 20% acidic pectin substances (of

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17 which 62% are methyl esterified), ~15% insoluble proanthocyanidins, and < 5% structural proteins ( 6 ) . Degradation of cell wall polysaccharides, which eliminates this physical barrier and opens up the cell, is a fundam ental step in improving the release of phenolics from grapes . Research has focused on the application of cell wall hydrolyzing enzymes, such as cellulases, glucanase, and pectinases, to release phenolics from the grape fruit and pomace. Kammerer et al. ( 8 ) reported that pectinases and cellulases could result in notably higher recovery rates of phenolics from Vitis vinifera L. grape pomace. Pectinases and macerating enzymes also were reported to promote anthocyanin extraction and improve the quality of red wines ( 9 ) . However, others have found that pectinases and macerating enzym es can cause a decrease in the total yield of anthocyanins and a loss of wine color, or the pectinases have no apparent benefit ( 10, 11 ) . Ce llulase treatment was not affective for phenolic release from grape ( Vitis vinifera L.) pomace ( 12 ) . Li et al. ( 13 ) reported that enzyme assisted aqueous extraction did not give as high a recov ery of citrus peel phenolics as solvent (72% ethanol) extraction. With so many contradictory findings, and considering the phenolic profiles from different grape species/varieties are not the same ( 14 16 ) , an i n depth study of phenolic release by enzymes in various grape species would be useful. Not only may hydrolysis of cell wall polysaccharides help release various phenolics, these enzymes may hydrolyze the polyphenols into low molecular weight phenolics, whi ch may increase the availability and bioactivity of these phenolics ( 12, 17 ) . Monomeric and some oligomeric polyphenols have been found to absorb into rat plasma and are directly involved in physiological functions ( 18 ) , while polymeric forms are poorly absorbed ( 19 ) . Therefore, it is important to evaluate the effect of enzyme

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18 hydrolysis of grape pomace on the magnitude of phenolic release, structure of phenolics after hydrolysis and antioxidant activities. Potential Health Benefits Muscadine grapes and wines are noted for their health benefit, antioxidant polyphenols and other nutrients. Cell culture studies have suggested that polyphenols from muscadine grapes can inhibit proliferation of many types of cancer cells ( blood, colon, an d prostate ) , and induce apoptosis in them ( 20 22 ) . Work continues and will continue to support muscadine grape products as healthful food s. Antimicrobial and Antib iofilm A ctivity of P olyphenols Another area of interest for muscadine polyphenols is their ability to act as antimicrobial agents, and also as part of its antimicrobial nature, antibiofilm agents. Most bacteria can form complex, matrix containing multicellular communities known as biofi lms, which protect them from environmental stresses such as antibiotic exposure. Biofilms contribute to the pathogenesis of many forms of microbial infection. It has been estimated that up to 80% of microbial infections in the body involve biofilms. These infections include those in the urinary tract, middle ear, mouth, heart, and lungs (particularly in cystic fibrosis patients), as well as those associated with medical devices such as catheters, prostheses and artificial heart valves ( 23 25 ) . Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm associated infections. Recent studies indicated that polyphenols such as ellagic acid ( 26 ) , proanthocyanidins ( 27 ) , and tannic acid ( 28 ) have strong antibiofilm properties, reducing formation of biofilm by bacteria, thereby killing the bacteria.

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19 Muscadine grapes are rich sources of these polyphenols, which contribute to this species strong resistant to pests and diseases, including Pierce's disease. It is these anti disease polyphenols that show promise as antimicrobial agents and bacterial biofil m inhibitors, especially as natural food preservatives, potential antibiotic replacement s and as natural sanitizers for processing equipment where bacteria a nd biofilms prominently reside. Unfortunately, the majority of polyphenol compounds are lost when p rocessors discard the thousands of tons of peel and seeds as pomace to landfills. This pomace may offer the grower s /processor s value added products in both the functional food and supplemental markets, and food preservative markets. It is estimated that nu traceutical ingredients will increase by 7.2% annually to approximately $24 billion by 2015 ( 29 ) while food preservative markets globally will steadily incre ase to over $2.5 billion by 2018 ( 30 ) . Muscadine grape ingredients having these properties could certainly infiltrat e these markets and provide the grower/ processor value added markets for their waste streams. Acrylamide I nhibition Activity of P olyphenols Acrylamide is a byproduct of the Maillard reaction and it is formed in a variety of heat ( 31 ) . There have been extensive studies on the formation mechanism of acrylamide. Antioxidants ha ve been proposed as one possible mechanism to reduce acrylamide formation ( 32 ) . Particularly, anti oxidants such as phenolic compounds, flavonoids, vitamins and phenolic extracts from various spices have been reported to inhibit acrylamide formation ( 33 35 ) . The above findings were confirmed by Totlani et al. ( 36 ) , using epicatechin which quenches 3 deoxy 2 -

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20 hexosulose (a key source of C 6 to C 1 sugar fragments) and consequ ently inhibits Maillard product formation. Muscadine grape ( Vitis rotundifolia Michx.) contains a large variety of antioxidant phenolic compounds. It is hypothesized that muscadine grape polyphenols may also reduce acrylamide formation because of their strong antioxidant activity. Finally, information about these nutrients in muscadines is only limited to few major cultivars used for processing ( 5, 37 ) . There are over 50 cultivars of muscadin es with little nutraceutical, antimicrobial or acrylamide inhibition information about them. One may assume that the major cultivars used in processing are representative of those other cultivars; however, the literature has shown that antioxidant and polyphenol characteristics and levels of fruit and vegetable are often cultivar dependent. For i nstance, the ellagic acid and myricetin content in Carlos skin were 3 times higher than in skin ( 37 ) . Access to information about the health, antimicrobial or acrylamide inhibition nature of muscadine cultivars would be very useful to growers and processors; allowing them to make decisions on whi ch cultivars offer the qualities for processing while potentially adding valu e to a waste stream in the form of functional ingredients. Research Question and Objectives industry could use the nutraceutical information of various muscadine varieties ; and thereby utilizing these winery waste s (pomace) to produce value added products while adding economic benefit for muscadine growers and processors

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21 To answer this question the following objectives were performed: Objective 1: To produce data on fruit quality and nutraceutical properties for muscadine grapes by analyzing 58 genetic ally differen t varieties over two consecutive years ( 2012 and 2013), which could be added to a searc hable data base for growers and processors . Objective 2: To s tudy the enzymatic releas e of muscadine grape polyphenols on content and partial phenolic identification . Objective 3: To i solate the polyphenols fro m muscadine grape and examine the ir antimicrobial and antibiofilm effect s on foodborn e pathogens, t he ir the synergism with antibiotics. Objective 4: To i solate the polyphenols fro m mus cadine grape and investigate the ir i nhibitory role on the formation of ca r ce n ogenic acrylamide, the ir re lationship, the correlation with antioxidant activity and Millard reaction, and the possible inhibition mechanism.

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22 CHAPTER 2 FRUIT QUALITY AND NUTRACEUTICAL PROPERTIES OF MUSCADINE GRAPES ( VITIS ROTUNDIFOLIA MICHX. ) Background Muscadine grapes and wines are noted for their health benefit due to high nutraceutical con tent and other nutritive value, especially in the pomace which is often released and used as animal feed, fertilizer or placed in landfills. Interestingly, the greatest concentration of muscadine nutraceuticals and other potential food and p harmaceutical by products are the pomace. Each year, thousands of tons of pomace are released as waste by wineries and juic ing facilities which account for one of the largest single waste in landfills. In addition, with over 50 cultivars of muscadine, there is sparse information on the levels of nutraceuticals in these cultivars. Most studies have only looked at a few muscadine varieties such as, because these are the most commonly grown for wine and juice production . Wh at are the fruit quality, polyphenol profiles and antioxidant properties of other muscadine cultivars? Very little information exists about these properties for these cultivars and this is the basis for the research . A more com prehensive look at muscadine cultivars would n cultivars. Also , n utraceutical ingredients from grape pomace could potentially infiltrate the $20 billion suppleme nt and functional food markets while adding economic benefit for muscadine growers and processors. This project aims to supply the information on fruit quality, polyphenol profiles and antioxidant properties by developing data for growers and processors of muscadines. Once the data is developed , a database could be established and any

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23 work outside of this proposal performed on the he alth benefits nature of muscadine cultivars can be incorporated, thereby compiling information that growers and processors can access. Materials and Method s Grape M aterials Fully ripened Muscadine grape s ( Vitis rotundifolia Michx.) (52 cultivars and 6 breeding lines ) were harvested from the Center for Viticulture and Small Fruit Research (latitude 30.65 N, longitude 84.60 W) at Florida A&M University in 2012 and 2013 . The collected samples were shipped to the University of Florida on the same day and st ored in a cold room (4 °C ). Grape skins and seeds were separated manually from berri es and freeze dried in a freeze drier (Advantage, The Virtis Company, NY, USA) . The freeze dried samples were stored in vacuum packaged polyethylene pouches at 20 °C until analyzed . Three commercial grape varieties ( Red Seedless, Green Seedless, and Red Seed) were used as controls and purchased in 2012 and 2013 from a Wal Mart Store, Gainesville , FL . Chemicals Epicatechin, Epicatechin gallate, Trans resveratrol, Ellagic aicd, Quercetin and Cyanidin 3, 5 diglucoside, 2,2 diphenyl 1 picrylhydrazyl (DPPH), and 6 hydroxy 2, 5, 7, 8 tetramethylchroman 2 carboxylic acid (Trolox) were obtained from Sigma Aldrich (St. Louis, MO). All other chemicals and solvents were purchased from Fisher Scientific Co. (Pittsburgh, PA).

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24 Sample P reparation Freeze dried grape skins (20 g) were ground with a stainless steel grinder (Omni Mixer 17105, OCI Instruments, CT, USA) for 1 min , and then placed on a sieve was collected ( 38 ) . The powdered samples were stored at 20 °C and used for subsequent analysis. Freeze dried grape seeds (20 g) were crushed and then defatted with hexane at a ratio of 1:10 (w/v). After 24 h of extraction at room temperature (shaking every 6 hr ), the hexane extract was filtered usi Scientific, Pittsburgh, PA) under vacuum. The residue was evenly distributed over a tray and kept in the hood for hexane to evaporate. The final defatted grape seed powder was ground again in the stainless steel 0.25 mm) was collected. The samples were also stored at 20 °C and used for subsequent analysis. Phenolic C ompounds E xtraction Powder ( 0. 5 g) from each sample above was extracted with 1 0 mL of 70% methanol. The extraction flasks were vortexed for 30 s, sonicated for 10 min, kept at room temperature (22 °C) for 60 min, and sonicated for an additional 5 min. The extracts were transferred to tubes , centrifuged at 2820 x g , 0 °C for 10 min (J LITE ® JLA 16.250, Beckman Coulter Inc., CA, USA) and the supernatant was collected in separate glass tubes. Residue was re extracted by the same procedu re. The collected supernatant ( 2 0 mL) was filtered (0.45 m) and used for further analysis.

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25 Colo r A nalysis The surface colo r of muscadine grape s was measured by a machine vision system. The machine vision system consisted of a fluorescent light box (42.5 cm (w) x 61 cm (l) x 11.4 cm (h)) and a digital Nikon D200 colo r camera (Nikon D200 Digital Camera, Nikon Corp., Japan) connected to a computer with a firewire connection. The Camera settings were 36 mm focal length, ISO 100 sensitivity, 1/3 s F/11 shutter speed, 1.0 EV exposure compensation, and direct sunlight white balance. Machine vision system used the average daylight illumina nt D65 mode with a colo r temperatur e of 6504 K. A software program was used to cap ture images, and to obtain colo r results based on L * (lightness), a * (redness), and b * (yellowness) values ( 39 ) . Grapes were placed in the light box and the digital camera captured a picture of the grapes for each variety . Machine vision system was calibrated using a standard red plate ( L * = 48.62, a * = 49.04, and b* = 25.72 ) from Labsphere (North Sutton, NH). Average L*, a*, b* values of the grapes surface were calculated using a color analysis program . Physicochemical A nalysis The weight of mu scadine grapes was measured using an analytical balance Mettler PM 400 (Mettler Instrument Corp., Highstown, NJ , USA ). The size s of muscadine grapes were measured by diameter using a V ernier caliper . The pH and soluble solid s ( °Brix ) were measu red using a pH meter EA920 (Or ion Research; Boston, MA, USA) and an ABBE Mark II refractometer (Leica Inc.; Buffalo, NY, USA). Titratable acid ity was determined using a previous method ( 40 ) . A 10 mL sample of juice was titrated with 0.1 N sodium hydroxide to an end point of pH 8.2 using a TPS digital pH meter ( Or ion Research; Boston, MA, USA ). The titrated volume of 0.1 N NaOH was

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26 recorded, and the acidity was calculated as tartaric acid content (g/100 mL o f juice) by using the following formula: The °Brix to acid ratio for each sample was calculated by dividing the °Brix value by % acidity. Phenolic C ontent and A ntioxidant A ctivity A nalysis Analysis of total phenolics Total phenolic content in grape juice, seeds or skin was determined by the method of Singleton et al. ( 41 ) using an ultraviolet visible Beckman Coulter DU 640 spectrophotometer (Beckman Instruments, CA, USA). A mixture of 100 juice or properly Ciocalteau reagent (2N), 2 CO 3 were introduced in a tube. After reacting for 30 min in a 40 °C water bath, absorbance was measured at 760 nm. Gallic acid (GA ) was used as standard and expressed as gallic acid equivalents ( mg gallic acid (GAE)/ mL juice or mg GAE /g dry matter (DM)) using a calibration curve. The linearity range of the calibration 2 = 0.9996). Analysis of total anthoc yanins Total anthocyanin content in muscadine grape skins was measured using the pH differential spectrophotometric method described by Lee et al. ( 42 ) . The anthocyanin extract was dissolved in 0.025 M potassium chloride buffer, pH 1.0 and 0.4 M sodium acetate buffer, pH 4.5 with a pre determined dilution factor. The absorbance was measured at 510 nm and 700 nm. The absorbance (A) of the diluted sample was then calculated as follows:

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27 A = (A 510nm A 700nm ) pH 1.0 (A 510nm A 700nm ) pH 4.5 The monomeric anthocyanin concentration in th e original sample was calculated as cyanidin 3,5 diglucoside equivalents according to the following formula: where MW (611) of cyanidin 3,5 diglucoside is used because the anthocyanin content was calculated in cyanidin 3,5 diglucoside equivalents (mg CAE/g DM); the molar absorptivity e was 30,175; the dilution factor (DF) of 1000 is the factor to convert g to mg; and A was absorbance. Anthocyanin content was mg/L, which was then converted to mg/g dried sample. Identification of individual phenolics The obtained extracts were subj ected to chromatographic analysi s on a Hitachi HPLC system with a Zorbax Stablebond Analytical SB C18 column (4.6 mm , 250 mm, acid aqueous solution) and mobile phase B (60% methanol with 0.5% formic acid). The following linear gradient was used: 0 5 min : 5% B, 5 10 min: 15% B, 10 20 min: 25% B, 20 30 min: 50% B, 30 40 min: 70% B, 40 50 min: 90% B. The flow rate was 0.9 mL/min. Injection volume was 20 µl with the UV detector set to an absorbance wavelength of 280 nm. The retention times for each of the di fferent components in the analyzed pool were compared to the following standards: gallic acid, catechin, epicatechin, epicatechin gallate, trans resveratrol, ellagic acid, and quercetin. Standards for pentagalloyl glu cose and ellagic acid hexoside were not available. Thus, a second HPLC system was employed to confirm the identification of these by ESI MS detection in the Selective reaction monitoring (SRM) mode. The LC MS

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28 analyses were performed on a Thermo Finnigan Surveyor HPLC system (Thermo Finnigan, St . Jose, CA, USA) equipped with a TSQuantum controlled by XCalibur data analysis software (version 1.3, Thermo Finnigan). The column and mobile phase used were the same as described above in HPLC analysis. The MS acquisition was with ESI interface in negati ve ionization mode at the following conditions: Sheath gas (N2) pressure, 42 arb; auxiliary gas (N2) pressure, 20 arb; spray voltage 3.96 kV; capillary temperature, 414 °C; collision gas pressure, 1.5 mTorr; collision energy, 22 V. These compounds were id entified on the basis of their mass fragmentation data compared with and identical to those reported by Sand h u et al. ( 5 ) . Analysis of antioxidant activities by DPPH Assay The DPPH assay was based on the slightly modified method of Brand W illiams et al. ( 43 ) . The properly of DPPH (0.0025 g/100 mL CH 3 OH). After 60 min reaction at room temperature in the dark, the absorbance at 515 nm was recorded to determine the concentration of the remaining DPPH. The percentage inhibition of DPPH in the test sample and known concentrations of Trolox were calculated by the following formula: %Inhibition = 100 × (A 0 A )/ A 0 , where A 0 was the beginning absorbance at 515 nm, obtained by measuring the same volume of solvent, and A was the final absorbance of the test sample at 515 nm. The calibration curve between %Inhibition and known concentration of Trolox solutions was then established. The radical scavenging activities of the test samples were expressed as Trolox equivalent antioxidant capacity ( /mL juice or l TE/g DM) from their percent inhibition. Trolox standard solutions were prepared at a concentration 2 =0.9999).

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29 Statistical A nalyses All measurements were performed in quadruplicate . Polyphenol extractions and analyses were performed in duplicate. Data were subjected to ANOVA and differences among samples were tested by pos t hoc comparison test (Student Newman Keuls) at p = 0.05 . The principal component analysis (PCA) ( 44 ) was done to detect clustering formation and estab lish relations hips between samples concerning fruit quality, phenolic compound s or antiox idant properties. Microsoft Excel 2010 and SPSS 21 .0 for Window s (SPSS Inc, Chicago, IL) were used for the above analysis and Pe coefficient ca lculation s . Results and Discussion Color A nalysis Four major co lors were determined among the investigated muscadine varieties (Table 2 1) . They were dark, red, brown, and orange. The detailed color description of each variety was listed in Table 2 1, L * (20.2), a * (9.7), b *(7.4) values. A p revious study also classified the color of muscadine grapes ( 45 ) , however, except for the overall description, the L * , a * , b * values of these muscadine grapes were only given in this stu dy which contributed to an exact d escription of color. Physicochemical A nalysis Weight and size . Fruit weight was significantly different among muscadine grape varieties , ranging from 2.9 3 g ( Fry Seedless ) to 22.3 2 g ( Pam ) with a media n weight of 12.12 g , and average weight of 11.6 2 g (Table 2 1) . Fruit size was measured by diameter, and grape diameter s showed a significant ( r = 0.9 43 ) relation to their weight,

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30 ranging from 1.6 0 cm ( Fry Seedless ) to 3.6 with a median size of 2.70 cm , and average size of 2.64 cm (Table 2 1 , 2 4 ). A good variety for fresh mark et consumption would be a large berry. Therefore, the lager varieties 1 1 would be considered good cho ices for table grapes . pH and acidity . The organic acids play an important role in flavor perception, and their concentration can be measured easily to determine maturity standards. The pH of juice ranged from 3. 0 1 ( Lommis ) to 3.8 4 ( Supreme ) with a median pH of 3.51 and average pH of 3.50 for the 2012 season (Table 2 1). F or the 2013 season, the pH of juice r anged from 2.90 ( Lommis ) to 3.57 ( Alachua ) with a median pH of 3.30 and average pH of 3.29. As expected, grape titratable acidity for the 2012 s eason was lower than the 2013 season on average, which ranged from 0.27% ( Noble ) to 0.83% ( Rosa ) with the median acidity of 0.47% and average acidity of 0.44% for the 2012 season; while it ranged from 0.29% ( Noble ) to 0.86 % ( Rosa ) with the m edian acidity of 0.54% and average acidity of 0.53% for the 2013 season. However, t here was no significant correlation ( r = 0.425) betw een pH and titratable acidity for the grape juice s (Table 2 4) . Overall, the average acidity was higher in the 2013 season grapes than 2012 indicating the 2013 grapes were less mature . This probably is close ly related to the weather of the grape s during veraison and maturation, which was very dry during the 2012 season, while very wet for the 2013 season. Drier wea ther (l ess than 100 mm of rainfall) between veraison and ma turation is an important indi cator for the selection o f superior wine producing areas. Since this would help the maturation and also contribute to richer nutrients and phenolic compound accumulation in grape berries ( 46 ) .

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31 Soluble solids ( °Brix ) and °Brix/acid ratio. The soluble solids concentration (SSC) represented as °Brix value ranged from 10. 92 ( Tara ) to 23.9 1 °Brix ( Dixie Land ) with median SSC of 15.35 and average SSC of 15.03 among the varieties evaluated. (average 21.6°Brix) than most of the muscadine grapes. However, their °Brix/acid ratio (average 33.0) was lo wer than most muscadine grapes (> 50%) , which ranged from 14.02 to 57.06 with a median ratio of 34.85 and average ratio of 34.46. Nelson et al. ( 47, 48 ) in their extensive study on the maturity of Perlette , Thom p son Seedless and Cardinal grapes, reveal ed a good positive correlation between °Brix /acid ratio and flavor preferences, and suggested that °Brix /acid ra tio should be used as the basis for determining fruit ripeness. Based on this evaluation, there should be a good market for muscadine grapes. Phenolic C ompounds and A ntioxidant A ctivity A nalysis Total phenols for the different muscadine juices varied from 0.26 ( Gold Isle ) to 1 .28 mg GA/mL ( Fry ), and showed significant ( r = 0. 908 ) cor relation to their antioxidant activities, which varied from 0.97 ( Doreen ) to 6.78 mM Trolox/mL ( 026 1 2 ) (Table 2 1 2 4 ) . T he total phenolic content and antioxidant activities of all muscadine varieties, based on dry weight, were higher in seeds than skins, and 80% of them were higher when compared to the commercial grapes. On average, about 70% and 29% of polyphenols are located in the muscadine seeds and skin, respectively (Figure 2 3) . The pulp contains very low amount (less than 1%) of polyphenols. For seeds, total phenolic conten Sugar Pop ) to 72.0 ( Florida Fry ) mg GA/g DM, and antioxidant activity ranged from 178.2 ( Sugar

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32 Pop ) to 619.7 ( Florida Fry ) µmol Trolox/g DM (Table 2 2) . For skins, total phenolic content on average rang ed from 10.1 ( Scartlett Janebell ) mg GA/g DM, and antioxidant activity ranged from 83.6 ( Scartlett Janebell ) µmol Trolox/g DM (Table 2 3) . The main compounds identified in seeds included gallic acid, catechin, epicatechin, epic atechin gallate, pentagalloylglucose, and ellagic acid; while skins contained gallic acid, anthocyanins, epicatechin, ellagic acid, resveratrol, and quercetin (Table 2 2, 2 3) . Significant season and varietal differences were found in seed and skin total p henolic content but not in their phenolic profiles (Figure 2 1, 2 2) . Overall, total phenolic content showed significant correlation to their total antioxidant activity ( r = 0.9 64 for seed and r = 0.870 for skin, Table 2 5, 2 6) . On average, the muscadine seeds and skin contributed about 70% and 29% of antioxidant activities , respectively (Figure 2 4). The pulp contributed very low amo unt (less than 1%) of antioxidant activity . Principal C omponent A nalys i s Fruit weight and size, or total polyphenols and antioxidant activity formed a cluster on the component plot, suggesting that they are positively correlated ( r = 0.943 and r = 0.908, respectively) and they had similar impacts on overall variance (Figure 2 5 ). Acidity and pH were distr ibuted oppositely indicating they are negative ly correlated ( r = 0.425). Year was distributed far away from the other characteristics indicating the year had little effect on them in general. Similarly, the total polyphenols and antioxidant activity of grape seeds or skin formed a cluster on the component plot, suggesting that they are positively correlated (Figure 2 6, 2 7). Total and individual anthocyanin s also formed a cluster on the component plot. Year showed little effect on other characteristic s.

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33 Samples with similarities cluster on the score plot of principal component analysis and segregate from samples of different properties. T he first three principal components accounted for 36%, 33%, and 22% of t he total variance for muscadine grape fruit quality . The extensive distribution of mus cadine varieties indicated there were significant difference s among them. No obvious separation was observed for muscadine grapes and the market grapes (Figure 2 8). Simi la r trends we re found in the score plots of principal component analysis of grape seeds and skin polyphenols (Figure 2 9, 2 10). Summary Overall, data for a database related to 58 muscadine and nutraceutical properties were developed in this study. Ac cess to this information for the 58 muscadine cultivars through a database would be very useful to breeders, growers and processors; allowing them to make decisions on which cultivars offer the quality for growing and processing while potentially providing information about a waste stream that could produce functional ingredients.

