Postharvest Storage and Nutraceutical Evaluation of Muscadine Grapes (Vitis rotundifolia Michx.)

Transcription

1 University of Arkansas, Fayetteville Theses and Dissertations Postharvest Storage and Nutraceutical Evaluation of Muscadine Grapes (Vitis rotundifolia Michx.) Derek Barchenger University of Arkansas, Fayetteville Follow this and additional works at: Part of the Horticulture Commons Recommended Citation Barchenger, Derek, "Postharvest Storage and Nutraceutical Evaluation of Muscadine Grapes (Vitis rotundifolia Michx.)" (2014). Theses and Dissertations This Thesis is brought to you for free and open access by It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of For more information, please contact

2 Postharvest Storage and Nutraceutical Evaluation of Muscadine Grapes (Vitis rotundifolia Michx.)

3 Postharvest Storage and Nutraceutical Evaluation of Muscadine Grapes (Vitis rotundifolia Michx.) A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Horticulture By Derek W. Barchenger Oklahoma State University Bachelor of Science in Agricultural Sciences and Natural Resources in Horticulture, 2012 May 2014 University of Arkansas This thesis is approved for recommendation to the Graduate Council. Dr. John R. Clark Thesis Director Dr. Renee T. Threlfall Committee Member Dr. Luke R. Howard Committee Member Dr. M. Elena Garcia Committee Member

4 ABSTRACT A major limiting factor in muscadine grape (Vitis rotundifolia Michx.) commercialization is deterioration during storage. One solution for extending market seasons and preventing market saturation for fresh muscadines could be the release of new cultivars with improved postharvest storability. Three studies were conducted; Study 1: The effect of fungicide treatments on muscadine genotype postharvest storage and nutraceutical content; Study 2: An evaluation of a diverse range of genotypes for postharvest storage potential and nutraceutical concentrations; and Study 3: The impact of postharvest storage and berry segment on the nutraceutical composition of Supreme. Research on table grapes has shown that field fungicide applications increase storability, but little is known of their effect on muscadines. The effect of field applications of fungicides on composition attributes during postharvest storage was evaluated on five muscadine cultivars (Nesbitt, Southern Jewel, Summit, Supreme, and Tara) and five breeding selections from the University of Arkansas Fruit Breeding Program. There were two field treatments (no fungicide and fungicide). For the fungicide treatment, alternating applications of two fungicides were applied at 14 d intervals during berry development. Fruit was harvested and composition attributes including berry volume, titratable acidity (TA), ph, soluble solids (%), soluble solids to titratable acidity ratio (SS/TA), color (L*, Chroma, and hue), firmness (force to penetrate berry skins), weight loss (%), and unmarketable fruit (%) were evaluated every 7 d for three weeks. An additional eight cultivars and selections from non-fungicide-treated vines were subjected to postharvest storage potential evaluations. These included AM 02, AM 03, AM 18, AM 26, AM 28, Delicious, Fry, and Ison, as well as the genotypes from the non-fungicide-sprayed vines

5 of genotypes in Study 1 (AM 01, AM 04, AM 15, AM 27, Nesbitt, Southern Jewel, Summit, Supreme, and Tara ) to broaden the evaluation and comparison among genotypes for postharvest storage potential, and aid in the development of an Arkansas muscadine storage protocol. Additionally, a third study was conducted with Supreme to determine the retention of nutraceutical compounds during storage on the berry segments of flesh (pulp and skin), seeds, and whole berries and to evaluate the variability in nutraceutical compounds among different vine of a single genotype. The storage attributes of force to penetrate the berry skin, percent weight loss, and percent unmarketable were all improved with fungicide applications, while the difference in percent volume change was minimal. Field fungicide applications had no effect on muscadine berry color (Chroma, hue angle, and L*) or berry composition (ph, TA, SS/TA, and soluble solids). There were some effects of field fungicide applications impacted some nutraceutical levels, however results varied. Weight loss, percent unmarketable, and force have potential for routine measurements in postharvest evaluation of muscadines, while percent volume change was found to be less useful. Titratable acidity, ph, soluble solids, and SS/TA can be useful measurement for ripeness, but not for storability. Chroma and hue angle are not indicators of storability, while L* shows potential, and nutraceuticals largely do not change during storage. The genotypes AM 04, AM 26, AM 27, AM 28, Southern Jewel, and Supreme were identified as having the highest storage potential, while AM 01, AM 15, AM 18, and Tara had the least storage potential. The genotypes AM 03, AM 04, AM 27, and Ison were identified as having the highest overall nutraceutical content, while AM 18, AM 28, Supreme, and Tara had the lowest overall nutraceutical content. Furthermore, muscadine grape nutraceuticals and antioxidant capacity vary by berry segment and vine. The primary contributors as sources

6 of variation were found to be genotype, year, and storage time.

7 ACKNOWLEDGEMENTS Many thanks are necessary to the people who have contributed towards the success of this project, beginning with my advisor Dr. John R. Clark. His advice, guidance, and friendship are invaluable to my education and experience. It was an honor to work underneath him. I would also like to thank the members of my committee, Dr. Renee T. Threlfall for her attentive guidance with my project, as well as Dr. Luke R. Howard, and Dr. M. Elana Garcia for their time, support, and suggestions offered. Furthermore, this work would not have been possible without the use of Dr. Luke R. Howard and Dr. Renee T. Threlfall s laboratories. I would also like to thank Dr. Ed Gbur for the statistical aid and guidance. Also, I would like to thank Sandra Sleezer for her assistance in all aspects of this project, and Cindi Brownmiller for her guidance with my nutraceutical and antioxidant capacity measurements. I greatly appreciate the assistance offered by the staff of the Fruit Research Station in Clarksville. Particular thanks need to be given to Jeff Iness and Dan Chapman for the fungicide application support and to David Gilmore and Kay Buck for the invaluable education and experience I gained working with them.

8 TABLE OF CONTENTS Introduction 1 Objectives 4 Hypothesis 5 Literature Review 5 Genotypic Effects on Storage and Composition 5 Postharvest Physiology 9 General Storage 10 Storage Effects of Sulfur Dioxide 12 Controlled Atmosphere Storage 13 Fungicide Treatments 14 Berry Composition 17 Berry Segments 18 Total Phenolics 20 Anthocyanins 22 Ellagitannins 24 Flavonols 25 Resveratrol 26 Oxygen Radical Absorbance Capacity 29 Literature Cited 30 Chapter 1: The Effect Of Field Fungicide Applications On Muscadine Genotype Postharvest Storage And Nutraceutical Content 38 Abstract 38 Introduction 40 Materials and Methods 42 Grapes and Vineyard 43 Fungicide Applications 43 Harvest and Transport 44 Storage Study 44 Berry and Composition Analysis 45 Nutraceutical Analysis 47 Experimental Design 50 Experimental Analysis 51 Results 51 Initial Attributes 51 Berry Storage Attributes 53 Berry Composition 62 Berry Color 74 Nutraceutical Content 80 Correlations 89 Discussion 91 Berry Storage Attributes 91 Berry Composition 95 Berry Color 98 Nutraceutical Content 100

9 Storage Protocol 106 Literature Cited 107 Appendix A 114 Chapter 2: Evaluation Of A Diverse Range Of Muscadine Grape Genotypes For Postharvest Storage Potential And Nutraceutical Concentrations 130 Abstract 130 Introduction 132 Materials and Methods 134 Grapes and Vineyard 134 Harvest and Transport 135 Storage Study 135 Berry and Composition Analysis 136 Nutraceutical Analysis 138 Experimental Design 141 Experimental Analysis 141 Results 142 Initial Attributes 142 Berry Storage Attributes 142 Berry Composition 149 Berry Color 156 Nutraceutical Content 158 Bronze and Black Storage Performance 169 Correlations 169 Discussion 171 Berry Storage Attributes 171 Berry Composition 177 Berry Color 180 Nutraceutical Content 183 Bronze and Black Storage Performance 188 Storage Protocol 188 Literature Cited 190 Appendix A 197 Chapter 3: The Effect Of Storage Time On Nutraceutical Content Of Supreme Muscadine Berry Segments 212 Abstract 212 Introduction 214 Materials and Methods 219 Grapes and Vineyard 219 Harvest and Transport 219 Composition Analysis 220 Postharvest Storage 220 Nutraceutical Analysis 221 Experimental Design 224 Experimental Analysis 225 Results 225 Initial Attributes 225

10 2012 Nutraceutical Analysis 225 Combined Nutraceutical Analysis 237 Correlations 246 Discussion 246 Literature Cited 252 Appendix A 258 Conclusions 263

11 LIST OF TABLES Chapter 1 Table 1.1. Initial values for force to penetrate berry skin, titratable acidity, ph, soluble solids, L*, Chroma, and hue angle averaged across year and fungicide treatment. 52 Table 1.2. F-test significance from ANOVA for berry weight loss, percent unmarketable berries, force required to penetrate the berry skin, and percent volume change of the berry during 3 weeks of storage. Highest-order interactions are italicized and shaded. 54 Table 1.3. F-test main effect significance for year on weight loss, percent unmarketable, force required to penetrate the berry skin, and percent volume change of the berry during three weeks of storage, averages across week and fungicide. 57 Table 1.4. F-test significance from ANOVA for ph, percent titratable acidity, percent soluble solids, and SS/TA ratio for four sources of variation of muscadine grapes during 3 weeks of storage. Highest-order interactions are italicized and shaded. 66 Table 1.5. Soluble solids to titratable acidity ratio for muscadine genotypes. 72 Table 1.6. Soluble solids to titratable acidity ratio of muscadine genotypes stored at 2 C for 3 weeks. 72 Table 1.7. Soluble solids to titratable acidity ratio of fungicide and no fungicide treatments. 75 Table 1.8 F-test significance from ANOVA for berry Chroma, hue, and L* values of the four sources of variation of muscadine grapes during 3 weeks of storage. Highest-order interactions are italicized and shaded. 75 Table 1.9. L* values of fungicide and no fungicide treatments during storage at 2 C for 3 weeks 76 Table 1.10 F-test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and resveratrol concentrations for the three sources of variation. Highest-order interactions are italicized. 82 Table Total phenolic concentations (mg/ 100g) of fungicide- and no fungicidetreated musadines. 88 Table Resveratrol concentations (mg/100 g) of fungicide- and no fungicidetreated musadines. 88 Table Total ellagitannin concentations of fungicide- and no fungicide-treated musadines. 90 Appendix A. Table A.1. Daily maximum and minimum temperatures and rainfall recorded at the Fruit Research Station; Clarksville, AR (lat N and long W) (2012 and 2013). 114 Table A.2. Interaction means of the postharvest attributes of percent weight loss, percent unmarketable, force to penetrate berry skin, and percent volume change for year, genotype, fungicide treatment, and week of storage at 2 C for 0-3 weeks. 115 Table A.3. Interaction means of the composition attributes of percent titratable acidity, percent soluble solids, ph, and soluble solids to titratable acidity of fungicide and no fungicide treated muscadine genotypes stored at 2 C for 0-3 weeks. 119 Table A.4. Interaction means of the berry color attributes of Chroma, Hue angle, and L* values of fungicide and no fungicide treated muscadine genotypes stored at 2 C for 0-3 weeks. 123

12 Table A.5. Interaction means of the berry nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) of fungicide and no fungicide treated muscadine genotypes. 127 Table A.6. Study 1 multivariate correlation coefficients among muscadine berry storage quality, composition, color, and nutraceutical content for 2012 and Chapter 2 Table 2.1. Initial mean values for force to penetrate berry skin, composition and berry color for muscadine genotypes averaged across year (2012 and 2013). 143 Table 2.2. F-test significance from ANOVA for muscadine berry weight loss, percent unmarketable berries, force required to penetrate the berry skin, and percent volume change of the berry during 3 weeks of storage at 2 C. Highest-order interactions are italicized and shaded (2012 and 2013). 143 Table 2.3. F-test significance from ANOVA for berry ph, titratable acidity, soluble solid content, and soluble solids to titratable acidity ratio during 3 weeks of storage. Highestorder interactions are italicized and shaded (2012 and 2013). 151 Table 2.4. F-test significance from ANOVA for Chroma, hue angle, and L* values during 3 weeks of storage at 2 C. Highest-order interactions are italicized and shaded (2012 and 2013). 157 Table 2.5. F-test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and resveratrol concentrations during. Highestorder interactions are italicized and shaded (2012 and 2013). 161 Table 2.6 Main effect of muscadine genotype means on total ellagitannin concentrations of the berry during three weeks of storage, averaged across years (2012 and 2013). 163 Table 2.7. Main effect of year means on total ellagitannin concentrations of the muscadine, averaged across genotypes (2012 and 2013). 164 Table 2.8. Main effect of genotype on total phenolic concentrations of the muscadine berries, averaged across years (2012 and 2013). 167 Table 2.9. F-test main effect significance for year on total phenolic concentrations averaged across muscadine genotypes (2012 and 2013). 167 Appendix A Table A.1. Average monthly maximum and minimum temperatures and total rainfall recorded at the Fruit Research Station; Clarksville, AR (lat N and long W) (2012 and 2013). 197 Table A.7. Interaction means of the postharvest attributes of percent weight loss, percent unmarketable, force to penetrate berry skin, and percent volume change for 198 year, genotype, and week of storage at 2-3 C for 0-3 weeks. Table A.8. Interaction means of the composition attributes of percent titratable acidity, percent soluble solids, ph, and soluble solids to titratable acidity for year, genotype, and week of storage at 2 C for 0-3 weeks. 202 Table A.9. Interaction means of the berry color attributes of Chroma, Hue angle, and L* values for year, genotype, and week of storage at 2 C for 0-3 weeks. 206 Table A.10. Interaction means of the berry nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) for year and genotypes. 210

13 Table A.11. Study 2 multivariate correlation coefficients among muscadine berry storage quality, composition, color, and nutraceutical content for 2012 and Chapter 3 Table 3.1. The initial nutraceutical concentrations of Supreme muscadine berry segments averaged across vines for 2012 and Table 3.2. F-test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and trans-resveratrol concentrations of Supreme muscadine berries stored for 6 weeks at 2 C in Highest-order interactions are italicized and shaded. 228 Table 3.3. F-test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and trans-resveratrol concentrations of Supreme muscadines at harvest in 2012 and Highest-order interactions are italicized and shaded. 238 Table 3.4. Main effects of year, muscadine berry segment, and vine means on total ellagitannin concentrations of Supreme berry segments (2012 and 2013). 241 Table 3.5. Two-way interactions of year by vine, year by muscadine berry segment, and segment by vine means on ORAC levels of Supreme (2012 and 2013). 241 Table 3.6. Multivariate correlation coefficients among nutraceutical concentrations of Supreme muscadines at harvest averaged across years (2012 and 2013). 247 Appendix A. Table A.1. Daily maximum and minimum temperatures and rainfall recorded at the Fruit Research Station; Clarksville, AR (lat N and long W) (2012 and 2013). 258 Table A.12. Interaction means of the berry segment nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) for year and week of storage at 2 C for 6 weeks in Table A.13. Interaction means of the berry segment nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) for year at harvest. 262

14 LIST OF FIGURES Chapter 1 Fig Percent berry weight loss of fungicide- and no fungicide-treated muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean. 55 Fig Percent unmarketable fungicide and no fungicide-treated berries of muscadine genotypes stored at 2 C for 3 weeks Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean. 58 Fig Force to penetrate skin of muscadine genotypes stored at 2 C for 3 weeks averaged across fungicide treatment. Each standard error bar is constructed using 1 standard error from the mean. 60 Fig Force to penetrate berry skin of fungicide- and no fungicide-treated muscadine genotypes average across weeks. Each standard error bar is constructed using 1 standard error from the mean. 61 Fig Percent change in volume of muscadine genotypes stored at 2 C for 3 weeks averaged across fungicide treatment. Values at week 0 (date of harvest) were excluded. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean. 63 Fig Percent change in volume of muscadine genotypes with fungicide and no fungicide treatments. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean. 64 Fig Percent titratable acidity of fungicide- and no fungicide-treated muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. Titratable acidity measured as tartaric acid. 67 Fig ph of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. 68 Fig ph of fungicide and no fungicide-treated muscadine genotypes Each standard error bar is constructed using 1 standard error from the mean. 68 Fig Percent soluble solids of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. 70 Fig Percent soluble solids of fungicide- and no fungicide-applied muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 71 Fig L* values of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. 76 Fig L* values of fungicide and no fungicide-treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 78 Fig Chroma of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. 79 Fig Hue angle of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. 81 Fig Total anthocyanin content of fungicide- and no fungicide-treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the

15 mean. 83 Fig Oxygen radical absorbance capacity of fungicide- and no fungicide-treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 85 Fig Total flavonol concentrations of fungicide and no fungicide-treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 86 Fig Total ellagitannin concentration of fungicide- and no fungicide-treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 90 Chapter 2 Fig Percent berry weight loss of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 145 Fig Percent unmarketable of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 146 Fig Force to penetrate skin of muscadine genotypes stored at 2 C for 3 weeks Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 148 Fig Percent change in volume of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 149 Fig ph of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 151 Fig Titratable acidity (%) of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 152 Fig Soluble solids (%) of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 154 Fig Soluble solids to titratable acidity ratio of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 155 Fig L* values of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 157 Fig Hue angle of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 159 Fig Chroma of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 160 Fig Total anthocyanin concentrations of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 162 Fig Total flavonol concentrations of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 165 Fig Resveratrol concentrations of muscadine genotypes. Each standard error bar

16 is constructed using 1 standard error from the mean (2012 and 2013). 168 Fig Oxygen radical absorbance capacity (ORAC) of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and ). Fig Storage performance of black and bronze muscadine genotypes, averaged across year (2012 and 2013) stored for 3 weeks at 2 C. Each standard error bar is constructed using 1 standard error from the mean. 170 Fig Muscadine genotype AM 01 at date of harvest (left) and after 3 weeks of storage (right), demonstrating the browning that occurred, likely caused by chilling injury (2013). 172 Fig Muscadine genotype AM 04 after 3 weeks of storage, demonstrating shriveling that occurred, likely caused by berry leakage (2012). 175 Chapter 3 Fig Total anthocyanin concentrations of Supreme muscadine berry segments stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 229 Fig Total ellagitannin concentrations of Supreme muscadine berry segments stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 230 Fig Oxygen radical absorbance capacity (ORAC) of Supreme muscadine berry segments stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 232 Fig Total flavonols of Supreme muscadine berry segments from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. 233 Fig Total flavonols of Supreme muscadine berry segments stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 233 Fig Total phenolics of Supreme muscadine berries stored at 2 C for 6 weeks, averaged across berry segment in Each standard error bar is constructed using 1 standard error from the mean. 235 Fig Total phenolics of Supreme berry muscadine segments from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. 235 Fig Resveratrol concentrations of Supreme muscadine berries from three different vines of Supreme stored at 2 C for 6 weeks in Values are averaged across berry segments. Each standard error bar is constructed using 1 standard error from the mean. 235 Fig Resveratrol concentrations of Supreme muscadine berry segments from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. 236 Fig Resveratrol concentrations of Supreme muscadine berry segments stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 236 Fig Total anthocyanin concentrations of Supreme muscadine berry segments at harvest in 2012 and Each standard error bar is constructed using 1 standard

17 error from the mean. 239 Fig Total ellagitannin concentrations of Supreme muscadine berry segments at harvest averaged across year (2012 and 2013). Each standard error bar is constructed using 1 standard error from the mean. 239 Fig Total flavonol concentrations of Supreme muscadine berry segments at harvest in 2012 and Each standard error bar is constructed using 1 standard error from the mean. 242 Fig Total phenolics of Supreme muscadine berry segments at harvest in 2012 and Each standard error bar is constructed using 1 standard error from the mean. 244 Fig Resveratrol concentrations of Supreme muscadine berry segments at harvest in 2012 and Each standard error bar is constructed using 1 standard error from the mean. 245

18 Introduction Native to the southeastern United States, the muscadine (Vitis rotundifolia Michx.) grape is commonly grown for its unique flavor, high nutraceutical content, and insect and disease resistance, which is often a limiting factor in the production of many bunch grapes (Vitis spp.) (Conner, 2009; Silvia et al., 1994; Striegler et al., 2005; Walker et al., 2001). Differentiation within Vitis between the subgenera Euvitis and Muscadina is thought to have occurred during the Mesozoic Era, prior to and during the breakup of Pangea (Olien, 1990; Olien, 2001). It is commonly believed that V. vinifera L. and other grape species descended from muscadines during the Quaternary Ice Ages, resulting in one less chromosome pair (2x = 2n = 40 for V. rotundifolia and 2x = 2n = 38 for V. vinifera) (Conner, 2009; Olien, 2001; Olien, 1990). Andre Michaux initially taxonomically described V. rotundifolia in France in 1803, from samples sent to him from the United States, and John Kunkel Small later described muscadines in 1913 (Olien, 2001). Muscadines have been under cultivation for over 400 years, originally in the North Carolina Colony followed soon after by surrounding colonies and states (Conner, 2009). Dr. Calvin Jones released the first cultivar, Scuppernong, in 1810, which was the dominant cultivar in production for over 100 years (Goldy, 1992; Olien 2001). As a result of this popularity, bronze muscadines are often mistakenly classified as scuppernongs, while dark colored muscadines are referred to as muscadines, bull grapes, bullet grapes, Southern fox grapes, or bullace grapes. Breeding efforts with muscadines began in the late 1800s and continued through the late 20 th century (Goldly, 1992; Olien 2001). However, muscadine breeding has been limited since the 1980s due to breeding program reductions, and it is thought that there is 1

19 substantial potential for further improvements of muscadines as a fresh fruit in a modern breeding program (J. R. Clark, personal communication). Muscadines have been described as having a pleasant aroma and taste and are prized by some as a delicacy (Degner and Mathis, 1980). This native grape is commonly grown in small vineyards and home plantings, ranging from North Carolina and Florida to Eastern Oklahoma and Texas. Though commercial shipping for retail marketing is practiced, the potential for expansion in this area exists (Perkins Veazie et al., 2012). Approximately 2023 ha of muscadines are planted across the United States, with Arkansas having approximately 202 ha of muscadines currently in production (Perkins Veazie et al., 2012). The berries are produced in small loose clusters that do not ripen evenly, but are removed from the vines as single berries rather than in clusters as with bunch grapes and are subsequently marketed in packages of these berries (Perkins Veazie et al., 2012). The fruit readily abscises from the rachis when ripe, lending itself to mechanical harvesting (Ballinger and Nesbitt, 1982a; Smit et al., 1971). Muscadine fruit range in color from greenish and bronze through pink, purple, and black. Muscadines have historically been commonly grown for wine, jelly, and juice, but more recently an increase in production for the fresh market has occurred (Perkins Veazie et al., 2012; Striegler et al., 2005). The recent recognition that the berries are important sources of essential vitamins, minerals, and antioxidants has increased consumer demand (Perkins Veazie et al., 2012; Striegler et al., 2005). Additionally, alternative crops, including muscadines, are being explored by many growers in the South as a means of increasing profits or diversifying farm operations (Conner, 2009). Three of the major limiting factors on fresh market production are uneven ripening, short harvest season, and 2

20 high perishability of the fruit (James et al., 1999; Morris, 1980; Perkins Veazie et al., 2012). The harvest season for muscadines in Arkansas usually extends from mid August through September (James et al., 1997; Perkins Veazie et al., 2012). One solution for extending the market season and to prevent market saturation for fresh muscadines is the development of new cultivars that maintain high quality appearance and taste, as well as high nutraceutical content during long periods of storage (Himelrick, 2003; James et al., 1997; James et al., 1999; Lane and Flora, 1980; Lutz, 1938; Morris, 1980; Perkins Veazie et al., 2012; Starnes Saunders et al., 1981; Smit et al., 1971; Takeda et al., 1983a; Walker, 2001). The University of Arkansas Division of Agriculture began its fruit breeding program in 1964 (J. R. Clark personal communication). This effort, founded and directed for many years by Dr. James N. Moore, has been one of the most productive fruit breeding efforts in the United States in recent years. Over 50 fruit cultivars have been commercialized from the program, including blackberries, table grapes, peaches, nectarines, strawberries, and blueberries. These Arkansas cultivars provide growers with the opportunity for increased profits. The muscadine breeding program at the University of Arkansas was first put into motion in 2005, by collecting open pollinated seeds from several cultivars. The first crosses were made among cultivars in Crossing has continued through The primary location of the breeding effort is the Division s Fruit Research Station near Clarksville, with integral testing at the Southwest Research and Extension Center near Hope. To date in Clarksville, 117 selections have been made and approximately 10,000 seedlings planted (J. R. Clark personal communication.). An expanded number of selections will be made in the coming years, which will result in cultivars released for commercial production. The forthcoming developments will provide expanded options for muscadine 3

21 growers. The major objectives of the Arkansas muscadine breeding program are to develop large fruit size, crisp texture, excellent flavor, edible skin, dry stem scar, selffruitful flowers, high productivity, improved winter hardiness, disease resistance, and improved postharvest storability (J. R. Clark personal communication). Since the implementation of a muscadine breeding program at the University of Arkansas, selections have been made based on improved texture and dry stem scar. Although increased crispiness and a greater percentage of dry stem scars has been observed, it is unknown whether there has been a true improvement in postharvest quality of muscadines (J. R. Clark, personal communication). Nutraceutical levels in muscadines vary among genotypes and little is known about the nutraceutical content of the University of Arkansas breeding selections (J. R. Clark, personal communication; Marshall et al., 2012). Additionally, little is known about the affect of field fungicide applications on storability and nutraceutical content of muscadines, and if nutraceutical levels, in whole berries and berry segments (seed and flesh plus skin), change during storage. Objectives The objectives of the following experiments were as follows: 1. To determine the effect of field applications of fungicides on the storage performance and nutraceutical content of muscadine grapes. 2. Identify superior post harvest storage and handling muscadine genotypes in the breeding program. 3. To determine the effect of storage time on nutraceutical content of berry flesh and seed segments of Supreme muscadine. 4