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20 Table 2 1 . Muscadine grape evaluation: fruit quality and n utraceutical properties in j uice Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Africa Queen Dark grayish reddish brown (20.56 _ 8.52 _ 6.61) 2012 10.39 2.56 3.48 14.21 0.38 37.39 0.84 2.90 2013 12.49 2.68 3.39 15.30 0.41 37.32 0.79 3.98 Alachua Dark grayish reddish brown (19.01 _ 9.76 _ 6.95) 2012 12.92 2.98 3.49 15.22 0.45 33.82 0.62 2.07 2013 9.89 2.44 3.57 15.90 0.36 44.17 0.52 2.96 Albemarl Dark grayish reddish brown (22.65 _ 8.71 _ 6.09) 2012 7.02 2.26 3.37 17.56 0.47 37.36 0.78 3.59 2013 7.26 2.12 3.29 16.37 0.57 28.72 0.69 3.66 Black Beauty Dark grayish reddish brown (21.44 _ 9.75 _ 6.55) 2012 16.07 3.4 3.53 16.71 0.55 30.38 0.44 1.67 2013 16.41 3.04 3.27 16.47 0.50 32.94 0.65 2.96 Black Fry Dark grayish reddish brown (20.48 _ 8.20 _ 6.41) 2012 11.22 2.64 3.48 15.43 0.58 26.60 0.81 4.25 2013 13.62 2.74 3.28 16.43 0.52 31.60 0.66 2.50 Early Fry Strong brown (44.52 _ 17.51 _ 37.54) 2012 14.12 2.93 3.44 14.76 0.56 26.36 0.66 2.47 2013 14.20 2.82 3.31 17.23 0.61 28.25 0.82 3.90 Carlos Grayish reddish orange (52.00 _ 20.29 _ 33.98) 2012 7.45 2.42 3.40 15.62 0.41 38.10 0.55 1.41 2013 8.02 2.38 3.25 15.40 0.35 44.00 0.42 1.61 Delight Dark grayish reddish brown (19.52 _ 9.11 _ 6.85) 2012 6.54 2.24 3.30 17.01 0.77 22.09 1.05 5.77 2013 6.23 2.21 3.29 16.24 0.70 23.20 1.03 5.54 Dixie Strong brown (43.25 _ 22.40 _ 33.55) 2012 6.66 2.38 3.27 20.54 0.36 57.06 0.48 2.44 2013 5.98 2.20 3.21 17.80 0.44 40.45 0.51 2.20 Dixie Red Dark red (36.97 _ 28.70 _ 25.36) 2012 11.02 2.66 3.65 16.04 0.44 36.45 0.45 2.81 2013 12.80 2.76 3.34 16.27 0.56 29.05 1.23 6.53

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35 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Doreen Strong brown (42.98 _ 19.07 _ 31.71) 2012 12.76 2.67 3.35 18.45 0.38 48.55 0.29 0.97 2013 14.21 2.70 3.17 16.97 0.43 39.47 0.38 1.99 Dixie Land Strong yellowish brown (50.55 _ 16.73 _ 44.70) 2012 11.32 2.74 3.61 23.91 0.43 55.60 0.52 2.99 2013 12.89 2.94 3.30 19.03 0.50 38.06 0.82 4.29 Cowart Dark grayish reddish brown (20.29 _ 8.37 _ 6.55) 2012 9.85 2.51 3.60 14.83 0.52 28.52 0.52 2.27 2013 9.49 2.46 3.31 14.33 0.59 24.29 0.67 4.11 Fry Seedless Dark reddish brown (23.15 _ 17.90 _ 10.24) 2012 2.93 1.64 3.28 17.46 0.53 32.94 0.59 3.70 2013 2.96 1.60 3.12 16.37 0.61 26.84 0.62 3.29 Digby Dark reddish orange (41.63 _ 30.15 _ 32.92) 2012 11.17 2.64 3.76 14.91 0.38 39.24 0.63 3.10 2013 12.69 2.56 3.50 15.20 0.50 30.40 0.95 4.10 Florida Fry Strong brown (46.59 _ 15.10 _ 40.88) 2012 13.08 2.89 3.39 22.04 0.75 29.39 0.60 2.01 2013 17.62 3.02 3.32 16.30 0.52 31.35 0.64 3.04 Farrer Dark grayish reddish brown (21.95 _ 10.20 _ 7.65) 2012 12.36 2.60 3.69 15.46 0.37 41.78 0.47 2.58 2013 11.50 2.54 3.09 14.30 0.45 31.78 0.96 3.81 Gold Isle Dark red (31.05 _ 35.66 _ 18.65) 2012 9.14 2.57 3.11 14.52 0.72 20.17 0.70 4.53 2013 8.43 2.44 3.14 16.23 0.70 23.19 0.26 1.39 Darlene Strong brown (45.05 _ 22.84 _ 31.97) 2012 18.82 3.30 3.50 14.15 0.41 34.51 0.40 2.19 2013 17.03 2.96 3.31 15.53 0.56 27.73 0.62 3.30 Fry Strong yellowish brown (47.47 _ 15.62 _ 40.54) 2012 17.17 3.20 3.55 14.96 0.44 34.00 0.54 2.79 2013 16.60 3.02 3.45 17.43 0.48 36.31 1.28 5.78

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36 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Hunt Dark grayish reddish brown (21.32 _ 9.31 _ 6.35) 2012 6.43 2.26 3.22 11.61 0.65 17.86 0.57 2.14 2013 6.59 2.02 3.04 11.87 0.80 14.84 0.35 1.89 Higgins Brownish orange (51.91 _ 17.33 _ 40.58) 2012 2013 12.31 12.97 2.58 2.64 3.35 3.45 15.62 17.03 0.55 0.50 28.40 34.06 0.48 0.79 2.61 3.83 Grany Val Strong yellowish brown (53.90 _ 10.63 _ 43.06) 2012 13.49 2.85 3.57 16.23 0.36 45.08 0.31 1.91 2013 14.83 2.80 3.48 16.87 0.32 52.72 0.42 2.10 Ison Dark grayish reddish brown (17.73 _ 7.28 _ 5.93) 2012 13.24 2.94 3.39 16.45 0.50 32.90 0.91 5.35 2013 11.37 2.48 3.31 15.93 0.65 24.51 0.47 2.00 Jumbo Dark grayish reddish brown (21.93 _ 7.69 _ 6.19) 2012 15.28 3.16 3.70 14.96 0.43 34.79 0.97 5.99 2013 14.17 3.08 3.36 15.50 0.48 32.29 0.81 2.97 Janet Strong yellowish brown (55.53 _ 11.79 _ 46.49) 2012 14.44 3.06 3.63 14.83 0.47 31.55 0.33 1.96 2013 14.76 2.72 3.45 15.93 0.47 33.89 0.58 3.17 Janebell Strong yellowish brown (48.72 _ 15.69 _ 42.52) 2012 11.89 2.76 3.42 15.04 0.47 32.00 0.72 2.60 2013 11.83 2.52 3.22 16.90 0.42 40.24 0.42 1.91 Lommis Dark red (29.96 _ 35.71 _ 19.24) 2012 7.89 2.30 3.01 12.06 0.86 14.02 0.73 2.99 2013 8.69 2.34 2.90 14.27 0.74 19.28 0.40 1.77 Magnolia Strong brown (40.50 _ 24.20 _ 32.72) 2012 10.34 2.75 3.39 13.78 0.32 43.06 0.30 1.35 2013 13.18 2.58 3.47 16.77 0.40 41.93 0.44 2.01 Nesbitt Dark grayish reddish brown (18.20 _ 10.61 _ 7.78) 2012 12.52 2.86 3.40 12.76 0.44 29.00 0.47 2.45 2013 12.29 2.64 3.25 14.30 0.55 26.00 0.57 2.49

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37 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Pineapple Brownish orange (46.30 _ 18.87 _ 40.20) 2012 12.02 2.65 3.45 16.82 0.39 43.13 0.40 2.04 2013 12.48 2.64 3.21 15.73 0.35 44.94 0.92 5.79 Southern Home Dark grayish reddish brown (19.76 _ 10.94 _ 7.83) 2012 6.45 2.18 3.29 14.53 0.71 20.46 0.97 4.82 2013 7.83 2.12 3.31 13.63 0.77 17.70 1.07 6.58 Later Fry Strong brown (40.78 _ 20.68 _ 34.53) 2012 14.57 3.10 3.67 18.46 0.50 36.92 0.77 3.68 2013 13.38 2.62 3.47 16.73 0.47 35.60 0.82 4.97 Pride Dark grayish reddish brown (20.73 _ 10.92 _ 7.81) 2012 4.48 2.00 3.51 16.87 0.37 45.59 0.44 1.94 2013 4.67 1.86 3.10 15.87 0.36 44.08 0.35 2.33 Sugargate (dark) Dark grayish reddish brown (18.96 _ 10.33 _ 7.97) 2012 11.06 2.58 3.66 17.82 0.50 35.64 0.76 5.24 2013 11.06 2.50 3.44 16.20 0.54 30.00 0.85 5.21 Sugargate (brown) Strong brown (42.22 _ 23.43 _ 35.22) 2012 13.39 2.94 3.35 17.41 0.50 34.82 0.61 2.73 2013 13.78 2.99 3.28 16.98 0.48 35.38 0.65 2.89 Noble Dark grayish reddish brown (20.23 _ 9.71 _ 7.36) 2012 4.74 2.00 3.49 15.03 0.27 55.67 0.63 2.87 2013 4.14 1.80 3.27 15.60 0.29 53.79 0.49 2.31 Sterling Dark reddish orange (38.90 _ 27.29 _ 31.97) 2012 12.62 2.98 3.67 19.06 0.44 43.32 0.40 1.70 2013 10.77 2.50 3.49 18.23 0.41 44.46 0.36 1.39 Senoria Dark reddish orange (41.47 _ 26.95 _ 33.39) 2012 9.76 2.40 3.58 18.76 0.36 52.11 0.32 1.76 2013 9.99 2.48 3.24 18.43 0.36 51.19 0.51 2.58 Sugar Pop Strong brown (44.61 _ 19.29 _ 39.03) 2012 12.87 2.92 3.36 12.44 0.41 30.34 0.44 2.33 2013 10.32 2.44 3.31 14.07 0.53 26.55 0.71 4.10

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38 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Southern Land Dark grayish reddish brown (20.65 _ 10.96 _ 8.06) 2012 8.12 2.46 3.54 13.63 0.48 28.40 0.49 2.38 2013 9.66 2.42 3.37 15.60 0.60 26.00 0.75 3.61 Summit Dark reddish orange (39.22 _ 28.47 _ 31.49) 2012 12.07 2.78 3.63 15.92 0.43 37.02 0.53 3.06 2013 12.06 2.62 3.34 16.93 0.56 30.23 1.25 5.71 Pam Strong yellowish brown (51.20 _ 17.54 _ 43.99) 2012 22.32 3.64 3.79 14.11 0.48 29.40 0.43 1.85 2013 16.18 2.98 3.38 14.30 0.54 26.48 0.68 2.94 Regale Dark grayish reddish brown ( 18.87 _ 9.75 _ 7.71) 2012 5.9 2.22 3.14 13.23 0.60 22.05 0.59 3.19 2013 7.27 2.08 3.04 13.47 0.57 23.63 0.67 2.85 Rosa Dark red (31.22 _ 34.29 _ 20.36) 2012 7.96 2.44 3.20 14.64 0.83 17.64 0.49 2.70 2013 9.65 2.42 3.08 14.50 0.75 19.33 0.50 2.58 Scarlett Moderate reddish brown (29.99 _ 28.85 _ 20.13) 2012 17.05 3.08 3.59 17.02 0.38 44.79 0.31 1.66 2013 16.18 2.90 3.43 16.20 0.47 34.47 0.61 3.08 Red Seeded Table Dark red (29.34 _ 31.01 _ 21.98) 2012 9.01 2.32 3.70 15.60 0.40 39.00 0.38 1.09 2013 9.22 2.42 3.79 16.60 0.38 43.68 0.39 1.16 Scupernong Strong brown (37.86 _ 27.62 _ 30.87) 2012 12.06 2.80 3.69 21.53 0.48 44.85 0.42 2.69 2013 12.57 2.68 3.36 19.53 0.53 36.85 0.60 2.63 Sweet Jenny Brownish orange (48.16 _ 22.67 _ 39.51) 2012 17.34 3.36 3.61 14.84 0.41 36.20 0.49 3.52 2013 15.51 2.96 3.40 15.93 0.47 33.89 0.66 4.33 Supreme Dark grayish brown (16.32 _ 9.78 _ 8.64) 2012 11.35 2.81 3.84 20.62 0.45 45.82 1.05 6.33 2013 15.47 2.88 3.27 20.17 0.50 40.34 0.59 3.20

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39 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Tara Brownish orange (47.74 _ 25.38 _ 44.50) 2012 11.49 2.80 3.61 13.33 0.34 39.21 0.28 1.64 2013 11.31 2.62 3.47 14.00 0.36 38.89 0.46 2.13 Triumph Strong brown (39.33 _ 28.55 _ 36.03) 2012 12.02 2.93 3.47 14.04 0.32 43.88 0.37 2.40 2013 13.07 2.62 3.26 15.77 0.37 42.62 0.43 1.98 Welder Strong brown (41.00 _ 21.24 _ 35.25) 2012 4.36 1.92 3.69 19.23 0.38 50.61 0.68 3.35 2013 4.29 1.80 3.16 16.83 0.31 54.29 0.59 2.62 Watergate Dark red (29.72 _ 31.48 _ 21.22) 2012 10.53 2.84 3.62 18.45 0.38 48.55 0.45 2.71 2013 10.61 2.46 3.21 16.70 0.43 38.84 0.71 3.92 040 22 9 Dark grayish reddish brown (20.36 _ 8.72 _ 6.41) 2012 12.51 2.82 3.61 13.46 0.57 23.61 0.69 4.25 2013 14.48 2.88 3.21 14.53 0.68 21.37 0.72 3.69 026 1 2 Dark grayish reddish brown (20.80 _ 11.60 _ 6.67) 2012 21.43 3.46 3.61 10.92 0.48 22.75 0.57 3.29 2013 18.23 3.14 3.33 13.57 0.62 21.89 1.03 6.78 026 1 8 Dark grayish reddish brown (21.53 _ 7.82 _ 6.22) 2012 17.96 3.56 3.32 14.63 0.49 29.86 0.85 5.12 2013 15.11 2.84 3.41 15.10 0.46 32.83 0.53 2.32 Majesty Dark grayish reddish brown (17.98 _ 7.93 _ 6.18) 2012 18.95 3.54 3.71 15.52 0.51 30.43 0.61 3.65 2013 20.02 3.22 3.39 15.17 0.41 37.00 0.46 2.17 028 22 5 Dark grayish reddish brown (21.81 _ 7.56 _ 6.26) 2012 5.04 2.10 3.66 16.96 0.45 37.69 1.11 6.46 2013 4.71 2.02 3.27 15.27 0.48 31.81 1.17 5.94 C1 1 1 Strong yellowish brown (51.98 _ 19.23 _ 38.73) 2012 15.44 3.00 3.47 13.78 0.46 29.96 0.42 2.65 2013 17.87 3.02 3.20 14.1 0.55 25.64 0.58 2.36

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40 Table 2 1. Continued Grape Varieties Machine Vision Color (L*_ a* _ b* value) Year Weight (g) Diameter (cm) pH SSC 1 ( 0 Brix) Acidity 2 (%) 0 Brix/acid Ratio 3 Total Phenols 4 Antioxidant activities 5 Green Seedless Thompson Dark yellow (61.51 _ 1.88 _ 48.34) 2012 8.21 2.16 3.63 21.93 0.62 35.37 0.64 3.75 2013 8.13 2.20 3.66 16.27 0.45 36.16 0.31 0.53 Red Seedless Crimson Dark red (30.56 _ 27.85 _ 11.84) 2012 6.88 1.98 3.65 21.32 0.69 30.90 0.89 5.16 2013 5.66 1.92 3.94 21 .93 0.39 56.23 0.25 1.77 Ripe Muscadine grapes (52 cultivars) and the breeding lines (6) were harvested from the Center for Viticulture and Small Frui t Research at Florida A&M University in 2012 and 2013. Ripe Red Seedless, Gre en Seedless, and Red Seed (3 controls varieties) were purchased from Wal Mart Store, Gainesville in 2012 and 2013. 1 SSC: Soluble Solids Concentration; 2 Acidity: Titratable acidity (g/100 mL of juice); 3 Ratio: SSC/Acidity; 4 To tal Phenols: (mg GA/mL juice); 5 Antioxidant activities: (mmol TE/mL juice).

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41 Table 2 2. Muscadine grape evaluation: g rape seed polyphenols (mg/g) and antioxidant activity (mmol/g) Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Africa Queen 2012 0.41 1.02 3.35 2.37 0.51 0.29 32.62 229.89 2013 0.40 1.06 2.73 2.33 0.36 0.25 27.57 298.74 Alachua 2012 0.25 5.28 6.09 6.62 1.90 0.35 70.23 603.91 2013 0.16 5.28 6.83 7.65 2.13 0.38 77.82 659.60 Albemarl 2012 0.41 1.28 3.61 1.55 1.15 0.14 28.56 198.26 2013 0.84 1.61 3.95 1.10 0.75 0.15 29.42 291.64 Black Beauty 2012 0.41 1.24 5.95 9.58 0.49 0.23 63.26 580.05 2013 0.84 1.79 6.83 8.52 0.21 0.38 64.11 594.28 Black Fry 2012 0.90 1.07 4.18 4.81 0.42 0.10 41.75 407.37 2013 1.27 1.53 5.09 4.44 0.18 0.18 44.94 489.89 Carlos 2012 0.23 3.12 6.83 2.79 1.13 0.38 58.82 504.00 2013 0.21 2.91 6.02 2.65 1.03 0.33 51.28 478.00 Cowart 2012 1.27 3.93 3.38 2.16 0.72 0.23 43.92 388.23 2013 1.13 1.38 5.09 6.17 1.07 0.24 60.16 576.07 Darlene 2012 0.41 1.97 3.39 2.75 0.48 0.12 38.04 344.02 2013 0.26 1.73 3.25 2.16 0.53 0.08 32.96 294.02 Digby 2012 0.84 5.40 3.56 4.83 0.27 0.37 51.10 438.85 2013 0.55 6.60 2.79 1.94 0.53 0.43 44.01 379.69 Delight 2012 1.50 2.16 1.95 1.12 1.51 0.60 35.18 338.44 2013 1.37 1.62 2.21 1.45 1.22 0.73 35.14 336.97

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42 Table 2 2. Continued Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Dixie 2012 2.13 4.61 3.48 3.94 0.77 0.49 57.57 484.17 2013 1.27 3.93 3.41 2.34 0.83 0.52 44.12 447.27 Dixie Land 2012 1.70 2.85 3.51 5.06 0.28 0.47 55.92 484.45 2013 2.13 2.97 4.24 5.33 0.26 0.45 51.72 511.56 Dixie Red 2012 1.50 2.11 1.51 2.91 0.84 0.63 38.77 343.44 2013 1.70 2.09 3.50 4.31 0.45 0.42 42.34 343.60 Doreen 2012 1.27 2.82 3.28 2.83 0.71 0.28 46.48 421.00 2013 1.27 1.84 3.60 3.05 0.75 0.55 42.81 423.45 Early Fry 2012 0.84 5.86 3.35 2.81 0.62 0.98 58.75 493.05 2013 1.50 3.47 6.83 4.03 0.66 0.71 70.21 598.59 Farrer 2012 2.27 3.10 5.35 2.04 0.76 0.33 55.74 487.26 2013 1.84 1.07 6.83 5.30 0.85 0.19 62.27 527.47 Florida Fry 2012 2.60 3.33 5.09 6.68 0.88 0.52 71.59 590.39 2013 2.84 3.13 5.16 6.02 0.74 0.38 72.32 649.08 Fry 2012 0.25 3.89 6.83 4.44 1.35 0.35 67.56 576.17 2013 0.20 4.11 6.42 4.02 1.15 0.45 66.16 547.51 Fry Seedless 2012 2013 Gold Isle 2012 1.70 4.00 1.61 4.26 1.19 0.27 47.13 456.65 2013 1.84 3.36 1.61 3.62 1.27 0.20 40.31 403.70

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43 Table 2 2. Continued Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Grany Val 2012 0.08 2.15 3.35 4.84 0.40 0.24 39.53 354.03 2013 0.08 0.39 5.09 3.92 0.28 0.09 33.71 351.27 Higgins 2012 1.70 3.60 4.09 5.01 2.22 0.74 60.45 538.23 2013 1.93 2.65 3.42 5.96 2.27 0.55 58.24 567.93 Hunt 2012 1.27 2.19 3.61 5.92 1.57 0.30 51.16 432.97 2013 1.27 1.77 4.09 6.93 1.68 0.38 56.70 525.22 Ison 2012 2.17 2.21 4.95 7.17 0.85 0.89 64.95 503.48 2013 1.70 2.03 5.09 6.85 0.76 0.65 71.07 631.49 Janebell 2012 0.84 2.10 3.35 5.58 1.14 1.28 60.17 443.41 2013 2.27 2.79 6.83 7.38 0.76 1.16 68.72 644.65 Janet 2012 1.41 1.70 3.09 4.04 1.20 0.40 43.37 363.47 2013 1.84 1.14 3.50 5.65 0.72 0.45 52.59 503.96 Jumbo 2012 1.27 2.82 3.35 9.52 0.94 0.29 64.36 518.47 2013 2.13 2.08 3.50 10.00 2.26 0.37 72.49 665.13 Later Fry 2012 2.40 1.95 3.35 0.81 2.08 0.75 39.58 299.52 2013 1.70 3.66 2.57 1.57 1.90 0.54 47.33 437.58 Lommis 2012 1.27 1.95 1.61 7.26 1.79 0.18 55.44 467.65 2013 0.41 2.46 1.46 6.08 1.50 0.14 49.29 406.53 Magnolia 2012 2.93 2.85 3.35 4.75 1.41 1.36 67.62 546.08 2013 2.60 1.49 4.09 5.92 1.17 1.17 57.14 546.06

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44 Table 2 2. Continued Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Nesbitt 2012 1.62 0.56 2.95 2.44 0.18 0.12 29.84 233.45 2013 1.70 0.92 3.35 2.93 0.01 0.33 36.78 393.43 Noble 2012 0.41 2.11 1.45 1.81 0.80 0.66 25.39 233.25 2013 0.18 2.14 1.61 3.20 1.05 0.44 32.83 355.31 Pam 2012 2.07 1.29 3.35 3.35 1.37 1.20 54.44 460.35 2013 1.84 1.36 3.50 6.66 1.80 0.95 60.86 538.52 Pineapple 2012 1.24 1.99 3.35 2.67 1.17 0.42 37.39 284.15 2013 1.21 2.05 2.33 2.43 0.78 0.42 31.84 256.03 Pride 2012 0.61 0.90 1.35 2.85 0.85 0.49 27.98 236.75 2013 0.84 0.90 1.55 3.23 1.02 0.54 32.86 334.34 Regale 2012 1.17 0.75 4.28 6.48 2.12 1.38 59.26 544.82 2013 1.60 0.20 4.50 8.62 2.43 1.50 66.62 655.74 Rosa 2012 0.86 2.91 1.49 4.45 1.82 0.27 47.82 483.17 2013 0.84 1.87 1.14 5.06 1.61 0.31 43.72 412.22 Scartlett 2012 0.61 2.24 2.61 1.82 1.16 0.76 36.61 321.18 2013 0.50 1.86 2.90 2.19 1.46 0.70 38.39 373.66 Scupernong 2012 1.90 2.47 4.94 4.40 1.71 2.26 70.49 661.07 2013 2.27 3.19 5.24 5.23 1.97 2.21 77.12 651.54 Senoria 2012 2.00 1.41 3.35 6.86 2.11 0.79 58.84 443.21 2013 1.70 1.70 4.57 9.00 2.60 1.20 69.74 539.27