22 4. To develop a postharvest evaluation protocol for Arkansas muscadine genotypes for potential commercial utilization. Hypotheses 1. Field fungicide applications will increase postharvest storage quality and decrease nutraceutical content and antioxidant capacity of muscadine grapes. 2. Recently developed Arkansas genotypes with improved skin and flesh texture will store better than other commercially grown cultivars. 3. Nutraceutical content and antioxidant capacity will be unaffected by storage time. 4. The greatest nutraceutical content will be found in the muscadine berry seeds and the least will be found in the berry flesh. Literature Review Genotypic Effects on Storage and Composition Studies have been conducted on the genotypic interaction on both muscadine storability and berry composition. Ballinger and Nesbitt (1982b) studied the quality of the black genotypes Nesbitt, Noble, NC 67A015 15, NC 67A015 18, NC 67A015 26, NC 67A015 27, NC 67A015 35, and the bronze genotypes Carlos and NC 67A after storage with sulfur dioxide (SO2) generators. The authors used three types of commercially available SO2 generating sheets, a quick release, single stage type, a slow release, single stage type, and a combination of the above two units. The berries were stored for 7 weeks at 0 C with 70 to 80% relative humidity (RH). They found that Carlos, Noble, NC 67A015 26, NC 67A015 35, and, NC 67A were not suited for long term storage for fresh marketing with SO2 generators because of their high susceptibility to SO2 injury including unacceptable appearance, excessive decay, and off flavors. The cultivar Nesbitt 5

23 and the selections NC 67A015 15, NC 67A015 18, and NC 67A were found to be acceptable for long term storage with SO2 generators. It was determined that NC 67A was best suited for long term storage with SO2 because of low susceptibility to injury and flavor was not adversely affected. James et al. (1997) studied the shelf life of Granny Val and Fry for fresh market use. The fruit were stored using three treatments including use of SO2 generators enclosed in a polyethylene bag, berries enclosed in a polyethylene bag with no SO2 generator, and a control without a bag at 0 C with slow release SO2 generators. The fruit was evaluated at 0, 4, and 6 weeks for changes in mass (largely due to moisture loss), percent decay, ph, titratable acidity (TA), and Brix (soluble solids content or SSC). They found that ph, TA, and SSC remained constant throughout storage. Granny Val showed signs of shriveling after 2 weeks of storage, and Fry maintained acceptable fruit quality for 4 weeks. Lamikanra (1987) evaluated the protein content of the cultivars Carlos, Welder, and Higgins, comparing his findings to the protein levels of V. vinifera cultivars. He found that Welder grape juice had a higher protein concentration than that of Carlos or Higgins juices, but all three cultivars yielded juice with relatively higher concentrations of protein than all V. vinifera juices evaluated. Magee et al. (2002) determined the effect of field fungicide application on resveratrol content of fruit of muscadines that were susceptible (S), intermediate (I), or resistant (R) to five of the major berry diseases (bitter rot [Greeneria uvicola Ellis], black rot [Guignardia bidwellii Ellis], angular leaf spot [Mycosphaerella angulata Jenkins], and Pierce s disease [(Xylella fastidiosa Wells et al.]). The authors used the bronze cultivars Carlos (I), Summit (R), and Higgins (S) and the black cultivars Cowart (S) and Noble(R). 6

24 The berries were grown in a vineyard with a fungicide spray program that used sequential applications of fungicides at d intervals from early bloom to just prior to harvest. They found that Noble received the highest, or most severe, foliar disease score, while Cowart and Summit received the lowest, or least severe. The fruit from Higgins from untreated vines had the highest bitter rot, black rot, and Macrophoma rot scores, while fruit from Noble had the lowest rot scores. It was found that fungicide treatments reduced the berry disease score on Higgins by almost 50%, and bitter rot disease scores of Higgins, Carlos, Summit, and Cowart were lower from fungicide treated vines. The authors concluded that though resveratrol concentration and disease incidence varied among cultivars and treatments, fungicide applications during the growing season reduced the incidence of fruit and foliar disease and increased resveratrol concentrations in the skin. Lee and Talcott (2004) evaluated the influence of fruit maturity on ellagic acid derivatives and other antioxidant polyphenolics in the muscadine cultivars Albemarle, Carlos, Cowart, Doreen, Fry, Georgia Red, Nesbitt, and Noble. The polyphenolics were extracted from the skin and pulp by homogenizing with 25 ml of 100% methanol, filtered through filter paper, and the solvent was removed at 40 C under a stream of N. The juice was analyzed directly following centrifugation and filtration. The polyphenolics were separated and quantified by high performance liquid chromatography (HPLC) using solvent programs to identify phenolic acid, free ellagic acid, and ellagic acid derivatives. Total soluble phenolics were analyzed using Folin Ciocalteu assay and expressed in gallic acid equivalents (GAE), while antioxidant activity was determined using an Oxygen Radical Absorbance Capacity (ORAC) assay. The authors found the free form of ellagic acid 7

25 (aglycone) and two ellagic acid glycosides in all eight cultivars following extraction and separation by HPLC. It was found that the skin of ripe Cowart and Doreen contained the highest concentrations of ellagic acid and glycosides (1900 and 1620 mg/l, respectively). Ellagic acid levels were considerably lower in the juice ( mg/l), when compared to the skin ( mg/l) and pulp ( mg/l). Anthocyanins, quantified only in the black cultivars, were expressed in cyanidin equivalents. As expected, the authors found that anthocyanin concentration increased in the skin as the fruit ripened, with lower concentrations found in the pulp. The skin of ripe Cowart, Nesbitt, and Noble (3250 mg/l, 5230 mg/l, and 4140 mg/l, respectively) contained the highest concentrations of anthocyanins. Among the cultivars evaluated, ripe Georgia Red contained the highest levels of total phenolics of both skin and juice and (29.1 µmol Trolox Equivalents/mL), but had lower anthocyanin, ellagic acid, and ellagic acid glycoside content. The data presented by the authors suggested a diversity of phytochemical compounds present in the cultivars evaluated, and within segments of fruit with the skin generally containing the highest levels. Striegler et al. (2005) studied the quality and nutraceutical potential of Black Beauty, Carlos, Cowart, Doreen, Early Fry, Fry, Granny Val, Ison, Jumbo, Late Fry, NC67A015 17, NC67A015 26, Nesbitt, Scarlett, Southern Home, Sterling, Sugargate, Summit, Supreme, and Tara. The cultivars with the highest SSC were Ison and Supreme in 2002 and Southern Home in The fruit skins of Ison had the highest total anthocyanins, while the seeds of Supreme had the highest total phenolic and ORAC levels. Threlfall et al. (2007) measured the composition and nutraceutical content of juices from the cultivars Black Beauty, Carlos, Granny Val, Ison, Southern Home, Summit, and 8

26 Supreme. They found that Southern Home had the highest SSC, while Supreme had the lowest. Total phenolics were not associated with color as might have been expected; the bronze cultivars Summit and Granny Val had the highest concentrations of total phenolics and Carlos, another bronze cultivar, had the lowest. Among the black cultivars, Ison had the highest total anthocyanin concentration and Supreme the lowest. Juice from the cultivars Southern Home and Carlos had the highest ORAC concentrations while the juice from Nesbitt had the lowest. Marshall et al. (2012) found that high levels of phenolics and resveratrol only exist in some cultivars and are not ubiquitous for muscadine juice or pulp, while ellagic acid was found in the skin of all cultivars tested. Postharvest Physiology The fresh market for muscadines could be greatly expanded to other parts of the country, if short or even long term storage life could be improved (Starnes Saunders et al., 1981). Unlike some fruits, muscadines do not continue to accumulate sugars after they are harvested. As there is no starch reserve to convert into sugar in the berry, it is crucial to harvest muscadines at the correct ripeness to ensure quality of the stored berries (Lane, 1978). Flora and Lane (1979) studied the chemical and composition factors affected by ripeness and harvest date of Cowart muscadine grapes. The fruit was density separated in 10, 12.5, and 15% sucrose solutions. It was determined that SSC, TA, and color were closely related to ripeness. Carroll and Marcy (1982) determined the chemical and physical changes during maturation of Carlos and Noble muscadines. They found that berry and seed weight, percent moisture, SSC, TA, and ph were all significantly correlated with berry maturity. 9

27 Starnes Saunders et al. (1981) researched the postharvest physiology and senescence of muscadines. They found that deterioration of the stored fruit occurred due to both senescence and pathogen attack. They found that muscadines could be held in cold storage at 1 C and 85% RH for up to 2 weeks without visible signs of tissue deterioration, and muscadines stored for longer periods deteriorated rapidly upon removal from cold storage. The authors concluded that the major limitations of storage life of muscadines were poor preharvest cultural practices, poor postharvest handling and storage, cultivars with high wet stem scar percentages, and poor transportation methods. Savoy and Hatton (1980) found that muscadines with dry or clear stem scars stored at 1.1 C and 95% RH maintained optimum postharvest quality. Ballinger and McClure (1983) stored the fruit of Carlos light sorted into four ripeness classes at 0 C for 7 weeks. They found that each increase in ripeness was related to a greater amount of decay development during and after storage. Lanier and Morris (1979) evaluated the sensory preference and composition of berries from Carlos sorted into five maturity classes based on density separation in 8, 9, 10, and 11% NaCl brine solutions. The authors found that the panelist s sensory preference, SSC, and berry weight increased, color improved, and acidity decreased with increasing fruit maturity. General Storage Lutz (1938) evaluated factors that influenced the quality of 84 V. labrusca L. genotypes and six muscadine genotypes in storage. The fruit were given either a wash treatment or were not washed, and placed in baskets. The baskets were then stored at different temperatures ranging from 0 to 26.7 C and at a range of 72 96% RH. During the washing treatment, the baskets of muscadines were submerged in a 0.5% hydrochloric acid 10

28 solution for 5 min, drained for 1 min, and then submerged in water for 5 min, and the berries were then stored wet. Measurements of respiration rates, SSC of the juice, and SSC to acid ratio were also taken. The author found that decay decreased as temperature decreased and fruit stored at 0 C maintained the highest quality especially for long storage (up to 6 weeks). Fruit stored at 2.2 and 4.4 C maintained satisfactory quality for moderate lengths of storage. Relative humidity of 80% to 85% resulted in the least amount of decay and shriveling in storage. It was also found that more decay occurred on fruit that were washed; however the manner of washing and lack of drying could have contributed to these findings. Takeda et al. (1983a) determined the storage potential of Fry and Southland muscadines for the fresh market. They measured several variables including, hydrocooling, washing in a chlorinated solution, and density separation. The authors found that berry weight, TA, SSC, and ph did not change during storage for the cultivars or treatments. They found that firmness decreased under all storage conditions. It was concluded that fruit should be harvested at full ripe, as underripe fruit were unmarketable because of off flavors, and overripe fruit were unmarketable because of decay during storage. With both cultivars, washed fruit stored better than unwashed fruit, and the number of wet stem scars played an important role in the length of storage. They also concluded that muscadines, even in ideal situations, do not store for longer periods than two weeks due to undesirable decay. Takeda et al. (1983b) studied the physical and chemical changes in muscadines during postharvest storage. The berries were stored at 20, 4.5, and 0 C to determine the biochemical parameters that changed during storage. It was determined that SSC, TA, 11

29 individual sugars, and organic acids did not change during storage at the three temperatures tested, while decay increased rapidly in the fruit stored at 20 and 4.5 C. They also found that total phenolics increased while pectin content decreased during storage, with more change occurring in the fruit stored at higher temperatures. Silva et al. (1994) evaluated the quality changes in muscadine grapes during cold storage using either polypropylene trays with holes, 2.7 mil flexible polyethylene bags without holes, or 2.7 mil flexible polyethylene bags with holes. Eight experienced panelists evaluated the berries for appearance, color, toughness, firmness, acidity, sweetness, and flavor. Measurements were taken to determine weight change, color, penetration force, compression force, SSC, TA, ph, and percent visible rot. They found that berries stored in polyethylene trays and flexible polyethylene bags without holes had an acceptable shelf life of around 21 d when held at 5 C, while the other treatments resulted in lower unacceptable quality berries. Walker et al. (2001) conducted an experiment to determine the effect of storage on quality attributes of muscadines at different maturities using polyethylene bags to evaluate extended storage. Berries were stored in clear, vented, clamshell containers that were either placed in 0.03 mm multilayered polyolefin polyethylene bags or not wrapped and stored for 6 weeks at 2 C with 89% RH. At 0, 2, 4, and 6 weeks, whole berries were blended and evaluated for ph, TA, and SSC. Additionally, sliced berries were used to measure firmness during storage. As storage time increased, decay increased and firmness decreased. Fruit stored in clamshells that were wrapped in bags had less decay, were less firm, and had less weight loss than those stored unwrapped. They found that the differences in the lack of loss of weight could have been due to the fruit leaking juice from cracks or splits that was retained in the bag and weighed with the grapes. 12

30 Storage Effects of Sulfur Dioxide Studies have been conducted on extending the shelf life or storage life of table grapes (V. labrusca, and V. vinifera) with the use of SO2 to decrease decay (Harvey et al., 1988; Litcher et al., 2008; Marois et al., 1986; Nelson and Ahmedullad, 1976). Treatments of SO2 have been used to effectively prevent decay in bunch grape (V. vinifera) storage by hindering the growth of decay causing fungi, namely Botrytis cinerea Pers.: Fr, Pennicillium sp., Aspergillus sp., Fusarium sp., Melaconium sp., Alternaria sp., and yeasts for over a century (Harvey et al., 1988; Litcher et al., 2008; MacLean et al., 2009; Marois et al., 1986; Morris et al., 1992; Nelson and Ahmedullad, 1976; Smit et al., 1972). The effect of SO2 on muscadine grapes in storage has also been studied (Ballinger and Nesbitt, 1982a; Ballinger and Nesbitt, 1982b; James et al., 1997; James et al., 1999; Lane, 1978; Lane and Flora, 1980; MacLean et al., 2009; Morris et al., 1992; Smit et al., 1971; Conner and Maclean, 2012). It has been found that SO2 can be an effective tool in slowing decay on muscadines in storage, but frequently with some negative or no affects (MacLean et al., 2009; Morris et al., 1992; Smit et al., 1971). Bleaching, white spots, and offflavors were found to occur in muscadines with the use of varying levels of SO2 and with varying times of fumigation, especially in those berries with wet stem scars and cracks or splits. Conversely, in other studies little to no negative effects of SO2 occurred during storage on muscadine grapes (Ballinger and Nesbitt, 1982b; Conner and Maclean, 2012; James et al., 1999; Lane and Flora, 1980). As a result it can be inferred that the influence of the SO2 storage treatment on the quality of grapes is cultivar specific, and with muscadines not reliably beneficial. Controlled Atmosphere Storage 13

31 Studies have been conducted to determine the effect of controlled atmosphere (CA), sometimes called modified atmosphere (MA), storage on the postharvest quality of muscadine grapes (Basiouny, 1998; Himelrick, 2003; Mercer and Smittle, 1990; Smittle, 1990). Controlled atmosphere storage usually involves regulating the amount of CO2, O2, and N, as well as the temperature, air circulation, and the RH in cold storage to prevent decay, reduce chilling injury, extend the storage or shelf life, and maintain high sensory quality of the fruit. It was found that a temperature of C, 90 95% RH, O2 concentrations of 5%, CO2 concentrations of 15%, N concentrations of 80%, and air circulation of 25 cfm/ton resulted in the maximum storage life of Fry and Granny Val muscadine grapes (Mercer and Smittle, 1990; Smittle, 1990). Recent research has found some important drawbacks to CA storage. Controlled atmosphere storage is very commodity specific; the ideal atmosphere for a certain cultivar of muscadines may not be beneficial to a different cultivar or a different crop. This limits what commodities can be stored in a single cold room at a particular time. Additionally, some commodities can only tolerate a particular CA at a certain temperature, or only for a limited time, and fine tuning and constant adjustment of CA conditions can cause additional costs to the producer (Himelrick, 2003). Fungicide Treatments Several diseases affect muscadine fruit during storage. Some diseases are the result of infection of the fruit during storage, while others are a result of pre or post harvest factors before storage (Lane, 1978). Some of the fungi that have been isolated on muscadines postharvest include Alternaria sp., Aspergillus sp., Botrytis. sp., Fusarium sp., Penicillum sp., and Melaconium sp. (Lane, 1978; Smit et al., 1971; Takeda et al., 1983b). It is 14

32 widely accepted that fungicides can significantly reduce losses due to disease in the yield and quality of muscadine grapes. Harvey (1955) found that field fungicide application of Captan (Ntrichloromethylthio 4 cylohecene 1,2 dicatboximide), B 622 (2,4 dichloro 6 ochloroanilino syntriazine), and Crag 5400 (a, a trithiobis N dimethylthioformamide) significantly reduced postharvest decay in Emperor (V. labrsuca). Lane (1978) determined the effect of vineyard fungicide treatments on the shelf life of Cowart, Hunt, Magnolia, and Nevermiss muscadine grapes. Alternating field applications of the fungicides Benlate 50 WP (methyl 1 (butylcarbamoyl) 2 benzimidazole carbamate (6)) and Manzate D 80 WP ([1,2 ethaznediybis (carbamodithio)(2 )]) were made at 18 d intervals from mid May to mid August. The berries were then harvested, and either stored at ambient temperatures (23 27 C during the day, and C at night) for 48, 72, and 96 h, or 5 C and 60% RH for 168 and 216 h. Significant differences were found in the number of sound fruit between the treated and control berries for all cultivars at ambient temperatures for 72 h except Cowart, where the samples deteriorated regardless of the treatment. After 96 h at ambient temperatures all samples except Manzate D 80 WP treated samples of Nevermiss, had more than 50% unsound fruit. Conversely, after storage at 5 C for 168 h and then exposed to shelf life at ambient temperatures for 48 h, it was found that all fungicide treated samples in all cultivars except Cowart had significantly more sound fruit (Lane, 1978). Takeda et al. (1982) studied the effects of prestorage fungicide dip treatments and storage temperatures on decay and composition of Fry. The authors sorted commercially 15

33 harvested fruit removing any damaged or decayed berries. Sound fruit was packaged into pint sized clamshells and either dipped in a Botran + Captan (2,6 Dichloro 4 nitroaniline + N trichloromethylthio 4 cylohecene 1,2 dicatboximide) fungicide mixture or left undipped. The clamshells were then placed in vented cardboard boxes and stored at either 20, 4.5, or 0 C. The authors found that little or no change occurred in ph, SSC, or TA for all treatments. It was determined that storage temperature influenced deterioration of muscadines, with the berries stored at 20, 4.5, and 0 C having 25%, <10%, and >10% decay after storage, respectively. It was found that a decrease in pectin and an increase in total phenolic occurred in the berries during storage. Furthermore, they found that treatment of a fungicide dip was ineffective in managing decay during storage, although microbial spoilage was the major factor contributing to postharvest deterioration. It was concluded that muscadine berries cannot be stored longer that 14 d with the postharvest handling techniques in the study. Smith and Magee (2002) applied the fungicides, Nova 40W (myclobutanil: a butyla (4 chlorophenyl) 1H 1,2,4,triazole 1 propanenitrile), Abound (azoxystrobin: methyl (E) 2 {2 [6 (2 cyanophenoxy) pyrimidin 4 yloxy]phenyl} 3 methoxyacrylate*), and Elite (tebuconazole: a [2 (4 chlorophenyl)ethyl] alpha (1,1 dimethylethyl) 1H 1,2,4 triazole 1 ethanol) sequentially to Doreen and Summit every 10 d beginning at early bloom and ending at pre harvest intervals (PHIs) of 64, 42, 28, 14, 7, 4, 2, 1, and 0 d to determine spray schedule effects on foliage and berry diseases and the relationship between disease incidence and berry resveratrol content. They found that all fungal diseases were reduced by fungicide treatments, and there were no differences in the number of asymptomatic berries among the nine PHIs. They found that Doreen showed less disease symptoms than 16

34 Summit. The authors also found that resveratrol content of berry skins from fungicidetreated vines was significantly lower than those from untreated vines. Berry Composition Muscadines have experienced a resurgence of consumer interest as recent research into the compositional and nutraceutical characteristics have shown that muscadines are good sources of valuable antioxidants, vitamins, minerals, and dietary fiber that have positive effects on health (Carroll et al., 1968; Ector et al., 1996; Lamikanra, 1988; Magee et al., 2002; Threllfall et al., 2007). Research has shown that the muscadine grape possesses one of the highest antioxidant levels among fruit crops (Greenspan et al., 2005). Some of these components of muscadines have been shown to have anti cancer, anti mutagen, and anti inflammatory properties, and to reduce levels of glucose, insulin, and glycated hemoglobin in people with diabetes (Banini et al., 2006; Bralley et al., 2007; Greenspan et al., 2005; God et al., 2007; Yi, 2005). Muscadine grapes contain phenolic acids, flavonols, anthocayanins, ellagic acid, and numerous ellagic acid derivatives (Boyle and Hsu, 1990; Haung et al., 2009; Lee et al., 2005; Pastrana Bonilla et al., 2003; Stringer et al., 2009; Talcott and Lee, 2002). Ellagic acid and other antioxidants have been shown to demonstrate anticarcinogenic activity in the colon, lungs, and liver, as well as a reduction of birth defects in rats and mice, and two forms of colon cancer in humans (Ector, 2001; Yi et al., 2005). Polyphenolic concentrations usually increase in muscadines as fruit ripens (Lee et al., 2005) and are higher in wine than in unfermented juices from an identical grape press (Musingo et al., 2001; Talcott and Lee, 2002). Greenspan et al. (2005) conducted a study determining the anti inflammatory properties of muscadine grapes. Their results indicated that the anti inflammatory 17

35 properties of muscadines could be, in part, directly related to the high antioxidant content of the grape, specifically the berry skin. Smith (2013) studied the relationship between phytochemical content, berry quality, and disease control following full season or early season applications of fungicides on Carlos, Doreen, and Summit muscadines. The author found no significant differences in vine vigor, foliar diseases, or bitter rot scores among treatments where fungicide applications were stopped at varying preharvest intervals ranging from 0 to 56 d, or in disease scores between the full season and early season treatments. She found that fullseason fungicide applications resulted in significantly lower berry disease scores than control treatments, and SSC was highest in full season fungicide treatment and untreated vines. Overall, ellagic acid and resveratrol content was lower in berries from fungicidetreated vines than from untreated vines, with control treatments resulting in berries with almost 10 times as much resveratrol as those from the full season treatment. It was concluded that fungicide treatments reduced berry diseases with as few as four applications compared to 12 applications in the full season schedules, and fungicides that control berry diseases had an effect on berry quality. Berry Segments Studies have been conducted to understand the concentration of nutraceutical compounds in the different segments of muscadine fruit (Lee and Talcott, 2004; Marshall et al., 2012; Sandhu and Gu, 2010; Takeda et al., 1983b; Threlfall et al., 2005). Ellagic acid was found in higher concentrations in the skins (587 1,900 mg/kg) of the berry when compared to the pulp (not detected 455 mg/kg) or juice ( mg/kg)(lee and Talcott, 2004). Anthocyanin concentrations were found to be generally higher in the berry skins 18

36 ( mg/k) and juice ( mg/kg) than in the pulp ( mg/kg) or seed ( mg/kg) (Lee and Talcott, 2004; Threlfall et al., 2005). Phenolic concentrations were found to be generally higher in the skin (2,260 3,454 mg/kg) and seeds (2,294 9,534 mg/kg) than in the juice (979 2,160 mg/kg) or pulp (443 1,100 mg/kg) (Lee and Talcott, 2004; Takeda et al., 1983b; Threlfall et al., 2005). ORAC was measured and found present in seeds (893 1,100 μmol TE/g), skins ( μmol TE/g), pulp ( μmol TE/g), and juice ( μmol TE/g) (Lee and Talcott, 2004; Threlfall et al., 2005). Sandhu and Gu (2010) determined the antioxidant capacity and total phenolic content of muscadine seeds, skin, and pulp. They also profiled the phenolic compounds present. The authors manually separated the berries into seeds, skins, and pulp and stored the portions at 20 C. The seeds, skin, and pulp were freeze dried and ground into a powder, and then extracted with an acetone/water/acetic acid mixture (70:29.7:0.3v/v). Chromatographic analyses were performed as high performance liquid chromatographydiode array detection electrospray ion trap tandem mass spectrometry (HPLC DAD ESI MS n ). The authors found that, compared to V. vinifera, the presence of ellagic acid in muscadines is unique, and is in the form of free ellagic acid, ellagic acid glycosides, and ellagitannins. Additionally, they found that the anthocyanins present in muscadines are 3,5 diglucosides (as opposed to 3 glucosides in V. vinifera), and were identified as delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn), pelargonidin (Pg), and malvidin (Mv). The authors found that total phenolic content and antioxidant capacity were highest in the seeds ( mg GAE/g), followed by the skin ( mg GAE/g), and pulp ( mg GAE/g). Their results confirmed that HPLC DAD ESI MS n is a valuable tool for identification of phenolic compounds. 19

37 Marshall et al. (2012) determined stilbene, ellagic acid, flavonols, and phenolic content of the whole berry, skin, pulp, and juice of 21 muscadine cultivars. The authors identified reseveratol in the skins of 20 cultivars ( μg/g), in the pulp of Eudora (0.948 μg/g) and Janet (4.297 μg/g), and in the juice of none of the cultivars. It was found that ellagic acid was most abundant in the skins of all cultivars tested ( ,554.7 μg/g), but was found in similar concentration in the juice (24.2 to 56.8 μg/g) and the pulp ( μg/g). Flavonols were only present in the skins and not in juice or pulp. The authors found that total phenolics and total anthocyanins were highest in the whole berry with the bronze cultivars having significantly less than black cultivars. Total phenolic concentrations ranged from to 1,061.7 mg/100 g, while total anthocyanin concentrations ranged from none to mg/100 g. Total Phenolics Phenolics are a large family of secondary metabolites involved in plant response to abiotic and biotic stresses (Marshall et al., 2012). Phenolics are ubiquitous in Plantae and are the most abundant secondary metabolites found in plants (Amakura et al., 2000). Measurements of total phenolics by the Folin Ciocalteu metal reduction assay is a common index to provide an overall assessment of the content and chemical activity of compounds present, and aids in determining the antioxidant capacity of fruits and vegetables (Lee and Talcott, 2004). Pastrana Bonilla et al. (2003) determined the phenolic content and antioxidant capacity of the skin, seed, pulp, and leaves of 10 muscadine cultivars (five bronze and five black skinned). The fruit segments and leaves were extracted using HPLC analysis, and total phenolics were determined colorimetrically using Folin Ciocalteu reagent. Total anthocyanins were determined according to a ph differential method, using 20

38 a UV visible spectrophotometer. Antioxidant capacity was determined by the Troloc equivalent antioxidant capacity (TEAC) assay. The major phenolics in muscadine berry skins were identified by retention times and characteristic spectra. Ellagic acid, transresveratrol, and kaempferol were found in the skins, whereas (+) catechin, ( ) epicatechin, and gallic acid were found in the seeds. Ellagic acid was the most abundant phenolic compound in muscadine berries. The authors found that muscadine seeds had approximately five times higher phenolic concentrations than any other fruit segment, while the leaves had 15 times more antioxidant capacity than the fruit. It was also found that the pulp had very low levels of phenolics. Threlfall et al. (2007) determined the nutraceutical content of juice from five black cultivars (Black Beauty, Ison, Nesbitt, Southern Home, and Supreme) and three bronze cultivars (Carlos, Granny Val, and Summit). The juice was analyzed for total phenolics with the Folin Ciocalteu assay with gallic acid as the standard. The authors found that total phenolics of the juice were not associated with skin color. The bronze cultivars Summit and Granny Val had the highest concentrations of total phenolics while Carlos, another bronze cultivar, had the lowest. Stringer et al. (2009) evaluated the phenolic concentrations in the muscadine cultivars Carlos, Magnolia, Albermarle, and Noble and eight breeding selections. The total phenolic content was measured by the Folin Ciocalteu method at 700 nm, using gallic acid as a standard. The authors found that total phenolics were high in all cultivars and selections except Magnolia. Striegler et al. (2005) evaluated the total phenolics of several muscadine genotypes grown in southwestern Arkansas. The phenolics were extracted from whole berries, and analyzed using the Folin Ciocalteu assay, with catechin as the standard. It was determined that Nesbitt had the lowest total phenolic values. Marshall et al. (2012) 21

39 identified the total phenolic content of 21 muscadine cultivars by the Folin Ciocaleteau assay with gallic acid equivalents with absorbance at 760 nm. The authors found that total phenolics ranged from mg/100 g ( Fry ) to 1, mg/100 g ( Nesbitt ), with bronze cultivars having significantly less concentrations than black cultivars. Anthocyanins Anthocyanins are known to protect blood vessels in humans, and play a role in cancer prevention, though anthocyanin absorption appears to be low in humans (Marshall et al., 2012). Brown (1940) first researched the anthocyanin pigments of muscadines. He found that the anthocyanins present in muscadines are 3,5 diglucosides, as opposed to 3 glucosides in V. vinifera and V. aestivalis Michx. Ballinger et al. (1973) evaluated the anthocyanins of 10 black genotypes of muscadines for use in the wine industry. They found that all 10 clones contained the same anthocyanin compounds, Dp, Cy, Pt, Pn, and Mv. Flora (1978) assessed the effects of heat treatments on juice and cultivar on muscadine anthocyanin concentrations. It was found that heating resulted in relative increases extracted in the 3,5 diglucosides of Cy, Mv, and Pn while resulting in lower levels of Dp and Pt 3,5 diglucosides. It was found that total anthocyanins in the 11 cultivars tested ranged from 40 to 403 mg/100 g, with wide variations in the relative contents of individual anthocyanins, averaged across extractions. Delphinidin was the most prevalent anthocyanin identified in the pigment complex followed by Cy or Pt, Pn, and Mv. Goldy et al. (1987) analyzed and correlated the anthocyanin content of muscadine fruit, stems, tendrils, leaves, and leaf petioles using high performance liquid chromatography (HPLC) techniques. All five of the known 3,5 diglucoside forms of were present in the fruit, stems, tendrils, and leaf petioles, while only Dp, Cy, and Pt were identified in the leaves. The 22

40 authors found that tendril analysis was best for predicting fruit Cy (r = 0.60), Mv (r = 0.57), Pn (r = 0.66), and Pt (0.87), while stem analysis was best for predicting fruit Dp (r = 0.66). Lee and Talcott (2004) evaluated the influence of fruit maturity on anthocyanins and other antioxidant polyphenolics in eight muscadine cultivars. The polyphenolics were extracted from the skin and pulp by homogenizing with 25 ml of 100% methanol, filtered through filter paper, and the solvent was removed at 40 C under a stream of N. The juice was analyzed directly following centrifugation and filtration. Total phenolics were analyzed using Folin Ciocalteu assay and expressed in gallic acid equivalents (GAE), while antioxidant activity was determined using an ORAC assay. As expected, the polyphenolic compounds were concentrated in the epidermal tissues, which is often exceptionally thick in muscadines. Anthocyanins, quantified only in the black cultivars, were expressed in cyanidin equivalents. The authors identified Dp, Pt, Mv, Pn, and Cy as the anthocyanins present in the fruit. As expected, the authors found that anthocyanin concentration increased in the skin as the fruit ripened, with lower concentrations found in pulp. The skin of ripe Cowart, Nesbitt, and Noble (3250 mg/l, 5230 mg/l, and 4140 mg/l, respectively) contained the highest concentrations of anthocyanins. On the basis of abundance, anthocyanins were the major antioxidant present in the muscadine grape skin and juice, and anthocyanin concentrations were directly related to antioxidant capacity (r = 0.99). Striegler et al. (2005) evaluated the total anthocyanins of 19 muscadine cultivars and two selections. The anthocyanin content of the total phenolics was determined by a ph differential method, and absorbance was measured at wavelengths of 510 and 700 nm. 23

41 The authors found that Ison, Southern Home, and Nesbitt had the highest anthocyanin concentrations, while Supreme had the lowest. Stringer et al. (2009) evaluated the anthocyanin content of the cultivars Carlos, Magnolia, Albermarle, and Noble and eight breeding selections. Total anthocyanins content was determined using a modified ph differential method. The authors found that anthocyanin content showed a definite distinction between bronze and black fruit. The bronze genotypes Magnolia, Carlos, and NC76A showed significantly less anthocyanin, which was expected as anthocyanin content develops the fruit color. Sandhu and Gu (2010) first identified six anthocyanin compounds present in muscadines as Dp, Cy, Pt, Pn, Pg, and Mv. Marshall et al. (2012) analyzed the total anthocyanin concentrations of 21 muscadine cultivars with a modified Giusti and Wroland ph shift assay. The authors found that total anthocyanin concentrations ranged from the highest of mg/100 g in Nesbitt to not detected in the bronze cultivars. Ellagitannins Ellagic acid is not commonly found in other grape species, but is exhibited in relatively high concentrations in muscadines (Marshall et al., 2012; Stinger et al., 2009). Ellagitannins have been widely studied because of their antiproliferative and antioxidant properties, due to their ability to directly inhibit DNA binding of certain carcinogens and by reducing oxidative stress (Marshall et al., 2012). Concentrations of ellagic acid are generally higher in dark skinned berries than in bronze muscadines (Ector, 2001). Lee et al. (2005) isolated and identified several ellagic acid derivatives present in muscadine berries and determined their relative antioxidant properties (AOX). Using methanol, the authors extracted compounds from the berry skins and flesh. Ellagic acid 24

42 derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. The authors concluded that muscadines contain phenolic acids, flavonols, anthocyanins, and numerous ellagic acid derivatives. The authors found that AOX varied with elution and retention, while correlating with total phenolics (r = 0.90) and total ellagic acid (r = 0.99). Boyle and Hsu (1990) identified and quantified ellagic acid in muscadine juice from 11 cultivars (Cowart, Doreen, Higgins, Hunt, Magnolia, Pamilco, Regale, Roanoke, Scuppernong, Sterling, and Tarheel) in order to find an approach to eliminate the sediment that is occasionally produced in the juice. The authors used a combination of organic extraction and separation by HPLC to identify and separate the ellagic acid compounds. Ellagic acid was found to vary significantly among cultivars, with a range of 1.6 µg/ml ( Higgins ) to 23.1 µg.ml ( Hunt ). Stringer et al. (2009) determined the ellagic acid concentrations in the muscadine cultivars (Carlos, Magnolia, Albermarle, and Noble) and eight breeding selections. Ellagic acid was identified using liquid chromatographic UV Vis detection analysis at 255 nm. The authors found that ellagic acid content varied greatly from mg/kg in Carlos, to mg/kg found in CD8 67. Sandhu and Gu (2010) identified ellagic acid in muscadines in the forms of ellagic acid hexoside, ellagic acid xyloside, and ellagic acid rhamnoside. Marshall et al. (2012) determined the total ellagic acid concentrations of 21 muscadine cultivars using UV/VIS spectral interpretation, and retention time of authentic standards. The authors found that ellagic acid was found in all cultivars tested, ranging from μg/g ( Alachua ) to 5,554.7 μg/g ( Southland ). Flavonols 25

43 Muscadine berries have been found to contain high levels of flavonols. In humans, flavonols protect against carcinogensis, DNA mutations, colon cancer, and heart disease (Marshall et al., 2012). Flavonols are found in both V. vinifera and V. rotundifolia grapes, however the flavonol compound myricetin is unique to bronze muscadines (Marshall et al., 2012). Yi et al. (2005) identified the phenolic compounds in muscadine grapes. Flavonols were extracted using HLB cartridge and LH20 column, and the compounds were identified using HPLC. The authors identified total flavonols fractions ranging from 76.3% to 86.1%. Sandhu and Gu (2010) identified the flavonols present in muscadines as glycosides of quercetin, kaempferol, and myricetin. They also identified the flavonols myricetin hexoside, kaempferol hexoside, quercetin glucoside, and kaempferol rutinoside for the first time in muscadines. Marshall et al. (2012) determined the flavonol concentrations of 21 muscadine cultivars using UV/VIS spectral interpretation, and retention time of authentic standards. The authors found flavonols only in the skins of the berries and not in the pulp or juice. The most abundant flavonol contained in muscadine skins was myricetin, followed by quercetin, then kaempferol. The bronze cultivars, Janet, Sweet Jenny, and Triumph ( g/g, g/g, g/g, respectively) contained significantly higher levels of myricetin, while black cultivars contained myricetin at lower levels. Quercetin was also found greatest in Sweet Jenny, and Nesbit, with g/g, and g/g. Kaempferol was most abundantly present in Nesbit ( g/g), but was undetected in Black Fry and Black Beauty. Resveratrol 26

44 Stilbenes are synthesized in grape leaves in response to both biotic and abiotic induction treatments, and the capacity to produce stilbenes is correlated with the resistance of grape leaves to fungal infection (Creasy and Coffee, 1988; Marshall et al., 2012). Resveratrol (trans 3,5,4 trihydroxystilbene) is a phytoalexin, or stilbene, produced as a response to fungal infection (particularly by B. cinerea), stress including injury, and UV irradiation (Jeandet et al., 1995; Jeandet et al., 1991; Marshall et al., 2012; Magee and Smith, 2002). Resveratrol has long been confirmed in both red and white V. vinifera grapes skins and pulp, but not in seeds. Only within the last 20 years has resveratrol been measured in muscadines. The fleshy parts of both black and bronze muscadine berries have higher concentrations of resveratrol than was reported for V. vinifera and V. labrusca (Ector et al., 1996). Resveratrol concentrations in muscadines have been found to be µg/g in the skins, 0.10 µg/g in the flesh, and µg/g in the seeds (Ector et al., 1996). Resveratrol can potentially act as a chemopreventative of cardiovascular disease and coronary heart disease (Hudson et at., 2007; Jang et al., 1997; Lu and Sorreno, 1999; Magee and Smith, 2002). Resveratrol has lipid lowering action, inhibition of human low density lipoprotein oxidation and thus may delay atherosclerosis onset, inhibition of platelet aggregation in the blood, reduce cholesterol levels, and has shown to have cancer chemopreventative activity in all stages of carcinogenesis in cancers including prostate and breast cancer (Ector et al., 1996; Ector, 2001; Hudson et at., 2007; Jang et al., 1997; Lu and Sorreno, 1999; Magee et al., 2002; Threlfall et al., 1999). Jeandet et al (1995) conducted a study in France, on V. vinifera wine from grapes grown in both sprayed and unsprayed vineyards. They determined that conditions leading to the development of B. cinerea do enhance resveratrol production, but extensive grey 27

45 mold development may in fact destroy the induced phytoalexin. Their findings associated years with conditions ideal for infection, but low disease incidence with higher levels of resveratrol in wine. The authors suggest that B. cinerea infection is required for high levels of resveratrol in wine, but that extensive B. cinerea development before harvest may lower resveratrol content. Ector et al. (1996) determined the resveratrol concentrations in whole berries, berries with the seeds removed, and the seeds of both black and bronze cultivars of muscadines. The authors established that resveratrol is a natural constituent of bronzeand black skinned muscadines, and though black skinned muscadines have higher levels of resveratrol, no significant difference existed between the two color groups. Additionally, they found that a broad range in resveratrol concentration within each color group, and among cultivars. It was also determined that the seeds of muscadines contained the highest levels of the berry segments, ranging from 24.5 to 62.2 µg/g, but only made up approximately 23 30% of the resveratrol in the whole berries. Yi et al. (2005) identified the phenolic compounds in muscadine grapes. Nutraceutical compounds were extracted using HLB cartridge and LH20 column, and the compounds were identified using HPLC. The authors found that resveratrol made up % of the flavonol fraction identified. Magee et al. (2002) determined the effect of field fungicide applications on resveratrol content of muscadine berries from the bronze cultivars Carlos, Summit, and Higgins and the black cultivars Cowart and Noble. The berries were grown in a vineyard with a fungicide spray program that used sequential applications of Nova 40W (myclobutanil: a butyl a (4 chlorophenyl) 1H 1,2,4,triazole 1 propanenitrile), Captan 50 WP (N Trichloromethylthio 4 cyclohexan 1,2 dicrboximide), Abound (azoxystrobin: 28

46 methyl (E) 2 {2 [6 (2 cyanophenoxy) pyrimidin 4 yloxy]phenyl} 3 methoxyacrylate*), and Benlate 50 WP (Benomyl (methyl 1 (butylcarbamoyl) 2 Benzimidazolecarbamate) at d intervals from early bloom to just prior to harvest. The authors found that resveratrol concentrations varied by cultivar and treatment. It was determined that resveratrol concentrations in the berry skins were much higher from unsprayed vines compared to those from sprayed vines. However, the relationship between resveratrol content and disease score was not determined. Seed resveratrol concentrations were not affected by fungicide treatments. Overall, resveratrol concentrations of skins from fungicide treated vines were lower than the untreated vines. It was concluded that fungicide treatments reduced the fungal inoculum available on the fruit to elicit resveratrol production. Stringer et al. (2009) evaluated the resveratrol content of the muscadine cultivars Carlos, Magnolia, Albermarle, and Noble and eight breeding selections. Resveratrol was identified by HPLC analysis detection with a UV Vis at 310 nm. The authors found that resveratrol concentrations varied among genotypes with the selection CD8 67 exhibiting the least (2.48 mg/kg) and NC71A006 5 exhibiting the highest amount (48.57 mg/kg). Sandhu and Gu (2010) identified trans resveratrol 3 O ß glucoside to be high in the skins of muscadines on the basis of mass spectral data and a standard. Oxygen Radical Absorbance Capacity Measurements of peroxyl radical scavenging activity using ORAC assay is a common index that provides an overall assessment of the content and chemical activity of compounds present, and aids in determining the antioxidant capacity of fruits and vegetables (Lee and Talcott, 2004; Prior et al, 2003). Lee and Talcott (2004) evaluated the 29

47 antioxidant activity of eight muscadine cultivars of varying ripeness using an ORAC assay. As expected, the polyphenolic compounds were concentrated in the epidermal tissues, which is often exceptionally thick in muscadines. Among the cultivars evaluated, Georgia Red contained the highest levels of total phenolics of both skin and juice and ORAC (29.1 µmol Trolox Equivalents/mL) in contrast to its low anthocyanin, ellagic acid, and ellagic acid glycoside content. The data presented by the authors suggested a diversity of phytochemical compounds present among different muscadine cultivars and within segments of fruit, with the skin generally containing the highest levels. Additionally the authors determined that physiologically ripe fruit contained higher levels of all compounds evaluated, resulting in greater antioxidant capacity. Striegler et al. (2005) evaluated the ORAC concentrations of several muscadine genotypes. ORAC was measured using a modified method of a microplate reader using fluorecerin as the fluorescent probe. The ORAC values ranged from 2.52 to 3.37 µmol ml 1 TE, and it was determined that Ison had the highest while Nesbitt and Granny Val had the lowest ORAC concentrations. Threlfall et al. (2007) analyzed for ORAC using a modified method with a microplate reader and using fluorescein as the florescent probe. They found that ORAC concentrations range from 2.1 to 4.1 µmol TE/mL, with Southern Home and Carlos having the highest concentrations and Nesbitt having the lowest. Literature Cited Amakura, Y, Y, Umino, S. Tsuji, Y. Tonogai Influence of jam processing on radical scavenging activity and phenolic content of berries. J. Agri. Food. Chem. 48: Ballinger, W.E., E.P. Maness, W.B, Nesbitt, and D.E. Carroll Anthocyanins of black grapes of 10 clones of Vitis rotundifolia Michx. J. Food Sci. 38: Ballinger, W.E. and W. F. McClure The effect of ripeness on storage quality of Carlos muscadine grapes. Scientia Hort. 18:

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51 Lee, J H., J.V. Johnson, and S.T. Talcott Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC ESI MS. J. Agr. Food Chem. 53: Lee, J H., and S.T. Talcott Fruit maturity and juice extraction influences on ellagic acid derivatives and other antioxidant polyphenolics on muscadine grapes. J. Agr. Food Chem. 52: Litcher, A., Z. Yohanan, T. Kaplunov, and S. Lurie Evaluation of table grape storage in boxes with sulfur dioxide releasing pad with either an internal plastic liner or external wrap. HortTechnology 18: Lu, R., and G. Sorreno Resveratrol, a natural product derived from grapes, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells. J. Cell Physiol. 197: Lutz, J.M Factors influencing the quality of American grapes in storage. U. S. Dept. Agr. Tech. Bul. 606:1 27. Luvisi, D., H. Shorey, J. Smilanick, J. Thompson, B. Gump, J. Knutson. Sulfur dioxide fumigation of table grapes. Bulletin University of California, Davis, Division of Agriculture and Natural Resources. MacLean, D., P.J. Conner, J. Paulk, and L. Grant Postharvest control of decay organisms. The Southern Region Small Fruit Consortium. Prog. Rpt Magee, J.B., B.J. Smith, and A. Rimando Resveratrol content of muscadine berries is affected by disease control spray program. HortScience 37: Marois, J.J., A.M. Bledsoe, W.D. Gubler, and D.A. Luvisi Control of Botrytis cinerea on grape berries during postharvest storage with reduced levels of sulfur dioxide. Plant Dis. 70: Marshall, D.A., S.J. Stringer, and J.D. Spiers Stilbene, ellagic acid, flavanol, and phenolic content of muscadine grape (Vitis rotundifolia Michx.) cultivars. Pharmaceutical Crops. 3: Mercer, M.D., and D.A. Smittle Controlled atmosphere storage of muscadine grapes. Proc. Viticult. Sci. Symp. Fla. A and M Univ Morris, J.R Handling and marketing of muscadine grapes. FruitSouth 4: Morris, J.R., O.L. Oswald, G.L. Main, J.N. Moore, and J.R. Clark Storage of new seedless grape cultivar with sulfur dioxide generators. Amer. J. Enol. Viticult. 43:

52 Musingo, M.N., S.F. O'Keefe, O. Lamikanra, C.A. Sims, and R.P. Bates Changes in ellagic acid and other phenols in muscadine grape (Vitis rotundifolia) juices and wines during storage. Amer. J. Enol. Viticult. 52: Nelson, K.E., and M. Ahmedullah Packaging and decay control systems for storage and transit of table grapes for export. Amer. J. Enol. Viticult. 27: Olien, W.C The muscadine grape: botany, viticulture, history, and current industry. HortScience 25: Olien W.C Introduction to the muscadines, p In: F.M. Basiouny and D.G. Himelrick, (eds.). Muscadine Grapes. ASHS Press, Alexandria, VA. Pastrana Bonilla, E., C.C. Akoh, S. Sellappan, and G. Krewer Phenolic content and antioxidant capacity of muscadine grapes. J. Agr. Food Chem. 51: Perkins Veazie, P., S. Spayd, B. Cline, and C. Fisk Handling and marketing guide for fresh market muscadine grapes. SFRC E03: Prior, R.L., H. Hoang, L. Gu, X. Wu, M. Bacchiocca, L. Howard, M. Hampschwoodill, D. Haung, B. Ou, and R. Jacob Assays for hydrophilic and lipophilic antioxidant capacity [oxygen radical absorbance capacity (ORACF1)] of plasma and other biological and food samples. J. Agr. Food Chem. 51: Prior RL, S.A. Lazarus, G. Cao, H. Muccitelli, and J.F. Hammer stone Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp.) using highperformance liquid chromatography/mass spectrometry. J Agr. Food Chem. 49: Sandhu, A.K., and L.W. Gu 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 MSn. J. Agr. Food Chem. 58: Savoy, C.F. and T.T. Hatton Post harvested characteristics of some commercially picked muscadine varieties. Proc. Viticult. Sci. Symp. Fla. A and M Univ Silva, J.L., E. Marroquin, C.P. Hegwood, G.R. Silva, and J.O. Garner Jr Quality changes in muscadines for table grapes during refrigerated storage in various packaging systems. Proc. Viticult. Sci. Symp. Fla. A and M Univ. 17: Slinkard, K. and V.L. Singleton Total phenol analysis: automation and comparison with manual methods. Am. J. Enol. Viticult. 28: Smit, C.J.B., H.L. Cancel, and T.O.M. Nakayama Refrigerated storage of muscadine grapes. Amer. J. Enol. Viticult. 22:

53 Smith, B.J., Magee, J.B Limited fungicide applications affect berry rot severity and resveratrol content of muscadine grapes. Phytopathol. 92:577. (abstr.). Smith, B.J Fruit quality, phytochemical content, and disease severity of muscadine grapes affected by fungicide applications. Pharmaceutical Crops 4:21 27 Smittle D.A Requirements for commercial CA storage of muscadine grapes. Proc. Viticult. Sci. Symp. Fla. A and M Univ Starnes Saunders, M.S., F. Takeda, and T.T. Hatton Postharvest physiology and senescence in muscadines. Proc. Fla. State Hort. Soc. 94: Striegler, R.K., P.M. Carter, J.R. Morris, J.R. Clark, R.T. Threlfall, and L.R. Howard Yield, quality, and nutraceutical potential of selected muscadine cultivars grown in southwestern Arkansas. HortTechnology 15: Stringer, S.J., D.A. Marshall, and P. Perkins Veazie Nutraceutical compound concentrations of muscadine (Vitis rotundifolia Michx.) grape cultivars and breeding lines. Acta. Hort. 841: Takeda, F., M. Starnes Saunders, C.F. Savoy, and T.T. Hatton. 1983a. Storageability of muscadines for use as fresh fruit. Proc. Viticult. Sci. Symp. Fla. A and M Univ. 3: Takeda, F., M. Starnes Saunders, and J.A. Saunders. 1983b. Physical and chemical changes in muscadine grapes during postharvest storage. Amer. J. Enol. Viticult. 34: Takeda, F., M. Starnes Saunders, J.A. Saunders, and T.T. Hatton Effects of prestorage treatment and storage temperature on incidence of decay and chemical composition in muscadine grape. Proc. Fla. State. Hort. Soc. 95: Talcott, S.T., and J H. Lee Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. J. Agr. Food Chem. 50: Threlfall, R.T., J.R. Morris, and A. Mauromoustakos Effects of fining agents on transresveratrol concentration in wine. Austral. J. Grape Wine Res. 5: Threlfall, R.T., J.R. Morris, J.F. Meullenet, and R.K. Striegler Sensory characteristics, composition, and nutraceutical content of juice from Vitis rotundifolia (muscadine) cultivars. Amer. J. Enol. Viticult. 58: Threlfall, R.T., J.R. Morris, L.R. Howard, C.R. Brownmiller, and T.L. Walker Pressing effects on yield, quality, and nutraceutical content of juice, seeds, and skins from Black Beauty and Sunbelt grapes. J. Food Sci. 70:

54 Walker, T.L., J.R. Morris, R.T. Threlfall, G.L. Main, O. Lamikanra, and S. Leong Density separation, storage, shelf life, and sensory evaluation of 'Fry' muscadine grapes. HortScience 36: Yi, W., C.C. Akoh, and J. Fischer Study of anticancer activities of muscadine grape phenolics in vitro. J. Agr. Food Chem. 53:

55 Chapter 1 THE EFFECT OF FIELD FUNGICIDE APPLICATIONS ON MUSCADINE GENOTYPE POSTHARVEST STORAGE AND NUTRACEUTICAL CONTENT Abstract A major limiting factor in muscadine grape (Vitis rotundifolia Michx.) commercialization is deterioration during storage. Research on table grapes has shown that field fungicide applications increase storability, but little is known of their effect on muscadines. The effect of field applications of fungicides on composition attributes during postharvest storage was evaluated on five muscadine cultivars (Nesbitt, Southern Jewel, Summit, Supreme, and Tara) and four breeding selections from the University of Arkansas Fruit Breeding Program (based at the Fruit Research Station, Clarksville, AR). There were two field treatments (no fungicide and fungicide). For the fungicide treatment, alternating applications of two fungicides were applied at 14 d intervals during berry development. Storage and composition attributes including berry volume, titratable acidity (TA), ph, soluble solids (%), color (L*, Chroma, and hue), firmness (force to penetrate berry skins), storage weight loss (%), and unmarketable fruit (%) were evaluated every 7 d for 3 weeks. The nutraceutical measures of total anthocyanins, total ellagitannins, total flavonols, total phenolics, resveratrol, and the antioxidant capacity measurement of oxygen radical absorbance capacity (ORAC) were measured only at date of harvest. Overall, the postharvest storage quality attributes of weight loss, percent unmarketable, force, and volume change were significantly affected by genotype, year, fungicide treatment, and storage time. The berry color attributes of L*, Chroma, and hue and the berry composition attributes of TA, soluble solids, and ph were significantly affected by genotype and year, 38

56 but remained fairly constant across time of storage and fungicide treatments. The berry nutraceutical measurements of total anthocyanins, total ellagitannins, total flavonols, total phenolics, and the antioxidant capacity measurement of ORAC were significantly affected by genotype and year of the study. Additionally, total ellagitannins, total flavonols, and ORAC were affected by fungicide treatments. Overall, resveratrol was only affected by genotype; however in 2013, resveratrol was greater in muscadines in the no fungicide treatment, but was unaffected in AM 27, Southern Jewel, and Supreme were identified as having the highest potential for postharvest storage, while AM 01, AM 15, and Tara had the least potential. AM 27 was also identified as having the overall highest nutraceutical concentration and antioxidant capacity (sum of anthocyanins, total phenolics, flavonols, resveratrol, and ORAC), while AM 28 and Supreme had the lowest. It was determined that field fungicide applications can improve postharvest storage quality of muscadine grapes, but generally berry color and composition are unaffected. Furthermore, field fungicide applications resulted in varying differences in nutraceutical concentrations and antioxidant capacity. 39

57 Introduction Native to the southeastern United States, the muscadine (Vitis rotundifolia Michx.) grape is commonly grown for its unique flavor, high nutraceutical content, and pest and disease resistance, which is often a limiting factor in the production of bunch grapes (Vitis spp.) (Conner, 2009; Silvia et al., 1994; Striegler et al., 2005; Walker et al., 2001). This native grape is currently grown in small commercial vineyards and home plantings, ranging from North Carolina and Florida to eastern Oklahoma and Texas. Arkansas has approximately 230 ha of muscadine in production, making up 10% of the total U.S. production. The recent recognition that the berries are important sources of beneficial antioxidants has increased consumer demand (Perkins Veazie et al., 2012; Striegler et al., 2005). Additionally, alternative crops, including muscadines, are being explored by growers in the South as a means of increasing profits or diversifying farm operations (Conner, 2009). Three of the major limiting factors on fresh market production are uneven ripening, short harvest season, and high perishability of the fruit (James et al., 1999; Morris, 1980; Perkins Veazie et al., 2012). Many variables contribute to muscadine storability, including berry maturity, texture (crispness), weight loss, decay, shriveling, browning, leakage, and amount of dry stem scars. Muscadines harvested at physiologically ripe maturity have been shown to successfully store for 2 to 3 weeks (Perkins Veazie et al., 2012; Takeda et al., 1982). To maintain adequate quality, muscadines should be stored from 1 to 5 C with 85 95% relative humidity (RH) (Lutz, 1938; Sliva et al., 1994; Takeda et al., 1983; Walker et al., 2001). The use of sulfur dioxide (SO2) storage treatment on the quality of bunch grapes is cultivar specific, and with muscadines not reliably beneficial (Ballinger and Nesbitt, 1982a; 40

58 Ballinger and Nesbitt, 1982b; Conner and Maclean, 2012; James et al., 1997; James et al., 1999; Lane, 1978; Lane and Flora, 1980; MacLean et al., 2009; Morris et al., 1992; Smit et al., 1971). While it is well known that fungicide applications benefit other fruits and vegetables, including other Vitis species, little is known about the effect of field fungicide applications on storability and nutraceutical content of muscadines (Smith 2013). It has been shown that field fungicide applications improved the shelf life of Doreen, Hunt, Magnolia, Nevermiss, and Summit muscadine grapes, but Cowart was unaffected (Lane, 1978; Smith and Magee, 2002). Additionally, it has been found that a treatment with a fungicide pre storage was ineffective in managing decay during storage, although microbial spoilage was the major factor contributing to postharvest deterioration (Takeda et al., 1982). Field fungicide applications have been shown to increase resveratrol concentrations in V. vinifera wine grapes and reduce resveratrol concentrations in muscadines (Jeandet, 1995; Magee et al., 2002). The effects of field fungicide applications on other nutraceutical compounds in muscadines are unknown. Muscadine grapes contain phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives (Boyle and Hsu, 1990; Haung et al., 2009; Lee et al., 2005; Pastrana Bonilla et al., 2003; Stringer et al., 2009; Talcott and Lee, 2002). Ellagic acid and other antioxidants have been shown to demonstrate anticarcinogenic activity in the colon, lungs, and liver, as well as a reduction of birth defects in rats and mice, and two forms of colon cancer in humans (Ector, 2001; Yi et al., 2005). Polyphenolic concentrations usually increase in muscadines as fruit ripens (Lee et al., 2005) and are higher in wine than in unfermented juices extracted from berries with identical fruit pressing procedures 41

59 (Musingo et al., 2001; Talcott and Lee, 2002). Research has shown that the muscadine grape possesses one of the highest antioxidant levels among fruit crops (Greenspan et al., 2005). Some of these components of muscadines have been shown to have anti cancer, anti mutagen, and anti inflammatory properties, and to reduce levels of glucose, insulin, and glycated hemoglobin in people with diabetes (Banini et al., 2006; Bralley et al., 2007; Greenspan et al., 2005; God et al., 2007; Yi et al., 2005). Since the implementation of a muscadine breeding program at the University of Arkansas in 2005, selections have been made based on flower type, fruit size, time of ripening, hardiness, improved texture, and dry stem scar. Although increased crispiness and a greater percentage of dry stem scars has been observed, it is unknown whether there has been a true improvement in postharvest quality of muscadines. Nutraceutical levels in muscadines vary among genotypes (Marshall et al., 2012), and no information has been collected on the nutraceutical content of the University of Arkansas breeding selections (J.R. Clark, personal communication). The objectives of this study were to determine the effect of field applications of fungicides on the storage performance, berry composition, berry color, and nutraceutical concentrations of muscadine grapes and to develop a postharvest evaluation protocol for Arkansas muscadine genotypes for potential commercial utilization. It was hypothesized that field fungicide applications will increase postharvest storage quality and decrease nutraceutical content and antioxidant capacity of muscadine grapes and that commercial cultivars along with recently developed Arkansas genotypes with improved skin and flesh texture will vary in storage potential. Materials and Methods 42

60 Grapes and Vineyard Vines of nine muscadine genotypes (AM 01, AM 04, AM 15, AM 27, Nesbitt, Southern Jewel, Summit, Supreme, and Tara ) used for the study were grown at the University of Arkansas Fruit Research Station, Clarksville, AR (lat N and long W). Vines were of varying ages within each genotype, most of the cultivars were approximately six years old, while many of the selections were from younger vines three to four years old. The vines were grown in Linker fine sandy loam, in USD hardiness zone 7a, where average annual minimum temperatures reach C. Vines were spaced 6.1 m apart and rows were spaced 3.0 m apart. A single wire trellis was used, and vines were trained to a bilateral cordon. The vines were dormant pruned annually in February using spur pruning with spurs retained of two to four buds in length. Weeds were controlled with pre and postemergence herbicides as needed, and vines did not have any stress from weed competition. Vines were irrigated by drip irrigation as needed, beginning in early June (prior months received adequate rainfall) and continuing through the harvest period. Vines received N fertilization in March of each year at a rate of approximately 70 kg/ha. No insecticides or other pest control compounds were applied to the vines, other than those vines that received the fungicide treatments. The vines used in the study had full crops produced each year, and no crop reduction due to winter injury or other limitation occurred. Thus, the vines produced fruit under representative conditions. Daily maximum and minimum temperatures along with rainfall were recorded at the research location to characterize the environment the vines were subjected to and potential differences among years (Table A.1). Fungicide applications 43

61 The vines of nine muscadine cultivars and selections were used for fungicide treatments. Each genotype had a single vine treated while the other did not receive fungicide applications (berries from the fungicide treated vines were referred to as fungicide treated berries and berries from the no fungicide treated vines were referred to as no fungicide treated berries). A rotation of field fungicide applications of Abound (azoxystrobin: methyl (E) 2 {2 [6 (2 cyanophenoxy) pyrimidin 4 yloxy] phenyl} 3 methoxyacrylate*) and Rally (myclobutanil: a butyl a (4 chlorophenyl) 1H 1,2,4,triazole 1 propanenitrile) were applied with a backpack sprayer every 14 d beginning when the fruit was approximately 3 5 mm in diameter and after approximately 400 growing degree units were accumulated beginning 1 Jan. Harvest and Transport The muscadines were once over, hand harvested at the Fruit Research Station. Fruit was harvested either early in the morning or late in the afternoon and transported to the University of Arkansas Institute of Food Science and Engineering, Fayetteville, AR., in an air conditioned car on the same day. Harvest date/maturity was based on soluble solids of 18 22% in 2012 and 15 18% in 2013 (due to differences in summer temperature and precipitation), ease of release from the pedicel, and berry color. Both fungicide treated and non fungicide treated vines within the same genotype were harvested on the same day. Storage Study Berries were then hand sorted to remove any split, shriveled, or decayed fruit before packaging to simulate commercial standards. Only sound berries, showing no signs of unmarketability, were stored. The fruit was packaged into hinged standard vented clamshells (18.4 cm x 12.1 cm x 8.9 cm) (H116, FormTex Plastics Corporation, Houston, TX) 44

62 and stored in plastic harvest lugs in cold storage at 2 C with 85 89% RH. From the harvested fruit, six vented clamshell containers were filled to approximately 500 g. Three of these clamshells were used as storage replications for each treatment. Total clamshell weight was determined at date of harvest, and percent weight loss was calculated as percent weight decrease from this initial value. Weight loss and percent unmarketable fruit were evaluated on the storage clamshells every 7 d for up to 21 d. Storage performance was evaluated by removing all the fruit from each clamshell and counting the number of fruit that showed signs of unmarketability, which included individual or a combination of characteristics of browning, softness, mold, rot, leakage, splitting, and shriveling (Conner, 2013; Conner and Maclean, 2012; P. Perkins Veazie, personal communication). Both the unmarketable and marketable berries were returned to the appropriate clamshell each week, and storage measurements were discontinued once the percent unmarketable in all three clamshells reached 50%, or after 3 weeks of storage. Each week during storage, berries were sent to the University of Arkansas Cooperative Extension Service Plant Health Clinic, Fayetteville, AR for disease diagnostics. Reports from the Clinic were provided on the fungal species isolated. The remaining three clamshells were used as composition replications. For composition measurements, every 7 d three berries were removed from each of the three clamshells and used to measure berry volume, Chroma, hue, L*, soluble solids, titratable acidity (TA), ph, and firmness of the skin and flesh. Composition measurements were discontinued once the percent unmarketable in all three clamshells reached 50% or after 3 weeks of storage. Berry and Composition Analysis 45

63 The composition procedures used were modeled from previously reported protocols (Conner, 2013; Conner and Maclean, 2012; Giusti and Wrolstad, 2001; Haung et al., 2009; Sandhu and Gu, 2010; Striegler et al., 2005; Slinkard and Singleton, 1977; Prior et al., 2003; Threlfall et al., 2005; Threlfall et al., 2007; Walker et al., 2001). Berry volume was determined by measuring the height and width of three randomly selected berries from each replication using electronic calipers every 7 d for up to 28 d. The formula used for calculating the berry volume was: berry volume = 4/3π*berry height*berry width. Change in berry volume was determined by calculating the percent of volume difference of the berries during storage from the initial berry volume. Decrease in size during storage is shown with positive values, while an increase in size during storage is shown by negative values. Titratable acidity and ph were measured by an 877 Titrino Plus (Metrohm AG, Herisau Switzerland) with an automated titrimeter and electrode standardized to ph 2.0, 4.0, 7.0, and 10.0 buffers. Titratable acidity was determined using 6 g of juice diluted with 50 ml of deionized, degassed water by titration of 0.1 N sodium hydroxide (NaOH) to an endpoint of ph 8.2, and results were expressed as percent tartaric acid. Soluble solids were measured using a Bausch and Lomb, Inc. Abbe Mark II refractometer (Rochester, NY). Soluble solids, TA, and ph were measured from the juice of the whole berries, strained through cheesecloth to remove any solids. Exterior skin color measurements were determined on each of the three berries every 7 d using a Chroma Meter CR 300 series (Konica Minolta Holdings Inc., Ramsey, NJ). The Commission Internationale de I Eclairage (CIE) Lab transmission L value indicates how dark or light the skin is, with 0 being black and 100 being white. Hue angle describes 46

64 color in angles from 0 to 360 : 0 = red; 90 = yellow; 180 = green; 270 = blue; and 360 = back to red. Chroma is the aspect of color by which the skin colors appears different from gray of the same lightness and corresponds to intensity of the perceived color. Firmness, or the maximum force to penetrate skin and flesh tissues, was determined using the three whole berries per replication. A TA XT2 Texture Analyzer (Stable Micro Systems, Haslemere, UK) with a 2 mm diameter probe was used to penetrate the skin and mesocarp tissues (flesh) to a depth of 10 mm in each berry at a rate of 10 mm. s 1. Measurements are expressed as force in Newtons (N), and the data was analyzed using Texture Expert Version 1.17 (Texture Technologies Corp., Scarsdale, NY). Nutraceutical Analysis Three randomly selected berries from each composition replication of each treatment were used from the harvest date sample to measure oxygen radical absorbance capacity (ORAC), total phenolics, total anthocyanin content, total ellagitannins, total flavonols, and resveratrol content by high performance liquid chromatography (HPLC) with modified methods determined by Cho et al. (2004), Cho et al. (2005), Hager et al. (2008), and Prior et al. (2003). The berries were homogenized three times each for 1 min in alternating washes of 80 ml of extraction solution containing methanol/water/formic acid (MWF) (60:37:3 v/v/v) and acetone/water/acetic (70:29.5:0.5 v/v/v) to the smallest particle size using a Euro Turrax T18 Tissuemizer (Tekmar Dohrman Corp, Mason, OH). Homogenates were then centrifuged for 5 min at 10,000 rpm and filtered through Miracloth (CalBiochem, LaJolla, CA). The samples were taken to a final volume of 250 ml with extraction solvent and stored at 70 C until further. All samples were passed through 0.45 μm filters (Whatman PLC, Maidstone, UK) prior to HPLC analysis. 47

65 Total phenolics were measured using the Folin Ciocalteu assay (Slinkard and Singleton 1977) on a diode array spectrophotometer (8452A; Hewlett Packard, Palo Alto, CA), with a gallic acid standard and a consistent standard curve based on sequential dilutions. Samples were prepared with 1 ml 0.2N Folins reagent, 0.8 ml Na2CO3 (75g/L) and 0.2 ml of extracted sample with a reaction time of 2 h. Absorbance was measured at 760 nm, and results were expressed as gallic acid equivalents (GAE). For flavonoid analysis, subsamples (5 ml) of solvent extracts were evaporated to dryness using a SpeedVac concentrator (ThermoSavant, Holbrook, NY) with no radiant heat and suspended in 1 ml of aqueous 3% formic acid solution. Samples (1 ml) were analyzed using a Waters HPLC system equipped with a model 600 pump, a model 717 Plus autosampler and a model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Symmetry C18 column (Waters Corp, Milford, MA) preceded by a 3.9 mm 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid and methanol from 2% to 60% for 60 min at 1 ml min 1. The system was equilibrated for 20 min at the initial gradient prior to each injection. Detection wavelength was 510 nm for anthocyanins. Individual anthocyanin glycosides were quantified as delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn), pelargonidin (Pg), and malvidin (Mv) glucoside equivalents using external calibration curves of authentic standards obtained from Polyphenols (Sandnes, Norway). Total flavonols and anthocyanins were calculated as the sum of individual compounds and their derivatives. For total flavonol and ellagitannin analysis, samples (5 ml) of solvent extracts were evaporated to dryness using a SpeedVac concentrator with no radiant heat and suspended in 1 ml of aqueous 50% methanol solution. The samples were then analyzed 48

66 using a Waters HPLC system (Waters Corp, Milford, MA) equipped with a model 600 pump, model 717 plus autosampler and model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Aqua C18 column (Phenomenex, Torrance, CA) preceded by a 3.0 mm 4.0 mm ODS C18 guard column (Phenomenex). The mobile phase was a gradient of 20 g L 1 acetic acid (A) and 5 g L 1 acetic acid in water and acetonitrile (50:50 v/v, B) from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min. The system was equilibrated for 20 min at the initial gradient prior to each injection. A detection wavelength of 360 nm was used for flavonols and 280 nm for ellagitannins at a flow rate of 1 ml min 1. Flavonols and ellagitannins were expressed as mg rutin equivalents kg 1 fresh weight, and mg ellagic acid equivalents kg 1 fresh weight, respectively. Trans resveratrol (3,4,5 Trihydroxy trans stilbene, 5 [(1E) 2 (4 Hydroxyphenyl)ethenyl] 1,3 benzenediol) concentrations were confirmed using an analytical standard (ID: ; Sigma Aldrich Co. LLC, St. Louis, MO). For compound confirmation, a representative black and bronze genotype was analyzed with HPLC/MS. For HPLC/MS analysis the HPLC apparatus was interfaced to a Burker Esquire (Burker Corporation, Billerica, MA) LC/MS ion trap mass spectrometer. Mass spectral data were collected with the Bruker software, which also controlled the instrument and collected the signal at 360 or 510 nm. Typical conditions for mass spectral analysis in positive ion electrospray mode for anthocyanins and negative ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 ml min 1 and a temperature of 300 C. Data were collected in full 49

67 scan mode over a mass range of m/z at 1.0 s per cycle. Characteristic ions were used for peak assignment. The ORAC of extracts was measured using the method of Prior et al. (2003) modified for use with a FLUOstar Optima microplate reader (BMG Labtechnologies, Durham, NC) using fluorescein as fluorescent probe. Fruit extracts were diluted 1600 fold or more with phosphate buffer (75 mm, ph 7) prior to ORAC analysis. The assay was carried out in clear 48 well Falcon plates (VWR, St. Louis, MO). Each well had a final volume of 590 μl. Initially, 40 μl of diluted sample, Trolox (TE) standards (6.25, 12.5, 25, 50 μm) and blank solution (75 mm, ph 7 phosphate buffer) were added to each well using an automatic pipette. The FLUOstar Optima instrument equipped with two automated injectors was then programmed to add 400 μl of fluorescein (0.108 μm) followed by 150 μl of AAPH (31.6 mm) to each well. Fluorescence readings (excitation 485 nm, emission 520 nm) were recorded after the addition of fluorescein, after the addition of AAPH and every 192 s thereafter for 112 min to reach 95% loss of fluorescence. Final fluorescence measurements were expressed relative to the initial reading. Results were calculated based upon differences in areas under the fluorescein decay curve between the blank, samples, and standards. The standard curve was obtained by plotting the four concentrations of TE against the net area under the curve (AUC) of each standard. Final ORAC values were calculated using the regression equation between TE concentration and AUC and are expressed as μmol TE equivalents kg 1 fresh weight. Experimental Design The storage experiment was a designed split split plot with three replications of each genotype and fungicide treatment. The first split was storage (weeks 0, 1, 2, and 3) 50

68 and the second split was year (2012 and 2013). The nutraceutical experiment was a split plot design with three replications of each genotype and treatment, with the split being year (these measurements were only done on the harvest date, not at each storage date). A single vine was used as an experimental unit. Experimental Analysis The data were analyzed by analysis of variance (ANOVA) using JMP (version 11.0; SAS Institute Inc., Cary, NC). Tukey s Honest Significant Difference and Student s t Test was used for mean separations (p = 0.05). Associations among all dependent variables were determined using multivariate pairwise correlation coefficients of the mean values using JMP (version 11.0; SAS Institute Inc., Cary, NC). Results Initial Attributes The initial measurement of berry force, TA, ph, soluble solids, L*, Chroma, and hue angle are of particular importance. Averaged across years and fungicide treatments AM 04 and Nesbitt had the highest initial force (11.1 N for both genotypes), while AM 01, AM 15, and Tara had the least (8.9, 8.6, and 8.1 N, respectively) (Table 1.1). TA ranged from 0.5 to 0.6, while ph ranged from 3.4 (AM 01 and Southern Jewel ) to 3.8 (AM 15, Summit and Supreme ) and soluble solids ranged from 17.6 % (AM 04) to 21.8 % ( Supreme ), averaged across years and fungicide treatments (Table 1.1). AM 01 and AM 04 had the highest L* values (66.8 and 66.3, respectively), while AM 15, Southern Jewel, and Tara had the lowest (26.8, 26.7, and 27.7, respectively) (Table 1.1). The bronze genotypes Chroma values that ranged from 2.2 (AM 15) to 10.1 (AM 01) and hue angles ranging from 70.8 ( Summit ) to ( Tara ), while the black genotypes Chroma values ranged from

69 Table 1.1. Initial values for force to penetrate berry skin, titratable acidity, ph, soluble solids, L*, Chroma, and hue angle averaged across year and fungicide treatment. Genotype Berry skin color Force (N) Titratable acidity (%) ph Soluble solids (%) L* Chroma Hue angle AM 01 Br z AM 04 Bl AM 15 Br AM 27 Bl Nesbitt Bl Southern Jewel Bl Summit Br Supreme Bl Tara Br z Bronze = Br and black = Bl. 52

70 ( Southern Jewel ) to 15.0 (AM 27) and hue angles that ranged from 90.5 ( Supreme ) to ( Southern Jewel ) (Table 1.1). Berry Storage Attributes The postharvest fruit diseases present were identified as black rot (Guignardia [Phyllosticta] bidwellii [ampelicida]), myrothecium leaf spot (Myrothecium sp./spp.), and botrytis fruit rot, (Botrytis sp./spp.). Occasionally an unknown species of fruit fly (Drosophila sp.) was present, but only in fruit with wet or torn stem scars. The ANOVA F test indicated a significant four way interaction of year by week of storage by genotype by fungicide treatment for weight loss (P=0.0335) and unmarketable berries (P<0.0001) (Table 1.2). The three way interactions of year by week of storage by genotype and year by genotype by fungicide treatment had significant effects for both force to penetrate berry skin (P< and P=0.0029, respectively) and volume change during storage (P< and P=0.0006, respectively) (Table 1.2). Weight loss. The dependent variable weight loss, due to the four way interaction, requires close examination of numerous mean values to evaluate and explain the results (Fig. 1.1). For instance, after 3 weeks of storage, AM 15 fungicide treated fruit in 2012 and Nesbitt no fungicide treated fruit in 2012 had the greatest weight loss (7.1 and 6.5 %, respectively), while Nesbitt and Southern Jewel from all treatments in 2013 each had the least weight loss (2.2 %) (Fig. 1.1 and Table A.2). The variable response of genotypes for years, with the example of Nesbitt highlighted, provides some insight why the interaction was significant. If one looks closer at major trends in the data, it can be seen that overall weight loss values appear to be lower for most genotypes in 2013 compared to 2012 (Fig. 1.1). When the main effect of year is examined, overall mean weight loss in 2012 was 2.6% 53

71 Table 1.2. F test significance from ANOVA for berry weight loss, percent unmarketable fruit, force required to penetrate the berry skin, and percent volume change of the berry during 3 weeks of storage. Highest order interactions are italicized and shaded. Source Degrees of freedom Weight loss (%) Unmarketable (%) Force (N) Volume change (%) Year 1 < < < < Week 3 < < < Year*week 3 < < < < Genotype 8 < < < < Year*genotype 8 < < < < Week*genotype 24 < < < < Year*week*genotype 24 < < < < Fungicide < * Year* fungicide Week* fungicide < Year*week* fungicide < Genotype* fungicide 8 < < < < Year*genotype* fungicide < Week*genotype* fungicide < Year*week*genotype*fungicide <

72 Fig Percent berry weight loss of fungicide and no fungicide treated muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean. Treatment Week Weight loss (%) AM 01 AM 04 AM 15 AM 27 Fungicide Nesbitt Southern Jewel Summit Supreme Tara AM 01 AM 04 AM 15 AM 27 No fungicide Nesbitt Southern Jewel Summit Supreme Tara Year Genotype 55