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45 Table 2 2. Continued Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Southern Home 2012 1.41 4.75 6.35 7.05 2.17 2.78 88.84 714.82 2013 2.36 4.35 6.97 5.90 1.97 2.43 84.38 692.69 Southern Land 2012 2.30 2.04 5.02 3.77 2.78 2.32 62.99 526.69 2013 2.13 1.41 6.83 4.95 3.52 1.52 68.59 604.77 Sterling 2012 1.50 0.72 0.95 2.40 1.18 1.21 29.98 270.20 2013 0.84 1.01 2.35 2.80 1.14 1.18 31.22 276.37 Sugargate 2012 0.84 2.50 4.95 3.18 1.77 1.61 60.44 531.82 2013 1.70 1.30 6.83 3.76 1.80 1.84 67.68 521.47 Sugar Pop 2012 1.10 0.37 1.63 2.07 0.79 0.19 23.39 184.16 2013 0.97 0.34 1.61 2.51 0.58 0.21 21.55 172.33 Summit 2012 0.54 1.10 1.56 2.46 1.83 0.75 33.44 258.85 2013 0.60 1.53 1.61 2.76 0.95 0.45 31.38 284.72 Supereme 2012 1.84 0.74 4.95 4.64 0.36 0.22 47.54 421.30 2013 1.70 0.82 5.09 5.65 0.45 0.24 51.66 488.45 Sweet Jenny 2012 1.84 2.19 6.69 4.39 2.41 1.52 80.85 669.78 2013 2.13 3.25 8.57 4.19 2.71 1.75 93.95 798.35 Tara 2012 1.41 4.04 3.35 5.60 1.40 0.83 67.85 600.48 2013 1.93 3.40 5.24 6.97 1.20 0.96 73.50 651.64 Triumph 2012 0.84 0.86 3.35 2.14 1.15 0.32 32.34 317.51 2013 0.66 0.95 3.28 2.20 0.79 0.45 31.69 260.05

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46 Table 2 2. Continued Grape Varieties Year Gallic acid Catechin Epicatechin Epicat e chin gallate Pentagalloylglucose Ellagic acid Total Phenols 1 Antioxidant activities 2 Watergate 2012 1.79 1.58 3.35 4.10 2.22 1.66 62.02 477.46 2013 2.13 1.19 3.50 5.33 2.44 1.28 67.13 586.13 Welder 2012 1.15 1.36 1.61 1.31 0.71 0.29 23.36 236.04 2013 0.86 2.49 1.37 1.92 0.77 0.38 26.77 205.33 028 22 5 2012 1.27 2.07 0.73 1.32 0.74 0.38 24.71 229.93 2013 0.41 2.34 1.61 1.61 0.38 0.23 26.03 169.93 026 1 8 2012 0.76 2.00 2.95 2.78 0.52 0.44 35.92 334.12 2013 0.59 1.73 3.76 3.51 0.58 0.96 38.06 291.21 C1 1 1 2012 0.54 0.37 2.97 3.16 0.76 1.15 29.90 262.09 2013 0.28 0.48 3.35 2.88 0.35 0.78 29.36 240.03 026 1 2 2012 1.70 2.43 3.35 1.68 0.81 0.59 42.10 448.84 2013 0.84 1.19 3.35 2.50 0.74 0.47 35.42 350.93 040 22 9 2012 0.41 0.97 4.32 1.78 2.51 0.79 40.34 455.20 2013 0.84 0.86 3.50 2.83 2.99 0.74 48.15 473.03 Majesty 2012 0.51 0.31 2.95 2.22 0.85 0.49 26.24 228.80 2013 0.43 0.32 3.35 2.38 0.39 0.15 23.06 193.06 Red Seedless 2012 2013 Green Seedless 2012 2013 Red Seed 2012 0.15 1.03 0.00 0.13 0.00 0.14 42.15 303.67 2013 0.15 0.69 0.00 0.21 0.00 0.07 44.40 329.76

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47 Ripe Muscadine grapes (52 cultivars) and the breeding lines (6) were harvested from the Center for Viticulture and Small Fruit Research at Florida A&M University in 2012 and 2013. Ripe Red Seedless, Green Seedless, and Red Seed (3 controls varieties) were purchased from Wal Mart Store, Gainesville in 2012 and 2013. 1 Total Phenols: ( mg GA/g dry weight); 2 Antioxidant activities: (mmol TE/g dry weight).

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48 Table 2 3 . Muscadine grape evaluation: grape skin polyphenols (mg/g) and antioxidant activity (mmol/g) Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagi c acid hexos ide Ellagic acid Quercetin Total Ant h ocyanin s 1 Total Phenols 2 Antioxidant activities 3 Africa Queen 2012 1.09 2.42 1.42 0.32 0.15 0.10 0.30 0.05 5.65 21.10 150.52 2013 1.43 1.99 1.05 0.66 0.20 0.14 0.46 0.04 5.13 17.89 136.36 Alachua 2012 3.80 4.54 2.13 0.49 0.25 0.10 0.57 0.49 13.56 18.46 129.98 2013 3.72 4.72 2.62 0.47 0.21 0.11 0.44 0.49 12.96 16.43 112.90 Albemarl 2012 3.34 4.95 1.80 0.90 0.19 0.15 1.12 0.19 14.09 23.63 163.59 2013 3.86 4.56 2.05 0.88 0.14 0.14 1.02 0.25 13.94 25.79 186.03 Black Beauty 2012 0.52 2.80 0.55 0.19 0.19 0.10 0.52 0.03 7.65 13.58 99.00 2013 0.55 2.51 0.47 0.21 0.13 0.10 0.44 0.04 6.24 11.94 104.35 Black Fry 2012 1.08 2.79 1.38 0.39 0.29 0.17 0.70 0.05 11.42 30.79 194.12 2013 1.18 2.27 1.35 0.65 0.29 0.18 0.53 0.05 10.67 26.74 195.97 Carlos 2012 0.04 0.92 0.20 0.47 0.28 13.93 112.90 2013 0.06 0.78 0.18 0.38 0.28 9.58 81.44 Cowart 2012 1.60 0.64 0.94 0.69 0.22 0.11 0.48 0.05 7.67 19.31 141.57 2013 2.09 0.68 0.97 0.51 0.20 0.13 0.43 0.04 7.98 21.12 152.17 Darlene 2012 0.25 0.32 0.15 0.62 0.02 16.13 119.59 2013 0.26 0.23 0.13 0.55 0.02 13.18 103.08 Delight 2012 2.93 3.08 0.05 0.24 0.17 0.10 0.38 0.03 9.34 19.40 121.19 2013 2.89 3.02 0.05 0.26 0.18 0.10 0.40 0.05 8.78 19.20 119.02 Digby 2012 2.39 1.23 1.43 0.28 0.59 0.10 0.69 0.04 6.58 17.33 125.33 2013 1.32 1.62 1.68 0.44 0.54 0.17 0.66 0.04 6.23 21.35 155.76

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49 Table 2 3. Continued Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagic acid hexos ide Ellagic acid Quercetin Total Ant h ocyanins Total Phenols 1 Antioxidant activities 2 Dixie 2012 0.94 0.52 0.14 1.13 0.07 17.58 137.67 2013 1.22 0.52 0.12 1.04 0.07 23.88 190.16 Dixie Land 2012 0.81 0.23 0.10 1.15 0.42 22.57 187.11 2013 0.96 0.20 0.14 1.06 0.32 22.13 179.86 Dixie Red 2012 2.01 0.30 0.13 0.76 1.08 0.25 0.52 0.01 5.34 22.57 170.45 2013 1.98 0.38 0.20 0.42 0.99 0.24 0.53 0.03 4.10 14.50 117.20 Doreen 2012 0.50 0.65 0.10 0.66 0.03 11.92 100.18 2013 0.35 0.76 0.23 0.40 0.02 17.35 152.93 Early Fry 2012 0.38 0.44 0.19 0.79 0.43 20.96 183.90 2013 0.51 0.22 0.11 1.00 0.36 18.70 151.67 Farrer 2012 0.47 1.33 0.65 0.24 0.17 0.14 1.28 0.32 5.93 19.12 137.69 2013 0.58 1.21 0.57 0.40 0.18 0.11 0.91 0.28 5.76 18.93 134.32 Florida Fry 2012 0.21 0.16 0.10 0.74 0.32 13.74 108.76 2013 0.18 0.19 0.10 0.59 0.20 10.52 98.77 Fry 2012 0.45 0.17 0.10 0.46 0.21 17.65 139.91 2013 0.37 0.20 0.11 0.57 0.16 11.64 99.88 Fry Seedless 2012 0.72 2.22 2.29 0.12 0.66 0.81 0.14 0.25 7.32 25.00 189.41 2013 0.64 1.86 1.78 0.07 0.54 0.93 0.17 0.18 6.17 19.87 130.43 Gold Isle 2012 1.60 2.98 0.99 0.30 0.12 0.14 1.35 0.14 12.67 21.57 160.13 2013 1.68 3.08 1.09 0.69 0.11 0.20 1.24 0.08 13.89 26.30 207.38

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50 Table 2 3. Continued Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagic acid hexos ide Ellagic acid Quercetin Total Ant h ocyanins Total Phenols 1 Antioxidant activities 2 Grany Val 2012 0.15 0.29 0.11 0.76 0.24 11.82 111.67 2013 0.11 0.19 0.11 0.40 0.21 9.53 83.66 Higgins 2012 0.55 0.54 0.10 0.84 0.35 18.71 163.21 2013 0.31 0.85 0.17 0.38 0.28 13.25 159.66 Hunt 2012 2.50 3.24 2.40 0.51 0.19 0.18 0.45 0.03 14.82 24.44 180.46 2013 2.10 3.20 2.23 0.67 0.12 0.21 0.62 0.04 15.87 25.31 191.07 Ison 2012 1.17 2.23 1.06 1.22 0.22 0.10 1.11 0.02 12.63 36.83 265.66 2013 0.77 2.13 1.05 0.69 0.24 0.11 0.62 0.02 10.24 29.98 209.11 Janebell 2012 0.87 0.58 0.47 1.56 0.03 30.53 237.62 2013 0.68 0.48 0.38 1.16 0.03 29.42 204.80 Janet 2012 0.22 0.20 0.18 0.89 0.04 20.00 144.12 2013 0.21 0.20 0.13 0.66 0.03 10.98 112.67 Jumbo 2012 1.82 0.33 0.64 1.22 0.31 0.10 0.47 0.09 13.11 22.01 135.48 2013 1.65 0.31 0.59 1.16 0.18 0.11 0.74 0.07 12.75 11.82 130.14 Later Fry 2012 0.61 0.37 0.39 0.60 0.07 15.79 121.18 2013 0.79 0.46 0.19 0.59 0.08 18.36 168.74 Lommis 2012 1.76 0.74 1.07 0.65 0.10 0.16 1.09 0.03 4.78 23.04 166.03 2013 1.74 0.85 1.09 0.42 0.20 0.50 1.57 0.04 5.57 26.49 227.26 Magnolia 2012 2.28 1.16 0.42 0.13 1.15 0.11 0.70 0.03 6.76 18.07 143.29 2013 2.26 1.25 0.50 0.29 0.46 0.16 0.45 0.02 5.98 12.62 108.72

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51 Table 2 3. Continued Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagic acid hexos ide Ellagic acid Quercetin Total Ant h ocyanins Total Phenols 1 Antioxidant activities 2 Nesbitt 2012 2.14 3.50 1.56 0.70 0.10 0.10 0.50 0.03 10.07 17.51 128.18 2013 2.26 3.84 1.41 1.22 0.18 0.14 0.43 0.05 13.87 25.62 201.69 Noble 2012 2.39 2.09 3.24 0.23 0.23 0.10 0.68 0.30 13.26 29.16 157.13 2013 2.74 2.21 3.02 0.40 0.12 0.23 0.68 0.29 12.65 25.49 134.08 Pam 2012 1.12 0.40 0.21 0.75 0.39 38.23 262.56 2013 1.40 0.35 0.24 0.66 0.40 34.49 252.37 Pineapple 2012 1.06 0.47 0.18 0.92 0.03 28.19 196.23 2013 1.12 0.21 0.16 0.88 0.04 31.08 234.29 Pride 2012 1.70 2.09 1.09 0.89 0.23 0.10 0.62 0.05 17.74 22.34 142.74 2013 1.94 2.05 1.49 0.97 0.24 0.11 1.08 0.08 16.53 20.80 161.91 Regale 2012 2.79 1.33 1.87 0.67 0.23 0.14 0.25 0.04 14.74 29.39 227.17 2013 2.62 1.83 1.93 0.77 0.28 0.17 0.31 0.05 13.98 29.74 221.91 Rosa 2012 0.57 0.32 0.02 0.88 0.18 0.17 1.44 0.04 3.05 29.25 212.29 2013 0.68 0.55 0.09 0.87 0.28 0.15 1.20 0.05 2.89 26.68 201.88 Scartlett 2012 0.05 0.29 0.48 0.14 0.10 0.10 0.55 0.05 1.80 10.70 90.71 2013 0.05 0.67 0.50 0.16 0.12 0.11 0.62 0.06 1.67 9.43 76.38 Scupernong 2012 0.03 0.06 0.01 0.06 0.17 0.10 0.42 0.11 0.39 10.91 102.23 2013 0.04 0.04 0.02 0.10 0.20 0.10 0.57 0.14 0.36 10.16 96.17 Senoria 2012 0.04 0.03 0.08 0.41 0.80 0.10 1.07 0.21 0.41 18.11 165.84 2013 0.05 0.05 0.05 0.39 0.78 0.10 1.03 0.12 0.38 16.46 150.86

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52 Table 2 3. Continued Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagic acid hexos ide Ellagic acid Quercetin Total Ant h ocyanins Total Phenols 1 Antioxidant activities 2 Southern Home 2012 1.28 2.38 1.05 0.42 0.22 0.10 0.79 0.02 14.31 25.57 191.31 2013 1.61 2.53 1.21 0.51 0.23 0.12 0.73 0.03 12.65 19.26 175.75 Southern Land 2012 1.12 1.69 2.02 1.22 0.17 0.10 0.69 0.05 10.76 27.72 212.62 2013 1.02 2.14 1.81 0.59 0.19 0.14 1.06 0.03 9.45 28.44 230.50 Sterling 2012 0.06 0.05 0.02 0.45 0.58 0.10 0.60 0.03 0. 1 5 14.91 120.80 2013 0.05 0.05 0.01 0.42 0.71 0.15 0.31 0.04 0.17 10.76 95.76 Sugargate 2012 0.55 1.01 1.33 0.18 0.16 0.20 0.49 0.03 11.05 18.17 127.31 2013 0.56 1.72 1.63 0.25 0.18 0.28 0.32 0.06 12.56 21.48 158.89 Sugar Pop 2012 0.76 0.47 0.18 0.82 0.05 20.78 160.80 2013 0.63 0.29 0.18 0.51 0.05 17.22 161.02 Summit 2012 0.05 0.02 0.04 0.15 0.44 0.10 0.75 0.06 0.17 17.25 133.26 2013 0.07 0.02 0.03 0.22 0.36 0.13 0.50 0.05 0.18 19.45 160.39 Supereme 2012 1.78 2.85 1.17 0.68 0.18 0.10 0.71 0.04 13.76 25.58 168.58 2013 1.50 3.05 1.55 0.69 0.15 0.10 0.90 0.04 12.67 23.24 180.34 Sweet Jenny 2012 0.31 0.29 0.31 1.67 0.03 26.49 201.86 2013 0.15 0.26 0.18 1.38 0.04 17.22 150.72 Tara 2012 0.54 0.47 0.15 0.46 0.03 12.81 110.32 2013 0.67 0.42 0.17 0.26 0.04 18.39 155.02 Triumph 2012 0.20 0.75 0.17 0.69 0.03 15.30 138.73 2013 0.40 0.79 0.18 0.74 0.03 17.36 160.32

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53 Table 2 3. Continued Grape Varieties Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resveratrol Ellagic acid hexos ide Ellagic acid Quercetin Total Ant h ocyanins Total Phenols 1 Antioxidant activities 2 Watergate 2012 0.93 0.14 0.14 0.73 1.33 0.30 0.70 0.03 3.67 22.93 186.46 2013 0.95 0.21 0.26 0.62 0.99 0.25 0.50 0.04 3.83 18.16 151.73 Welder 2012 1.00 0.33 0.10 0.66 0.03 18.05 153.98 2013 0.72 0.48 0.13 0.75 0.03 17.39 163.79 028 22 5 2012 2.11 2.24 2.09 0.91 0.73 0.11 0.94 0.03 15.78 34.24 234.41 2013 1.95 2.59 1.27 1.22 0.85 0.17 0.71 0.04 15.27 31.83 243.35 026 1 8 2012 1.46 2.38 2.17 0.20 0.31 0.12 0.81 0.01 13.34 24.65 188.77 2013 1.09 2.72 1.66 0.16 0.24 0.11 0.66 0.02 13.65 25.84 190.80 C1 1 1 2012 0.21 0.72 0.23 1.10 0.13 19.75 176.29 2013 0.18 0.66 0.16 0.63 0.06 14.49 115.03 026 1 2 2012 3.53 1.96 0.90 0.37 0.24 0.10 0.66 0.22 12.09 23.70 190.62 2013 3.09 2.20 0.87 0.41 0.24 0.15 0.45 0.28 11.76 23.48 181.35 040 22 9 2012 2.03 3.01 0.78 0.26 0.47 0.10 1.04 0.32 10.98 22.22 176.98 2013 1.43 2.43 0.90 0.26 0.29 0.13 0.95 0.30 9.75 19.70 140.87 Majesty 2012 1.20 2.91 1.01 0.78 0.28 0.10 0.59 0.21 9.85 18.59 152.00 2013 1.10 2.16 1.38 0.77 0.27 0.10 0.63 0.19 10.34 20.81 160.52 Red Seedless 2012 0.06 0.04 0.05 0.05 0.10 0.15 0.38 0.20 2.54 17.42 133.28 2013 0.07 0.05 0.05 0.04 0.10 0.10 0.32 0.18 2.17 15.74 128.30 Green Seedless 2012 0.05 0.10 0.10 0.36 0.07 9.42 90.48 2013 0.04 0.15 0.10 0.32 0.08 8.31 66.87 Red Seed 2012 0.06 0.07 0.03 0.26 0.20 0.10 0.40 0.34 1.98 26.43 80.29 2013 0.08 0.09 0.04 0.25 0.19 0.15 0.37 0.31 2.24 27.95 83.49

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54 Ripe Muscadine grapes (52 cultivars) and the breeding lines (6) were harvested from the Center for Viticulture and Small Frui t Research at Florida A&M University in 2012 and 2013. Ripe Red Seedless, Green Seedless, and Red Seed (3 cont rols varieties) were purchased from Wal Mart Store, Gainesville in 2012 and 2013. 1 Total Antocyanin: (mg Cyn/g dry weight); 2 Total Phenols: ( mg GA/g dry weight); 3 Antioxidant activities: (mmol TE/g dry weight). : Not determined.

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55 Table 2 4 . Correlation matrix of grape fruit quality Correlation a Year Weight Diameter pH SSC Acidity Ratio Total phenols Antioxidant activities Color Year 1.000 .014 .179 .465 .017 .073 .068 .150 .080 .000 Weight 1.000 .943 .226 .136 .106 .107 .023 .004 .196 Diameter 1.000 .283 .134 .118 .091 .029 .008 .166 pH 1.000 .305 .425 .409 .113 .029 .087 SSC 1.000 .151 .568 .005 .029 .265 Acidity 1.000 .852 .331 .308 .253 Ratio 1.000 .322 .280 .316 Total phenols 1.000 .908 .287 Antioxidant activities 1.000 .298 Color 1.000 a . Determinant = .001

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56 Table 2 5 . Correlation matrix of grape seed polyphenols and antioxidant activit ies Correlation year Gallic acid Catechin Epicate chin Epicatechin gallate Pentagalloyl glucose Ellagic acid Total phenols Antioxidant activi ties year 1.000 .015 .085 .149 .118 .013 .037 .051 .122 Gallic_acid 1.000 .097 .216 .317 .285 .417 .509 .498 Catechin 1.000 .247 .137 .076 .104 .475 .442 Epicatechin 1.000 .436 .219 .350 .747 .731 Epicatechin gallate 1.000 .264 .187 .720 .714 Pentagalloylglucose 1.000 .584 .508 .484 Ellagic acid 1.000 .544 .480 Total phenols 1.000 .964 Antioxidant activit ies 1.000 a . Determinant = .001

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57 Table 2 6 . Correlation matrix of grape skin poly phenols and antioxidant activities . Correlation Year Delphinidin 3,5 diglucoside Cyanidin 3,5 diglucoside Pe tuni din 3,5 diglucoside Gallic acid Resvera trol Ellagic acid hexos ide Ellagic acid Quercetin Total ant h ocyanin s Total phenols Antioxidant activities Year 1.000 .012 .009 .000 .024 .061 .061 .121 .035 .014 .105 .031 Delphinidin 3,5 D 1.000 .798 .722 .185 .146 .088 .060 .031 .824 .314 .214 Cyanidin 3,5 D 1.000 .789 .124 .318 .056 .025 .051 .855 .328 .225 Petunidin 3,5 D 1.000 .119 .264 .111 .082 .033 .838 .414 .294 Gallic acid 1.000 .008 .060 .281 .099 .244 .577 .631 Resveratrol 1.000 .268 .035 .102 .278 .065 .065 Ellagic acid hexos ide 1.000 .007 .018 .082 .193 .209 Ellagic acid 1.000 .003 .035 .354 .456 Quercetin 1.000 .077 .008 .092 Total ant h ocyanin s 1.000 .442 .332 Total phenols 1.000 .870 Antioxidant_activities 1.000 a . Determinant = .001

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58 Figure 2 1 . Distribution of individual polyphenols from Muscadine grape seed 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Phenolics (mg/g) Median Average

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59 Figure 2 2 . Distribution of individual polyphenols from Muscadine grape skin 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Polyphenols (mg/g) Median Average

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60 Figure 2 3 . Distribution of total polyphenols from Muscadine 0.00 20.00 40.00 60.00 80.00 100.00 Polyphenols (mg/g) Median Average

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61 Figure 2 4 . Distribution of total antioxidant activity from Muscadine 0 150 300 450 600 750 900 Antioxidant activity (mM TE/g DM) Median Average

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62 Figure 2 5 . Component plot of principal component analysis of muscadine grape fruit quality based on principal components 1 , 2 and 3 .

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63 Figure 2 6 . Component plot of principal component analysis of muscadine grape seed polyphenols based on principal components 1 , 2 and 3

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64 Figure 2 7 . Component plot of principal component analysis of muscadine grape skin polyphenols based on principal components 1 , 2 and 3 .

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65 Figure 2 8 . Score plot s of principal component analysis of grape fruit quality based on principal components 1 , 2 and 3 . PC1: Antioxidant activity and Total phenols; PC2: Diameter and Weight; PC3: SSC and Ratio.