73 and in 2013 was 1.4% (and was significant in the ANOVA F test) (Table 1.3). If storage time (week) overall means are examined across years, fungicide treatment, and genotype, means for weeks (week 0, 1, 2, and 3 were 0%, 1.4%, 2.7%, 4.0%, respectively) were all significantly different, and the values for weight loss for weeks 2 and 3 were greater than the higher values for year effect (Table 1.3). For genotypes, the overall genotype means ranged from 1.6% for AM 27 to 2.4% for AM 01 with a number of significant differences among the means (Tables 1.5 and A.2). Main effect of fungicide treatment was not significant for weight loss, indicating minimal contribution of this source of variation to the interaction means (Table 1.3). Unmarketability. For dependent variable unmarketable berries, again the four way significant interaction provided for a challenging presentation and explanation of the major sources of variation. AM 01 no fungicide treated berries in 2013 and Nesbitt fungicidetreated fruit in 2012 had the greatest amount of unmarketable berries after 3 weeks of storage (94.9% and 81.7%, respectively), while AM 04 fungicide and no fungicide treated berries in 2013 and Summit fungicide treated berries in 2012 had the least amount of unmarketable berries after 3 weeks (12.6% and 14.5%, respectively) (Fig. 1.2 and Table A.2). To further examine the effects of each source of variation individually, again an examination of year shows that mean unmarketable fruit for 2012 was 17.1% and 2013 was 18.5%, with these means significantly different (P=0.0001) (Table 1.3). Fungicide main effect was greater, with treated vines having an average unmarketable value of 16.5% compared to 19.2% for untreated, again with high significance (P<0.0001) and of greater magnitude (thus more important in contribution) than year (Table 1.3). For storage time, large differences among week means were found, ranging from 0.0% for week 0, 9.3% for 56

74 Table 1.3. F test main effect significance for year on weight loss, percent unmarketable, force required to penetrate the berry skin, and percent volume change of the berry during three weeks of storage, averages across week and fungicide. Source Weight loss (%) Unmarketable (%) Force (N) Volume change (%) Year p value < < < < Treatment Fungicide No fungicide p value < Week d z 0.0 d 9.5 a 0.0 b c 9.3 c 7.1 b 2.6 a b 19.6 b 6.9 b 3.6 a a 42.4 a 6.3 c 1.9 ab p value < < < Genotype AM a z 25.0 ab 6.5 de 4.1 c AM ab 14.6 cde 9.9 a 2.4 c AM ab 20.2 abc 6.3 ef 3.5 c AM c 19.4 bcd 6.5 de 6.0 ab Nesbitt 2.1 ab 16.7 cde 8.8 b 8.0 a Southern Jewel 1.8 bc 14.7 cde 7.4 cd 0.7 abc Summit 1.8 bc 13.6 de 7.9 bc 0.3 bc Supreme 2.2 ab 10.4 e 8.3 bc 7.6 ab Tara 1.9 abc 25.7 a 5.4 f 5.7 ab p value < < < < z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. 57

75 Fig Percent unmarketable fungicide and no fungicide treated fruit of muscadine genotypes stored at 2 3 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean. Treatment Week Unmarketable (%) AM 01 AM 04 AM 15 AM 27 Fungicide Nesbitt Southern Jewel Summit Supreme Tara AM 01 AM 04 AM 15 AM 27 No fungicide Nesbitt Southern Jewel Summit Supreme Tara Year Genotype 58

76 week 1, 19.6% for week 2, and 42.4% for week 3, with all means significantly different from each other (P<0.0001) (Table 1.3). Further, there was a substantial range among genotypes for this variable, with main effect means ranging from a low of 10.4% for Supreme to 25.7% for Tara (Table 1.3). Some genotypes, such as AM 04, in 2013, and Summit in 2012 had lower increases in unmarketable fruit as length of storage increased, while many genotypes had major increases in unmarketable values particularly in week 3 (Tables 1.3 and A.2). Those genotypes that have less unmarketable berries during storage with consistent performance are valuable to identify examination of cultivar potential. Force. Force to penetrate berry skin, with increasing force indicating the firmness of the berry and ability to retain this characteristic in storage, had two three way interactions in the ANOVA with both year and genotype sources of variation in both interactions (Table 1.2). In examining the year by week by genotype interaction means, one can see that after 3 weeks of storage, AM 15 in 2012 and Tara in 2013 required the least amount of force to penetrate the berry skin (2.3 and 3.9 N, respectively), while AM 04 in 2013 required the most force to penetrate the berry skin (11.4 N) (Fig. 1.3 and Table A.2). The means for the year by genotype by fungicide interaction indicated both fungicide and no fungicidetreated AM 04 in 2013 required the highest force to penetrate the berry skin (11.9 and 11.7 N, respectively), while both fungicide and no fungicide treated Tara and AM 15 in 2012 required the least (4.9, 4.8, and 5.1 N, respectively) (Fig. 1.4 and Table A.2). In further examining contributions of the sources of variations to mean differences, year had almost a 2.0 N difference and was significant (P=0.0001) (Table 1.3), while fungicide main effect difference was only 0.2 N difference for treated verses untreated vines (P=0.0104) (Table 1.3). Main effect mean for force for weeks of storage ranged from 9.5 N for week 0 to 6.3 N 59

77 Fig Force to penetrate skin of muscadine genotypes stored at 2 3 C for 3 weeks averaged across fungicide treatment. Each standard error bar is constructed using 1 standard error from the mean. Force (N) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Week Genotype h b i d i d d f h 60

78 Fig Force to penetrate berry skin of fungicide and no fungicide treated muscadine genotypes average across weeks. Each standard error bar is constructed using 1 standard error from the mean. Force (N) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Treatment Fungicide No fungicide Genotype 61

79 for week 3, although the means did not differ for weeks 1, 2, and 3 (Table 1.3). Genotype main effect means ranged from a low of 5.4 N for Tara to 9.9 N for AM 04 (Table 1.3). These findings indicate that genotype and week were greater contributors as sources of variation. Volume Change. Volume change resulted from a decrease in berry size shown with positive numbers, while an increase in size shown by negative numbers. The same three way interactions for force were found for volume change (Table 1.2). For the year by week by genotype interaction, data after 3 weeks of storage for Nesbitt in 2013 indicated the greatest decrease in volume (21.1%), while AM 01 in 2012 had the greatest increase in volume ( 19.0%) after 3 weeks of storage (Fig. 1.5 and Table A.2). For the other three way interaction, Nesbitt fungicide and no fungicide treated berries in 2013 and Supreme no fungicide treated and AM 27 fungicide treated fruit in 2012 had the greatest volume change (13.3, 14.9, 14.1, and 13.9%, respectively), while AM 01 no fungicide treated and AM 15 fungicide treated fruit in 2012 had the least volume change ( 16.9 and 14.1%, respectively), across storage time (Fig. 1.6 and Table A.2). For main effects, fungicide did not impact volume change (Table 1.3), while year had a 2.1% mean difference with 2013 being significantly higher (Table 1.3). Week had a difference of 3.6% between week 0 and week 2, and genotype main effect means differed by 9.8% change, the greatest of any variables (Table 1.3). The ANOVA F test indicated a significant four way interaction of year by week of storage by genotype by fungicide treatments for the dependent variable TA (P=0.0040) (Table 1.4). Soluble solids content and ph had a significant F test for the three way Berry Composition 62

80 Fig Percent change in volume of muscadine genotypes stored at 2 3 C for 3 weeks averaged across fungicide treatment. Values at week 0 (date of harvest) were excluded. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean. Volume change (%) Year Week AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Genotype 63

81 Fig Percent change in volume of muscadine genotypes with fungicide and no fungicide treatments. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean. Volume change (%) Year Treatment Fungicide No fungicide AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Genotype 64

82 interaction for year by week of storage by genotype (P= and P=0.0011, respectively) and with year by genotype by fungicide treatments (P< and P=0.0002, respectively) (Table 1.4). There were significant two way interactions of year by genotype (P<0.0001), week of storage by genotype (P=0.0394), and year by fungicide treatments (P=0.0197) for the dependent variable SS/TA (Table 1.4). Titratable Acidity. Fungicide treated berries of Supreme at harvest in 2013 had the highest TA (0.64%), while no fungicide treated berries of Southern Jewel stored for 2 weeks and fungicide treated berries of AM 01 at date of harvest in 2012 had the lowest TA (0.47% and 0.48%, respectively) (Fig. 1.7 and Table A.3). In 2012, TA was 0.56% and in 2013 TA was 0.57% resulting in a significant difference (<0.0001) (Table 1.4) when averaged across genotype, week, and fungicide treatments. For each week of storage (0, 1, 2, and 3), TA was 0.57% when averaged across year, genotype, and fungicide treatments, with no significant differences. Based on the lack of differences in week, year, and fungicide treatment one can see that genotype was the greatest contributor as a source of variation for TA (Table 1.4 and Fig. 1.7). ph. The fruit of Supreme at date of harvest in 2013 had the overall highest ph (4.1), while the fruit of AM 15 stored for 3 weeks and Southern Jewel at date of harvest in 2012 had the lowest ph (3.3) across fungicide treatments (Fig. 1.8 and Table A.3). The fungicidetreated fruit of Supreme in 2013 had the highest ph (3.9), while the fungicide treated fruit of AM 15 and Southern Jewel in 2012 had the lowest ph (3.3) across storage time. (Fig. 1.9 and Table A.3). When averaged across genotype, year, and fungicide treatment, the ph for each week of storage (0, 1, 2, and 3) was 3.6. In 2012 the average ph was 3.6, while in 2013 it was 3.7, which resulted in a significant difference (<0.0001) (Table 1.4), but only a 65

83 Table 1.4. F test significance from ANOVA for ph, percent titratable acidity, percent soluble solids, and SS/TA ratio for four sources of variation of muscadine grapes during 3 weeks of storage. Highest order interactions are italicized and shaded. Source Degrees of freedom ph Titratable acidity (%) z Soluble solids (%) SS/TA y Year 1 < < < < Week Year*week Genotype 8 < < < < Year*genotype 8 < < < < Week*genotype Year*week*genotype Fungicide Year* fungicide Week* fungicide Year*week* fungicide Genotype* fungicide Year*genotype* fungicide < Week*genotype* fungicide Year*week*genotype*fungicide z Titratable acidity expressed as tartaric acid ysoluble solids to titratable acidity ratio 66

84 Year Tara Supreme Summit Fig Percent titratable acidity of fungicide and no fungicide treated muscadine genotypes stored at 2 3 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. Titratable acidity measured as tartaric acid. Treatment Fungicide No fungicide Titratable acidity (%) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Genotype Week

85 Year Tara Fig ph of muscadine genotypes stored at 2 3 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Genotype Week ph 68

86 0.1 difference in means. The lack of differences in week, year, and fungicide treatment indicated that genotype was the greatest contributor as a source of variation for ph (Table 1.4 and Figs. 1.8 and 1.9). Soluble Solids. Across fungicide treatments, Summit harvest, after 2 weeks of storage and after 3 weeks of storage, and AM 01 after 1 week of storage in 2012 had the highest soluble solids (26.8, 25.9, 25.1, and 25.1%, respectively) (Fig. 1.10). Across storage time and fungicide and no fungicide treatments, the fruit of Summit and AM 01 has the highest SSC (25.6, 25.6, 25.5, and 23.9, respectively) (Fig. 1.11). The difference in means of soluble solids for year was 3.5%, when averaged across fungicide treatments, genotype, and week of storage. The difference in soluble solids for fungicide treated and no fungicidetreated fruit was 0.1% averaged across genotypes and year, week, and genotype. For soluble solids, no differences among week means were found (Table 1.4), with means of 18.9% for week 0, 19.2% for week 1, 19.3% for week 2, and 19.2% for week 3. Based on minimal difference of week and fungicide treatment, one can see that genotype and year were the greatest contributors as sources of variation for soluble solids (Table A.3 and Figs and 1.11). Soluble Solids/Titratable Acidity Ratio. The berries of Supreme in 2013 had the highest SS/TA ratio (83.5), while Southern Jewel, Supreme, and AM 04 in 2013 had the lowest (43.3, 42.9, and 42.5, respectively), across storage time and fungicide treatments (Table 1.5). The fruit of Supreme at harvest had the highest SS/TA ratio (68.7), while the fruit of AM 15 after 3 weeks of storage had the lowest (47.8), across fungicide treatments and year (Table 1.6). Across all genotypes, both fungicide and no fungicide treated berries harvested in 2013 had higher SS/TA ratios (67.2 and 63.1, respectively) than the fungicide 69

87 Year Tara Fig Percent soluble solids of muscadine genotypes stored at 2 3 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Genotype Week Soluble solids (%) 70

88 Fig Percent soluble solids of fungicide and no fungicide applied muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. Treatment Fungicide No fungicide Year Tara Supreme Summit Southern Jewel Nesbitt AM 27 AM 15 AM 04 AM 01 Genotype Soluble solids (%) 71

89 Table 1.5. Soluble solids to titratable acidity ratio for muscadine genotypes. Year Genotype AM de z 49.9 e g AM g 66.8 bc AM e g 53.7 d f AM fg 60.4 cd Nesbitt 45.3 fg 66.7 bc Southern Jewel 43.3 g 62.7 cd Summit 54.5 d f 73.4 b Supreme 42.9 g 83.5 a Tara 47.9 e g 69.2 bc z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. Table 1.6. Soluble solids to titratable acidity ratio of muscadine genotypes stored at 2 3 C for 3 weeks. Week Genotype AM c i z 49.0 kl 52.3 g l 52.1 g l AM c h 53.4 f l 51.6 h l 55.1 e l AM i l 48.8 kl 52.1 g l 47.8 l AM e k 51.2 h l 52.3 g l 52.4 g l Nesbitt 50.4 j l 52.6 g l 60.2 b f 60.7 b f Southern Jewel 49.8 j l 49.0 kl 59.3 c g 53.8 f l Summit 64.9 a c 59.1 c g 64.7 a c 67.0 ab Supreme 68.7 a 63.5 a d 63.6 a d 57.1 d j Tara 61.8 a e 60.5 b f 58.0 c i 54.0 f l z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. 73

90 and no fungicide fruit harvested in 2012 (46.9 and 47.3, respectively) (Table 1.7). Across fungicide treatments, genotypes, and weeks of storage, the differences in year means of SS/TA ratio were The mean SS/TA was 57.0 for fungicide treated fruit, and 55.2 for the no fungicide treatments, with no significant differences. Week was a significant source of variation for SS/TA ratio (P=0.0172), week means of 57.7 for week 0, 54.1 for week 1, 57.1 for week 2, and 55.5 for week 3. Similar to soluble solids, genotype and year were the greatest contributors as sources of variation for SS/TA due to minimal differences in week and fungicide treatment. Berry Color The ANOVA F test indicated significant three way interactions of year by week of storage by genotype for Chroma (P<0.0001), hue (P<0.0001), and L* (P<0.0001) (Table 1.8). Additionally, the three way interactions of year by week of storage by fungicide treatment and year by genotype by fungicide treatments were significant (P= and P=0.0316, respectively) (Table 1.8). This data includes both black and bronze genotypes. L* Value. Across fungicide treatments, AM 01 after 1 week of storage in 2013 had the highest L* value (103.4), while AM 04 after 1, 2, and 3 weeks of storage in 2013 had the lowest (24.9, 24.8, and 24.4, respectively) (Fig. 1.12). Across all genotypes, at week 0 for fungicide and no fungicide treated in 2012 and 2013 had the highest (41.8, 42.2, 48.3, and 48.3, respectively), while both fungicide and no fungicide treated berries stored for 1, 2, and 3 weeks in 2012 had the lowest L* values (33.4, 33.0, 32.8, 33.5, 33.5, and 35.5, respectively) (Table 1.9). Across all weeks of storage, fungicide and no fungicide treated fruit of AM 01 in 2013 had the highest L* values (90.1 and 91.2, respectively), while fungicide and no fungicide treated AM 04 in 2013 had the lowest L* values (25.2 and 74

91 Table 1.7. Soluble solids to titratable acidity ratio of fungicide and no fungicide treatments. Treatment Year Fungicide No Fungicide b 47.3 b a 63.1 a Table 1.8 F test significance from ANOVA for berry Chroma, hue, and L* values of the four sources of variation of muscadine grapes during 3 weeks of storage. Highest order interactions are italicized and shaded. Degrees of Chroma Hue L* Source freedom Year 1 < < < Week < Year*week < Genotype 8 < < < Year*genotype 8 < < < Week*genotype 24 < < < Year*week*genotype 24 < < Fungicide Year* fungicide Week* fungicide Year*week* fungicide Genotype* fungicide Year*genotype* fungicide Week*genotype* fungicide Year*week*genotype*fungicide

92 Fig L* values of muscadine genotypes stored at 2 3 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. L* AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Week Genotype Table 1.9. L* values of fungicide and no fungicide treatments during storage at 2 3 C for 3 weeks Week Year Treatment Fungicide 41.8 a z 33.4 b 32.8 b 33.5 b 2012 No fungicide 42.4 a 33.0 b 33.5 b 33.5 b 2013 Fungicide 48.3 a 38.1 ab 39.8 ab 36.9 ab 2013 No fungicide 48.3 a 38.8 ab 39.7 ab 35.2 b z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. 76

93 25.1, respectively) (Fig. 1.13). For L* values, large differences among week means were found, ranging from 45.2 for week 0, 35.9 for week 1, 36.4 for week 2, and 34.8 for week 3, with week 0 means being significantly different from week 1, 2, and 3 means (P<0.0001). Across genotype, year, and week there were no differences in fungicide and no fungicidetreatment means. Across genotype, storage, and fungicide treatment, L* value means were greater in 2013 than in 2012 with a significant difference of 5.2. Like Chroma and hue angle, genotype and year were major contributors as sources of variation for L*, however week was also a major contributor as a source of variation. Chroma. The cultivar Southern Jewel at date of harvest in 2013 had the highest Chroma value (23.3), while AM 27 after 1 and 3 weeks of storage in 2013 had the lowest (1.8 and 1.9, respectively) (Fig and Table A.4). The difference in mean Chroma values for years was 1.0, across genotypes, fungicide treatments, and weeks of storage with Chroma values generally being greater in Chroma was highest at date of harvest and then decreased during storage, with means of 8.8 for week 0, 7.6 for week 1, 7.6 for week 2, and 7.4 for week 3, with week 0 means significantly different from the other weeks of storage (P<0.0001). The difference in Chroma value means for fungicide treatments across year, week, and genotype was 0.2, which was not significant. The major contributors as sources of variation for Chroma were genotype and year, which was most strongly illustrated by Southern Jewel having a Chroma value of 23.3 in 2013 and 6.7 in 2012 at week 0 (Fig 1.14 and Table A.4). Hue Angle. Hue angle was highest for Supreme at harvest and after 1 week and 3 weeks of storage in 2012 (360, 360, and 360 ), while Summit at date of harvest in 2012 lowest (24.4 ). The difference in year means for hue angle was 35.7, with the values in 77

94 Fig L* values of fungicide and no fungicide treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. L* AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Treatment Fungicide No fungicide Genotype 78

95 Fig Chroma of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. Hue angle AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Week Genotype 79

96 2013 being significantly higher (<0.0001) (Table 1.8 and Fig. 1.15). Hue angle means were not different during any week of storage, with means of for week 0, for week 1, for week 2, and for week 3. The difference in fungicide and no fungicide treatment means was 5.6, which was not a significant difference (Table 1.8). Similar to Chroma, the major contributors as sources of variation for hue angle were genotype and year. Nutraceutical Content The ANOVA F test indicated significant three way interactions of year by genotype by fungicide treatment for total anthocyanins (P<0.0001), ORAC (P<0.0001), and total flavonols (P<0.0001) (Table 1.10). The ANOVA F test also indicated a significant two way interaction of year by fungicide treatment for total ellagitannins (P=0.0078), total phenolics (P=0.0229), and resveratrol concentrations (P<0.0001) (Table 1.10). Additionally, there was a significant two way interaction of genotype by fungicide treatment for total ellagitannins (P=0.0180) (Table 1.10). Total Anthocyanins. The fungicide treated AM 04 and the no fungicide treated AM 27 in 2012 had the highest levels of the dependent variable total anthocyanins (127.8 and mg/100g, respectively), and anthocyanins were not detected in bronze genotypes (AM 01, AM 15, Summit, and Tara ) (Fig. 1.16). Across genotype and fungicide treatment, there was a significant difference in mean total anthocyanin concentrations of 19.3 mg/100 g, with total anthocyanins being greater in 2012 than in 2013 (P<0.0001) (Table 1.10). Although not significant, the difference in anthocyanins means across genotypes and year for fungicide treated and no fungicide treated was 2.4. mg/100g. Based on the lack of differences in fungicide treatments, genotype and year were major contributors as sources 80

97 Fig Hue angle of muscadine genotypes stored at 2 3 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean. Hue angle AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Year Week Genotype 81

98 Table F test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and resveratrol concentrations for the three sources of variation. Highest order interactions are italicized. DF z Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmol Trolox equivalents /g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) Year 1 < < < < Genotype 8 < < < < < < Year*fungicide 8 < < < < Fungicide < Year*fungicide Genotype*fungicide 8 < < Year*genotype*fungicide 8 < < < z Degrees of freedom. 82

99 Fig Total anthocyanin content of fungicide and no fungicide treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. Year Total anthocyanins (mg/100 g) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Treatment Fungicide No fungicide Genotype 83

100 of variation for total anthocyanin concentrations. ORAC. The fungicide treated AM 27 in both 2012 and 2013 had the greatest ORAC values (125.3 and µmol Trolox equivalents/g, respectively), while the no fungicidetreated Tara in 2012 had the lowest ORAC values (47.7 µmol Trolox equivalents/g) (Fig. 1.17). The difference in ORAC value means across genotype and year for fungicide and no fungicide treated was 6.5 µmol Trolox equivalents/g, with the overall fungicide treated fruit having significantly higher ORAC values (P<0.0001). Across fungicide treatments and genotype, the mean difference in ORAC for year was 5.9 µmol Trolox equivalents/g, with 2013 having significantly higher ORAC levels (P<0.0001) (Table A.5 and Fig. 1.17). Thus, year, genotype, and fungicide treatment were all identified as major contributors of sources of variation for ORAC levels (Fig. 1.17). Total Flavonols. No fungicide treated Summit in 2012 had the highest total flavonol concentrations (63.1 mg/100 g), while fungicide treated Supreme in 2012 had the lowest (4.9 mg/100 g) (Fig. 1.18). The fungicide and no fungicide treated berries of 2013 had the highest levels of total flavonols (9.2 and 7.9 mg/100 g), while the fungicide and no fungicide treated fruit in 2012 had the lowest total flavonol content (5.8 and 4.8 mg/100 g). Across genotype and fungicide treatments, the mean difference of total flavonols for year was 8.2 mg/100g, with 2012 having significantly higher levels overall (P<0.0001). The overall difference in total flavonol means for fungicide and no fungicide treatments was 4.1 mg/100g, with the fungicide treatment having significantly greater levels across year and genotype (P=0.0012). For the dependent variable total flavonols, year, genotype, and fungicide treatment are all major contributors of sources of variation (Table 1.10 and Fig. 1.18). 84

101 Fig Oxygen radical absorbance capacity of fungicide and no fungicide treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. Year ORAC (µmol Trolox equivelants/g) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Treatment Fungicide No fungicide Genotype 85

102 Fig Total flavonol concentrations of fungicide and no fungicide treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. 86

103 Total Phenolics. Total phenolic concentrations averaged for genotype across year and fungicide treatments ranged from mg/100 g ( Southern Jewel ) to mg/100 g ( Supreme ), with significant differences for genotypes (Table A.5). Fungicide treated berries in 2012 had the highest total phenolic concentration (574.3 mg/100g), while the fungicide and no fungicide treated berries in 2013 had the lowest (512.5 and mg/100 g, respectively) (Tables 1.11 and A.5). Though not significant, the difference in total phenolic means for fungicide treatments was 15.8 mg/100g, across years and genotype. Across genotype and fungicide treatment, the differences in means of years for the dependent variable of total phenolics was 54.7 mg/100g, with 2012 having significantly higher concentrations than 2013 (P=0.0019) (Table 1.11). Year and genotype were identified as major contributors of sources of variation for total phenolics, while fungicide had less contribution (Table 1.11). Resveratrol. Resveratrol concentrations, across year and fungicide treatment, ranged from 10.4 mg/100g (AM 27) to 3.8 mg/100 g (AM 15), with significant differences occurring for genotypes (<0.0001) (Table A.5). The no fungicide treated fruit in 2012 had the highest resveratrol concentrations (7.2 mg/100 g), while the fungicide treated fruit in 2013 had the lowest resveratrol concentrations (4.7 mg/100 g) (Table 1.12). The difference in mean resveratrol concentrations for year was 1.6 mg/100 g, across genotype and fungicide treatment with higher levels found in 2012, though not significant. Across year and genotype, the difference in means for the dependent resveratrol concentrations was 0.95 mg/100 g. In 2012 the difference in mean resveratrol concentration for fungicide and no fungicide treatments was 0.4 mg/100 g, while in 2013 the difference was

104 Table Total phenolic concentations (mg/100 g) of fungicide and no fungicide treated muscadines. Year Treatment Fungicide a z b No Fungicide ab b z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. Table Resveratrol concentations (mg/100 g) of fungicide and no fungicide treated muscadines. Year Treatment Fungicide 6.8 a z 4.7 b No Fungicide 7.2 a 6.2 a z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. 88

105 mg/100 g, with the no fungicide treatment having significantly higher levels (Table 1.12). The independent variables of year and genotype were identified as overall major contributors of sources of variation for resveratrol concentrations, while in 2013 fungicide was also a contributor to variation. Total Ellagitannins. Across years, the fungicide treated fruit of Summit and AM 04 had the highest total ellagitannin concentrations (13.6 and 13.5 mg/100g), while the no fungicide treated AM 01 had the lowest (3.1 mg/100 g) (Fig and Table A.5). The difference in means of years across fungicide treatments and genotypes for the dependent variable of total ellagitannins was 3.3 mg/100g, with 2013 having significantly higher concentrations than 2012 (<0.0001). The difference in means for the fungicide treatments across genotypes and year was 1.1 mg/100g, though not significantly different (Table 1.13). Based on interactions, it appears that fungicide treatment as a source of variation was dependent on year and genotype (Table 1.13 and Fig. 1.19). Like total phenolics, the major contributors as sources of variation for total ellagitannin concentrations were genotype and year. Correlations Though some strong correlations did occur, the dependent variables measured were generally minimally correlated (Table A.6). Force to penetrate the berry skin was negatively correlated with percent unmarketable (r= 0.73) (Table A.6). SS/TA was positively correlated with TA (r=0.73) and ph (r=0.74) (Table A.6). The dependent variable ph was strongly correlated with TA (r=0.99) and negatively correlated with total phenolics (r= 0.70), and total phenolic concentration was positively correlated with ORAC (r=0.77) (Table A.6). Chroma was negatively correlated with both hue angle (r= 0.88) and 89

106 Table Total ellagitannin concentations of fungicide and no fungicide treated muscadines. Year Treatment Fungicide 5.8 b z 9.2 a No Fungicide 4.8 b 7.9 a z Means followed by the same letter are not significantly different at α= 0.05; separated by Tukey s HSD. Fig Total ellagitannin concentration of fungicide and no fungicide treated muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean. Total ellagitannins (mg/100 g) AM 01 AM 04 AM 15 AM 27 Nesbitt Southern Jewel Summit Supreme Tara Treatment Fungicide No fungicide Genotype 90

107 total anthocyanins (r= 0.86), while hue angle was negatively correlated to total flavonols (r= 0.70) (Table A.6). Discussion Berry Storage Attributes Weight Loss and Unmarketable Berries. The average percent unmarketable berries and percent weight loss were generally similar in both years of the study, but varied by genotype. Both percent weight loss and percent unmarketable berries increased during storage and varied among genotypes, and these results were consistent with other studies (Ballinger and Nesbitt, 1982a; James et al., 1997; James et al., 1999; Lutz, 1938; Silva et al., 1994; Takeda et al., 1983). For example, James et al. (1997) found that after 2 weeks of storage Summit had 72% unmarketable berries and 4.9% weight loss, while Fry had 6.2% unmarketable berries and 4.3% weight loss. Overall, berries from fungicide treatments had less unmarketable berries, but had much less effect on weight loss; these results are consistent with data reported by Lane (1978). Percent unmarketable berries were greater in 2013, while the percent weight loss was greater in These differences were potentially due to the unusually hot and dry conditions of the 2012 growing season, which resulted in less fungal growth on the berries, but had lower quality berries (leaked and shriveled) (Table A.1). The postharvest fruit diseases present were identified as black rot (Guignardia [Phyllosticta] bidwellii [ampelicida] Ellis.), myrothecium leaf spot (Myrothecium sp./spp.), and botrytis fruit rot, (Botrytis sp./spp.). This is similar to reports by Lane (1978), Smit et al. (1971) and Takeda et al. (1983). Generally, fruit diseases were not a major cause of unmarketability until 3 weeks of storage. The primary factors involved in unmarketable 91

108 fruit were browning (especially in bronze genotypes), leakage from torn or wet stem scars, and shriveling, which is consistent with similar work reported by Perkins Veazie et al. (2012). Occasionally an unknown species of fruit fly (Drosophila sp) was present, but only in fruit with wet or torn stem scars and did not contribute to unmarketable berries. A major cause of unmarketability in the bronze was browning during storage (especially no fungicide treated AM 01 in 2013), likely caused by chilling injury. The abiotic disorder of chilling injury is common in many horticultural crops, including bananas (Musa paradisiaca L.), citrus (Citrus spp.), sweet potatoes (Ipomoea batatas L.), and tomatoes (Solanum lycopersicum L.) (Wang, 1990). Chilling injury can increase susceptibility to decay through providing a favorable medium for the growth of pathogens (Wang, 1990). The primary symptom of chilling injury identified in this study was brown discoloration of the skin, pulp, and vascular strands of fruit (Himelrick, 2003; Wang, 1990). Table grapes (V. vinifera) were successfully stored at 1 C without showing symptoms of chilling injury (Burg, 2004). Chilling injury has been reported in muscadines stored at or below 1.7 C (Himelrick, 2003; Smittle 1990), but is uncommon in muscadine grapes stored at 2 3 C. It was unexpected to find possible symptoms of chilling injury on the muscadine berries studied, as they were stored above the previously reported threshold of 1.7 C (Himelrick, 2003; Smittle 1990). This illustrates that chilling injury susceptibility might be genotype specific and creates the potential for opportunities of improvement through tolerance selection in fruit breeding programs. Leakage and shriveling are common problems in muscadines during storage, and are managed by removing berries with wet stem scars prior to storage and maintaining high RH during storage (Perkins Veazie et al., 2012; Smit et al., 1971). It has been shown 92

109 that use of plastic film packaging of lemons (Citrus limon L.) and bell peppers (Capsicum annuum L.) prevents water loss, resulting in less leakage and shriveling (Ben Yehoshua et al., 1983). Conversely, muscadines stored in polyethylene bags had less weight loss, but this did not prevent leakage and shriveling; due to the juice retention of the bags (Walker et al., 2001). Edible coatings have been shown to prevent weight loss during storage of fresh blueberries (Vaccinium corymbosum L.), but this is unknown on muscadines (Duan et al., 2011). Other table grapes (V. vinifera) had less leakage and shriveling during storage, as the berries were retained on the pedicel during marketing. It was uncommon that a single genotype performed well in both years of storage. In 2012, the genotypes with the least percent unmarketable were Summit, Southern Jewel and Supreme, while the genotypes AM 01, AM 04, and Nesbitt had the most. Conversely, in 2013, the genotypes AM 04, Nesbitt, and Supreme had the least percent unmarketable, while AM 01, AM 15, Summit, Southern Jewel, and Tara had the most (Fig. 1.2 and Table A.2). In 2012, the genotypes with the least amount of weight loss were AM 27, Summit, Southern Jewel, and Tara, while AM 01, AM 04, AM 15, and Nesbitt had the most. In 2013, the genotypes AM 27, Nesbitt, Southern Jewel, and Summit had the least weight loss, while AM 01, AM 04, AM 15, and Supreme had the most (Fig and Table A.2). This strongly shows the influence of uncontrollable environmental factors on storage performance of muscadines. Additionally, this emphasizes the importance of testing multiple years for postharvest performance in a muscadine storage protocol. Similar to our findings, Ballinger and Nesbitt (1982b) found Nesbitt to have acceptable postharvest storage quality. While James et al. (1999) found Summit to have the greatest percent 93

110 decay and weight loss, I found Summit to have intermediate quality during storage, which was supported by James et al. (1997). Force. Force to penetrate muscadine skin has been shown to be a useful characteristic to assess berry crispiness and texture as well as berry maturity (Conner, 2013); however, use of force to determine storability of muscadine grapes has shown results with no clear trend (Silva et al., 1994; Walker et al., 2001). It has been shown that muscadines require a force up to 13.9 N to penetrate the skin at harvest, which is nearly twice that of V. vinifera cultivars (Conner, 2013). Similarly, we found that muscadine grapes require up 13.2 N to penetrate the skin at harvest (Fig. 1.4 and Table A.2). Similar to the findings of Conner (2013) it was determined that Nesbitt was among the most firm cultivars. Berries stored in 2013 were generally firmer than the berries stored in 2012, further showing the significance of environmental influences on storage quality (Figs. 1.3, 1.4, and Table A.2). Force to penetrate berry skin was negatively correlated to percent unmarketable (r = 0.73), which potentially shows that berries requiring greater force to penetrate the berry skin store better as they were firmer. I found that berry firmness decreased during storage, but was occasionally lowest after 2 weeks of storage, similar results were reported by James et al. (1999); this could be due to water loss causing an increase of firmness at week 3 (Fig. 1.3 and Table A.2). It was found that the genotypes requiring the most force to penetrate the berry skin at harvest also required the most force to penetrate the berry skin after 3 weeks of storage (especially in 2013), showing force to be a strong indicator of storage performance (Fig. 1.3 and Table A.2). Though fungicide treated fruit were often more firm, it was determined that genotype and year were much more influential on berry firmness (Fig. 1.3 and Table A.2). 94

111 Volume Change. Volume change of muscadines during storage is widely unstudied. Unexpectedly, no relationship between storage time and volume change was found. Conversely, it has been found that a decrease in size occurred in mangoes (Mangifera indica L.) and citrus during storage due to water loss (Jha et al., 2006; McCornack, 1975). The muscadine berries stored in 2012 generally increased in volume during storage while the berries stored in 2013 generally decreased in volume during storage (Figs. 1.5 and 1.6). This may have been due to the substantial differences of growing season, as 2012 was extremely hot and dry resulting in lower quality berries, while 2013 had more moderate temperatures and more rainfall (Table A.1). There was also no relationship between fungicide treatments and berry volume (Fig. 1.6 and Table A.2). The lack of correlations found was potentially due to the extreme variation of berry size within each genotype. Berry Composition Muscadines have been shown to increase in soluble solids and decrease in TA during ripening (Carroll and Marcy, 1982; Flora and Lane, 1979; Johnson and Carroll, 1973; Lanier and Morris, 1979; Peynard and Riberau Gayon, 1971; Walker et al., 2001). Unlike other fruits, muscadines are nonclimacteric, and do not continue to accumulate sugars after harvest. Titratable Acidity. It was found that TA stayed relatively constant during storage (Fig 1.7 and Table A.3); this was consistent with the results of other studies (James et al., 1997; James et al., 1999; Takeda et al., 1983; Walker et al., 2001) and is contradictory to Silva et al., (1994), who reported that TA increased during storage and Lutz (1938), who reported that TA decreased during storage. Titratable acidity has also been shown to remain constant during storage of muskmelon (Cucumis melo L.), kiwifruit (Actinidia 95

112 deliciosa L.), mango, pineapple (Ananas comosus L.), strawberry (Fragaria x ananassa Weston.) and watermelon (Citrullus lanatus Thunb.) (Gil et al., 2006). The TA was lower than that of V. vinfera, which ranged from 0.6 to 1.5 % (Mascarenhas et al., 2012). There was no clear identifiable effect on TA of fungicide treatment (Fig. 1.7), which is contrary to Smith (2013), who found that TA was significantly lower in fungicide treated muscadines. There was a strong correlation between TA and ph (r = 0.99) (Table A.6), possibly showing that as berry acidity increased so did ph. Titratable acidity was much more affected by year and genotype, with 2012 generally having lower TA than 2013 (Fig. 1.7), which can be explained by the warmer growing season (Jackson, 1986). The cultivar Supreme generally had the highest percent TA and AM 01 and Southern Jewel had the lowest (Fig. 1.7 and Table A.3). Soluble Solids. Soluble solids were strongly impacted by year and genotype (Figs and 1.11, and Table A.3). Probably due to the extremely hot and dry conditions, soluble solids were higher in 2012, potentially due to the heat and dryness concentrating the sugars, than in 2013 (Figs and 1.11). Overall soluble solids remained constant during storage (Fig and Table A.3); this is consistent with most other studies (James et al., 1997; James et al., 1999; Takeda et al., 1983), but was contradictory to Silva et al. (1994) and Walker et al. (2001), who reported decreased soluble solids during storage, and Lutz (1938), who reported increased soluble solids during storage. Soluble solids have also been shown to remain constant during storage of muskmelon, kiwifruit, mango, pineapple, strawberry, and watermelon (Gil et al., 2006). Soluble solids was found to be similar to that reported for V. vinifera (Mascarenhas et al., 2012). Threlfall et al. (2007) found that Supreme had the lowest soluble solids of all genotypes evaluated, while I identified 96

113 Supreme as being midrange in soluble solids. In 2012, the no fungicide treated vines had slightly higher SSC than did the fungicide treated vines (Fig. 1.11), which supports the findings of Smith (2013). The differences in fungicide treatments for soluble solids were potentially due to variability that occurred among individual muscadine vines. ph. I found that ph was strongly affected by genotype and year (Figs. 1.8 and 1.9). The overall ph measured in 2013 was higher than in 2012 and ranged from 3.8 to 3.4 among the genotypes in 2012 and from 3.9 to 3.4 in 2013 (Fig. 1.8 and Table A.3), this is contradictory with Jackson (1986), who found that high ph is often associated with warmer temperatures during the growing season. I found that ph of muscadines remained constant during storage (Fig. 1.8), which was consistent with several other studies (James et al., 1997; James et al., 1999; Silva et al., 1994; Takeda et al., 1983; Walker et al., 2001). The ph reported for some V. vinifera cultivars was higher than found in muscadines in 2012 and 2013 (Mascarenhas et al., 2012). Additionally, stable ph during storage was found in kiwifruit, mango, pineapple, strawberry, and watermelon, while ph was shown to decrease in muskmelon (Gil et al., 2006). Soluble Solids/Titratable Acidity Ratio. Soluble solids to titratable acidity ratio was strongly influenced by year and genotype (Tables 1.7 and A.3). The overall average SS/TA measured in 2013 was higher than in 2012 (Tables 1.7 and 1.9), resulting in unusually high SS/TA. High temperatures during the growing season of 2012 produced muscadines with lower TA and higher SSC (Jackson, 1986). There were positive correlations between SS/TA and TA (r = 0.73) and ph (r = 0.73), possibly showing that SS/TA increased with increased acidity (Table A.6). Though there was a main effect with storage time, SS/TA did not change consistently during storage, which is contradictory to Lutz (1938), who reported an 97

114 increase of SS/TA during storage time. The SS/TA was higher than reported for some V. vinfiera cultivars (Mascarenhas et al., 2012). Berry Color The United States Department of Agriculture (USDA) currently has no standards in place to grade muscadine berries on the color attributes of L*, Chroma, and hue. The standards for color grades of muscadines state the fruit should be well colored, in the case of black and red varieties that 75% of the surface of the berry shows characteristic color for the variety, while no requirement exist for bronze genotypes except that Carlos, Fry or similar cultivars can show any amount of blush or bronze color on the berry (USDA, 2006). Additionally the USDA information states that black cultivar colors can include reddish purple, purple, and black; red cultivar colors include light pink, pink, red, dark red, and purple; and bronze cultivar colors include light green, straw, amber, and bronze with allowance for an amount of blush or pink color that may also be characteristic for certain cultivars (USDA, 2006). The effect of storage time on L*, hue angle, and Chroma values of fresh market muscadine grape berries is widely unstudied, although it is well studied in juice and wine. Variability of the berry color was strongly influenced by genotype in this study. L* Value. Generally, L* values remained relatively constant during storage; however, with the genotypes AM 04 in 2012 and AM 15, and Southern Jewel in 2013 L* values decreased after the date of harvest, but remained relatively stable during storage (Fig and Tables 1.9 and A.4). A decrease in L* value would represent a darkening of the fruit during storage, as L* measures lightness from completely opaque (0) to completely transparent (100). Silva et al. (1994) found that L* values increased during storage, though 98

115 the differences were not visibly discernable by panelists. Conversely, I found that generally, L* values remained stable or slightly decreased during storage (Fig and Table A.4). Similarly, Hernandez Herrero and Frutos (2014) found that the L* values for model juices of grape, plum, and strawberry stayed relatively constant during storage. Though the L* values of fungicide and no fungicide treated fruit varied among years, there were no significant differences within each year (Table 1.9). Possibly due to the more favorable growing season (Table A.1) resulting in overall lighter colored berries due to less sunburn (darkening of the berry skin due to sun exposure), the L* values were higher in 2013, than in 2012 (Figs and 1.13 and Tables 1.9 and A.4). Overall, L* values were strongly affected by genotype and year, while less affected by week of storage. For week 0, the average L* value for the bronze genotypes was 55.1 and 37.3 for the black genotypes. For weeks 1, 2, and 3 the average L values for the black genotypes was 26.6 and was 47.1 for the bronze genotypes, this illustrates the berries darkened after date of harvest, but remained relatively unchanged during storage. Chroma. I found that Chroma was influenced by storage time, year, and genotype. Walker et al. (2001) found that Chroma of the bronze cultivar Fry ranged from 12.1 to 14.2 based on maturity level. This is similar to the findings of my study. Conner and MacLean (2013) found Chroma values ranging from 2.4 to 22.8 and Threlfall et al. (2007) found Chroma values ranging from 8.0 to 52.8, both of which are consistent with my findings. Fungicide treatments were found to have no effect on Chroma values (Table A.4). I found Chroma generally had no clear pattern during storage (Fig and Table A.4), similarly Hernandez Herrero and Frutos (2014) found that the Chroma values of model juices of grape (V. vinifera), plum (Prunus spp.), and strawberry stayed relatively constant during 99

116 storage. Chroma values were generally highest for bronze genotypes, with the exception of Southern Jewel at harvest in 2013 (Fig and Table A.4), which is similar to the findings of Conner and Maclean (2012). Overall, Chroma values were higher in 2012 than in 2013 (Fig 1.14 and Table A.4), which could be explained by the milder growing season in 2013 (Table A.1). I found that the average Chroma for the bronze genotypes was 12.1, while the black genotypes had an average Chroma of 4.5, showing on average the bronze genotypes were less grey than the black genotypes. Hue Angle. I found hue values ranged from 48.8 to 360, with the darker genotypes having greater hue angles (Fig and Table A.4). Conversely, Conner and MacLean (2013) found hue values that ranged from 1.5 to 91.8 and Threlfall et al. (2007) found hue values ranging from 53.4 to 98.6, while Walker et al. (2001) found hue values that ranged from 76.5 to I found there was no clear relationship between week of storage and hue angle, with values remaining stable during storage. Similarly, Hernandez Herrero and Frutos (2014) found that the hue angles of model juices of grape, plum, and strawberry stayed relatively constant during storage. There was a negative correlation between hue angle and Chroma (r = 0.88) (Table A.6), illustrating that as hue angle increased, Chroma values decreased. Overall, hue values were greater in 2013 than in 2012 (Fig and Table A.4), which might be due to the milder growing season in 2013, resulting in less sunburn (darkening of the berry skin due to sun exposure) (Table A.1). The average hue angle for the black genotypes was 264.9, which falls between blue and green coloration, while the average hue angle was 91.8 for bronze genotypes, which is approximately yellow. Nutraceutical Content. 100

117 Among fruits, muscadines contain some of the highest levels of nutraceuticals; additionally, several of the compounds present are unique to muscadines (Marshall et al. 2012). Studies exploring the effect of field fungicide treatments on muscadine nutraceutical concentrations are limited. Total Anthocyanins. Total anthocyanin concentrations found were similar to those previously reported (Ballinger et al., 1973; Brown, 1940; Conner and MacLean 2013; Lee et al., 2005; Lee and Talcott, 2002; Goldy et al., 1987; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Sandhu and Gu 2010; Striegler et al., 2005; Stringer et al., 2009; Threlfall et al., 2007). Anthocyanins were not detected in any of the bronze genotypes in either year of the study (Fig and Table A.5). Additionally, I found a negative correlation between anthocyanin concentration and Chroma (r = 0.86), possibly showing a relationship that as Chroma decreases anthocyanin concentrations increase (Table A.6). I found greater total anthocyanin concentrations than those reported for other grape species (Hernandez Herrero and Frutos, 2014). Total anthocyanins found were lower than those reported for blackberry (Rubus sp. L.) and highbush blueberry, but greater than those reported for raspberry (Rubus sp. L.) and strawberry (Cordenunsi et al., 2002; Ehlenfeldt and Prior, 2001; Maatta Riihinen et al., 2004; Siriwoharn et al., 2004). Anthocyanin concentrations were generally higher in 2012 than in 2013 with the exceptions of no fungicide treated Nesbitt and Supreme, and fungicide treated Southern Jewel. The differences in total anthocyanins among years may be due to higher temperature and greater sun exposure and therefore greater color development and anthocyanin concentration in the 2012 growing season (Table A.1). The cultivar Nesbitt was identified as having among the highest levels of anthocyanins (Lee and Talcott, 2004), while I found Nesbitt to have some 101

118 of the lowest levels among black genotypes. The cultivar Supreme had among the lowest levels of total anthocyanins, which was also found by Threlfall et al. (2007) and Striegler et al. (2005). Among years, fungicide treatments did not consistently affect total anthocyanin concentrations, and within each year fungicide treatments had few significant differences (Fig. 1.16). Conversely, Nwankno et al. (2011) found that fungicide treatments enhanced the total anthocyanin concentrations of blackcurrants (Ribes nigrum L.). ORAC. Oxygen radical absorbance capacity is widely accepted as being a good estimation of antioxidant capacity of fruits, although its significance is often questioned, as it does not accurately represent the bioactivity of the antioxidants in the human body. The ORAC values I found were similar to those previously reported by Sandhu and Gu (2010) and Talcott and Lee (2002), but were considerably higher than those reported by Lee et al. (2005), Striegler et al. (2005), and Threlfall et al. (2007). I found ORAC values to be greater than or comparable to those found in apple (Malus domestica Borkh.), blackberry, highbush blueberry, plum, orange, red table grapes, strawberry, and white table grapes (Ehlenfeldt and Prior, 2001; Siriwoharn et al., 2004; Wang et al., 1996; Wu et al., 2004). ORAC was found to be higher overall in 2013 than in 2012 (Fig. 1.17), which could possibly be due to the extremely hot and dry growing season in 2012 (Table A.1). Overall, the berries from fungicide treated vines had higher ORAC values than the berries from no fungicide treated vines (Fig. 1.7 and Table A.5). This is contradictory to Nwankno et al. (2011), who found that fungicide treatments had no effect on antioxidant capacity of blackcurrants. It is hypothesized that the reason for this difference in fungicide treatments is due to the sterol inhibiting effect of myclobutanil, the active ingredient in Rally, potentially interfering with the sterol pathway of the muscadines (Fletcher, 1987). The genotype AM 27 had the 102

119 highest ORAC values both years of the study, while Supreme and Tara had the lowest (Fig and Table A.5). Conversely, Threlfall et al. (2007) reported Nesbitt having among the lowest ORAC levels, while Striegler et al. (2005) identified Supreme as having among the highest. Generally, genotypes performed proportionally the same among years of the study (Fig and Table A.5), which shows how significant genotype is on antioxidant capacity. I found a positive correlation between ORAC and total phenolic concentrations (r = 0.77), as expected since total phenolic concentrations are a major component of ORAC, and as total phenolic concentrations increase so does antioxidant capacity (Table A.6). It is important to note that some genotypes differ more than others with fungicide treatment; this shows that ORAC has potential as a possible characteristic goal to be included in breeding programs. Total Flavonols. Total flavonol concentrations found were lower than those reported by Marshall et al. (2012) and Talcott and Lee (2002). Overall, total flavonols were higher in 2012 than in 2013 (Fig and Table A.5). Genotypes performed proportionally similar among years of the study, with AM 15 and Summit having the highest concentrations both years. The bronze genotypes were found to be generally higher in total flavonols than the darker genotypes (Fig. 1.18), this is potentially due to the presence of myricetin in the bronze genotypes (Marshall et al., 2012). Additionally, I found a negative correlation between hue angle and total flavonol concentrations (r = 0.78) (Table A.6). This correlation represents decreasing hue angle being related to increasing total flavonol, which is supported by the data showing the bronze genotypes to have higher flavonol concentrations and lower hue angles (Figs and 1.18). Total flavonol concentrations were higher for the fungicide treated fruit overall, although this varied among genotypes 103

120 and years (Fig and Table A.5). I found total flavonols at higher levels than those found in strawberry, but lower levels than those found in blackcurrant, chokeberry (Aronia mitschurinii A.K.Skvortsov & Maitul), cranberry (Vaccinium oxycoccos L.), and raspberry (Cordenunsi et al., 2002; Hakkinen et al., 1999; Maatta Riihinen et al., 2004). Total Ellagitannins. Total ellagitannin concentrations found were lower than those reported for muscadines by Marshall et al. (2012) and Lee and Talcott (2004), but similar to those reported by Boyle and Hsu (1990), Lee et al. (2005), Pastrana Bonilla et al. (2003), Stringer et al. (2009), and Talcott and Lee (2002). Ellagitannin concentrations were higher than those reported for strawberry (Cordenunsi et al., 2002), but lower than those reported in blackberry and raspberry (Maatta Riihinen et al., 2004; Siriwoharn et al., 2004). Additionally, ellagitannins are absent in all other Vitis species (Marshall et al., 2012). I found total ellagitannins were higher in 2013 than in 2012, and overall there was no significant difference in fungicide treatments among years (Table 1.13). Similar to our findings, Marshall et al. (2012) found Tara to have the lowest total ellagitannin concentrations of the genotypes they studied. Ellagitannin concentrations varied greatly among genotypes and treatments with no consistent effect of fungicide treatments (Fig and Table A.5). These findings are contrary to those reported by Smith (2013), who found the total ellagitannins were greater in no fungicide treated berries. Total Phenolics. I found total phenolic concentrations similar to those previously reported for muscadines (Lee et al., 2005; Lee and Talcott, 2004; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Striegler et al., 2005; Stringer et al., 2009; Talcott and Lee, 2002; Threlfall et al., 2007). I found that total phenolics were negatively correlated with ph (r = 0.70), which supports the findings of Orak (2007) who determined that total phenolic 104

121 concentration was negatively correlated to ph in V. vinifera grapes. Similar phenolic concentrations have been reported in blackberry (Siriwohorn et al., 2004), while lower phenolic concentrations have been found in highbush blueberry and strawberry (Corenunsi et al., 2002; Ehlenfeldt and Prior; 2001). Total phenolics were higher in 2012 than in 2013, likely due to the less favorable growing conditions (Table 1.11). In 2012, total phenolics were higher for the fungicide treatments, while in 2013 no significant differences were found among fungicide treatments (Tables 1.11 and A.5). I found Summit to have among the highest levels of total phenolics, which was similar to the findings of Threlfall et al. (2007). Additionally, I found the cultivar Supreme to have the overall lowest total phenolics, while Striegler et al. (2005), found Supreme to have among the highest total phenolic levels. Resveratrol. I found resveratrol concentrations similar to those previously reported in muscadines (Ector et al., 1996; Magee et al., 2002; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Stringer et al., 2009). As I did, Marshall et al. (2012) found resveratrol in nearly every genotype evaluated. I found no clear relationship between berry color and resveratrol concentrations, conversely Ector et al. (1996) found resveratrol to be greater in black genotypes. Magee et al. (2002) found Summit to have among the highest levels of resveratrol, which is similar to my findings. Resveratrol concentrations were found to be equivalent to those in V. vinifera (Vincenzi et al., 2013). In 2012 no significant differences occurred among fungicide treatments, while in 2013 the no fungicide treated fruit had higher levels of resveratrol than the fungicide treated (Tables 1.12 and A.5). These results are similar to those reported by Smith (2013), who found resveratrol concentrations to be ten times greater in no fungicide treated berries. The differences in fungicide treatment 105