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66 Figure 2 9 . Score plot s of principal component analysis of grape seed polyphenols based on p rincipal components 1 and 2

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67 Figure 2 10 . Score plot s of principal component analysis of grape skin polyphenols based on principal components 1 , 2 and 3

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68 CHAPTER 3 ENZYME RELEASE OF PHENOLICS FROM MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX. ) SKINS AND SEEDS Background Muscadine grape ( Vitis rotundifolia Michx . ) is an important grapevine species native to southeastern United States and Mexico, and has been extensively cultivated since the 16th century. Muscadine grapes are rich sources of bioactive phenolics and other nutrients studied for their potential health benefits. The compounds identified in muscadine seeds included hydroxybenzoic acid, hydrolyzable tannins, fl avan 3 ols and condensed tannins, ellagic acid derivatives, and quercetin rhamnoside; the skin contained hydroxybenzoic acid, hydrolyzable tannins, flavonoids, including anthocyanin 3,5 diglucosides, ellagic acid derivatives, quercetin, myricetin, and kaem pferol glycosides ( 5 ) . Cell c ulture studies have suggested that phenolics from muscadine grapes have strong anticancer activities, such as inhibiting proliferation of colon and prostate cancer cells by inducing apoptosis ( 20, 21 ) . Therefore, muscadine grape pomace is a potential source of bioactive phenolics, which could be used in the food and pharmaceutical industries. In grape, p henolics in gener al can be classified as 1) cell wall phenolics, which are bound to polysaccharides by hydrophobic interactions, hydrogen bonds and covalent bonds, and 2) non cell wall phenolics, encompassing phenolics confined in the vacuoles of plant cel ls and phenolics associated with the cell ( 6, 7 ) . The cell wall of grape fruit is a complex network composed of about 30% neutral polysaccharides (cellulose, xyloglucan, arabinan, galactan, xylan and mannan), 20% acidic pectin substances (of which 62% are methyl esterified), ~15% insoluble proanthocyanidins,

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69 and < 5% structural proteins ( 6 ) . Degradation of cell wall polysaccharides, which eliminates this physical barrier and opens up the cell, is a fundamental step in improving the release of phenolics from grape fruit. Research has focused on the application of cell wall hydrolyzing enzymes, such as cellulases, glucanase, and pectinases, to release phenolics from gr ape fruit and pomace. Kammerer et al. ( 8 ) reported that pectinases and cellulases could result in notably higher recovery rates of phenolics from Vitis vinifera L. grape pomace. Pectinases and macerating enzymes also were reported to promote anthocyanin extraction and improve the quality of red wines ( 9 ) . However, others have found that pectinases and macerating enzymes can cause a decrease in the total yield of anthocyanins and a loss of wine color, or the pectinases have no apparent benefit ( 10, 11 ) . Cellulase treatment was reported not effective for phenolic release from grape ( Vitis vinifera L.) pomace ( 12 ) . Li et al. ( 13 ) reported that enzyme assisted aqueous extraction did not give as high a recovery of citrus peel phenolics as solvent (72% ethanol) extraction. With so m any contradictory findings, and considering the phenolic profiles from different grape species/varieties are not the same ( 14 16 ) , an in depth study of phenolic release by enzymes in various grape species would be useful. Not only may hydrolysis of cell wall polysaccharides help release various phenolics, these enzymes may hydrolyze the polyphenols into low molecular weight phenolics, which may increase the availability and bioactivity of these phenolics ( 12, 17 ) . Monomeric and some oligomeric polyphenols have been found to absorb into rat plasma and are directly involved in physiological functions ( 18 ) , while polymeric forms are poorly absorbed ( 19 ) . Therefore, it is important to evaluate the effect of enzyme

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70 hydrolysis of grape pomace on the magnitude of phenolic release, stru cture of phenolics after hydrolysis and antioxidant activities. Additionally, muscadine skins are much thicker compared to any other varietal grapes. The thick skin accounts for about 40% of the fresh weight of the grape, which gives muscadine grapes a nat ural resistance to disease, fungi, and insects, and are where much of the antioxidant power o f the muscadine grape is stored. Enzyme degradation of muscadine skin cell wall polysaccharides has the potential to enhance the release of these bioactive phenoli cs. To our knowledge, there has been little information on enzyme hydrolysis for the release of muscadine grape ( Vitis rotundifolia M ichx.) skin and seed phenolics ( 49 ) . This study comprehensively studied the enzymatic release of muscadine grape skin and seed phenolics by evaluating various combinations of solvent and enzyme, enzyme type (cellulase, pectinase, ß glucosidase), and hydrolysis time (1, 4, 8, 24 h) on the re lease of muscadine grape skin and seed phenolics and their antioxidant activities. Materials and methods Grape M aterials Fully ripened Muscadine grape ( Vitis rotundifolia Michx.) cv. Noble (red) and cv. Carlos (bronze) were harvested from the Center for Vi ticulture and Small Fruit Research (latitude 30.65 N, longitude 84.60 W) at Florida A&M University on September 2, 2011. The collected samples were shipped to the University of Florida on the same day and stored in a cold room (4 °C ). Grape skins and seeds were separated manually from berries and freeze dried in a freezer drier (Advantage, The Virtis Company, NY, USA)

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71 within the following three days. The freeze dried samples were stored in vacuum packaged polyethylene pouches at 20 °C until analysis. Sample P reparation Freeze dried grape skins (20 g) were ground with a stainless steel grinder (Omni Mixer 17105, OCI Instruments, CT, USA) for 1 min, and then placed on a sieve ( 38 ) . The powdered samples were stored at 20 °C and used for subsequent analysis. Freeze dried grape seeds (20 g) were crushed and then defatted with hexane at a ratio of 1:10 (w/v). After 24 h of extraction at room temperature (shaking every each 6 Scientific, Pittsburgh, PA) under vacuum. The residue was evenly distributed over a tray and kept in the hood to evaporate the hexane. The final defatted grape seed powder was ground again in the stainless 0.25 mm) was collected. The samples were also stored at 20 °C an d used for subsequent analysis. Chemicals Epicatechin gallate, Trans resveratrol, Ellagic aicd, Quercetin and Cyanidin 3, 5 diglucoside, 2,2 diphenyl 1 picrylhydrazyl (DPPH), and 6 hydroxy 2, 5, 7, 8 tetramethylchroman 2 carboxylic acid (Trolox) were obtained from Sigma Aldrich (St. Louis, MO). All other chemicals and solvents were purchased from Fisher Scientific Co. (Pittsburgh, PA).

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72 Enzymes Three different enzymes were selected on the basis of the structural composition Trichoderma reesei was purchased from Sigma Aldrich (St. Louis, MO). Pectinase (3500 U/g, E.C. 3.2.1.15) from Asperigillus niger and ß glucosidase (1000 U/g, E.C. 3.2.1.21) from Sweet almonds were purchased from MP Biomedicals LLC (Solon, OH). All enzyme reactions were performed in a 0.2 M sodium acetate buffer (pH 4.8). Extraction of P henolic C ompounds Different combinations of solvent or enzyme A total of 10 combinations of solvent or enzyme were used for the phenolic extraction experiments (Table 3 1). Freeze dried and ground skins or seeds (0.25 g) were weighed into a tube and extracted following the procedures in Table 1 with 2 or 5 mL of the listed solvents for 1 h at 50 °C or room temperature. To investigate the yield at each step, three extractions were performed per sample. To examine the enzyme release of phenolics from grape skin or seed, 0.25 g of sample was incubated with a mix ture of cellulase (100 U/g dry matter, 100 enzyme activity units/g dry weight sample), pectinase (7 U/g dry matter) and ß glucosidase (25 U/g dry matter) in 2 mL of 0.2 M Na acetate buffer (pH 4.8) for 1 h at 50 °C in a Microprocessor Controlled 280 Series water bath (Precision, Winchester, VA, USA). The selected hydrolysis conditions (pH 4.8 and 50 °C ) were based on evaluations of temperature and pH activity curves for opti mal enzyme concentration for each enzyme was selected in preliminary experiments by testing different concentrations of the enzymes and measuring total phenolic and

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73 antioxidant activities at pH 4.8 and 50 °C (data not shown). After 1 h of shaking, the sampl es were centrifuged at 1500 rcf in an Eppendorf Centrifuge 5702 (Brinkmann Instruments Inc., NY, USA) for 3 min, and the resulting supernatants collected, filtered residue wa s re extracted two more times following Table 3 1 protocols to obtain Extraction 2 and Extraction 3 samples. All supernatants were stored at 20 °C until analyzed. All samples from Extractions 1, 2 and 3 were analyzed in duplicate for total phenolics and th e Noble skin samples for anthocyanins. Enzyme type and incubation time To investigate enzyme type and incubation time, 0.25 g of freeze dried and ground skins or seeds were weighed into separate tubes and extracted with 2 mL selected solvents (50% ethanol, buffer, cellulase, pectinase, ß glucosidase, and a mixture of these en zymes) for 1, 4, 8, and 24 h at 50 °C . Ethanol (50%) was used as control. The enzyme concentrations were previously described. After 1 h of shaking, the samples were centrifuged at 1500 rcf for 3 min, and the resulting supernatants were collected, filtered after the enzyme hydrolysis was collected and subjected to a subsequent extraction at room temperature for 1 h by adding 5 mL 50% ethanol, shaking and repeating the centrifugation. This sup 2 samples. All supernatants were stored at 20 °C until analyzed. All extractions (Extraction 1 and 2) were analyzed for total phenolics and antioxidant activity in duplicate.

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74 Phenolic C onte nt and A ntioxidant A ctivity A nalysis Analysis of total phenolics Total phenolic content in grape seeds or skins was determined by the method of Singleton et al. ( 41 ) using an ultraviolet visible Beckman Coulter DU 640 fold diluted Ciocalteau reagent (2N), 20% Na 2 CO 3 were introduced in a tube. After reacting for 30 min in a 40 °C water bath, absorbance was measured at 760 nm. Gallic acid (GA) was used as standard and expressed as gallic acid equivalents (mg gallic acid (GAE)/g dry matter (DM)) using a 2 = 0.9996). Analysis of total anthocyanins Total anthocyanin content in cv. Noble skins was measured using the pH differential spectrophotometric method d escrib ed by Lee et al . ( 42 ) . The anthocyanin extract was dissolved in 0.025 M potassium chloride buffer, pH 1.0 and 0.4 M sodium acetate buffer, pH 4.5 with a pre determined dilution factor. The absorbance was measured at 510 nm and 700 nm. The absorbance (A) of the diluted sample wa s then calculated as follows: A = (A 510nm A 700nm ) pH 1.0 (A 510nm A 700nm ) pH 4.5 The monomeric anthocyanin concentration in the original sample was calculated as cyanidin 3,5 diglucoside equivalents according to the following formula:

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75 where MW (611) of cyanidin 3,5 diglucoside is used because the anthocyanin content was calculated in cyanidin 3,5 diglucoside equivalents (mg CAE/g DM); the molar absorptivity e was 30,175; the dilution factor ( DF) of 1000 is the factor to convert g to mg; and A was absorbance. Anthocyanin content was mg/L, which was then converted to mg/g dried sample. Identification of individual phenolics The obtained extracts were subjected to chromatographic analyses on a Hi tachi HPLC system with a Zorbax Stablebond Analytical SB C18 column (4.6 mm, 250 mm, 5 acid aqueous solution) and mobile phase B (60% methanol with 0.5% formic acid). The fo llowing linear gradient was used: 0 5 min: 5% B, 5 10 min: 15% B, 10 20 min: 25% B, 20 30 min: 50% B, 30 40 min: 70% B, 40 50 min: 90% B. The flow rate was 0.9 mL/min. Injection volume was 20 µl with the UV detector set to an absorbance wavelength of 280 n m. The retention times for each of the different components in the analyzed pool were compared to the following standards: gallic acid, catechin, epicatechin, epicatechin gallate, trans resveratrol, ellagic acid, and quercetin. Standards for pentagalloyl g lucose, ellagic acid hexoside and mono and digalloyl glucose were not available. Thus, a second HPLC system was employed to confirm the identification of these by ESI MS detection in the Selective reaction monitoring (SRM) mode. The LC MS analyses were pe rformed on a Thermo Finnigan Surveyor HPLC system (Thermo Finnigan, St. Jose, CA, USA) equipped with a TSQuantum controlled by XCalibur data analysis software (version 1.3, Thermo Finnigan). The column and mobile phase used were the same as described above in

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76 HPLC analysis. The MS acquisition was with ESI interface in negative ionization mode at the following conditions: Sheath gas (N2) pressure, 42 arb; auxiliary gas (N2) pressure, 20 arb; spray voltage 3.96 kV; capillary temperature, 414 °C; collision gas pressure, 1.5 mTorr; collision energy, 22 V. These compounds were identified on the basis of their mass fragmentation data compared with and identical to those reported by Sandu et al. ( 5 ) . Analysis of antioxidant activities by DPPH Assay The DPPH assay was based on the slightly m odified method of Brandwilliams et al. ( 43 ) . The 10 DPPH (0.0 025 g/100 mL CH 3 OH). After 60 min reaction at room temperature in the dark, the absorbance at 515 nm was recorded to determine the concentration of the remaining DPPH. The percentage inhibition of DPPH in the test sample and known concentrations of Trolox were calculated by the following formula: %Inhibition = 100 × (A 0 A)/ A 0 , where A 0 was the beginning absorbance at 515 nm, obtained by measuring the same volume of solvent, and A was the final absorbance of the test sample at 515 nm. The calibration curv e between %Inhibition and known concentration of Trolox solutions was then established. The radical scavenging activities of the test samples percentage inhibition. Trolox s tandard solutions were prepared at a concentration 2 =0.9999). Ratio of A ntioxidant A ctivity to T otal P henolics The following Noble seed extracts were selected in this test: 1) samples extracted with 50% ethanol for 8 h Extraction 1 (EtOH 8h 1), and 2) the resulting

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77 residue further extracted with 50% ethanol for 1 h Extraction 2 (EtOH 1h 2); 3) samples extracted with a mixture of cellulase, pectinase and ß glucosidase in buffer for 8 h Extraction 1 (Enzyme 8h 1), and 4) the resulting residue further extracted with 50% ethanol for 1 h Extraction 2 (Enzyme 1h 2) ( Table 3 2) . Each extract was separated by HPLC a nd fractions were collected every 5 min, starting around 3 min when the first peak appeared and terminated after 43 min. Eight fractions were then subjected to total antioxidant activity and phenolic assay, and ratio of antioxidant activities (DPPH values) to total phenolics was expressed as mmol Trolox per gram Gallic acid. To further investigate enzyme incubation treatment on grape phenolics, Noble seed samples : EtOH 4h 1 (extracted with 50% ethanol for 4 h Extraction 1) and EtOH 8h 1 were dried under n itrogen and then incubated with the mixture of cellulase, pectinase and ß glucosidase in 2 mL buffer for 4 or 8 h at 50 °C . EtOH 4h 1, EtOH 8h 1 and their hydrolyzed samples were separated by HPLC and fractions were collected and analyzed as previously desc ribed. Statistical A nalyses All extractions and analyses were performed in duplicate. Data were subjected to ANOVA and differences among samples were tested by post hoc comparison test (Student Newman Keuls) at p = 0.05 with Microsoft Excel 2010 and SPSS 21 .0 for Windows (SPSS Inc, Chicago, IL)

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78 Results and D iscussion Different C ombinations of S olvent or E nzy me on R elease of P henolics from G rape S kin and S eed s Total phenolics released by the 10 combinations of solvent or enzyme for Noble and Carlos skins showed significant differences while those from seeds showed little difference (Figure 3 1(a, c, d), since patterns for Noble and Carlos seeds were similar, onl y the Carlos data was presented). All first extractions released a majority of the total phenolics. The 50% ethanol (#2) was the most effective solvent for extracting total phenolics from grape skins and seeds, followed by combinations of enzyme and solven t (#3 ~ #10) while the least effective was water (#1). For Noble skins, combinations of buffer (enzymes) and 50% ethanol (#5, 6, 7, 8) yielded the highest total phenolics (average 34.83 mg GA/g DM) compared to the 50% ethanol control (#2), and was signific antly higher by 27% than the water control (#1). As expected, combinations of buffer (enzymes), water or 50% ethanol (#3, 4, 9, 10) yielded the medial total phenolics (average 30.98 mg GA/g DM). For Carlos skins, 50% ethanol buffer (enzymes) 50% ethanol co mbinations (#7, 8) yielded the highest total phenolics (average 43.38 mg GA/g DM) comparable to the 50% ethanol control (#2), and significantly higher by 45% than the water control (#1). Extracting first with buffer or enzymes (#5, 6) significantly decreas ed the total phenolic yield compared to their second extraction with buffer or enzymes (#7, 8). Furthermore, enzyme hydrolysis used in Extraction 1 (#4, 6) reduced the release of phenolics compared to no enzyme treatments (#3, 5). Phenolic extraction from Noble and Carlos grape seeds were similar to Carlos skin with 50% ethanol buffer (enzymes) 50% ethanol combinations (#7, 8), and 50% ethanol control (#2) yielding the

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79 highest total phenolics (about 70 mg GA/g DM) while the water control (#1) yielded the lo west total phenolics (about 35 mg GA/g DM). When compared with combinations of buffer and solvent (#3, 5, 7, 9), combinations of enzyme and solvent (#4, 6, 8, 10) did not result in an enhanced release of total phenolics from the studied skins and seeds ( Figure 3 1). This finding was consisten t with the report of Li et al. ( 13 ) that citrus peel phenolics released by enzymes is in fact due to water, and especially hot water. In contrast, enzyme treatments severely depressed the yield of total phenolics in Carlos skin (Fig 3 1c #4 vs . #3, #6 vs . #5). Further studies showed that t his was caused by cellulase (table 3 2). It was probably cellulase hydrolysis that caused disorder in the Carlos skin cell wall so as to retard phenolics extracted by solvents in Extraction 2. However, cellulase did not severely depress the yield of total phenolics in Noble skin. This was possible because the major phenolics in Noble skin were anthocyanins (in Carlos skins, they are non anthocyanins). Anthocyanins are water soluble vacuolar pigments, not bound to the grape skin polysaccharide matrix, and ar e instantly released from the skin during the early phase of the enzymatic treatments ( 50 ) . Figure 3 1(b) showed that treatments #4 and 6 in Noble skins yielded the highest total anthocyanins (average 23.15 mg CAE/g DM), which was 28% higher than the water control (#1) and 10% higher than the 50% ethanol control (#2). Thus, the release of total phenolics in Noble skin was not severely affected by enzyme treatments #4 and #6 while it was for Carlos skin. Enzymes or buffer used in Extraction 2 (#7, 8) yielded similar total phenolics compared to the 50% ethanol control (#2), and significantly higher levels than Extraction 1 (#5, 6). This was because most of the phenolics in treatments #7 and 8, as well as #2,

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80 were released in Extraction 1 by the 50% ethanol, and not influenced by the addition of enzyme in Extraction 2. Most often, enzyme hydrolysis used for phenolic extraction was performed initially and then extracted with other solvents ( 8, 12, 51 ) . However, our results showed that treating muscadine grape skins and seeds this way decreased the total phenolic yields. Enzyme T ype and I ncubation T ime on R elease o f P henolics from G rape S kin and S eed s Previously, Lee et al. ( 49 ) studied hydrolys glucosidase and tannase on ellagic acid derivatives of muscadine grapes ( Vitis rotundifolia glucosidase could effectively hydrolyze ellagic acid glycosides whereas tannase had little effect on ellagic acid precursors. In th is study, three different enzymes (cellulase, pectinase, and ß glucosidase) and incubation times were investigated as to their release of grape phenolics. Thus, muscadine grape skins and seeds were subjected to individual or mixed enzyme hydrolysis for different times followed by extraction with 50% ethanol. Noble skin . For enzyme Extraction 1, compared to the yield from buffer, cellulase hydrolysis had no significant influence on increasing the release of total phenolics (Table 2). Cellulase hydrolysis increased phenolic release as incubation time increased (1 to 8 h) but this was more related to the extension of incubation time than enzyme. However, longer incubation time (24 h) decreased the total phenolic yield. This was probably due to temperature ( 50 °C ) and additional side reactions during the long hydrolysis time ( 50, 52 ) . During the first hour of incubation, ß glucosidase hydrolysis significantly increased the release of total phenolics by 12%. However, longer

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81 incubation with ß glucosidase showed no additional benefit compared to buffer control. Pectinase was better than cellulase and ß glucosidase and significantly increased (by 72%) total phenolics at first (1 h), but then the total phenolic yield decreased with longer incubation time. It is possible that pectinase helps break down the structural polysaccharides of the cell wall quickly, thereby accelerating the release of phenolics; however, all released pheno lics then become exposed to the adverse conditions (temperature, cell enzymes, etc.) during the long incubation time and start to degrade ( 50 ) . Hydrolyzing with the mixture of enzymes also significantl y increased the release of total phenolics during the first 4 h of incubation, but the yield decreased after 24 h hydrolysis. This was mainly driven by pectinase while there was little contribution from cellulase and ß glucosidase. Even though the phenolic s yield was lower than the 50% ethanol control, enzyme hydrolysis did enhance or accelerate to some extent the release of total phenolics in Noble skins. Phenolics extracted by 50% ethanol almost reached maximum yield after 1 h extraction and were stable t ill 8 h, but beyond this time, the yield in total phenolics significantly decreased at 24 h hydrolysis. A possible reason was the long incubation time at a high temperature (50 °C ) degraded the phenolics ( 52 ) . Since the enzyme extraction was performed in buffer, an appreciable amount of non water soluble phenolics such as highly polymerized procyanidins may be retained in the residue. To recover these phenolics, Extraction 2 was performed with 50% ethanol for 1 h. Table 3 2 shows that as hydrolysis time for Extraction 1 increased, phenolic recovery in Extraction 2 for the 50% ethanol control increased, especially for the 24 h treatment. This may be the result of the precipitated phenolics in the residue

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82 re dissol ving in the solvent. For Extraction 2, more phenolics (about 40 ~ 60%) were recovered after enzyme treatment than the 50% ethanol control, especially the early incubation times. By combining the two extractions (1 and 2), enzyme treatment yielded about 90% of the phenolics from the 50% ethanol control (Table 3 2), similar to the rate of 87.9% reported by Li et al. ( 13 ) . However when compared with buffer, enzyme hydrolysis seemed to only accelerate the release of phenolics rather than enhance the release of total phenolics in Noble skins. The release of Noble skin anthocyanins by enzymes was similar to the release of their total phenolics (Table 3 2) and probably related to anthocyanins being the major phenolics in Noble skins. Cellulase hydrolysis slightly decreased the release of total anthocyanins whi le ß glucosidase and pectinase hydrolysis significantly increased (by 13% and 70%, respectively) the release of total anthocyanins at the early hydrolysis time (1 h). The effect of pectinase was consistent with the reports of Kammerer et al. ( 8 ) and Haight et al. ( 9 ) while the outcome of cellulase was not. ß glucosidase was re ported to degrade anthocyanins by deglycosylation and formation of unstable anthocyanin aglycones ( 11 ) . However in this study, this seems to have taken place after 4 h hydrolysis in view of the significant decrease in anthocyanins after this time. A previous report ( 49 ) glucosidase could effectively hydrolyze phenolic glycoside compounds of muscadine grapes. Therefore, ß glucosidase was used in this study to investigate whether it had the same consequence on the released muscadine grape skin and seed polyphenols. Results suggested that ß glucosidase also could effectively hydrolyze phenolic glycoside compounds of muscadine grapes

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83 (such as pentagalloyl glucose), and relea se low molecular weight phenolics with high antioxidant activity (such as gallic acid) (Figure 3 2). The use of ß glucosidase also confirmed the previous report ( 11 ) that it could degrade anthocyanins, but in this study the degradation of anthocyanins was not immediate. Major degradation took place after 4 h hydrolysis (Table 3 2). This was consistent with Arnous et al. ( 50 ) work that the enzyme catalyzed decay of the acylated anthocyanins most likely took place via a sequential enzyme catalyzed degradation as caused by both cinnamate esterase activity and ß glucosidase activity: as a first step, cinnamate esterase catalyzes the stripping of the acylated anthocyanins from its shielding hydroxycinnamic acid (p coumaric, caffeic, ferulic acids), which makes it possible for ß glucosidase to deglycosylate anthocyanins releasing glucose and the more sus ceptible aglycone form of anthocyanins. Carlos skin . Enzyme rele ase of phenolics in Carlos skin was signific antly different from Noble skin (Table 3 2). For enzyme Extraction 1, cellulase hydrolysis severely reduced (by 50%) the release of total phenolics in Carlos skins, even though the released phenolics increased slightly with increasing hydrolysis time. Pectinase or ß glucosidase at 1 h hydrolysis time increased (by 11%) the release of total phenolics in Carlos skins. However, with the increase in hydrolysis time, the release of total phenolics decreased. Hydrolysis at 1 h for the mixture of enzymes severely inhibited (by 34%) the release of total p henolics in Carlos skins, and with the increase in incubation time, the release of total phenolics decreased. This effect was mainly driven by cellulase while very little contribution occurred from pectinase or ß glucosidase. Compared to the 50% ethanol co ntrol, enzyme hydrolysis released only half or less total

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84 phenolics from Carlos skins. For Extraction 2, Carlos skins showed a similar trend as Noble skins, that is, about 40~60% phenolics were recovered from enzyme treatments compared to the 50% ethanol c ontrol, and the recovered phenolics decreased with increasing incubation time. When adding the two extractions (1 and 2) together, enzyme treatments yielded 48~65% of the phenolics extracted by the 50% ethanol control. When compared with buffer, enzyme hyd rolysis had very little or no influence on the release of phenolics in Carlos skins. Noble and Carlos seeds . Enzyme release of phenolics in Noble and Carlos seeds was similar (Table 3 2). For enzyme Extraction 1, enzyme hydrolysis showed little influence on the release of total phenolics. This is because the cell wall of grape seeds is hard and very resistant to chem ical and biological degradation ( 16, 53 ) . The maximum phenolic yield in Noble seeds was obtained at 4 h incubation while in Carlos seeds it took 8 h. Compared with the 50% ethanol control; enzyme hy drolysis only released about 35% of the total phenolics from Noble seeds and 48% from Carlos seeds. Besides the degradation issue of the grape seed cell wall, another factor for the low yield is that the major phenolics are highly polymerized procyanidins and are non water soluble. For Extraction 2, about 50~60% phenolics were recovered from enzyme treatments, and the recovered phenolics decreased with increasing hydrolysis time. For the two extractions (1 and 2), enzyme treatments yielded 70~80% phenolics when compared to the 50% ethanol control. Enzyme hydrolysis after 1 h yielded the maximum phenolics and the amount decreased with increasing hydrolysis time.