122 among years could be due to the hot and dry conditions during the growing season of 2012 (Table A.1), as resveratrol is produced in response to fungal infection, which occurs more readily in cooler, wetter conditions (Jeandet et al., 1995). Similar findings of increased resveratrol concentrations, of muscadines, resulting from no fungicide applications have been reported (Magee et al., 2002). Conversely, Jeandet et al. (1995) found that while resveratrol concentration in V. vinifera wine increased as B. cinerea infection increased, excessive grey mold development may destroy the induced phytoalexin. Storage Protocol A major component of this study was to determine the important parameters of storage performance of muscadine genotypes, and in so doing develop a storage protocol for the University of Arkansas muscadine breeding program. Overall, both percent unmarketable and percent weight loss increased during storage, showing importance as storage parameters. Force to penetrate the berry skin generally decreased during storage, also showing potential as an important postharvest storage parameter. Percent change in berry volume showed no clear pattern during storage, probably due to the variation in individual berries within each genotype limiting the usefulness of berry volume as a storage measurement. Composition parameters TA, ph, soluble solids, and SS/TA remained relatively constant during storage, therefore are not valuable postharvest storage measurements. Though no clear correlations were identified in this study, it has been shown that soluble solids can be useful in determining maturity, which has been shown to be related with storage performance (Ballinger and McClure, 1983; Carroll and Marcy, 1982). Berry color measurements Chroma and hue angle generally showed no clear pattern during storage, while L* showed a sharp decrease after date of harvest, it remained 106

123 relatively constant during storage. Therefore, it is potentially valuable to determine L* value at date of harvest and again after storage is complete to evaluate color change during storage. The retention of nutraceutical content (total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol) and antioxidant capacity (ORAC) was not evaluated in this study, but in a related study (Study 3), it was determined that all nutraceuticals remained relatively constant during storage with some significant differences among weeks, but no clear linear pattern of change. Literature Cited Ballinger, W.E., E.P. Maness, and W.B. Nesbitt Anthocyanins of black grapes of 10 clones of Vitis rotundifolia, Michx. J. Food Sci. 38: Ballinger, W.E. and W. F. McClure The effect of ripeness on storage quality of Carlos muscadine grapes. Scientia Hort. 18: Ballinger, W.E. and W.B. Nesbitt. 1982a. Postharvest decay of muscadine grapes (Carlos) in relation to storage temperature, time, and stem condition. Amer. J. Enol. Viticult. 33: Ballinger, W.E. and W.B. Nesbitt. 1982b. Quality of muscadine grapes after storage with sulfur dioxide generators. J. Amer. Soc. Hort. Sci. 107: Banini, A.E., L.C. Boyd, J.C. Allen, H.G. Allen, and D.L. Sauls Muscadine grape products intake, diet and blood constituents of non diabetic and type 2 diabetic subjects. Nutrition. 22: Ben Yehoshua, S., B. Shapiro, Z.E. Chen, and S. Lurie Mode of action of plastic film in extending life of lemon and bell pepper fruit by alleviation of water stress. Plant Physiol. 73: Boyle, J.A., and L. Hsu Identification and quantitation of ellagic acid in muscadine grape juice. Amer. J. Enol. Viticult. 41: Bralley, E.E., D.K. Hartle, P. Greenspan, and J.L. Hargrove Topical anti inflammatory activities of Vitis rotundifolia (muscadine grape) extracts in the tetradecanoylphorbol acetate model of ear inflammation. J. Medicinal Food. 10: Brown, W.L The anthocyanin pigment of the Hunt muscadine grape. J. Amer. Chem. Soc. 62:

124 Burg, S.P Postharvest physiology and hypobaric storage of fresh produce. 1 st ed. CABI Publishing, Wallingford, Oxfordshire, U.K. Carroll, D.E. and J.E. Marcy Chemical and physical changes during maturation of muscadine grapes (Vitis rotundifolia). Amer. J. Enol. Viticul. 33: Cho, M.J., L.R. Howard, R.L Prior, and J.R. Clark Flavanoid glycosides and antioxidant capacity of various blackberry, blueberry, and red grape genotypes determined by highperformance liquid chromatography/mass spectrometry. J. Sci. Food Agri. 84: Cho, M.J., L.R. Howard, R.L. Prior, and J.R. Clark Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high performance liquid chromatography/mass spectrometry. J. Sci. Food. Agr. 85: Conner, P.J A century of muscadine Grape (Vitis rotundifolia Michx.) breeding at the University of Georgia. Acta Hort. 827: Conner, P.J Instrumental texture analysis of muscadine grape germplasm. HortScience. 48: Conner, P.J., and D. Maclean Evaluation of muscadine grape genotypes for storage ability. HortScience. 47:S386. (abstr.). Conner, P.J., and D. MacLean Fruit anthocyanin profile and berry color of muscadine grape cultivars and Muscadinia germplasm. HortScience 48: Cordenunsi, B.R., J.R. Oliveira do Nascimento, M.I. Genovese, and F.M. Lajolo Influence of cultivar on quality parameters and chemical composition of strawberry fruits grown in Brazil. J. Agr. Food. Chem. 50: Duan, J., R. Wu, B.C. Strik, and Y. Zhao Effect of edible coatings on the quality of fresh blueberries (Duke and Elliot) under commercial storage conditions. Postharvest Biol. Technol. 59: Ector, B.J Compositional and nutritional characteristics, p In: F.M. Basiouny and D.G. Himelrick, (eds.). Muscadine Grapes. ASHS Press, Alexandria, VA. Ector, B.J., J.B. Magee, C.P. Hegwood, and M.J. Coign Resveratrol concentration in muscadine berries, juice, pomace, purees, seeds, and wines. Amer. J. Enol. Viticult. 1: Ehlenfeldt, M.K., and R.L. Prior Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbush blueberry. J. Agr. Food Chem. 49:

125 Flora, L.F. and R.P. Lane Effects of ripeness and harvest date on several physical and composition factors of Cowart muscadine grapes. Amer. J. Enol. Viticult. 30: Fletcher, R.A Plant growth regulating properties of sterol inhibiting fungicides, p In: S.S. Purohit (ed.). Hormonal regulation if plant growth and development. Springer Sci. and Business Media, Berlin, Germany. Gil, M.I., E. Aguayo, and A.A. Kader Quality changes and nutrient retention in freshcut versus whole fruits during storage. J. Agr. Food. Chem. 54: Giusti, M.M. and R.E. Wrolstad Characterization and measurement of anthocyanin by UV visible spectroscopy. Current Protocols Food Anal. Chem. F1.2.1 F God, J.M., P. Tate, and L.L. Larcom Anticancer effects of four varieties of muscadine grape. J. Medicinal Food. 10: Goldy, R.G., W. E. Ballinger, E. P. Maness, and W. H. Swallow Anthocyanin content of fruit, stem, tendril, leaf, and leaf petiole in muscadine grape. J. Amer. Soc. Hort. Sci. 112: Greenspan, P., A. Ghaffar, J.L. Hargrove, D.K. Hartle, E.P. Mayer, J.D. Bauer, S.H. Pollock, and J.D. Gangemi Antiinflammatory properties of the muscadine grape (Vitis rotundifolia). J. Agr. Food Chem. 53: Hakkinen, S.H., S.O. Karenlampi, L.M. Heinonen, H.M. Mykkanen, and A.R. Torronen Content of edible flavonols quercetin, myricetin, and kaempferol in 25 edible berries. J. Agr. Food. Chem. 47: Hager, T.J., L.R. Howard, R. Liyanage, J.O. Lay, and R.L. Prior Ellagitannin composition of blackberry as determined by HPLC ESI MS and MALD TOF MS. J. Agr. Food. Chem. 56: Hernandez Herrero, J.A., and M.J. Frutos Colour and antioxidant capacity stability in grape, strawberry, and plum peel model juices at different phs and temperatures. Food. Chem. Himelrick, D.G Handling, storage, and postharvest physiology of muscadine grapes. Small Fruits Rev. 2:4: Huang, Z., R.D. Pace, P. Williams, and B. Wang Identification of anthocyanins in muscadine grapes with HPLC ESI MS. Food Sci. Tech. 42: Jackson D.I Factors affecting soluble solids, acid, ph, and color in grapes. Amer. J. Enol. Viticult. 37:

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127 Magee, J.B., B.J. Smith, and A. Rimando Resveratrol content of muscadine berries is affected by disease control spray program. HortScience 37: Marshall, D.A., S.J. Stringer, and J.D. Spiers Stilbene, ellagic acid, flavanol, and phenolic content of muscadine grape (Vitis rotundifolia Michx.) cultivars. Pharmaceutical Crops 3: Mascarenhas, R.d.J., S.d.M. Silva, M.A.C.d. Lima, R.M.N. Mendonca, and H.J. Holschuh Characterization of maturity and quality of Brazilian apirenic grapes in the Sao Francisco river Valley. Cie ncia e Tecnologia de Alimentos. 32: McCornack, A.A Postharvest weight loss of Florida citrus fruits. Proc. Fla. State Hort Soc. 88: Morris, J.R Handling and marketing of muscadine grapes. FruitSouth, 4(2): Morris, J.R., O.L. Oswald, G.L. Main, J.N. Moore, and J.R. Clark Storage of new seedless grape cultivar with sulfur dioxide generators. Amer. J. Enol. Viticult. 43: Musingo, M.N., S.F. O'Keefe, O. Lamikanra, C.A. Sims, and R.P. Bates Changes in ellagic acid and other phenols in muscadine grape (Vitis rotundifolia) juices and wines during storage. Amer. J. Enol. Viticult. 52: Nwankno, A.J., S.L. Gordon, S.R. Verrall, R.M. Brennan, and R.D. Hancock Treatment of fungicides influences phytochemical quality of blackcurrant juice. Ann. Appl. Biol. 160: Pastrana Bonilla, E., C.C. Akoh, S. Sellappan, and G. Krewer Phenolic content and antioxidant capacity of muscadine grapes. J. Agr. Food Chem. 51: Perkins Veazie, P., S. Spayd, B. Cline, and C. Fisk Handling and marketing guide for fresh market muscadine grapes. SFRC E03: Peynard, E. and P. Ribercau Gayun The grape, p In: A.D. Hulme (ed.). The bio chemistry of fruits and Their Products. Vol. 2. Academic Press, Waltham, MA. Prior, R.L., H. Hoang, L. Gu, X. Wu, M. Bacchiocca, L. Howard, M. Hampschwoodill, D. Haung, B. Ou, and R. Jacob Assays for hydrophilic and lipophilic antioxidant capacity [oxygen radical absorbance capacity (ORACF1)] of plasma and other biological and food samples. J. Agr. Food Chem. 51: Prior RL, S.A. Lazarus, G. Cao, H. Muccitelli, and J.F. Hammer stone Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium Spp) using highperformance liquid chromatography/mass spectrometry. J Agr. Food Chem 49:

128 Sandhu, A.K., and L.W. Gu 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 MSn. J. Agr. Food Chem. 58: Savoy, C.F. and T.T. Hatton Post harvest characteristics of some commercially picked muscadine varieties. Proc. Viticult. Sci. Symp. Fla. A and M. Univ Silva, J.L., E. Marroquin, C.P. Hegwood, G.R. Silva, and J.O. Garner Jr Quality changes in muscadines for table grapes during refrigerated storage in various packaging systems. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 17: Siriwoharn, T., R.E. Wrolstad, C.E. Finn, and C.B. Pereira Influence of cultivar, maturity, and sampling on blackberry (Rubus L. hybrids) anthocyanins, polyphenolics, and antioxidant properties. J. Agr. Food Chem. 52: Slinkard, K. and V.L. Singleton Total phenol analysis: automation and comparison with manual methods. Amer. J. Enol. Viticult. 28: Smith, B.J Fruit quality, phytochemical content, and disease severity of muscadine grapes affected by fungicide applications. Pharmaceutical Crops 4: Smit, C.J.B., H.L. Cancel, and T.O.M. Nakayama Refrigerated storage of muscadine grapes. Amer. J. Enol. Viticult. 22: Smith, B.J., and J.B. Magee Limited fungicide applications affect berry rot severity and resveratrol content of muscadine grapes. Phytopathology. 92:577. (abstr.). Smittle, D.A Requirements for commercial CA storage of muscadine grapes. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 13: Striegler, R.K., P.M. Carter, J.R. Morris, J.R. Clark, R.T. Threlfall, and L.R. Howard Yield, quality, and nutraceutical potential of selected muscadine cultivars grown in southwestern Arkansas. HortTechnology 15: Stringer, S.J., D.A. Marshall, and P. Perkins Veazie Nutraceutical compound concentrations of muscadine (Vitis rotundifolia Michx.) grape cultivars and breeding lines. Acta. Hort. 841: Takeda, F., M. Starnes Saunders, C.F. Savoy, and T.T. Hatton Storageability of muscadines for use as fresh fruit. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 3: Takeda, F., M. Starnes Saunders, J.A. Saunders, and T.T. Hatton Effects of prestorage treatment and storage temperature on incidence of decay and chemical composition in muscadine grape. Proc. Fla. State. Hort. Soc. 95:

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130 Appendix A: Meteorological data, interaction means, and correlations. Table A.1. Average monthly maximum and minimum temperatures and total rainfall recorded at the Fruit Research Station; Clarksville, AR (lat N and long W) (2012 and 2013). Year Month Maximum temperature ( C) Minimum temperature ( C) Precipitation (mm) 2012 January February March April May June July August September October November December January February March April May June July August September October November December

131 Table A.2. Interaction means of the postharvest attributes of percent weight loss, percent unmarketable, force to penetrate berry skin, and percent volume change for year, genotype, fungicide treatment, and week of storage at 2 C for 0 3 weeks. Year Genotype Fungicide treatment Week of storage Weight loss (%) Unmarketable (%) Force (N) Volume change (%) 2012 AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 z No fung AM 01 No fung AM 01 No fung AM 01 No fung AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 No fung AM 04 No fung AM 04 No fung AM 04 No fung AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fung AM 15 No fung AM 15 No fung AM 15 No fung AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fung AM 27 No fung AM 27 No fung AM 27 No fung Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fung

132 Year Genotype Fungicide treatment Week of storage Weight loss (%) Unmarketable (%) Force (N) Volume change (%) 2012 Nesbitt No fung Nesbitt No fung Nesbitt No fung S. Jewel Fungicide S. Jewel Fungicide S. Jewel Fungicide S. Jewel Fungicide S. Jewel No fung S. Jewel No fung S. Jewel No fung S. Jewel No fung Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fung Summit No fung Summit No fung Summit No fung Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fung Supreme No fung Supreme No fung Supreme No fung Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fung Tara No fung Tara No fung Tara No fung AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 No fung

133 Year Genotype Fungicide treatment Week of storage Weight loss (%) Unmarketable (%) Force (N) Volume change (%) 2013 AM 01 No fung AM 01 No fung AM 01 No fung AM 04 No fung AM 04 No fung AM 04 No fung AM 04 Fungicide AM 04 No fung AM 04 No fung AM 04 No fung AM 04 No fung AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fung AM 15 No fung AM 15 No fung AM 15 No fung AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fung AM 27 No fung AM 27 No fung AM 27 No fung Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fung Nesbitt No fung Nesbitt No fung Nesbitt No fung S. Jewel Fungicide S. Jewel Fungicide S. Jewel Fungicide S. Jewel Fungicide S. Jewel No fung

134 Year Genotype Fungicide treatment Week of storage Weight loss (%) Unmarketable (%) Force (N) Volume change (%) 2013 S. Jewel No fung S. Jewel No fung S. Jewel No fung Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fung Summit No fung Summit No fung Summit No fung Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fung Supreme No fung Supreme No fung Supreme No fung Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fung Tara No fung Tara No fung Tara No fung z No fung= no fungicide. y S. Jewel= Southern Jewel. 118

135 Table A.3. Interaction means of the composition attributes of percent titratable acidity, percent soluble solids, ph, and soluble solids to titratable acidity of fungicide and no fungicide treated muscadine genotypes stored at 2 C for 0 3 weeks. Year Genotype Fungicide treatment Week of storage TA ph SSC (%) z (%) SS/TA y 2012 AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 x No fung AM 01 No fung AM 01 No fung AM 01 No fung AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 No fung AM 04 No fung AM 04 No fung AM 04 No fung AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fung AM 15 No fung AM 15 No fung AM 15 No fung AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fung AM 27 No fung AM 27 No fung AM 27 No fung Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fung

136 Year Genotype Fungicide treatment Week of storage TA (%) z ph SSC (%) SS/TA y 2012 Nesbitt No fung Nesbitt No fung Nesbitt No fung Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel No fung Southern Jewel No fung Southern Jewel No fung Southern Jewel No fung Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fung Summit No fung Summit No fung Summit No fung Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fung Supreme No fung Supreme No fung Supreme No fung Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fung Tara No fung Tara No fung Tara No fung AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 No fung

137 Year Genotype Fungicide treatment Week of storage TA (%) z ph SSC (%) SS/TA y 2013 AM 01 No fung AM 01 No fung AM 01 No fung AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 No fung AM 04 No fung AM 04 No fung AM 04 No fung AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fung AM 15 No fung AM 15 No fung AM 15 No fung AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fung AM 27 No fung AM 27 No fung AM 27 No fung Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fung Nesbitt No fung Nesbitt No fung Nesbitt No fung Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel No fung

138 Year Genotype Fungicide treatment Week of storage TA (%) z ph SSC (%) SS/TA y 2013 Southern Jewel No fung Southern Jewel No fung Southern Jewel No fung Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fung Summit No fung Summit No fung Summit No fung Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fung Supreme No fung Supreme No fung Supreme No fung Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fung Tara No fung Tara No fung Tara No fung z Titratable acidity measured as tartaric acid y Soluble solids to titratable acidity ratio x No fung= no fungicide. 122

139 Table A.4. Interaction means of the berry color attributes of Chroma, Hue angle, and L* values of fungicide and no fungicide treated muscadine genotypes stored at 2 C for 0 3 weeks. Year of Genotype Treatment Week of Chroma Hue L* study storage angle 2012 AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 No fungicide AM 01 No fungicide AM 01 No fungicide AM 01 No fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 No fungicide AM 04 No fungicide AM 04 No fungicide AM 04 No fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fungicide AM 15 No fungicide AM 15 No fungicide AM 15 No fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fungicide AM 27 No fungicide AM 27 No fungicide AM 27 No fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fungicide Nesbitt No fungicide

140 Year of Genotype Treatment Week of Chroma Hue L* study storage 2012 Nesbitt No fungicide Nesbitt No fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel No fungicide Southern Jewel No fungicide Southern Jewel No fungicide Southern Jewel No fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fungicide Summit No fungicide Summit No fungicide Summit No fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fungicide Supreme No fungicide Supreme No fungicide Supreme No fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fungicide Tara No fungicide Tara No fungicide Tara No fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 Fungicide AM 01 No fungicide AM 01 No fungicide AM 01 No fungicide

141 Year of Genotype Treatment Week of Chroma Hue L* study storage 2013 AM 01 No fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 Fungicide AM 04 No fungicide AM 04 No fungicide AM 04 No fungicide AM 04 No fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 Fungicide AM 15 No fungicide AM 15 No fungicide AM 15 No fungicide AM 15 No fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 Fungicide AM 27 No fungicide AM 27 No fungicide AM 27 No fungicide AM 27 No fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt Fungicide Nesbitt No fungicide Nesbitt No fungicide Nesbitt No fungicide Nesbitt No fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel Fungicide Southern Jewel No fungicide Southern Jewel No fungicide Southern Jewel No fungicide Southern Jewel No fungicide

142 Year of Genotype Treatment Week of Chroma Hue L* study storage 2013 Summit Fungicide Summit Fungicide Summit Fungicide Summit Fungicide Summit No fungicide Summit No fungicide Summit No fungicide Summit No fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme Fungicide Supreme No fungicide Supreme No fungicide Supreme No fungicide Supreme No fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara Fungicide Tara No fungicide Tara No fungicide Tara No fungicide Tara No fungicide

143 Table A.5. Interaction means of the berry nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) of fungicide and no fungicide treated muscadine genotypes. Year Genotype Fungicide Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmolte/g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) 2012 AM 01 Fungicide AM 01 No fung AM 04 Fungicide AM 04 No fung AM 15 Fungicide AM 15 No fung AM 27 Fungicide AM 27 No fung Nesbitt Fungicide Nesbitt No fung S. Jewel Fungicide S. Jewel No fung Summit Fungicide Summit No fung Supreme Fungicide Supreme No fung Tara Fungicide Tara No fung AM 01 Fungicide AM 01 No fung AM 04 Fungicide AM 04 No fung AM 15 Fungicide AM 15 No fung

144 Year Genotype Fungicide Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmolte/g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) 2013 AM 27 Fungicide AM 27 No fung Nesbitt Fungicide Nesbitt No fung S. Jewel Fungicide S. Jewel No fung Summit Fungicide Summit No fung Supreme Fungicide Supreme No fung Tara Fungicide Tara No fung

145 Table A.6. Study 1 multivariate correlation coefficients among muscadine berry storage quality, composition, color, and nutraceutical content for 2012 and *Significant at P=0.05. WL z UM y VC x TA w ph SS v L* Chr u Hue t For s Anth q Ella p ORAC o Fla n TP m WL z UM y -0.1 VC x TA w ph * SS v L* Chr u Hue t * For s * Anth q * Ella p ORAC o Fla n TP m * 0.5 Res l z WL=weight loss of berries (%) y UM=umarketable berries (%) x VC=volume change (%) w TA=titratable acidity (%) measures as tartaric acid v SS=soluble solids (%) u Chr=Chroma t Hue=hue angle s For=force to penetrate berry skin (%) q Anth=total anthocyanins (mg/100 g) p Ella=total ellagitannins (mg/100 g) o ORAC=oxygen radial absorbance capacity (µmol Trolox equivalent/ g) n Fla=total flavonols (mg/100 g) m TP=total phenolics (mg/100 g) l Res=trans-resveratrol (mg/100) 129

146 Chapter 2 EVALUATION OF DIVERSE MUSCADINE GRAPE GENOTYPES FOR POSTHARVEST STORAGE POTENTIAL AND NUTRACEUTICAL CONCENTRATIONS Abstract A major limiting factor in muscadine grape (Vitis rotundifolia) commercialization is deterioration during storage. One solution for extending market seasons and preventing market saturation for fresh muscadines could be the release of new cultivars with improved postharvest storability. Storage and composition attributes including berry volume, titratable acidity (TA), ph, soluble solids (%), berry color (L*, Chroma, and hue), firmness (force to penetrate berry skins), storage weight loss (%), and unmarketable berries (%) were evaluated on eight muscadine cultivars and nine breeding selections grown at the University of Arkansas Fruit Research Station, Clarksville, AR in 2012 and The objective of the study was to identify muscadine genotypes with potential for commercialization and postharvest storability. Muscadine genotypes were hand harvested and evaluated for postharvest performance and composition every 7 d for 3 weeks. The nutraceutical measurements of total anthocyanins, total ellagitannins, total flavonols, total phenolics, resveratrol, and the antioxidant capacity measurement of oxygen radical absorbance capacity (ORAC) of muscadines were measured at date of harvest. Overall, the postharvest storage quality attributes of weight loss, percent unmarketable, firmness, and volume change were significantly affected by genotype, year, and storage time. The berry color attributes, Chroma and hue, and the berry composition attributes of TA, soluble solids, and ph were significantly affected by genotype and year, but remained fairly constant across time of storage. The berry color attribute of L* was affected by genotype, 130

147 year, and week of storage showing a decrease, or darkening, after date of harvest. The berry nutraceutical concentrations of total anthocyanins, total ellagitannins, total flavonols, total phenolics, resveratrol, and the antioxidant capacity measurement of ORAC were significantly affected by genotype and year of the study. Storage attributes varied by genotype and year; overall AM 04, AM 26, AM 28, and Southern Jewel were identified as having the highest potential for postharvest storage, while the genotypes AM 01, AM 15, AM 18, and Nesbitt had the least potential. Nutraceutical content varied by genotypes; overall AM 03, AM 04, AM 27, and Ison had the highest nutraceutical content (sum of anthocyanins, total phenolics, flavonols, resveratrol, and ORAC), while AM 18, AM 28, Supreme, and Tara had the lowest. It was determined that postharvest storage potential, berry composition, berry color, and nutraceutical content were genotype specific, but commercially viable genotypes were identified. 131

148 Introduction Muscadine grapes (Vitis rotundifolia Michx.) are indigenous to the southeastern United States. Muscadines have been under cultivation for over 400 years, originally in the North Carolina Colony followed by surrounding colonies and states (Conner, 2009). This native grape is presently grown in small commercial vineyards and home plantings, ranging from North Carolina and Florida to eastern Oklahoma and Texas. Arkansas has approximately 230 ha of muscadines in production, making up 10% of total US production. The recent recognition that muscadines are sources of beneficial antioxidants has increased consumer demand (Perkins Veazie et al., 2012; Striegler et al., 2005). Additionally, alternative crops, including muscadines, are being explored by growers in the South as a means of increasing profits or diversifying farm operations (Conner, 2009). Muscadine grapes are produced in small loose clusters that do not ripen evenly. The berries are removed from the vines as single berries rather than in clusters as with bunch grapes and are subsequently marketed in packages (often clamshells) (Perkins Veazie et al., 2012). Though commercial shipping for retail marketing is practiced, the potential for expansion in this area exists (Perkins Veazie et al., 2012). Major limiting factors on freshmarket muscadine production include uneven ripening, short harvest season, and high perishability of the fruit (James et al., 1999; Morris, 1980; Perkins Veazie et al., 2012). Storage protocols to assist the muscadine grape industry in providing quality fresh market grapes are limited (Walker et al., 2001). Several variables contribute to muscadine storage performance, including berry maturity, texture (crispness), weight loss, decay, shriveling, browning, leakage, and percentage of dry stem scars (where the berry releases from the pedicel). Muscadines 132