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85 Enzyme T ype and H ydrolysis T ime on A ntioxidant A ctivities from G rape S kin and S eeds The DPPH method which has been extensively used to measure the free radical scavenging activity of pure antioxidant compounds extracted from fruits, plants, or food materials was used in this study to characterize the total antioxidant activity. Enzyme ty pe and hydrolysis time on grape skin and seed antioxidant activities were very similar to the patterns seen for their total phenolics (Table 3 3). This is in agreement with previous reports that antioxidant activities of grapes have good correlation with t heir phenolic content ( 46 ) . Although enzyme hydrolysis yielded lower phenolics from muscadine skins and seeds than the 50% ethanol control, the total antioxidant activities for these same samples, especially grape seeds, were comparable and even higher than the 50% ethanol control. For example, total antioxidant activity for This higher activity f or enzyme hydrolysis indicated that this treatment might modify the structure of some phenolics since total phenolics were lower for this treatment. Antioxidant A ctivity a fter E nzyme H ydrolysis To investigate the antioxidant activities of grape phenolics after enzyme hydrolysis, several Noble seed samples were separated into eight fractions by HPLC, and then the ratio of antioxidant activity to total phenolics for each fraction was tested. Figure 3 2 shows the chromatographic profiles of phenolics released by 50% ethanol (a, b) and enzymes (c, d). Low molecular weight phenolics, especially gall ic acid, mono and di galloyl glucose, epicatechin, and ellagic acid and its conjugates were the major

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86 phenolics present after enzyme hydrolysis; while phenolics extra cted by the 50% ethanol treatment had more galloylated forms, such as epicatechin gallate and pentagalloyl glucose. This indicated that enzyme hydrolysis changed the galloylated form of muscadine grape ( V. rotundifolia ) phenolics (epicatechin gallate and p entagalloyl glucose) to low molecular weight phenolics, releasing phenolic acids such as gallic acid and mono and di galloyl glucose. This finding was consistent with the re port of Chamorroa et al. ( 12 ) that the use of tannase and pectinase in grape pomace ( V. vinifera ) changed the galloylated form of epicatechin to its f ree form, releasing gallic acid and increasing the antioxidant activity; however, it contradicted the report by Kammerer et al. ( 8 ) that the use of pectinolytic and cellulolytic en zymes in grape pomace ( V. vinifera ) decreased the yield of gallic acid. The increase in level of phenolic acids after enzyme treatment indicates the possibility of enhancing antioxidant potency, since phenolic acids are supposed to have greater antioxidan t power ( 54 ) . Indeed, this study showed that gallic acid had the highest antioxidant activity per unit among all the tested standards (data not shown). The ratio of antioxidant activity to total phenolics (Figure 3 3a) also showed that Enzyme 8h 1 fractions (#1_14.0 m mol/g, #2_15.4 mmol/g) and Enzyme 1h 2 fraction (#2_15.9 mmol/g) were significantly higher than EtOH 8h 1 fractions, which ranged from 6.1 mmol/g (#8) to 11.6 mmol/g (#5). Furthermore, fractions #1 and 2 were the major phenolics released by enzyme hydrolys is. Since these two fractions have high antioxidant activities this explains why enzyme hydrolysis released significantly lower total phenolics in Noble seeds, but had comparable or even higher total antioxidant activities than the 50% ethanol control.

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87 To further explain the increase in antioxidant activities of grape phenolics after enzyme hydrolysis, the 50% ethanol control extract for Noble seed was dried under nitrogen and then hydrolyzed by the mixture of enzymes. Figure 3 2 shows that enzyme hydrolys is did increase phenolic acid content, especially gallic acid, by changing the galloylated form of the phenolics (a vs . e; f vs . g). The ratio of antioxidant activity to total phenolics showed that EtOH 4h 1 Enzyme fraction 2 (15.6 mmol/g) and EtOH 8h 1 En zyme fraction 2 (14.5 mmol/g) were significantly higher than EtOH 4h 1 and EtOH 8h 1 fractions, which ranged from 6.4 mmol/g to 13.3 mmol/g (Figure 3 3b). This demonstrated that enzyme hydrolysis could modify released phenolics to more potent antioxidant c ompounds. Summary The results obtained in this study demonstrated that combinations of solvent and enzyme, and treatment order greatly affected the release of phenolics from muscadine grape skins and seeds. Pre treating muscadine skin and seed samples with enzymes, which is the most common method of hydrolysis, decreased total phenolic yield compared with solvent (50% ethanol) alone. For different muscadine varieties, enzymatic release of phenolics from skins was significantly different while for seeds, the y were similar. One hour pectinase hydrolysis significantly increased the release of total phenolics in Noble skins while no apparent outcome was observed for cellulase and ß glucosidase hydrolysis; cellulase hydrolysis severely inhibited the release of to tal phenolics in Carlos skins while pectinase or ß glucosidase hydrolysis increased the release of total phenolics. Therefore, both enzyme type and grape varieties should be considered to achieve an effective enzyme method for releasing phenolics from grap e

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88 skins. Since adverse conditions (temperature, cell enzymes, etc.) could degrade phenolics affecting their total yield, long incubation times should be avoided. Enzyme hydrolysis was found to shorten extraction time. Most importantly, enzyme hydrolysis co uld modify the galloylated form of phenolics to low molecular weight phenolics, releasing higher antioxidant activity in the form of phenolic acids, especially gallic acid. Further studies are needed to explore the specific pathway of enzyme modification f or phenolics to their more potent antioxidant compounds. This will help convert the complex polyphenols into their simple phenolic acids, which might increase the bioavailability of these phenolics. For this reason, enzyme release of phenolics might have a pplicability in the food and nutraceutical industries.

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89 Table 3 1 . Treatment conditions for 10 combinations of solvent and enzyme. Condition Extraction 1 Extraction 2 Extraction 3 # 1 Water (2 mL) A Water (5 mL) Water (5 mL) #2 50% ethanol (2 mL) 50% ethanol (5 mL) 50% ethanol (5 mL) #3 Buffer (2 mL) B Water (5 mL) Water (5 mL) #4 C + P + ß (2 mL) C Water (5 mL) Water (5 mL) #5 Buffer (2 mL) 50% ethanol (5 mL) 50% ethanol (5 mL) #6 C + P + ß (2 mL) 50% ethanol (5 mL) 50% ethanol (5 mL) #7 50% ethanol (5 mL) Buffer (2 mL) 50% ethanol (5 mL) #8 50% ethanol (5 mL) C + P + ß (2 mL) 50% ethanol (5 mL) #9 Water (5 mL) Buffer (2 mL) 50% ethanol (5 mL) #10 Water (5 mL) C + P + ß (2 mL) 50% ethanol (5 mL) A : Samples using solvents at 2 mL were extracted in a 50 o C water bath, while those using 5 mL were extracted at room temperature. B: 0.2 M sodium acetate buffer (pH 4.8). C : Mixture of cellulase (C), pectinase (P) and ß glucosidase (ß) in a final volume o f 2 mL 0.2 M sodium acetate buffer (pH 4.8).

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90 Table 3 2 . Ethanol , enzyme type and incubation time on extraction of total phenolics and anthocya nins from grape skins and seeds Extraction 1 A Extraction 2 B Total (Extraction 1+2) Treatment Incubation time (h) Incubation time (h) In cubation time (h) 1 4 8 24 1 1 1 1 1+1 4+1 8+1 24+1 Noble Skins (Total Phenolics , mg GA/g DM ) 50% Ethanol 22.98 a 23.13 a 22.63 a 16.99 a 7.40 c 8.47 c 8.40 b 10.93 a 30.38 a 31.60 a 31.03 a 27.92 a Buffer 9.89 d 15.50 c 16.88 b 16.49 a 15.19 a 12.51 a 11.29 a 10.09 a 25.08 c 28.01 b 28.17 b 26.58 a Cellulase 9.96 d 16.07 c 17.56 b 15.75 a 14.98 a 12.88 a 10.54 a 9.25 b 24.94 c 28.95 b 28.10 b 25.00 ab Pectinase 17.03 b 16.25 c 16.17 c 15.03 ab 11.99 b 11.02 b 9.99 ab 9.18 b 29.02 ab 27.27 b 26.16 c 24.21 b ß glucosidase 11.06 c 15.36 c 16.45 bc 16.12 a 14.60 a 12.67 a 10.39 a 9.34 ab 25.66 c 28.03 b 26.84 c 25.46 ab C+P+ ß C 16.79 b 17.48 b 16.31 c 13.90 b 11.35 b 11.31 b 10.23 a 9.07 b 28.14 b 28.79 b 26.54 c 22.97 b Noble Skins (anthocyanins , mg Cyn/g DM ) 50% Ethanol 16.90 a 16.21 a 16.66 a 11.27 a 5.03 c 5.98 b 5.98 c 7.01 a 21.93 ab 22.19 a 22.64 a 18.27 a Buffer 9.25 c 12.81 c 15.20 b 10.96 a 10.66 a 8.20 a 7.32 a 5.74 b 19.91 b 21.01 ab 22.52 a 16.70 b Cellulase 8.14 d 12.01 c 14.51 b 10.24 ab 10.66 a 8.03 a 6.38 b 3.82 c 18.80 b 20.04 b 20.89 ab 14.06 cd Pectinase 15.70 a 14.76 b 12.92 c 10.27 ab 7.82 b 6.64 b 6.19 b 4.73 bc 23.52 a 21.40 ab 19.11 b 15.00 c ß glucosidase 10.43 b 13.25 bc 12.12 c 11.15 a 8.79 b 8.08 a 6.75 b 5.34 b 19.22 b 21.33 ab 18.87 b 16.49 b C+P+ ß 16.27 a 14.26 b 13.27 c 8.06 c 7.17 bc 6.15 b 5.58 c 3.59 c 23.44 a 20.41 b 18.85 b 11.65 d Carlos Skins (Total Phenolics) 50% Ethanol 31.35 a 31.40 a 31.93 a 23.75 a 8.99 c 9.37 a 10.37 a 13.58 a 40.34 a 40.77 a 42.30 a 37.33 a Buffer 15.02 c 15.73 b 14.09 b 12.49 b 11.06 a 9.63 a 7.69 b 6.68 b 26.08 b 25.36 b 21.78 b 19.17 b Cellulase 7.57 e 8.55 d 8.44 d 8.65 d 11.60 a 7.16 c 6.32 c 5.09 c 19.17 c 15.71 e 14.76 c 13.74 d Pectinase 16.70 b 12.93 c 12.83 c 11.33 c 10.15 b 8.41 b 7.03 b 5.45 c 26.85 b 21.34 d 19.86 b 16.78 c ß glucosidase 16.72 b 14.53 bc 12.93 c 11.30 c 10.06 b 8.67 ab 7.85 b 6.14 b 26.78 b 23.20 c 20.78 b 17.44 c C+P+ ß 9.85 d 9.00 d 8.66 d 7.89 d 9.91 b 7.33 c 6.31 c 5.00 c 19.76 c 16.33 e 14.97 c 12.89 d Noble Seeds (Total Phenolics) 50% Ethanol 51.77 a 51.66 a 47.93 a 37.99 a 12.94 c 13.33 b 13.80 b 14.57 b 64.71 a 64.99 a 61.73 a 52.56 a Buffer 18.66 b 22.82 b 20.45 b 19.10 b 28.41 ab 21.54 a 16.16 a 15.61 a 47.07 b 44.36 b 36.61 b 34.71 b Cellulase 16.23 b 22.81 b 21.86 b 19.73 b 29.88 a 18.69 ab 15.67 a 14.12 b 46.11 b 41.50 c 37.53 b 33.85 b Pectinase 16.17 b 22.41 b 21.19 b 18.61 b 31.28 a 19.76 ab 16.28 a 12.97 c 47.45 b 42.17 c 37.47 b 31.58 c ß glucosidase 16.28 b 22.68 b 20.86 b 17.88 b 27.75 b 20.88 a 16.05 a 12.93 c 44.03 c 43.56 b 36.91 b 30.81 c C+P+ ß 17.33 b 22.06 b 20.56 b 18.29 b 27.09 b 20.86 a 15.41 a 12.93 c 44.42 c 42.92 bc 35.97 b 31.22 c Carlos Seeds (Total Phenolics) 50% Ethanol 43.92 a 46.47 a 45.00 a 35.15 a 11.15 c 12.19 b 12.11 c 15.05 a 55.07 a 58.66 a 57.11 a 50.20 a Buffer 18.47 c 25.26 b 25.33 b 19.65 b 21.95 ab 17.29 a 15.53 ab 12.89 b 40.42 c 42.55 b 40.86 b 32.54 b Cellulase 21.24 b 23.38 c 25.71 b 19.42 b 19.94 b 17.57 a 16.66 a 13.62 b 41.18 c 40.95 b 42.37 b 33.04 b Pectinase 21.78 b 23.76 c 24.12 b 18.51 b 23.37 a 17.67 a 14.83 b 13.12 b 45.15 b 41.43 b 38.95 c 31.63 b ß glucosidase 20.53 bc 24.76 bc 24.17 b 18.88 b 22.76 a 17.84 a 15.50 ab 13.73 b 43.29 b 42.60 b 39.67 bc 32.61 b C+P+ ß 20.41 bc 23.81 c 24.48 b 18.99 b 21.26 ab 17.38 a 16.65 a 13.49 b 41.67 c 41.19 b 41.13 b 32.48 b A : Extraction 1 extracted with 2 mL listed solvents and incubated for 1, 4, 8, 24 h at 50 o C ; 50% ethanol and buffer used as controls. B : Extraction 2 residue of Extract 1 (samples Incubated for 1, 4, 8, 24 h) was extracted with 5 mL of 50% ethanol for 1 h at room temperature. C : Mixture of cellulase (C), pectinase (P) and ß glucosidase (ß) in a final volume of 2 mL 0.2 M Na acetate buffer (pH 4.8). a,b,c,d,e : Mean values within a short column (each kind of sample) with different superscript letters are significantly diffe rent.

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91 Table 3 3 . Effect of ethanol, enzyme type and incubation time on grape skins and seeds antio xidant activities (DPPH values) Extraction 1 A Extraction 2 B Total (Extraction 1+2) Treatment Incubation time (h) Incubation time (h) Incubation time (h) 1 4 8 24 1 1 1 1 1+1 4+1 8+1 24+1 Noble Skins 50% Ethanol 120.27 a 133.58 a 133.05 a 95.28 b 42.94 d 59.11 c 57.83 b 71.05 a 163.21 b 192.69 a 190.88 a 166.33 a Buffer 67.29 cd 97.03 b 97.73 bc 103.79 a 95.46 ab 92.11 a 62.65 b 61.21 b 162.75 b 189.14 a 160.38 b 165.00 a Cellulase 53.15 d 96.47 b 107.67 b 103.38 a 103.05 a 98.31 a 71.74 a 59.15 b 156.20 b 194.78 a 179.41 ab 162.63 a Pectinase 107.41 b 101.92 b 88.92 c 93.40 b 85.22 b 85.56 b 70.54 a 57.00 b 192.63 a 187.48 a 159.46 b 150.40 b ß glucosidase 84.37 c 83.24 c 103.34 b 96.23 ab 95.72 ab 80.64 b 61.43 b 58.33 b 180.09 ab 163.88 b 164.77 b 154.56 b C+P+ ß C 106.70 b 104.61 b 104.86 b 96.07 ab 70.42 c 67.02 c 59.80 b 61.60 b 177.12 ab 171.63 ab 164.66 b 157.60 ab Carlos Skins 50% Ethanol 206.92 a 207.79 a 198.61 a 130.34 a 94.78 ab 99.01 a 111.01 a 133.66 a 301.70 a 306.80 a 309.62 a 264.00 a Buffer 92.69 c 112.56 b 85.70 bc 80.78 b 102.16 a 82.80 b 73.35 b 70.50 b 194.85 b 195.36 b 159.05 b 151.28 b Cellulase 54.54 e 95.49 c 75.14 c 72.37 bc 110.00 a 80.19 b 62.64 c 50.59 c 164.54 c 175.68 c 137.78 c 122.96 c Pectinase 102.33 b 90.98 c 88.83 bc 85.87 b 89.49 b 78.83 b 69.97 b 61.06 b 191.82 b 169.81 c 158.80 b 146.93 b ß glucosidase 105.29 b 98.18 c 97.18 b 83.59 b 91.77 b 85.23 b 71.38 b 58.23 bc 197.06 b 183.41 b 168.56 b 141.82 b C+P+ ß 72.86 d 75.10 d 69.73 c 62.18 c 84.66 b 63.53 c 57.50 c 57.49 bc 157.52 c 138.63 d 127.23 c 119.67 c Noble Seeds 50% Ethanol 413.61 a 394.05 a 366.97 a 276.76 b 126.59 c 168.20 c 175.98 c 194.89 a 540.20 b 562.25 ab 542.95 a 471.65 a Buffer 249.53 b 324.03 b 325.13 b 288.12 b 319.67 a 267.49 a 227.88 a 188.42 a 569.20 a 591.52 a 553.01 a 476.54 a Cellulase 240.61 b 317.82 b 325.33 b 258.71 c 343.65 a 236.76 b 215.64 ab 180.18 ab 584.26 a 554.58 b 540.97 a 438.89 b Pect inase 234.57 b 316.37 b 336.52 b 270.74 b 334.68 a 248.35 a 197.19 b 193.87 a 569.25 a 564.72 ab 533.71 ab 464.61 a ß glucosidase 232.37 b 324.86 b 324.34 b 304.48 a 325.70 a 257.65 a 228.22 a 183.70 ab 558.07 ab 582.51 a 552.56 a 488.18 a C+P+ ß 263.08 b 322.67 b 335.44 b 273.01 b 283.17 b 261.82 a 188.25 bc 173.46 b 546.25 b 584.49 a 523.69 ab 446.47 b Carlos Seeds 50% Ethanol 351.23 a 369.21 a 369.07 a 280.45 a 143.52 b 150.46 c 153.79 b 201.13 ab 494.75 b 519.67 a 522.86 ab 481.58 a Buffer 234.75 b 292.31 b 306.10 c 262.66 a 261.72 a 225.85 a 195.89 a 185.45 b 496.47 b 518.16 a 501.99 b 448.11 b Cellulase 225.47 b 280.64 b 313.61 c 227.33 b 242.82 a 231.64 a 215.30 a 191.47 b 468.29 c 512.28 a 528.91 ab 418.80 c Pectinase 262.05 b 285.66 b 320.15 c 230.59 b 264.43 a 236.72 a 206.19 a 210.90 a 526.48 a 522.38 a 526.34 ab 441.49 b ß glucosidase 243.72 b 269.49 c 327.68 c 218.64 b 249.57 a 216.63 ab 211.96 a 218.47 a 493.29 b 486.12 b 539.64 a 437.11 b C+P+ ß 259.64 b 306.95 b 344.12 b 223.93 b 244.76 a 200.79 b 204.78 a 193.88 b 494.40 b 507.74 ab 548.90 a 417.81 c A : Extraction 1 extracted with 2 mL listed solvents and Incubated for 1, 4, 8, 24 h at 50 o C ; 50% ethanol and buffer used as controls. B : Extraction 2 residue of Extract 1 (samples Incubated for 1, 4, 8, 24 h) was extracted with 5 mL of 50% ethanol for 1 h at room temperature. C : Mixture of cellulase (C), pectinase (P) and ß glucosidase (ß) in a final volume of 2 mL 0.2 M Na acetate buffer (pH 4.8). a,b,c,d,e : Mean values within a short colum

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92 Figure 3 1 . Total phenolics and an thocyanins of grape skin and seed extracted by 10 combinations of solvent and enzyme. Combination of solvents were presented in Table 1. Extraction 1, 2, and 3 were measured respectively, and then added to obtain the total values. Post hoc comparison test (Student Newman Keuls) at p = 0.05 . c a b b a a a a b b 0 10 20 30 40 50 #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 GA (mg/g DM) Combinations of solvent and enzyme (a) Noble Skin (total phenolics) 1st 2nd 3rd Total c b b a ab a b ab b b 0 10 20 30 #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 Cyn (mg/g DM) Combinations of solvent and enzyme (b) Noble Skin (anthocyanins) 1st 2nd 3rd Total b a c d b c a a b b 0 10 20 30 40 50 60 #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 GA (mg/g DM) Combinations of solvent and enzyme (c) Carlos Skin (total phenolics) 1st 2nd 3rd Total d a d c b b a a c c 0 20 40 60 80 100 #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 GA (mg/g DM) Combinations of solvent and enzyme (d) Carlos Seed (total phenolics) 1st 2nd 3rd Total

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93 Figure 3 2 . HPLC chromatogram (280 nm) of the predominant phenolic compounds present in Noble seeds by different treatments. (a): Extraction 1, extracted with 50% ethanol for 8 h; (b): Extraction 2, extracted with 50% ethanol for 1 h; (c): Extraction 1, extracted with enzymes for 8 h; (d): Extraction 2, after enzyme hydrolysis and then extracted with 50% ethanol for 1 h; (e): extract (a) dried under nitrogen and then hydrolyzed with enzymes for 8 h; (f): Extraction 1, extracted with 50% ethanol for 4 h; (g): extract (f) dried under nitrogen and then hydrolyzed with enzymes for 4 h .

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94 1 Figure 3 3 . The ratio of antioxidant activities (DPPH values) to total phenols for different 2 fractions of Noble seeds by various treatments. Treatments showed in (a) and (b) same 3 as in Figure 2. Fractions were collected every 5 min within the retention time. Data not 4 sho wn in the figure means the phenolics in that fraction were too low to detect. Post hoc 5 comparison test (Student Newman Keuls) at p = 0.05. 6

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95 CHAPTER 4 7 ANTIOXIDANT, ANTIBACTERIAL AND ANTIBIOFILM PROPERTIES OF 8 POLYPHENOLS FROM MUSCADINE GRAPE ( VITIS ROTUNDIFOL IA MICHX.) 9 POMACE AGAINST SELECTED FOODBORNE PATHOGENS 10 Background 11 Foodborne disease is a common, costly yet preventable public health 12 problem. The United States Centers for Disease Control and Prevention (CDC) 13 estimates that one in six Americans (or 48 million people) get sick, 128,000 are 14 hospitalized, and 3,000 die fr om foodborne diseases each year ( 55 ) . The most 15 commonly identified foodborne pathogens include Staphylococcus aureus , Salmonella 16 typhimurium , Shigella sonnei , and Escherichia coli O157: H7 ( 55 ) . The increasing 17 incidence of methicillin resistant Staphylococcus aureus (MRSA) ( 56 ) , multiantibiotic 18 resistant Salmonella typhimurium (57) , multidrug resistant Shigella sonnei (58) and 19 Escherichia coli (59) are worldwide problems in both food, and clinical or medical 20 settings. For example, although Staphylococcus aureus is not always pathogenic, it is a 21 common cause of food poisoning, skin infections (e.g. boils), and respiratory disease 22 (e.g. sinusitis) ( 60 ) . Furthermore, the National Institutes of Health estimates that 80% of 23 all bacterial infections occurred when bacteria are at the biofilm mode of growth ( 61 ) . 24 Treatment of these infections is complicated by intrinsic resistance to conventional 25 antibiotics. Therefore there is an urgent need for new compounds or innovative 26 treatment strategies. 27 Muscadine grape ( Vitis rotundifolia Michx.) is indigenous to the southeastern 28 United States and contains a large variety of antioxidant phenolic compounds. 29 Muscadine phenolic compounds are well known for their nutraceutical benefit, such as 30 anticancer acti vities ( 20, 21 ) an d improving cardiovascular health ( 62 ) . Recent studies 31

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96 also confirmed that muscadine grape polyphenols and wine have antibacterial activity 32 against foodborne pathogenic bacteria E. coli O157:H7 ( 63, 64 ) , Salmonella 33 typhimurium (65) , Listeria monocytogenes (66) , and Streptococcus (67) . However, few 34 studies have been published about the antibacterial activity of specific muscadine 35 polyphenols against these bacteria, the correlation between antioxidant and 36 antibacterial activity of polyphenol s, the treatment use of muscadine polyphenols 37 with/without antibiotics, and their inhibition to biofilm formation and e radicating biofilm 38 capability. 39 With increasing demands or requirements for natural food preservatives and 40 antibiotics, this work was a co mprehensive study to examine the antioxidant, 41 antibacterial and antibiofilm activities of muscadine grape pomace polyphenols against 42 foodborne pathogens. Polyphenols time kill curves and synergism with antibiotics also 43 were investigated. Ultimately, it was hoped that muscadine polyphenols might have 44 potential as antimicrobial and/or antibiofilm agents for use in the food and healthcare 45 industries. 46 Materials and Methods 47 Bact erial Strains 48 Three Staphylococcus aureus strains (ATCC 35548, ATCC 12600 U, and ATCC 49 29247), one Salmonella strain ( S. typhimurium ), one Shigella strain ( S. sonnei ATCC 50 25931), and one Escherichia coli O157:H7 strain (204P) were used for the antimicrobial 51 activity study. All the ATCC bacterial st rains tested were purchased from American 52 Type Culture Collection (ATCC, Manassas, VA, USA). S. typhimurium and E. coli 53

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97 O157:H7 (204P) strains were obtained from Dr. Tung 54 University, Auburn, AL, USA. 55 Antibiotics and Chemica ls 56 Ampicillin and streptomycin were purchased from Fisher Scientific (Fair Lawn, 57 NJ, USA). Nalidixic acid was purchased from Sigma Aldrich (St Louis, MO, USA). 58 Plant derived phenolic standards, including gallic acid, caffeic acid, catechin, ellagic 59 acid, and quercetin were purchased from Sigma Aldrich (St Louis, MO, USA). All the 60 above commercially purchased chemicals were dissolved in sterile distilled water to a 61 diphenyl 1 62 picrylhyd razyl (DPPH), and 6 hydroxy 2, 5, 7, 8 tetramethylchroman 2 carboxylic acid 63 (Trolox) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium carbonate 64 and HPLC grade organic solvents were purchased from Fisher Scientific Co. 65 (Pittsburgh, PA, USA). 66 G rape Materials, and Extraction, Separation, and Identificat ion of Grape Phenolic 67 Compounds 68 69 Fully ripened Muscadine grape ( Vitis rotundifolia Michx.) cv . Noble (red) and cv . 70 Carlos (bronze) were harvested from the Center for Viticulture and Small Fruit Re search 71 (latitude 30.65 N, longitude 84.60 W) at Florida A&M University on August 24, 2012. 72 Harvested samples were shipped to the University of Florida on the same day and 73 stored at refrigeration (4 °C). Grape skin and seeds were separated manually from 74 ber ries and freeze dried in a freeze drier (Advantage, The Virtis Company, NY, USA) 75 within the following three days. The freeze dried samples were stored in vacuum 76 packaged polyethylene pouches at 20 °C until analyzed. 77

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98 Plant extracted phenolic compounds (Fig ure 4 1) were prepared in house at the 78 Food and Environmental Toxicology Laboratory from Muscadine grape skin and seeds. 79 Phenolic compounds from grape were extracted and isolated by a method adapted from 80 previous study ( 38 ) Freeze dried grape skin and seeds (defatted) were ground 81 sufficiently with a stainless steel grinder (Omni Mixer 17105, OCI Instruments, CT, 82 83 30 mL of 70% methanol. The ext raction flasks were vortexed for 30 s, sonicated for 10 84 min, kept at room temperature (22 °C) for 60 min, and sonicated for an additional 5 min. 85 The extracts were transferred to tubes and centrifuged at 2820 x g , 0 °C for 10 min (J 86 LITE ® JLA 16.250, Beckman Coulter Inc., CA, USA), and the supernatant was collected 87 in separate glass tubes. Residue was re extracted with the same procedure. The 88 collected supernatant (60 mL) was evaporated in a rotary evaporator (Büchi, 89 Labortechnik AG, Flawil, Switzerland) unde r reduced pressure at 40 °C to remove 90 solvent. The concentrates obtained after evaporation were re dissolved in 5 mL of 91 distilled water and sonicated for 5 min. 92 Solid phase extraction technique adapted from previous study ( 68 ) was used to 93 separate fractions of phenolic compounds. Briefly, two C18 Sep Pak cartridges 94 (Whatman ® Chromatography, ODS 5) w ere connected and preconditioned by 95 sequentially passing 12 mL ethyl acetate, 12 mL absolute methanol, and 12 mL of 0.01 96 N aqueous hydrogen chloride (HCl) through the cartridges. The prepared re dissolved 97 extract (0.5 mL) was loaded onto cartridges. Each c artridge was rinsed separately with 98 12 mL of 0.01N aqueous HCl (Fraction 1, F1), 12 mL of 10% methanol (F2), and 12 mL 99 of 30% methanol (F3) for Carlos skin extract; for Noble skin extract, the cartridges were 100

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99 rinsed with 4 mL of 0.01N aqueous HCl (F1), 15 mL of 10% methanol (with 0.1% HCl) 101 (F2), and 12 mL of methanol (with 0.1% HCl) (F3); and finally for Carlos and Noble seed 102 extracts, the cartridges were rinsed with 12 mL of 0.01N aqueous HCl (F1), 12 mL of 103 30% methanol (F2), and 12 mL of 50% methanol (F3) . To obtain a sufficient volume for 104 subsequent study, four individual separations of each extract was performed and then 105 combined. The collected fractions were evaporated in a rotary evaporator under 106 reduced pressure at 40 °C to remove the solvent. The con centrates obtained after 107 evaporation were dissolved in distilled water and stored at 4 °C until analyzed. 108 The above described extracts and their fractions were subjected to 109 chromatographic analyses by HPLC (Hitachi, L 7000 series, Japan) with an analytical 110 SB 111 Mobile phase was 0.5% formic acid in water (solvent A) and 0.5% formic acid in 60% 112 methanol (solvent B) under the following gradient: 0 3 min: 5% B, 3 8 min: 30% B, 8 113 25 min: 50% B, 25 30 min: 70% B, 30 35 min: 80% B, 35 47 min: 100% B, 47 51min: 114 5% B. The flow rate was 0.9 mL/min. Injection volume was 20 µl. Detection was 115 accomplished with a UV detector (Hitachi, L 7400, Japan) set at an absorbance 116 wavelength of 280 nm. The re tention times for each eluted components in the analyzed 117 samples were compared to those of the following standards: gallic acid, caffeic acid, 118 catechin, epicatechin, epicatechin gallate, trans resveratrol, ellagic acid, quercetin, and 119 cyanidin 3,5 diglucos ide. 120 Standards for caffeic acid hexoside, mono and digalloyl glucose, pentagalloyl 121 glucose, ellagic acid hexoside, xyloside and rhamnoside, delphinidin 3,5 diglucoside, 122 and peonidin 3,5 diglucoside were not available. Thus, a second HPLC system was 123

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100 emplo yed to confirm the identification of these by ESI MS detection in the Selective 124 reaction monitoring (SRM) mode. The LC MS analyses were performed on a Thermo 125 Finnigan Surveyor HPLC system (Thermo Finnigan, St. Jose, CA, USA) equipped with 126 a TSQuantum contr olled by XCalibur data analysis software (version 1.3, Thermo 127 Finnigan). The column and mobile phase used were the same as described above in 128 HPLC analysis. The MS acquisition was with ESI interface in negative ionization mode 129 at the following conditions: Sheath gas (N2) pressure, 42 arb; auxiliary gas (N2) 130 pressure, 20 arb; spray voltage 3.96 kV; capillary temperature, 414 °C; collision gas 131 pressure, 1.5 mTorr; collision energy, 22 V. These compounds were identified on the 132 basis of their mass fragmentation data compared with and identical to those reported by 133 the literature ( 5 ) . 134 Phenolic Content and Antioxidant Activity Assay 135 Total phenolic content of each extract or fraction was determined by the Folin 136 Ciocalteu colorimetric method ( 41 ) with an ultraviolet visible Beckman Coulter DU 640 137 spectrophotometer (Beckman Instruments, Fullerton, CA, USA) and expressed as mg 138 gallic acid equivalents per gram dry matter. The unitary antioxidant activity of each 139 extract or fraction was evaluated using the DPPH radical scavenging capacity assay ( 43 ) 140 and antibacterial activities of the phenolics (experimental compounds) were determined 141 by agar disc diffusion method ( 69 ) , and pathogenic bacteria for the test were listed 142 above. Isolated colonies of test bacterial strains grown overnight on tryptic soy agar 143 plates (TSA; Difco, Becton Dickson, Sparks, MD, USA) were cultured in Mueller Hint on 144 broth (MHB; Difco, Becton Dickinson) at 37 °C for 18 h. The turbidity of bacterial 145 suspensions was adjusted to approximately 2.0 × 10 8 CFU/mL (Optical Density at 600 146

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101 nm = 0.2). Bacterial suspensions were evenly swab inoculated on surfaces of Mueller 147 Hin ton agar (MHA; Difco, Becton Dickinson). Then each sterile blank disc (6 mm in 148 diameter, Difco, Becton Dickinson) was placed on the surface of the test MHA plates, 149 4 2, in triplicate per 150 compound), an d incubated at 37 °C for 24 h. Commercial antibiotics (ampicillin, nalidixic 151 152 as the experimental compounds were used as positive and negative controls, 153 respectively. The size of the zones of inhibition was measured and the antibacterial 154 activity was expressed in terms of the average diameter of the zone of inhibition in 155 156 absence of antibacterial activity. 157 The MIC was determined using an Alamar blue assay adapted from previous 158 study ( 69 ) 159 bacteria at a density of 5 x 10 6 CFU/mL were incubated in flat bottom, polystyrene, non 160 tissue culture treated 96 well microtiter plates at 37 °C without shaking for 20 h. A fter 20 161 162 added to the wells and the plates were shaken gently and incubated for 1 h at 37 °C. 163 Plates were gently shaken again, and absorbance at 570 nm and 600 nm were obtained 164 in a Microplate Reader (ELX808 Universal, Bio Tek Instruments, USA). Controls 165 included media alone (blank), diluted media plus experimental compounds plus AB 166 (negative), and media plus cells plus AB (positive). Percent reduction of AB was 167 calculated using 168 and 600 nm for each experimental well divided by the difference in positive control). 169

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102 Assays were performed in triplicate, and the average % reduction was used to 170 determine MIC. The AB MIC wa s defined as the lowest concentration of the 171 172 after the addition of AB ( 70 ) . 173 To investigate the correlation of Alamar blue reduction to CFU/mL, CFU/mL was 174 obtained from the same wells used to obta in AB absorbances. Before adding AB to all 175 fold) in 0.1% 176 177 after incubation at 37 °C for 24 178 correlations were calculated between Alamar blue reduction and CFU/mL. 179 Time kill and Synergy Assay 180 Time kill assays were performed using a method adapted from previous 181 study ( 71 ) . Briefly, bacteria were cultured to exponential phase (1.0 × 10 8 CFU/mL, 182 OD 600 = 0.15) in MHB and challenged with antibiotics or experimental compounds at 4 × 183 MIC or 500 mg/L. Synergy between antibiotics and phenolic compounds was te sted at 184 0.9 x MIC. Bacterial viability was monitored every hour (0 6 h) by plating cultures onto 185 TSA, and counting colonies after incubation at 37 °C for 24 h as described previously. 186 To detect bacterial lysis following the challenge with antibacterial a gents, the culture 187 turbidity of early exponential phase cultures was monitored every hour (0 6 h) and 188 finally 24 h by absorbance measurements at 600 nm at 37 °C. Synergy between 189 antibiotics and phenolic compounds was defined as follows, using log 10 CFU/mL at 24 190 h: synergy was >2 log 10 kill, additivity was >1 to 2 log 10 kill, indifference was 1 log 10 kill 191 to 2 log 10 growth, and antagonism was >2 log 10 growth compared to the most active 192

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103 log 10 CFU/ mL kill at 24 h from 193 baseline ( 72 ) . 194 Antibiofilm Activity Assay 195 The planktonic cell susceptibility to polyphenols and polyphenols inhibiting biofilm 196 formation were determined using the Alamar blue assay adapted from previous 197 study ( 69 ) 198 diluted 2,500 to 5 mg/L) with bacteria at a density of 5 x 10 6 CFU/mL were incubated in 199 round bottom, polystyrene, non tissue culture treated 96 well microtiter plate s at 37 °C 200 without shaking for 20 h. After 20 h, visual MICs were identified by the dot at the bottom 201 of the well (the well before dots appear is defined as MIC point); then, the content of 202 each well was aspirated and transferred to new flat bottom plates; 203 was subsequently added to the new plate wells and analyzed as the previous procedure 204 to determine the MICs of planktonic cells. After this, the original round bottom plate 205 terile physiological 206 saline to remove non 207 208 determine the Minimum Biofilm Inhibitory Concentration (MBIC). 209 To determine the reduction capability of polyphenols on preformed biofilm, 210 assays were performed in flat bottom plates with bacteria incubated at a density of 5 x 211 10 6 212 h to allo w biofilm formation. Then, two fold dilutions (diluted 2,500 to 5 mg/L) of 213 experimental compounds in MHB were prepared external to the plates. After 24 h 214 incubation, well contents were aspirated from all experimental and control wells; and 215

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104 wells were gentl 216 the appropriate diluted compounds was added. Preformed biofilms were exposed to 217 218 to the wells and anal yzed previously. 219 Statistical Analysis 220 Experimental results were expressed as means ± standard error (SE) of three 221 independent replicates. Data were subjected to ANOVA and significant differences were 222 tested by post hoc comparison test (Student Newman Keuls) at P < 0.05. Microsoft 223 Excel 2010 and SPSS 21 .0 for Windows were used for this analysis and for calculating 224 225 Results 226 Muscadine Grape Phenolic Compounds and Antioxidant Activity 227 Phe nolic compounds from Muscadine grape (Noble and Carlos) skin and seeds 228 were separated into three fractions, respectively, and chromatograms of identified 229 phenolics from Noble skin and seed fractions were presented in Figure 4 1. Caffeic acid 230 hexoside, hydr olyzable tannins, and gallic acid were the major phenolics in fraction 1 of 231 Noble skin polyphenols (Figure 4 1a). Anthocyanins were the major phenolics in the 232 corresponding fraction 2 (Figure 4 1b), and ellagic acid and its conjugates were in 233 fraction 3 (F igure 4 1c). Polyphenol fractions from Carlos skin were different from Noble 234 skin, with richer ellagic acid and conjugates in fraction 3, but almost no anthocyanins in 235 fraction 2 (data not shown). However, polyphenol fractions from Noble and Carlos seed 236 sh owed similar profiles, with hydroxybenzoic acid, hydrolyzable tannins, and little 237 flavan 3 ols distributed in fraction 1 (Figure 4 1d), major flavan 3 ols and condensed 238

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105 tannins distributed in fraction 2 (Figure 4 1e), and ellagic acid and its conjugates 239 di stributed in fraction 3 (Figure 4 1f). Overall, anthocyanins (fraction 2, 22.6 mg/g) were 240 the major phenolics for Noble skin, while it was ellagic acid and its conjugates (fraction 241 3, 20.5 mg/g) for Carlos skin; flavan 3 ols and condensed tannins (fraction 2, 38.8 and 242 40.8 mg/g, respectively) were the major phenolics for Noble and Carlos seeds (Table 4 243 1). 244 Antioxidant activity of phenolic compounds varied greatly among tested samples 245 (Table 4 1). For the polyphenol standards, gallic acid showed the strongest antioxidant 246 activity per unit, and ranked in order as: Gallic acid > Ellagic acid > Quercetin > 247 Catechin > Caffeic acid. For the polyphenol extracts, fraction 3 showed the strongest 248 antioxidant activity per unit, and ranked in order as: fraction 3 (ellagi c acid and 249 conjugates) > fraction 1 (hydroxybenzoic acid and hydrolyzable tannins) > fraction 2 250 (anthocyanins or flavan 3 ols and condensed tannins). As expected, antioxidant activity 251 for the non separated grape skin or seed extracts varied among their cor responding 252 activities of the fractions. 253 Antibacterial Act ivity of Phenolic Compounds 254 Antibacterial activity of phenolic compounds was initially screened by the 255 standard agar disc diffusion method, and then subjected to the MIC test. Table 4 2 256 showed that the inhibition of bacterial growth was quite drug and species dependent. 257 Muscadine grape skin and seed polyphenol extracts exhibited a broad spectrum of 258 antibacterial activity against Gram positive bacteria (S. aureus) , but exhibited little to no 259 antibacte rial efficacy against Gram negative bacteria (Salmonella typhimurium, S. 260 sonnei ATCC 25931 , E. coli O157:H7 204P ). Inhibition by seed polyphenols (Carlos 261

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106 Seed vs. Noble Seed) against S. aureus was positively dependent on concentration 262 while skin polyphenol s (Carlos Skin vs. Noble Skin) was negatively dependent. 263 Although Carlos Skin was used at higher polyphenol concentration than Carlos Seed, its 264 inhibition was significantly weaker than the later. These indicated that the antibacterial 265 activity of muscadine grape phenolic compounds were not only concentration 266 dependent, but also phenolic specific dependent. Gallic acid exhibited good 267 antibacterial activity on tested S. aureus strains while ellagic acid showed slightly less 268 acid, catechin, and quercetin exhibited little to no 269 antibacterial efficacy against S. aureus at this concentration. None of these polyphenol 270 standards was found to show inhibition on Gram negative bacteria in this study. The 271 272 varied significantly with zon es of inhibition ranging from 0 to 43.8 mm. In general, 273 ampicillin and streptomycin showed strongest inhibition than nalidixic acid in this study. 274 Muscadine grape polyphenols were further demonstrated to have good 275 antibacterial activity against Gram posi tive bacteria (S. aureus) as shown by an MIC of 276 74 367 mg/L for skin polyphenols and 67 173 mg/L for seed polyphenols (Table 4 277 3). Inhibition for different phenolic fractions was significantly different. In general, the 278 non separated skin and seed poly phenols exhibited comparable or even better inhibition 279 than some of their corresponding fractions. This indicated there might be a synergistic 280 effect between different phenolics in inhibiting bacteria growth. Muscadine grape 281 polyphenols also showed good an tibacterial activity against Gram negative bacteria (S. 282 sonnei ATCC 25931 ) with an MIC of 112 735 mg/L, while exhibiting fair antibacterial 283 efficacy against S. typhimurium and E. coli O157:H7 204P with an MIC of 224 1,976 284

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107 mg/L and 304 1,540 mg/L, res pectively. The phenolic concentration required to inhibit 285 Gram negative bacteria ranged from 1 to 16 times higher than that required for Gram 286 287 commercial polyphenol standa rds on all tested bacterial strains. The inhibition of 288 antibiotic controls on all study strains, even though within the same species, varied 289 290 contrast, muscadine grape polypheno ls displayed consistent antibacterial activity within 291 one species, such as S. aureus (Table 4 3) and Salmonella typhimurium (data not 292 completely shown). This indicated that muscadine grape polyphenols had a broad 293 spectrum of antibacterial activity and poss ibly could be used to treat these antibiotic 294 resistant strains. 295 Evaluation of Bacterial Killing and Synergist ic Effect by Phenolic Compounds 296 In order to further evaluate the bactericidal activity of phenolic compounds, the 297 killing kinetics of these compou nds were assessed against S. aureus ATCC 35548 298 alongside established antibiotics. At 4 x MIC, Noble skin and seed polyphenols caused 299 a reduction in cell viability of 4.7 and 4.9 Log 10 CFU/mL after 6 h in MHB, respectively, 300 while Streptomycin caused a reduc tion of 6.1 Log 10 CFU/mL (Figure 4 2a). Gallic acid 301 and Ellagic acid caused only a slight bacteriostatic effect at 500 mg/L. Significant 302 synergism between antibiotics and polyphenols at sub MIC were observed. Ampicillin 303 (150 mg/L), with a high MIC of 2,500 mg/L, when added with Gallic acid (500 mg/L), 304 Ellagic acid (500 mg/L), Noble skin (0.9 x MIC), and Noble seed (0.9 x MIC) caused a 305 reduction in cell viability of 0.5, 3.9, 5.1 and 4.8 Log 10 CFU/mL, respectively (Figure 4 306 2b). While Streptomycin added with Gallic acid, Ellagic acid, Noble skin, and Noble 307

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108 seed caused a reduction in cell viability of 1.6, 4.0, 5.0 and 6.2 Log 10 CFU/mL, 308 respectively (Figure 4 2c). Although both Gallic acid and Ellagic acid had little effect on 309 bacterial viability individually, when combined with antibiotics, Ellagic acid showed 310 considerably stronger bactericidal activity than Gallic acid, especially after 2 h. Synergy 311 of Streptomycin and Noble Seed (at 0.9 x MIC) was better than Streptomycin or Noble 312 seed (at 4 x MIC) alone, an d was the strongest bactericidal activity among all the 313 treatments. These indicated that combining grape polyphenols with antibiotics could 314 maximize the bactericidal activity while redu cing the usage of antibiotics. 315 Bacterial lysis showed that, at 4 x MIC, Noble skin and seed polyphenols caused 316 a reduction in culture turbidity over a 6 h period while Streptomycin exhibited an 317 increase (Figure 4 2d). This indicated Noble skin and seed polyphenols were 318 bactericidal and killed bacterial with concomitant cell l ysis while Streptomycin was 319 nonlysis but bactericidal. Antibiotics combined with Noble seed polyphenols exhibited 320 stronger bacterial lysis ability than skin polyphenols (Figure 4 2e, 2f). Overall, there was 321 a positive correlation ( r = 0.84) between bacteri al number (Log 10 CFU/mL) and culture 322 turbidity (OD 600 ) at 6 h. After 24 h, as the turbidity of the control continued to increase, 323 all treatments except Gallic acid caused a reduction in culture turbidity. This suggests 324 that with plenty of time, phenolic co mpounds promoted a loss of culture turbidity as well 325 as a loss of bacterial viability. 326 Antibiofilm Activity of Phenolic Compounds 327 To investigate the resistance of biofilm versus planktonic grown strains, Alamar 328 blue MICs of Nalidixic acid against a variety of pathogenic bacteria and MBICs against 329 their preformed biofilm were studied (Table 4 4). Alamar blue MBICs increased at least 330

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109 four fold relative to planktonic MICs. These data are consistent with previous reports of 331 many fold increases in drug resistanc e of biofilm versus planktonic grown strains ( 70, 332 73 ) . 333 A variety of antibiotics and phenolic compounds with different mechanisms were 334 further studied on planktonically grown S. aureus susceptibility, inhibition of biofilm 335 formation and resistance of preformed biofilm (Table 4 5). Visual and Alamar blue MICs 336 for planktonically grown strains were identical with Ellagic acid as an exception. Thus, 337 MICs can be generally determined visually or spectrophotometrically. MBICs for 338 inhibiting biofilm formation were within two 2 fold dilutions lower than MICs, while 339 MBICs of preformed biofilm were at least 16 fold higher than MICs. For example, the 340 MIC of Carlos seed polyphenols against planktonic grown S. aureus ATCC 35548 was 341 40 mg/L, against biofilm formation was 20 mg/L, and against preformed biofilm was as 342 high as 641 mg/L. These findings indicated that grape polyphenols were able to inhibit 343 bacteria biofilm formation at a sub MIC concentratio n without killing the bacteria; 344 however, to eradicate the preformed biofilm a high concentration o f phenolic 345 compounds is needed. 346 Discussion 347 This study demonstrated that muscadine grape skin and seed polyphenols had 348 potent bactericidal activity against planktonic cells and biofilm formation of Gram 349 positive bacteria, S. aureus . The fact that polyphenols acted to cause cell lysis and 350 therefore c ompromise the integrity of the bacterial membrane may explain why these 351 compounds have the ability to eradicate S. aureus biofilms; potent antibiofilm activity is 352 a property that is often associated with membrane perturbing agents ( 74 ) . The limited 353

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110 activity of grape polyphenols against Gram negative bacteria was probably because of 354 the limited ingress across the outer membrane ( 75 ) . However, another intrinsic 355 mechanism that has been insufficiently studied is efflux, and multidrug efflux pumps 356 have been shown to be associated with the insusceptibility of E. coli to various bio cidal 357 agents ( 76 ) . 358 A positive correlation ( r = 0.72) between antioxidant (Table 4 1) and antibacterial 359 (Table 4 2) activities of polyphenol standards against S. aureus on agar medium was 360 found. Also a weak po sitive correlation ( r = 0.53) between antioxidant (Table 4 1) and 361 antibacterial (Table 4 3) activities of muscadine polyphenols against S. aureus was 362 observed. These findings suggested that the stronger the antioxidant activity per unit 363 polyphenols, the be tter the antibacterial activity against S. aureus . However, strong 364 antibacterial activity against S. aureus 365 activity since no antioxidant activity was demonstrated for antibiotics (ampicillin, nalidix 366 acid, and streptomycin) (data not shown). Similar to a previous finding ( 16 ) , there was a 367 positive correlation ( r = 0.77) between total polyphenol content and total antioxidant 368 activity for these grape extracts and fractions. Nonetheless, higher phe nolic levels do 369 not necessarily correlate with increased inhibition against S. aureus but rather the type 370 and concentration of compo unds present in these extracts. 371 Interestingly, compared to grape phenolic extracts, commercial phenolic 372 standards Gallic aci d and Ellagic acid exhibited stronger antioxidant activity per unit, 373 11.1 and 9.8 mmol/g, respectively (Table 4 1); and also showed good antibacterial 374 activity on tested S. aureus strains using the disc diffusion method (Table 4 2). 375 However, further Alamar blue MIC test (Table 4 3) and time killing study (Figure 4 2) 376

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111 suggested that Gallic acid and Ellagic acid possess negligible antibacterial efficacy 377 against S. aureus . This contradictory finding indicated the very different antibacterial 378 mechanism of Galli c acid and Ellagic acid on a solid surface versus in a liquid. Other 379 phenolic standards Caffeic acid, Catechin, and Quercetin exhibited little to no inhibition 380 on the tested strains in all assays. Therefore, it seems that antibacterial activities of 381 commer cial phenolic standards were weaker compared to the muscadine grape 382 phenolic extracts. One possible reason was that the antibacterial activities of these 383 phenolics were lost during their processing or these phenolics existed as ineffective 384 isomer forms. An other possible reason was the synergism between multiple phenolics 385 increased their antibacterial activities. These findings can be further supported by other 386 387 activity agai nst Gram positive and Gram negative bacteria ( 77 ) ; while plant extracted 388 whole polyphenols exhibited good antibacterial activity against several food borne 389 pathogenic bacteria ( 78 ) , for instance, the mean MICs against S. aureus ranged from 98 390 to 389 mg/L and against E. coli r anged from 450 to 1519 mg/L. Also, in this study, whole 391 extracted skin and seed polyphenols exhibited comparable or even better inhibition than 392 their corresponding fractions. Therefore, whole extracted polyphenols were used in the 393 following studies. 394 This s tudy also investigated the correlation between Alamar blue (AB) percent 395 reduction by bacteria metabolic activity ( 70 ) , and the existing bacteria number per well 396 on Carlos seed polyphenols and Ellagic acid at various concentrations. Results showed 397 that percent reduction of AB had a significant correlation ( r = 0.94 and 0.92, 398 respectively) with existing bacteria number (Figure 4 3). Carlos seed polyphenols at 40 399

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112 mg/L inhibited the bacteria number at initial level (6 Log 10 CFU/mL) with a 24.9% 400 reduction of AB after 20h incubation , while at 160 mg/L (4 x MIC) killed the bacteria with 401 no reduction of AB. Ellagic acid at 2,500 mg/L reduced bacteria number by 1.4 Log 10 402 CFU/mL with a 39.2% reduction of AB after 20h incubation. These confirmed that the 403 colorimetric AB assay is a reliabl e, reproducible means of determining bacteria antibiotic 404 susceptibility. A previous study also showed that the AB assay was a good means of 405 determining biofilm antibiotic susceptibility and had good to excellent correlation with 406 two other biofilm susceptib ility methods, XTT reduction and viable counts ( 70 ) . 407 The antibacterial mechanisms of phenolics are attracting more and more 408 research in recent years. The membrane interaction of phenolics is a prevailing theory 409 ( 77 ) . Phenolics are partially hydrophobic; this character may allow them to interact with 410 the lipid bilayer of bacterial cytoplasmic membrane and lipopolysaccharide interfaces 411 more effectively by decreasing membrane stability ( 79 ) . For example, Epicatechin 412 gallate could rapidly enter the cytoplasmic membrane, causing an immediate reduction 413 in bilayer fluidity ( 80 ) . The cytoplasmic membrane constitutes the selec tive barrier 414 controlling cellular ingress and egress of materials and houses many of the enzymes 415 involved in bioenergetic functions, cell wall synthesis and macromolecule secretion. 416 Penetration of phenolics into this phospholipid bilayer will have a profou nd outcome on 417 a wide range of essential cell functions. Since intercalation of these chemical structures 418 could induce membrane disruption that permits solute equilibration across the bilayer, 419 resulting in cell death, but more subtle interactions at sub inh ibitory concentrations 420 induce re organization of membrane architecture with implications for the capacity of the 421 target bacteria to cause disease in the susceptible host ( 79 ) . Phenolics also were found 422

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113 to prevent the formation of biofilms in this study (Table 4 ( 81, 423 82 ) . This may further support the membrane interactive mechanism since potent 424 antibiofilm activity is a property that is often associated with membrane perturbing 425 agents ( 74 ) . Thus, phenolics exhibit at least two biol ogical activities: inhibition of 426 bacterial growth at high concentrations and/or induce bacteria membrane disruption and 427 prevention of biofilm formation at lower concentrations. In view of these activities, 428 phenolics may be useful in a synergistic approach with conventional antibiotics against 429 antibiotic resistant forms of pathogens (e.g. multidrug resistant clinical isolates MRSA) 430 in clinical medicine. Our time killing study (Figure 4 2) indicated that muscadine Noble 431 seed phenolics combined with Ampicillin or Streptomycin at subinhibitory concentrations 432 greatly reduced S. aureus ATCC 35548 (MRSA) cell viability by 4.8 or 6.2 Log 10 433 CFU/mL, respectively. 434 Summary 435 Naturally occurring molecules, predominantly secondary metabolites, are the 436 source of the majority of drugs in clinical use today and they continue to be an important 437 basis of new therapeutics for bacterial infectious diseases ( 83 ) . Phenolics are 438 secondary metabolites in muscadine grapes and play important roles in the strong 439 440 ( 84 ) . Evidence is emerging that phenolics may be useful in the control of common oral 441 infections, such as dental caries and periodon tal disease ( 85, 86 ) . In this case, 442 phenolics can be incorporated into rinse and toothpaste formulations. Muscadine 443 phenolics also have been proven to possess strong anticancer activities, such as 444 inhibiting proliferation of prostate ( 20 ) and colon ( 21 ) cancer cells by inducing apoptosis. 445

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114 Importantly, phenolics are widely distributed in edible grapes ( 46 ) ; they have minimal 446 toxicity ( 87 ) . Therefore, even though outstanding bacte ricidal properties cannot be 447 achieved because of a relatively high concentration required in this study and the partial 448 metabolism of these by intestinal bacteria ( 88 ) , phenolic agents still have promise as 449 supplements for nutr aceutical value and conventional antibacterial chemotherapies by 450 inhibiting the growth of foodborne pathogens in the body. The broad ability of 451 muscadine phenolics to have strong antibacterial activity, to inhibit biofilm formation, 452 and to synergistically work with antibiotics may stimulate a muscadine by product 453 market as natural food preservatives, potential antibiotic replacements and/or as natural 454 sanitizers for processing equipment where foodborne pathogens reside. 455 456 457 458 459 460 461 462 463 464 465 466 467 468

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115 Table 4 1 . Phenolic content and antioxidant activity of experimental compounds 469 1 Phenolic content of extracts was expressed as mg gallic acid equivalents per gram dry matter. 470 2 Antioxidant activity was det ermined by DPPH assay and expressed as mmol Trolox equivalents per 471 gram polyphenols. 472 3 Fractions 1 3 for Carlos and Nobel skins and seeds were eluted from tandem C18 Sep Pak cartridges 473 with different solvents: Skins, F1 0.01 N HCl; F2 10% MeOH; F3 30% MeOH and Seeds, F1 0.01 N 474 HCl; F2 30% MeOH; F3 50% MeOH. 475 Results were expressed as means of triplicate determinations ± SE; different superscr ipt letters within 476 columns were significantly different ( P < 0.05). 477 478 479 Experimental compound Phenolic content 1 Antioxidant activity per unit 2 (mg/g) (mmol/g) Polyphenol Standards Gallic Acid 11.1 ± 0.2 a Caffeic Acid 5.1 ± 0.2 e Catechin 5.3 ± 0.3 e Ell agic Acid 9.8 ± 0.6 ab Quercetin 9.4 ± 0.4 b Polyphenol Extracts 3 Carlos Skin (CK) Polyphenols 39.0 ± 2.1 c 6.3 ± 0.5 d CK F1 9.1 ± 0.8 f 7.4 ± 0.3 cd CK F2 8.7 ± 0.7 f 5.9 ± 0.2 de CK F3 20.5 ± 1.4 e 8.7 ± 0.6 bc Noble Skin (NK) Polyphenols 35.9 ± 2.5 d 5.3 ± 0.3 e NK F1 5.5 ± 0.3 h 7.9 ± 0.5 c NK F2 22.6 ± 1.8 e 5.0 ± 0.4 e NK F3 7.5 ± 0.3 g 8.9 ± 0.8 bc Carlos Seed (CS) Polyphenols 50.8 ± 3.4 b 10.1 ± 0.4 ab CS F1 5.1 ± 0.2 h 8.4 ± 0.2 bc CS F2 38.8 ± 1.2 c 7.4 ± 0.3 cd CS F3 7.1 ± 0.4 g 10.8 ± 0.5 a Noble Seed (NS) Polyphenols 58.2 ± 3.5 a 9.2 ± 0.3 b NS F1 7.6 ± 0.6 g 8.6 ± 0.2 bc NS F2 40.8 ± 3.1 c 6.9 ± 0.1 d NS F3 8.9 ± 0.6 f 11.1 ± 0.5 a

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116 Table 4 2 . Antibacterial activity of experimental compounds against selected foodborne 480 pathogens using the disc diffusion method 481 482 Results were expressed as means of triplicate determinations ± SE; Results with different superscript 483 lowerc ase letters in columns and different superscript uppercase letters in rows were significantly different 484 ( P 485 effective and recorded as 0. 486 * Partial inhibition with in a zone and not included in the statistical analysis. 487 488

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117 Table 4 3 . Minimum inhibitory concentration (MIC) of experimental compounds against 489 human pathogenic bacteria 490 491 1 MICs were determined by the Alamar blue assay. MIC defined as the lowest experimental compound 492 493 min after addition of AB. Results with different superscr ipt lowercase letters in columns and different 494 superscript uppercase letters in rows were significantly different ( P < 0.05). MIC values > 2,500 mg/L 495 were not included in the statistical analysis. Test strains were inoculated at 5 x 10 6 CFU/mL in each well . 496 2 Fractions 1 3 for Carlos and Nobel skins and seeds were eluted from tandem C18 Sep Pak cartridges 497 with different solvents: Skins, F1 0.01 N HCl; F2 10% MeOH; F3 30% MeOH and Seeds, F1 0.01 N HCl; 498 F2 30% MeOH; F3 50% MeOH. 499 500

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118 Table 4 4 . Minimum inhibitory concentration (MIC) of Nalidixic acid against pathogenic 501 bacteria and Minimum Biofilm Inhibitory Concentration (MBIC) against their preformed 502 biofilm reduction 503 504 1, 2 MIC or MBIC defined as the lowest nalidixic acid concentratio 505 (average of three experiments) and a purplish well 60 min after addition of AB. 506 * Significant differences between MICs of nalidixic acid versus MBICs ( P < 0.05). 507 # Significant differences between MBICs of nalidix ic acid against biofilms within the same S. aureus 508 species ( P < 0.05). 509 510

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119 Table 4 5 . Minimum inhibitory concentration (MIC) of experimental compounds against 511 S. aureus ATCC 35548, and Minimum Biofilm Inhibitory Concentration (MBIC) against 512 its biofilm formation and preformed biofilm reduction 513 514 1 Visual MIC was identified by the dot at the bottom of the well. The well before dots appear defined as 515 MIC point. 516 2, 3, 4 Planktonic cell MIC or MBIC de fined as the lowest experimental compound concentration resulting in 517 tion of AB (average of three experiments) and a purplish well 60 min after addition of AB. 518 * Significant differences between MICs (visual and planktonic cell) of experimental co mpounds versus 519 MBICs (biofilm formation and reduction) ( P < 0.05). 520 # Significant differences between MBICs of experimental compounds against biofilm formation versus 521 preformed biofilm reduction ( P < 0.05). 522

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120 Figure 4 1 . Phenolic compounds and their structures found in muscadine grape skin and seed. (a), (b), and (c) are Noble skin fractions 1, 2, and 3 respectively; (d), (e), and (f) are Noble seed fractions 1 , 2, and 3 respectively

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121 Figure 4 2 . Evaluation of killing (a, b, c) and lytic (d, e, f) action of experimental compounds and their synergism with antibiotics against S. aureus ATCC 35548 in MHB. (Means of three independent replicates; MICs of Noble Skin, Noble Seed, and Streptomycin were 113, 88, and 20 mg/L, respectively. 2 4 6 8 10 0 1 2 3 4 5 6 Log 10 CFU/mL Time (h) (a) Control Gallic Acid (500 mg/L) Ellagic Acid (500 mg/L) Noble Skin (4 x MIC) Noble Seed (4 x MIC) Streptomycin (4 x MIC) 2 4 6 8 10 0 1 2 3 4 5 6 Log 10 CFU/mL Time (h) (b) Control Ampicillin (150 mg/L) Ampicillin (150 mg/L) + Gallic Acid (500 mg/L) Ampicillin (150 mg/L) + Ellagic Acid (500 mg/L) Ampicillin (150 mg/mL) + Noble Skin (0.9 x MIC) Ampicillin (150 mg/L) + Noble Seed (0.9 x MIC) 2 4 6 8 10 0 1 2 3 4 5 6 Log 10 CFU/mL Time (h) (c) Control Streptomycin (0.9 x MIC) Streptomycin (0.9 x MIC) + Gallic Acid (500 mg/L) Streptomycin (0.9 x MIC) + Ellagic Acid (500 mg/L) Streptomycin (0.9 x MIC) + Noble Skin (0.9 x MIC) Streptomycin (0.9 x MIC) + Noble Seed (0.9 x MIC) 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 24 Absorbance OD 600 Time (h) (d) 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 24 Absorbance OD 600 Time (h) (e) 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 24 Absorbance OD 600 Time (h) (f)

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122 Figure 4 3 . Percent reduction of Alamar blue (bars) and Log 10 CFU/mL ( ) for phenolic compounds on S. aureus ATCC 35548. ( Means of three independent replicates; Test strains were inoculated at 5 x 10 6 CFU/mL in each well).* Values differ significantly ( P < 0.05) from values without phenolic compounds. * * * * * 0 2 4 6 8 10 0 20 40 60 80 100 160 80 40 20 10 5 0 Log 10 CFU/mL % Reduction of Alamar blue Phenolic Compound Concentration (Carlos Seeds, mg/L) (a) r = 0.94 % Reduction AB CFU/mL * * 6 7 8 9 10 20 40 60 80 100 2500 1250 625 312.5 156.2 78.1 0 Log 10 CFU/mL % Reduction Alamar blue Phenolic Compound Concentration (Ellagic Acid, mg/L) (b) r = 0.92 % Reduction AB CFU/mL

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123 CHAPTER 5 MUSCADINE GRAPE ( VITIS ROTUNDIFOLIA MICHX.) POLYPHENOLS RE DUCE ACRYLAMIDE FORMATION IN BOTH ASPARAGINE/GLUCOSE AND POTATO CHIP MODEL SYSTEMS Background Acrylamide is a bypr oduct of the Maillard reaction and it is formed in a variety of heat treated commercial starchy foods. It is known to be to xic and ( 31 ) . T here have been extensive studies on the formation mechanism of a crylamide . Antioxidants have b een proposed as one pos sible mechanism to reduce acrylamide formation ( 32 ) . Muscadine grape ( Vitis rotundifolia Michx.) is indigenous to the southeastern United States and contains a large variety of antioxidant phenolic compounds. It is hypothesized that muscadine grape polyphenols may also reduce acrylamide formation because of their strong antioxidant activity. In this study, m uscadine grape polyphenols and pure phenolic compounds ( such as Gallic acid, Ellagic acid, Epicatechin, and their mixture) w ere investig ated on acrylamide f ormation in an equimolar asparagine/ glucose model system ( 2 mM) during heating (180 °C for 30 min). GC ECD was used with bromine derivatization of the sample for acrylamide quantification. Antioxidant activities of phenolic compounds also were investigated and correlated with acrylamide inhibition. Finally, a potato chips system was introduced to study the inhibition of muscadine grape polyphenols on acrylamide formation in heated starchy food products and also a simulated physiological system was introduced to study the trapping effect of polyphenols on acrylamide in vivo .

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124 Materials and Method Chemicals Caffeic acid, Gallic acid, Catechin, Epicatechin, Epicatechin gallate , Ellagic aicd, Quercetin, 2,2 diphenyl 1 picrylhydrazyl (DPPH), and 6 hydroxy 2, 5, 7, 8 tetramethylchroman 2 carboxylic acid (Trolox) were obtained from Sigma Aldrich (St. Louis, MO). Acrylamide standard ( 99.8%), L asparagine, D (+) glucose, p hosphate buffered saline (PBS, pH 7.4) and all other chemicals and solvents were purchased from Fisher Scientific Co. (Pittsburgh, PA). Grape M aterials Fully ripened ripened Muscadine grape s ( Vitis rotundifolia Michx.) were harvested from the Center for V iticulture and Small Fruit Research (latitude 30.65 N, longitude 84.60 W) at Florida A&M University in 2013 . The collected samples were shipped to the University of Florida on the same day and stored in a cold room (4 °C ). Grape skins and seeds were separated manually from berri es and freeze dried in a freeze drier (Advantag e, The Virtis Company, NY, USA). The freeze dried samples were stored in vacuum packaged polyethylene pouches at 20 °C until needed . A c ommercial table grape Red Seed was used as a varietal control and purchased in 2013 from Wal Mart Store, Gainesville, FL . Sample P reparation Freeze dried grape skins (20 g) were ground with a stainless steel grinder (Omni Mixer 17105, OCI Instruments, CT, USA) for 1 min, and then placed on a siev e ( 16 ) . The powdered samples were stored at 20 °C a nd used for subsequent analysis.

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125 Freeze dried grape seeds (20 g) were crushed and then defatted with hexane at a ratio of 1:10 (w/v). After 24 h of extraction at room temperature (shaking every 6 hr ), the hexane extract was filtered using Whatman #4 filter paper (0.4 Scientific, Pittsburgh, PA) under vacuum. The residue was evenly distributed over a tray and kept in the hood to evaporate the hexane. The final defatted grape seed powder was ground again in the stainless steel grinder and the powder passing through the 20 °C and used for subsequent analysis. Phenolic C ompounds E xtraction Freeze dried grape skin and seeds (defatted) were ground sufficiently with a stainless steel grinder (Omni Mixer 17105, OCI Instruments, CT, USA) to a fine powder 0. 5 g) from each sample was extracted with 1 0 mL of 70% methanol. The extraction flasks were vortexed for 30 s, sonicated for 10 min, kept at room temperature (22 °C) for 60 min, and sonicated for an additional 5 min. The extracts were transferred to tubes , centrifuged at 2820 x g , 0 °C for 10 min (J LITE ® JLA 16.250, Beckman Coulter Inc., CA, USA), and the supernatant was collected in separate glass tubes. Residue was re extracted with the same procedu re. The collected supernatant ( 2 0 mL) was filtered (0.45 m) and used for further analysis. Phenolic C ompounds A nalysis Total phenolic content in grape seeds or skin was determined by the method of Singleton et al. ( 41 ) using an ultraviolet visible Beckman Coulter DU 640 spectrophotometer (Beckman Instruments, CA, USA). A mixture of properly diluted ex

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126 20% Na 2 CO 3 were introduced in a tube. After reacting for 30 min in a 40 °C water bath, absorbance was measured at 760 nm. Gallic acid (GA) was used as standard and expressed as gallic acid equivalents (mg gallic acid (GAE ) /g dry matter (DM)) using a g/mL (R 2 = 0.9996). A ntioxidant A ctivities A nalysis The DPPH assay was based on the slightly mo dified method of Brandwilliams et al. ( 43 ) . The properly of DPPH (0.0025 g/100 mL CH 3 OH). After 60 min reaction at room temperature in the dark, the absorbance at 515 nm was recorded to determine the concentration of the remaining DPPH. The percen t inhibition of DPPH in the test sample and known concentrations of Trolox were calculated by the following formula: %Inhibition = 100 × (A 0 A)/ A 0 , where A 0 was the beginning absorbance at 515 nm, obtained by measuring the same volume of solvent, and A was the final absorbance of the test sample at 515 nm. The calibration curve between %Inhibition and known concentration of Trolox solutions was then establish ed. The radical scavenging activities of the test samples percent inhibition. Trolox standard solutions were prepared at a concentration ranging 2 =0. 9999). Chemical M odel M aillard R eactions The role of polyphenols in acrylamide formation was first investigated in chemical model systems ( 89 ) . The comp ositions of different model reactions are listed in Table 5 1. The reaction mixtures (1 mL 2 mM Asparagine + 1 mL 2 mM Glucose + 0.5 mL

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127 polyphenols at different concentration (50, 100, 200 g/mL) or 0.5 mL 100 g/mL polyphenols of different grape varieties) were added in to screw cap glass reaction vials ( PYREX ® VISTA, 1 0 mL capacity , Fisher Scientific Co., Pittsburgh, PA) and heated in a Reacti Therm III heating module at 180 5 °C for 60 min . After they were hea ted, reaction mixtures were immediately cooled down in an ice bath and then redissolved in 5 mL distilled water for further analysis. For comparison of chemical profiles, reaction mixtures from the asparagine glucose models with/without polyphenols (model reactions E and F ) were subjected to GC analysis after bromination and a single syringe driven filtering step. Fried P otato C hip M odel M aillard R eactions The role of polyphenols in acrylamide formation was then investigated in a potato chip model system ( 32 ) . Potat o chips (2 mm in thickness ) w ere prepared and immersed in a 0.025 % , 0.05%, and 0.1% solution of Carlos skin polyphenols for 60 min at room temperature; meanwhile, water was used for immersion of the control samples. The potato strips were drained for 2 min prior to frying, which was carried out in canola oil at 175 °C for 5 min with an electric fryer (Philips). After they were fried and cooled, the potato chips were ground to a paste, which (10 g) was then extracted with 100 mL 0.1% formic acid solution wit h mixing on a wrist action shaker for 20 min . The extracts were refrigerated to e asily remove the oily top layer and f ilter ed syringe filter to collect the supernatant followed by bromination and analysis. Simulated P hysiological C onditions T rapping R eactions E xperiments was conducted in a buffer system that simul ated physiological conditions: c ommercial ly available PBS as dry powder was packed in a foil pouch ( 90 ) .

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128 After the dry powder was dissolved in 1 L of deionized water, 0.01 M PBS was obtained that contained 0.138 M NaCl and 0.0027 M KCl, pH 7.4, at 25 °C . The buffer (5 mL) solutions containing epicatechin and a crylamide in molar ratios of 1:1, 5 :1, 10:1, and 15:1 , respectively, were in cubated in a water bath at 37 °C . The PBS solution containing only acrylamide was used as a control. T ime points were set at 1 and 2 days. At each specific ti me point, samples were taken from the incubator and immediately chilled in an ice bath before bromination . Acrylamide B romination A 5 mL of prepared sample was pipetted into a 20 mL vial containing 0.75 g of potassium bromide. The pH of solution was adjusted between 1 and 3 wit h concentrated bromic acid. Two mL of saturated bromine water was added and shake n . If color disappears, more bromine water was added to the sample. The sample vial wrapped with aluminum foil in order to exclude light was stored in the dark at 4°C overnigh t. Aft er reacting overnight, the excess bromine was remov ed by adding 1M sodium thiosulfate solution drop by drop, until the solution became colorless. S odium sulfate (2 g) was added and the solution shake n vigorously. The s ample was transferred to a 60 mL separatory funnel. The reaction vial was rinsed three times with 1 mL of water and transferred to the separatory funnel. T he a queous solution was extracted with 5 mL of ethyl a cetate, shake n for 2 min and the bottom layer (aqueous layer) transferred into a small Erlenmeyer flask (50 ml). The organic phase was collected into a small Erlenmeyer flask (25 ml) and the aqueous solution was transferred back into the separatory funnel for a repeat of the e thyl acetate extraction. The organic phase poured through a column of sodium sulfate to remove excess water was collected in a 15 mL

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129 graduated test tube. The column was rinsed three times with 1 mL o f e thyl acetate and these were added to the test tube. The f inal extract ion volume was brought to 15 mL with ethyl acetate. Two ml of sample was fil tered thru a 0.45µm syringe filter into GC vials for GC analysis. GC ECD A nalysis The analysis of acrylamide by GC ECD was performed using a DB Wax fused silica capillary [3 0 m × 0.32 mm i.d., 0.5 column in an HP 5890 GC (HNU Systems, Newton, MA) equipped with a n electron capture d etector (ECD) . The oven program consisted of an initial temperature of 14 0 ° C for 1 min, foll owed by a temperature ramp to 2 0 0 ° C at 15 ° C/min . The temperature was held at 20 0 ° C for 10 min. Injector a nd detector temperatures were 220 ° C and 30 0 ° C, respectively, and carrier gas ( nitrogen ) was used in constant pressure mode (average linear flow rate 40 ml/min ). Data of retention times and peak areas were automatically documented using Turbochrom Workstation 6.1.1. (Perkin Elmer, MA, USA). The quantification of acrylamide was performed by external calibration method. HPLC ESI MS n A nalysis An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector and a fluorescent detector (Agilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Bil lerica, MA). Maillard reaction products were filtered through 0.45 Stablebond SB C18 column (250 mm x Technologies, Palo Alto, CA) was used for separation of polyphenols . Mobile phase was

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130 0.5% formic acid in water (solvent A) and 0.5% formic acid in 60% methanol (solvent B) under the following gradient: 0 3 min: 5% B, 3 8 min: 30% B, 8 25 min: 50% B, 25 30 min: 70% B, 30 35 min: 80% B, 35 47 min: 100% B, 47 51min: 5% B. The flow rate was 0.9 mL/min. The detection wavelength was 28 0 nm . Electrospray ioniza tion in negative mode was performed using nebulizer 4 5 psi, drying gas 11 L/min, drying temperature 350 °C , and capillary 4000 V. The full scan mass spectra of the polyphenols were measured from m/z 5 0 to m/z 2000. Auto MS2 was conducted with 10 0% compound stability and 10 0% trap drive level. Data were collected and calcu lated using Chemstation software (Version B. 01.03, Agilent Technologies, Palo Alto, CA). Color A nalysis The surface colo r of reacted solutions was measured by a machine vision system. Machine vision system, consisted of a fluorescent light box (42.5 cm (w ) x 61 cm (l) x 11.4 cm (h)) and a digital Nikon D200 colo r camera (Nikon D200 Digital Camera, Nikon Corp., Japan) connected to a computer with a firewire connection. The Camera settings were 36 mm focal length, ISO 100 sensitivity, 1/3 s F/11 shutter spee d, 1.0 EV exposure compensation, and direct sunlight white balance. The m achine vision system used the average daylight illuminant D65 mode with a colo r temperatur e of 6504 K. A software program was used to cap ture images, and to obtain colo r results based on L * (lightness), a * (redness), b * (yellowness) values ( 39 ) . Samples were placed in the light box and the digital camera captured a picture of samples . The m achine vision system was calibrated using a standard yellow plate ( L * = 87.09 , a * = 7.71 , and b* = 70.75 ) from Labsphere (North Sutton, NH). Average L*, a*, b* values of the solutions surface were calculated using a color analysis program .

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131 Statistical A nalyses All tests were performed in tri plicate . Polyphenol extractions and analyses were performed in duplicate. Data were subjected to ANOVA and differences among samples were tested by post hoc comparison test (Student Newman Keuls) at p = 0.05 with Microsoft Excel 2010 and SPSS 21 .0 for Windows (SPSS Inc, Chica go, IL) Results and Discussion Effect of P henolic C ompounds C oncentration on the F ormation of A crylamide Chemical model reactions were carried out to investigate the potential of phenolic compounds in reducing the formation of acrylamide under thermal conditions (1 8 0 °C, 60 min). Because the samples in this study were not in the form of complex matrices that might hinder the analysis of acrylamide , GC ECD was selected for quantitative and qualitative analyses. Results showed that phenolic compounds dose dependently reduced the content of acrylamide , although not in a linear manner (Figure 5 1). Moreover, an obvious significant ( P < 0.05) inhibitory effect was observed even at a level of addition as low as 50 g/mL . For example, the Noble skin polyphenols almost completely inhibit ed acrylamide formation at 50 g/mL. These data demonstrated phenolic compounds as an effective inhibitor of acrylamide formation under heating conditions. The inhibition by different phenolic compounds varied greatly, with a decr easing order of Noble skin polyphenols > Carlos skin polyphenols > Noble seed polyphenols > Carlos seed polyphenols > Standards mixture > Ellagic acid > Catehchin > Epicatechin > Epicatechin gallate > Caffic acid > Gallic acid > Quercetin. The complex grap e polyphenols and the pure standards mixture e xhibited higher inhibition indicating phenolic compounds have a synergistic influence on inhibiting acrylamide formation.

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132 The muscadine grape skin polyphenols have higher inhibiting rate s than seed polyphenols probably because the skin is rich in ellagic aicd while the seed is rich in catechin, epicatechin, and gallic acid. Effect of M uscadine G rape P henolic C ompounds on the F ormation of A crylamid e The inhibition by different varietal muscadine grape seed and skin polyphenols on acrylamide formation were higher than 90% at 10 0 g/mL (Figure 5 2). Muscadine grape skin polyphenols (average 98.6%) exhibited higher inhibition than seed polyphenols (average 92.9%), but no t significantly different within the varieties. Contrary to the muscadine grape, showed higher inhibition for the seed polyphenols than skin. Correlation b etween A ntibrowning A ctivity and I nhibiting A crylamide F ormation of P henolic C ompounds The effects of phenolic compounds on color development were evaluated in the asparagine glucose Maillard model . Since visual perception of color is an integration of differen t dimensions of the color space, c olor change is better reflecte d by monitoring parameters that are indicative of the different dimensions, not merely by measuri ng the by a spectrophotometric method. Results from colorimetric analysis showed that the addition of phenolic compounds to the asparag ine glucose model affected all of the three coor dinates of the color space, as reflected by changes in the L * (lightness), a * (redness), and b * ( yellow ness) values. Moreover, the effects were in a concentration dependent manner. In terms of lightness ( L * value) (Figure 5 3A ), when the phe n olics concentration increased from 50 to 200 g/mL, the lightness ( L * value) of the reaction systems

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133 decreased gradually with the except ion of the . The increased in lightness ( L * value) because it contains quercetin which resulted in a n orange solution and affected the analysis. The addition of phenolic compounds did affect the various reaction matrices in view of the differences between control and 50 g /mL treatments (Figure 5 3) . Nevertheless, it the correlation study between color development and concentration. The redness ( a * value) was not significantly affected by the concentratio n of phenolic compounds. T he a * value for Noble ski n and seed polyphenol treatments were higher than the control because they are naturally red in color ( Figure 5 3B). The yellow ness ( b * value) of the reaction syst ems increased gradually with concentration, except for the ment ( Figure 5 3C). Apart from an effect on organoleptic properties, it was recently reported that browning, which is associated with the advanced and final stages of the Maillard reaction, was significantly correlated with the content of acrylamide in foo d models ( 91 ) . Arribas Lorenzo and Morales ( 92 ) found that browning was nearly 2 fold less when pyridoxamine was added to a glucose asparagine model and subjected to heat treatme nt at temperatures ranging from 120 to 180 °C. This antibrowning activity correlated significantly with the effect of pyridoxamine to inhibit acrylamide formation in their study. A similar phenomenon was also found in our study. The inhibition of acrylamid e formation by phenolic compounds showed negative correlation with the lightness ( L * value) (Table 5 1). This indicated that the more potent inhibition of acrylamide by phenolic compounds was accompanied with a higher antibrowning effect in the asparagine glucose models.

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134 Correlation between A ntioxidant A ctivity and I nhibiting A crylamide F ormation of P henolic C ompounds The antioxidant activities of tested phenolic compounds were evaluated and ranked as: Gallic acid (11.1 mmol Trolox /g) > Carlos seed polyphenols (10.9 mmol /g) > Noble seed polyphenols (10.8 mmol /g) > Ellagic acid (9.8 mmol /g) > Quercetin (9.4 mmol /g ) > Phenolic mixture (8.2 mmol /g) > Carlos skin polyphenols (8.1 mmol /g) > Noble skin polyphenols (7.1 mmol /g) > Epicatechin ga llate (6.0 m mol/g) > Epicatechin (5.6 m mol/g) > Catechin (5.3 m mol/g) > Caffeic acid (5.1 m mol/g) (Figure 5 4). However, there was no significant correlation between antioxidant activities of phenolic compounds and acrylamide inhibitio n. This indicates the inhibition m echanism of antioxidants against carcinogenic acrylamide f ormation may need to be further elucidated. Several researchers also investigated the effect of various antioxidants and antioxidative extracts on acrylamide formation, but the data wer e discordant ( 93 97 ) . Some studies claimed mitigation while others showed no effect or even a n increase. It can be a ttributed to the ability of an tio xidants with different structural or functional groups to react with acryl amide precursors, with interme diates of the reaction or with acrylamide itself, leading to either reducing or promoting effect s ( 98 ) . The fact that the same kind of antioxidant, or extract and its representative components behaves differently in differ ent studies might be due to the different reaction conditions among the studi es, concentrations of the anti oxidant, as well as preparative methods of the extract.

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135 Inhibitory M echanism of P henolic C ompounds and P roposed P athwa y To study the inhibitory mechanism of phenolic compounds, a chemical model reaction was introduced (Table 5 2). Resulted showed that only both asparagine and glucose at high temperature could produce acrylamide. E picatechin could inhibit the formation of ac rylamide, and when the concentratio n increased, the inhibition increased (Model E H). Interesting, at high temperature (180 °C ) , epicatechin was found to directly decrease the level of acrylamide (Model K). As expected, the acrylamide level in Model L was lower than the sum total of Model s G and K. The solutions from chemical model reaction G before and after heating were subjected to HPLC MS/MS analysis. Result showed that, after heating, the epicatechin disappeared while several new compounds were produced (Figure 5 5). Figure 5 6 shows a representative MS/MS spectr um registered with the negative ionization mode for one of the m/z 432 analytes together with the proposed fragmentation pathway . It shows one molecular epicatechin ( m/z 289) combined with two molecules acrylamide ( m/z 71 ) . MS/MS data therefore suggest that the adducts derived from the reaction between epicatechin and asparagine (models G ) probably have two acrylamide s substituting at both C 6 and C 8 on the A ring of epicatechin. Totlani et al. ( 99 ) reported that epicatechin (EC) in aque ous glucose glycine model systems functioned as a carbonyl trapping agent of C2, C3, and C4 sugar fragments or key transient precursors of the Maillard reaction (i.e., glyoxal, methylglyoxal, acetol, erythrose, etc.). The authors suggested that EC underwent electrophilic aromatic substitution reactions wi th these carbonyl compounds. This mechanism was further confirmed by NMR analysis of a EC methylglyoxal adduct

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136 product, which indicated that the C 6 or C 8 position of the flavanol (A ring) and the carbonyl bearing carbon of the aldehyde compound were the bonding sites ( 100 ) . EC exerts a trapping effect on sugar fragments or 3 deoxy 2 hex osulose intermediates and consequently inhibits Maillard products (including acrylamide) formation. Muscadine G rape P henolic C ompounds R educing A crylamide in P otato C hip The inhi bition of muscadine grape polyphen ols on acrylamide formation was further inve stigated with a potato chip model. Result s showed that, muscadine grape skin polyphenols significantly reduced the acrylamide level of fried chips (Figure 5 7). When the polyphenols concentration increased from 0 to 0.1%, the acrylamide level decreased from cant inhibition rate of 60.3%. This finding agreed with the report of Zhang et al. ( 32 ) who showed that nearly 74.1% and 76.1% of acryla mide in potato crisps and F rench fries was reduced when antioxidant from bamboo leaves (polyphenols) were died at a ratio of 0.1% and 0.01% (w/w), respectively. Acrylamide T rapping C apability of P henolic C ompounds in S imulated P hysiological C onditions The above studies have confirmed that phen olic compounds could inhibit acrylamide formation in food products . However, complete removal of acrylamide from food remains a challenge for the food industry. Thus , human s are still susceptible to exposure of acrylam ide up on consuming high temperature processed carbohydrate rich foods. In this regard, acrylamide trapping by phenolic compounds at simulated physiological conditions was evaluated. A p revious study showed that , after acrylamide was incubated with niacin (niacin: acrylamide , 20: 1, molar ratio) for 2 days under

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137 simulated physiological conditions, the acrylamide content was reduced by 21%, while incubation for 6 days in the same molar ratio resulted in 71% reduction ( 90 ) . However, in this study, Figure 5 8 showed that the phenolic compounds (epicatechin) exhibited no effect for trapping acrylamide. This indicated that traping acrylamide with epicatechin probably need s the high temperature. Summary Acrylamide has been found to occur in many cooked (fried or roasted) starchy foods and is of concern as a possible carcinogen. This study indicated that muscadine grape polyphenols could significant ly reduce acrylamide format ion in chemical and potato chip model system s . The reduced rates of grape polyphenols were higher than used as a natural food additive to mitigate ac rylamide formation in heat treated commercial starchy foods.

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138 Table 5 1 . Correlation between color development and inhibition of acrylamide formation by phenolic compounds L* a* b* %Inhibition (Ellagic acid) 0.823 0.597 1.000** %Inhibition (Epicatechin) 0.979* 0.838 0.971* %Inhibition (Standards mixture) 0.946* 0.973* 0.783 %Inhibition (Noble skin polyphenols) 0.840 0.978* 0.962* %Inhibition (Noble seed polyphenols) 0.255 0.928* 0.979* *. Correlation is significant at the 0.05 level (2 tailed). **. Correlation is significant at the 0.01 level (2 tailed).

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139 Table 5 2 . Aqueous c hemical m odel r eactions for m echanism s tudy a Reactant Reaction Concentration (mM) A B C D E F G H I J K L Asparagine 0.80 0.80 0.80 0.80 0.80 0.80 0.80 Glucose 0.80 0.80 0.80 0.80 0.80 0.80 0.80 Epicatechin 0.35 0.17 0.35 0.70 0.35 0.35 0.35 0.35 Acrylamide 0.01 0.01 0.01 Produced Acrylamide (µg) 0 0 0 1.76 3.54 2.37 1.74 1.11 0 0 1.04 2.72 a All of the model reactions were carried out in 2.5 mL of water and heated at 180 ± 5 °C for 60 min.

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140 Figure 5 1 . Effect of different concentrations of phenolic compounds on the formation of acrylamide in chemical models containing 0.8 mM each of asparagine and glucose in 2.5 mL of water and heated at 180 ± 5 °C for 60 min. 0 20 40 60 80 100 0 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL %Inhibition of Acrylamide Formation Concentration (µg/mL) Caffeic acid Ellagic acid Gallic acid Catechin Epicatechin Epicatechin gallate Quercetin Mixed Std. Carlos seed polyphenols Carlos skin polyphenols Noble seed polyphenols Noble skin polyphenols

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141 Figure 5 2 . Effect of different varietal muscadine seed and skin phenolic compounds (100 µg/mL) on the formation of acrylamide in chemical models containing 0.8 mM each of asparagine and glucose in 2.5 mL of water and heated at 180 ± 5 °C for 60 min. 89.2 92.8 95.9 90.1 95.5 94.6 91.0 91.0 93.7 95.5 92.9 95.7 97.3 96.4 99.3 99.1 98.4 98.2 99.2 100.0 98.9 99.1 98.6 93.8 80 85 90 95 100 105 Alachua seed Carlos seed Doreen seed Fry seed Grany Val seed Ison seed Pam seed Supereme seed Majesty seed Noble seed Average-Seed Table red seed-control Alachua skin Carlos skin Doreen skin Fry skin Grany Val skin Ison skin Pam skin Supereme skin Majesty skin Noble skin Average-Skin Table red skin-control %Inhibition of Acrylamide Formation

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142 Figure 5 3 . Concentration study on the effect of phenolic compounds on color development in acrylamide producing chemical models. (A) Changes in L * value, (B) changes in a * value, and (C) changes in b * value. 80 85 90 0 50 100 200 L* Value Concentration (µg/mL) (A) Epicatechin Ellagic acid Mixed standards Noble seed Noble skin 0 5 10 0 50 100 200 a* Value Concentration (µg/mL) (B) Epicatechin Ellagic acid Mixed standards Noble seed Noble skin 15 20 25 0 50 100 200 b* Value Concentration (µg/mL) (C) Epicatechin Ellagic acid Mixed standards Noble seed Noble skin

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143 Figure 5 4 . Correlation analysis between antioxi dant activity (DPPH method) and inhibition of acrylamide by phenolic compounds at 50 µg/mL. y = 0.0111x + 7.6105 R² = 0.0228 0 2 4 6 8 10 12 0 20 40 60 80 100 Antioixdant Activity (mmol/g) %Inhibition of Acrylamide Formation

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144 Figure 5 5 . HPLC chromatograms (280 nm) of chemical model reaction G (0.8 mM asparagine + 0.8 mM glucose + 0.35 mM epicatechin) before heating and heated at 180 ± 5 °C for 60 min.

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145 Figure 5 6 . MS/MS spectrum demonstrating the possible ex istence of epicatechin acrylamide adduct (m/z 432) and proposed pathway for the scavenging of amide source to inhibit acrylamide formation by epicatechin.

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146 Figure 5 7 . Relationship between acrylamide levels and different immersion solution s of muscadine (Carlos skin) polyphenols for chips before frying (immersion time: 60 min, n = 6). Acrylamide levels were quantified with GC and standards. Error bars designate standard deviation (SD) and different letters designate signif icant differences via P < 0.05). a b c d 0 500 1000 1500 2000 2500 Control 0.025% 0.050% 0.100% Acrylamide level (µg/kg) Concentration of Muscadine Polyphenols Solution

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147 Figure 5 8 . Activity of epicatechin in direct trapping of acrylamide in simulated physiological conditions. 0 0.4 0.8 1.2 1.6 Control 1 5 10 15 Acrylamide Concentration (µg/mL) Epicatechin: Acrylmide (mol) Day 1 Day 2

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148 CHAPTER 6 CONCLUSIONS Fruit quality and nutraceutical properties were established for muscadine grapes ( Vitis rotundifolia Michx.) by analyzing 58 varieties over 2 seasons. Fruit quality (color, weight, size, pH, titratable acidity, soluble solids content, °Brix/acid ratio), phenolic compound content and profiles , and antioxidant activities wer e significantly different among the 58 cultivars. This data could be developed into a muscadine database, which would be very useful to growers and processors; allowing them to make decisions on which cultivars offer the quality for processing while potentially adding value to a waste stream by producing functional ingredients. The enzymatic extraction study showed that pre treated muscadine skin and seeds with enzymes decreased total phenolic yi eld compared with solvent (50% ethanol) alone. Enzyme release of phenolics from skins of different muscadine varieties was significantly different while release from seeds was similar. Enzyme hydrolysis was found to shorten extraction time. Most importantl y, enzyme hydrolysis modified the galloylated form of polyphenols to low molecular weight phenolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity. Therefore, the e nzymatic method could provide a better way to extrac t polyphenols for the food industry, which not only is environmentally friendly, but also could increase the amount of bioactive phenolics available. In the antimicrobial study, result showed that antioxidant activity for different polyphenol s varied grea tly ranging from 5 .0 11.1 m mol Trolox/g. Antioxidant and antibacterial activity for polyphenols showed a positive correlation. Muscadine polyphenols exhibited a broad spectrum of antibacterial activity against tested

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149 foodborne pathogens. Muscadine polyphen ols at 4 × MIC caused nearly a 5 log 10 CFU/mL drop in cell viability for S. aureus in 6 h with lysis, while at 0.5 x MIC, they inhibited its biofilm formation, and at 16 × MIC, they eradicated biofilms. Muscadine polyphenols showed synergy with antibiotics and maximally caused a 6.2 log 10 CFU/mL drop in cell viability at sub inhibitory concentration. Antimicrobial and antibiofilm products made from muscadine by product show promise as natural food preservatives, potential antibiotic replacements and as natu ral sanitizers for processing equipment where bacteria and biofilm re side. Acrylamide has been found to occur in many cooked starchy foods and is of concern as a possible carcinogen. This study indicated that muscadine grape polyphenols could significantl y reduce acrylamide formation in chemical and potato chip model system s . The reduced rates of grape polyphenols were higher than 90% at 100 natural food additive to mitigate acr ylamide formation in heat treated commercial starchy foods. Overall, this project established important information on fruit quality and nutraceutical properties for muscadine grapes and illustrated several potential utilization s of grape pomace for value added products . This is a good example of the technology transfer between re search and industry application, with potential benefit to grape growers, processors and the public.

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150 LIST OF REFERENCES 1. Andersen, P. C.; Crocker, T. E.; Breman, J., The muscadine grape. University of Florida, IFAS Extension 2010, HS763 , 1 18. 2. Olien, W. C.; Hegwood, C. P., Muscadine a classic southeastern fruit. HortScience 1990, 25 , 726 831. 3. Talcott, S. T.; Lee, J. H., Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. Journal of Agricultural and Food Chemistry 2002, 50 , 3186 3192. 4. Flora, L., Influence of heat, cultivar and maturity on the anthocyanidin 3,5 diglucos ides of muscadine grapes. Journal of Food Science 1978, 43 , 1819 1821. 5. Sandhu, A. K.; Gu, L., Antioxidant capacity, phenolic content, and profiling of phenolic compounds in the seeds, skin, and pulp of Vitis rotundifolia (muscadine grapes) as determined by HPLC DAD ESI MS n. Journal of agricultural and food chemistry 2010, 58 , 4681 4692. 6. Lecas, M.; Brillouet, J. M., Cell wall composition of grape berry skins. Phytochemistry 1994, 35 , 1241 1243. 7. Pinelo, M.; Arnous, A.; Meyer, A. S., Upgrading of gra pe skins: Significance of plant cell wall structural components and extraction techniques for phenol release. Trends in food science & technology 2006, 17 , 579 590. 8. Kammerer, D.; Claus, A.; Schieber, A.; Carle, R., A novel process for the recovery of po lyphenols from grape ( Vitis vinifera L.) pomace. Journal of Food Science 2005, 70 , C157 C163. 9. Haight, K. G.; Gump, B. H., The use of macerating enzymes in grape juice processing. American journal of enology and viticulture 1994, 45 , 113 116. 10. WIGHTMA N, J. D.; WROLSTA D, R. E., glucosidase activity in juice processing enzymes based on anthocyanin analysis. Journal of food science 1996, 61 , 544 548. 11. Wrolstad, R. E.; Wightman, J. D.; Durst, R. W., Glycosidase activity of enzyme preparations used in fruit juice processing. Food technology 1994, 48 , 90 98. 12. Chamorro, S.; Viveros, A.; Alvarez, I.; Vega, E.; Brenes, A., Changes in polyphenol and polysac charide content of grape seed extract and grape pomace after enzymatic treatment. Food Chemistry 2012, 133 , 308 314.

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159 BIOGRAPHICAL SKETCH Changmou Xu was born in Jiangxi, China. He received his B.S. degree in food science and engineering at Jiangxi Agricultural University in 2007, and enrolled into the rsity. He had three publ ications as the first author from his m ng with a M.S. degree with highest honors from China Agricultural University in 2010, he entered the food s cience doctoral program at the University of Florida under the supervision of Dr. Maurice R. Marshall, and with a minor in food and resource economics . During his doctoral period, Changmou published two papers as the first author. Upon his completion of the Ph. D. degree in August 2014 , Changmou plans to continue his research as a post doc at University of Florida and looks forward to any challenges that life will bring .