149 harvested at physiological ripeness have been shown to successfully store for 2 to 3 weeks (Lutz, 1938; Perkins Veazie et al., 2012; Takeda et al., 1982). To maintain adequate quality, muscadines should be stored from 1 to 5 C with 85 95% relative humidity (RH) (Lutz, 1938; Silva et al., 1994; Takeda et al., 1983; Walker et al., 2001). The use of sulfur dioxide (SO2) storage treatment on the quality of table grapes is genotype specific, and with muscadines not reliably beneficial (Ballinger and Nesbitt, 1982a; Ballinger and Nesbitt, 1982b; James et al., 1997; James et al., 1999; Lane, 1978; Lane and Flora, 1980; MacLean et al., 2009; Morris et al., 1992; Smit et al., 1971; Conner and Maclean, 2012). Muscadine grapes contain phenolic acids, flavonols, anthocyanins, and ellagitannins (Boyle and Hsu, 1990; Haung et al., 2009; Lee et al., 2005; Pastrana Bonilla et al., 2003; Stringer et al., 2009; Talcott and Lee, 2002). Polyphenolic concentrations usually increase in muscadines as the berries ripen (Lee et al., 2005) and are higher in wine than in unfermented juices (Musingo et al., 2001; Talcott and Lee, 2002). It has been shown that the muscadine grape possesses one of the highest antioxidant levels among fruit crops (Greenspan et al., 2005). Some of these components of muscadines have been shown to have anti cancer, anti mutagen, and anti inflammatory properties, and to reduce levels of glucose, insulin, and glycated hemoglobin in people with diabetes (Banini et al., 2006; Bralley et al., 2007; Greenspan et al., 2005; God et al., 2007; Yi et al., 2005). Since the implementation of a muscadine breeding program at the University of Arkansas in 2005, selections have been made based on improved texture and dry stem scar. Although increased crispiness and a greater percentage of dry stem scars has been observed, it is unknown whether there has been a true improvement in postharvest quality of muscadines. Nutraceutical levels in muscadines vary among genotypes and no 133

150 information has been collected on the nutraceutical content of the University of Arkansas breeding selections (J.R. Clark, personal communication; Marshall et al., 2012). The objectives of this study were to develop a postharvest evaluation protocol for Arkansas muscadine genotypes for potential commercial utilization and to identify superior post harvest storage and handling muscadine genotypes in the University of Arkansas Fruit Breeding Program. It was hypothesized that commercial cultivars along with recently developed Arkansas genotypes with improved skin and flesh texture will vary in storage potential. Materials and Methods Grapes and Vineyard The berries of 17 genotypes (AM 01, AM 02, AM 03, AM 04, AM 15, AM 18, AM 26, AM 27, AM 28, Delicious, Fry, Ison, Nesbitt, Southern Jewel, Summit, Supreme and Tara ) were evaluated for postharvest storability and nutraceutical content. Vines used for the study were grown at the University of Arkansas Fruit Research Station, Clarksville, AR (lat N and long W). Vines were of varying ages within each genotype, most of the cultivars were approximately six years old, while many of the selections were from younger vines three to four years old. The vines were grown in Linker fine sandy loam, in USD hardiness zone 7a, where average annual minimum temperatures reach C. Vine spacing was 6.1 m apart and rows were spaced 3.0 m apart. A single wire trellis was used, and vines were trained to a bilateral cordon. The vines were dormant pruned annually in February using spur pruning with spurs retained of two to four buds in length. Weeds were controlled with pre and postemergence herbicides as needed and vines did not have any stress from weed competition. Vines were irrigated by trickle 134

151 irrigation as needed, beginning in early June (prior months received adequate rainfall) and continuing through the harvest period. Vines received N fertilization in March of each year at a rate of approximately 70 kg/ha. No insecticides or other pest control compounds were applied to the vines. The vines used in the study had full crops produced each year, and no crop reduction due to winter injury or other limitation occurred. Thus, the vines produced fruit under full crop conditions. Daily maximum and minimum temperatures along with rainfall were recorded at the research location to characterize the environment the vines were subjected to and potential differences among years (Table A.1). Harvest and Transport The muscadines were once over, hand harvested at the Fruit Research Station. Fruit was harvested either early in the morning or late in the afternoon and transported to University of Arkansas Institute of Food Science and Engineering, Fayetteville, AR., in an air conditioned car on the same day. Harvest date/maturity was based on soluble solids of 18 22% in 2012 and 15 18% in 2013, due to differences in summer temperature and precipitation, ease of release from the pedicel, and berry color. Storage Study Berries were hand sorted to remove any split, shriveled, or decayed fruit before packaging to simulate commercial standards. Only sound berries showing no signs of unmarketability were used for the study. The fruit was packaged into hinged standard vented polyethylene clamshells (18.4 cm x 12.1 cm x 8.9 cm) (H116, FormTex Plastics Corporation, Houston, TX) and stored in plastic harvest lugs in cold storage at 2 C with 85 89% RH. 135

152 From the harvested fruit, six vented clamshell containers were filled to approximately 500 g. Three of these clamshells were used as storage replications for each genotype. Weight loss and percent unmarketable berries were evaluated on the storage clamshells every 7 d for up to 21 d. Storage performance was evaluated by removing all the berries from each clamshell, and counting the number of berries with signs of unmarketability, which included characteristics of browning, softness, mold, rot, leakage, splitting, and shriveling (Conner, 2013; Conner and Maclean, 2012; P. Perkins Veazie, personal communication). Both the unmarketable and marketable berries were returned to the appropriate clamshell each week, and storage measurements were discontinued once the percent unmarketable in all three clamshells reached 50%, or after 3 weeks of storage. Each week during storage, berries were sent to the University of Arkansas Cooperative Extension Service Plant Health Clinic, Fayetteville, AR for disease diagnostics. Reports from the Clinic were provided on the fungal species isolated. The remaining three clamshells were used as composition replications 1, 2, and 3. For composition measurements, every 7 d three berries were removed from each of the three composition clamshells and used to measure berry volume, Chroma, hue, L*, soluble solids (%), titratable acidity (%) (TA), ph, and firmness of the skin and flesh (N). Berry and Compositional Analysis The composition procedures used were modeled from previously reported protocols (Conner, 2013; Conner and Maclean, 2012; Striegler et al., 2005; Slinkard and Singleton, 1977; Threlfall et al., 2005; Threlfall et al., 2007; Walker et al., 2001). Berry volume was determined by measuring the height and width of three randomly selected berries from each replication using digital calipers. The formula used for 136

153 calculating the berry volume was: berry volume = 4/3π*berry height*berry width. Change in berry volume was determined by calculating the percent of volume difference of the berries during storage from the initial berry volume. Decrease in size during storage was shown with positive values, while an increase in size during storage was shown by negative values. Titratable acidity and ph were measured by an 877 Titrino Plus (Metrohm AG, Herisau Switzerland) with an automated titrimeter and electrode standardized to ph 2.0, 4.0, 7.0, and 10.0 buffers. Titratable acidity was determined using 6 g of juice diluted with 50 ml of deionized, degassed water by titration of 0.1 N sodium hydroxide (NaOH) to an endpoint of ph 8.2, and results were expressed as percent tartaric acid. Soluble solids were measured using a Bausch and Lomb Inc. Abbe Mark II refractometer (Rochester, NY). Soluble solids, TA, and ph were measured from the juice of the whole berries, strained through cheesecloth to remove any solids. Exterior skin color measurements were determined on each of the three berries every 7 d using a Chroma Meter CR 300 series (Konica Minolta Holdings Inc., Ramsey, N J). The Commission Internationale de I Eclairage (CIE) Lab transmission L value indicates how dark or light the skin is, with 0 being black and 100 being white. Hue angle describes color in angles from 0 to 360 : 0 = red; 90 = yellow; 180 = green; 270 = blue; and 360 = back to red. Chroma is the aspect of color by which the skin colors appears different from gray of the same lightness and corresponds to intensity of the perceived color. Firmness, or the maximum force to penetrate skin and flesh tissues, was determined using three whole berries per replication. A TA XT2 Texture Analyzer (Stable Micro Systems, Haslemere, UK) equipped with a 2 mm diameter probe used to penetrate the 137

154 exocarp and mesocarp tissues (flesh) to a depth of 10 mm in each berry at a rate of 10 mm. s 1. Measurements were expressed as force in Newtons (N), and the data was analyzed using Texture Expert Version 1.17 (Texture Technologies Corp., Scarsdale, NY). Nutraceutical Analysis Three randomly selected berries from each composition replication of each treatment were used from the harvest date sample to measure oxygen radical absorbance capacity (ORAC), total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol content by high performance liquid chromatography (HPLC) with modified methods determined by Cho et al. (2004), Cho et al. (2005), Hager et al. (2008), and Prior et al. (2003). The berries were homogenized three times each for 1 min in alternating washes of 80 ml of extraction solution containing methanol/water/formic acid (MWF) (60:37:3 v/v/v) and acetone/water/acetic (70:29.5:0.5 v/v/v) to the smallest particle size using a Euro Turrax T18 Tissuemizer (Tekmar Dohrman Corp, Mason, OH). Homogenates were then centrifuged for 5 min at 10,000 rpm and filtered through Miracloth (CalBiochem, LaJolla, CA). The samples were taken to a final volume of 250 ml with extraction solvent and stored at 70 C until analysis. Prior to HPLC analysis, the samples were filtered through 0.45 μm filters (Whatman PLC, Maidstone, UK). Total phenolics were measured using the Folin Ciocalteu assay (Slinkard and Singleton 1977) on a diode array spectrophotometer (8452A; Hewlett Packard, Palo Alto, CA), with a gallic acid standard and a consistent standard curve based on sequential dilutions. Samples were prepared with 1 ml 0.2N Folins reagent, 0.8 ml Na2CO3 (75g/L) and 0.2 ml of extracted sample with a reaction time of 2 h. Absorbance was measured at 760 nm, and results were expressed as gallic acid equivalents (GAE). 138

155 For flavonoid analysis, subsamples (5 ml) of supernatant were evaporated to dryness using a SpeedVac concentrator (ThermoSavant, Holbrook, NY) with no radiant heat and suspended in 1 ml of aqueous 3% formic acid solution. Samples (1 ml) were analyzed using a Waters HPLC system equipped with a model 600 pump, a model 717 Plus autosampler and a model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Symmetry C18 column (Waters Corp, Milford, MA) with a 3.9 mm 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid and methanol from 2% to 60% for 60 min at 1 ml min 1. Prior to each injection, the system was equilibrated for 20 min at the initial gradient. Detection wavelength was 510 nm for anthocyanins. Individual anthocyanin diglycosides were quantified as delphinidin (Dp), cyanidin (Cy), petunidin (Pt), peonidin (Pn), pelargonidin (Pg), and malvidin (Mv) diglycoside equivalents. Total anthocyanins were calculated as the sum of individual compounds and their derivatives. For total flavonol and ellagitannin analysis, samples (5 ml) of supernatant were evaporated to dryness using a SpeedVac concentrator with no radiant heat and suspended in 1 ml of aqueous 50% methanol solution. The samples were analyzed using a Waters HPLC system (Waters Corp, Milford, MA) equipped with a model 600 pump, model 717 plus autosampler and model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Aqua C18 column (Phenomenex, Torrance, CA) with a 3.0 mm 4.0 mm ODS C18 guard column (Phenomenex). The mobile phase was a gradient of 20 g kg 1 acetic acid (A) and 5 g kg 1 acetic acid in water and acetonitrile (50:50 v/v, B) from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min. Prior to each injection, the system was equilibrated for 20 min at the initial gradient. A detection 139

156 wavelength of 360 nm was used for flavonols and 280 nm for ellagitannins at a flow rate of 1 ml min 1. Flavonols and ellagitannins were expressed as mg rutin equivalents kg 1 fresh weight. Although, resveratrol is produced in two forms, trans and cis resveratrol, this study focused only on the trans isomer. Trans resveratrol (3,4,5 Trihydroxy trans stilbene, 5 [(1E) 2 (4 Hydroxyphenyl)ethenyl] 1,3 benzenediol) concentrations were confirmed using an analytical standard (ID: ; Sigma Aldrich Co. LLC, St. Louis, MO). For flavonol and ellagitannin confirmation, a representative bronze and black genotype were analyzed using mass spectrometry (MS). For HPLC/MS analysis the HPLC apparatus was interfaced to a Burker Esquire (Burker Corporation, Billerica, MA) LC/MS ion trap mass spectrometer. Mass spectral data were collected with the Bruker software, which also controlled the instrument and collected the signal at 360 or 510 nm. Typical conditions for mass spectral analysis in negative ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 ml min 1 and a temperature of 300 C. Data were collected in full scan mode over a mass range of m/z at 1.0 s per cycle. Characteristic ions were used for peak assignment. The ORAC of muscadine extracts was measured using the method of Prior et al. (2003) modified for use with a FLUOstar Optima microplate reader (BMG Labtechnologies, Durham, NC) using fluorescein as a fluorescent probe. Muscadine extracts were diluted 1600 fold with phosphate buffer (75 mm, ph 7) prior to ORAC analysis. The assay was carried out in clear 48 well Falcon plates (VWR, St. Louis, MO), each well having a final volume of 590 μl. Initially, 40 μl of diluted sample, Trolox (TE) standards (6.25, 12.5, 25,

157 μm) and a blank solution of phosphate buffer were added to each well. The FLUOstar Optima instrument equipped with two automated injectors was programmed to add 400 μl of fluorescein (0.108 μm) followed by 150 μl of 2,2 azobis(2 amidino propane) dihydrochloride (AAPH) (31.6 mm) to each well. Fluorescence readings (excitation 485 nm, emission 520 nm) were recorded after the addition of fluorescein and AAPH and every 192 s for 112 min to reach 95% loss of fluorescence. Results were based upon differences in areas under the fluorescein decay curve between the blank, samples, and standards, and expressed relative to the initial reading. The standard curve was obtained by plotting the four concentrations of TE against the net area under the curve of each standard. Final ORAC values were calculated using the regression equation between TE concentration and the net area under the curve and expressed as μmol TE equivalents kg 1 fresh weight. Experimental Design The postharvest experiment was a designed split split plot with three replications of each genotype. The first split was storage (weeks 0, 1, 2, and 3) and the second split was year (2012 and 2013). The nutraceutical experiment was a split plot design with three replications of each genotype, and the split being year, (these measurements were only done on the harvest date, not at each storage date). Experimental Analysis The data was analyzed by analysis of variance (ANOVA) using JMP (version 11.0; SAS Institute Inc., Cary, NC). Tukey s Honest Significant Difference and Student s t Test were used for mean separations (p = 0.05). Associations among all dependent variables were determined using multivariate pairwise correlation coefficients of the mean values using JMP (version 11.0; SAS Institute Inc., Cary, NC). 141

158 Results Initial Attributes From a production and marketing perspective, the initial measurement of berry force, TA, ph, soluble solids, L*, Chroma, and hue angle are of particular importance. Averaged across year Nesbitt, AM 04, AM 28, and AM 03 had the highest initial force (11.5, 10.6, 10.5, and 10.0 N, respectively), while Ison, Delicious, Fry, and Tara had the least (7.4, 7.7, 7.8, and 7.8 N, respectively) (Table 2.1). TA ranged from 0.3 to 0.5, while ph ranged from 3.3 ( Ison ) to 3.9 (AM 02 and AM 26) and soluble solids ranged from 16.9% (AM 18) to 22.5% ( Summit ), averaged across years (Table 2.1). The bronze genotypes AM 03, AM 01, and AM 15 had the highest L* values (71.9, 66.8, and 66.6, respectively, while the black genotypes AM 28, AM 27, and Supreme had the lowest (25.9, 26.3, and 26.6, respectively) (Table 2.1). The bronze genotypes had Chroma values that ranged from 9.4 (AM 01) to 15.6 ( Fry ) and hue angles that ranged from 70.0 ( Summit ) to (AM 15), the black genotypes had Chroma values ranged from 2.0 (AM 27) to 14.2 ( Southern Jewel ) and hue angles that ranged from (AM 04) to (AM 02) (Table 2.1). Berry Storage Attributes The postharvest berry diseases present were identified as black rot (Guignardia [Phyllosticta] bidwellii [ampelicida] Ellis.), myrothecium leaf spot (Myrothecium sp./spp.), and botrytis fruit rot, (Botrytis sp./spp.). Occasionally an unknown species of fruit fly (Drosophila sp.) was present, but only in berries with wet or torn stem scars. The ANOVA F test indicated significant (P<0.0001) three way interactions of year by week of storage by genotype for weight loss, unmarketable berries, force to penetrate berry skin, and volume change (Table 2.2). 142

159 Table 2.1. Initial mean values for force to penetrate berry skin, composition and berry color for muscadine genotypes averaged across year (2012 and 2013). Genotype Berry color Force (N) Titratable acidity ph Soluble solids L* Chroma Hue angle (%) (%) AM 01 Br z AM 02 Bl AM 03 Br AM 04 Bl AM 15 Br AM 18 Bl AM 26 Br AM 27 Bl AM 28 Bl Delicious Bl Fry Br Ison Bl Nesbitt Bl Southern Jewel Bl Summit Br Supreme Bl Tara Br z Bronze=Br and black=bl. Table 2.2. F test significance from ANOVA for muscadine berry weight loss, percent unmarketable berries, force required to penetrate the berry skin, and percent volume change of the berry during 3 weeks of storage at 2 C. Highest order interactions are italicized and shaded (2012 and 2013). Degrees of freedom Weight loss (%) Unmarketable (%) Force (N) Volume change (%) Year 1 < < < Week 3 < < < Year*week 3 < < < < Genotype 16 < < < < Year*genotype 16 < < < < Week*genotype < < Year*week*genotype 48 < < < <

160 Weight Loss. For the dependent variable percent weight loss, large differences among overall week means were found, ranging from 1.4% for week 1, 2.7% for week 2, and 4.0% for week 3, with all means being significantly different from each other (P<0.0001) (Table A.7) indicating that longer storage resulted in more weight loss. After 3 weeks of storage, Nesbitt and AM 18 in 2012 had the highest percent weight loss (6.5% and 6.2%, respectively), while AM 28 and Southern Jewel in 2013 had the lowest (1.9% and 2.0%, respectively) (Fig. 2.1 and Table A.7). Overall weight loss values appear to be lower for most genotypes in 2013 compared to 2012 (Fig. 2.1). The variable response of genotypes for years, with the example of Nesbitt highlighted, provides some insight why the interaction was significant. Unmarketable Berries. For percent unmarketable berries, large differences were found among week means were found, ranging from 9.9% for week 1, 20.6% for week 2, and 42.7% for week 3, all means significantly different from each other (P<0.0001) (Table A.7), again highlighting the increasing difficulty in increased storage time to maintain marketable fruits. After 3 weeks of storage, AM 01 in 2013 had the highest percent unmarketable (95.0 %), while AM 26 in 2013 had the lowest (8.8%) (Fig. 2.2 and Table A.7). When examining major trends in the data, it can be seen that overall unmarketable values appear to be relatively similar among most genotypes in 2013 compared to 2012, with a few exceptions (Fig 2.2). The overall difference in means between years for percent unmarketable after 3 weeks of storage is 1.9%, with 2013 having slightly higher percent unmarketable (Fig. 2.2). The genotypes AM 01, AM 04, AM 26, AM 28, and Nesbitt performed quite differently among years, and likely contributed the greatest to the significant interaction (Fig. 2.2.). 144

161 Fig Percent berry weight loss of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Weight loss (%) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

162 Fig Percent unmarketable of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Unmarketable (%) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

163 Force. Large differences were found in force to penetrate the berry skin among weeks of storage, ranging from 9.1 N for week 0, 6.9 N for week 1, 6.6 N for week 2, and 6.2 N for week 3, all means significantly different from each other (P<0.0001) (Table A.7). However, it was noteworthy the major reduction after one week of storage, but minimal practical reductions in force after additional storage. Generally force to penetrate the berry skin decreased during storage, however weeks 1 or 2 often had the lowest force in some genotypes, especially in 2012 (Fig. 2.3 and Table A.7). After 3 weeks of storage AM 04 in 2013 had the greatest firmness (11.2 N), while AM 15 in 2012 had the least (2.2 N) (Fig. 2.3 and Table A.7). The overall difference in means between years was 1.9 N. The overall trend in the data shows that the force to penetrate the berry skin appears to be greater for most genotypes in 2013 compared to 2012, providing some insight why the interaction was significant (Fig. 2.3 and Table A.7). And again, differences among genotypes among years and weeks indicate that some genotypes likely offer specific postharvest performance advantages. Volume Change. Volume change resulted from a decrease in berry size shown with positive numbers, with an increase in size shown by negative numbers. The same significant three way interaction for weight loss, unmarketable, and force was also identified for volume change (Table 2.2). Differences were found in volume change during storage ranging from 2.9% for week 1, 0.5% for week 2, and 5.1% for week 3, all means significantly different from one another (P=0.0011). The mean difference in volume change for years was 10.4%, with the overall trend showing more increase in size during storage in 2012, and more decrease in size in 2013 (Fig 2.4 and Table A.7). AM 02, AM 28 after 2 weeks of storage, and Supreme after 1 week of storage in 2012 showed the greatest 147

164 Fig Force to penetrate skin of muscadine genotypes stored at 2 C for 3 weeks Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Week Force (N) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Genotype Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Year

165 Fig Percent change in volume of muscadine genotypes stored at 2 C for 3 weeks. Values at week 0 (date of harvest) were excluded. Decrease in size shown with positive values, while an increase in size shown by negative values. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Volume change (%) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

166 increase in volume ( 35.4%, 31.9%, and 31.7%, respectively), while Ison and Nesbitt stored for 3 weeks in 2013, showed the greatest decrease in volume (21.2% and 22.5%, respectively) (Fig. 2.4 and Table A.7). Many genotypes that decreased in size in 2012, often increased in size in 2013, offering some insight why the interaction was significant. Berry composition The ANOVA F test indicated a significant three way interaction of year by week of storage by genotype for ph (P=0.0046), and titratable acidity (TA), soluble solids content, and soluble solids to titratable acidity ratio (SS/TA) (P<0.0001) (Table 2.3). ph. For the dependent variable ph, no significant differences among week means were found (P=0.3220) (Fig. 2.5 and Table 2.3). Though significant, the mean differences in year for ph was minimal (0.08) with ph being slightly higher in 2013 compared to 2012 (P<0.0001) (Tables 2.3 and A.8). After 2 weeks of storage, AM 02 in 2013 had the highest ph (4.0), while Ison at date of harvest in 2012 had the lowest (3.1) (Fig. 2.5 and Table A.8). When looking at the overall trends in the data, it is challenging to identify a clear linear change of week of storage or the difference in year and ph, leaving the variability within and among genotypes as a possible contributor of significance of the interaction (Fig. 2.5) Titratable Acidity. If one looks at major trends in the data, it can be seen that overall TA values appear to be lower for most genotypes in 2013 compared to 2012 (Fig. 2.6). Among years of the study, TA means of 0.3% for 2013 and 0.5% in 2012 were found (P<0.0001) (Table 2.3). When averaged across years and genotypes, minimal differences were found among weeks of storage, ranging from 0.43% for week 0, 0.43% for week 1, 0.43% for week 2, and 0.44% for week 3, with only week 3 being significantly different from the others (P=0.0218). Overall, AM 04 after 2 weeks of storage and Supreme after 1 150

167 Table 2.3. F test significance from ANOVA for berry ph, titratable acidity, soluble solid content, and soluble solids to titratable acidity ratio during 3 weeks of storage. Highestorder interactions are italicized and shaded (2012 and 2013). Degrees of freedom ph Titratable acidity (%) z Soluble solids (%) SS/TA y Year 1 < < < < Week Year*week < < Genotype 16 < < < < Year*genotype 16 < < < < Week*genotype < < < Year*week*genotype < < < z Titratable acidity measured as tartaric acid. y Soluble solids to titratable acidity ratio. Fig ph of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). ph AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

168 Fig Titratable acidity (%) of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Titratable acidity (%) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

169 week of storage in 2012 had the highest TA% (0.61% and 0.62%, respectively), while AM 28 at date of harvest and after 1 week of storage in 2013 had the lowest (0.21% and 0.21%, respectively) (Fig. 2.6 and Table A.8). The variable response of genotypes for years and weeks, with the examples of AM 28 and Ison highlighted, provides some insight why the interaction was significant (Fig. 2.6 and Table A.8). Soluble Solids. When considering the major trends in the data, it appears that soluble solids values were lower for most genotypes in 2013 when compared to 2012 (Fig. 2.7 and Table A.8). The difference in mean soluble solids among years was 3.7%, with significant differences among years (P<0.0001) (Table A.8). Averaged across genotypes and years, the differences in mean soluble solids are minimal, ranging from 18.9% for week 0, 19.1% for week 1, 19.2% for week 2, and 19.3% for week 3, with only week 3 being significantly different than week 0 (P=0.0220) (Table A.8). Overall, Summit at initial date of harvest in 2012 had the highest soluble solids (27.3%), while AM 28 in 2013 had the lowest soluble solids during storage (14.1% for week 0, 14.3 % for week 1, 14.2 % for week 2, and 14.6% for week 3) (Fig. 2.7 and Table A.8). The variable response of genotypes among years provides some insight why the interaction was significant (Fig. 2.7). Soluble Solids to Titratable Acidity Ratio. Differing from soluble solids when considering the major trends in the data, it appears that SS/TA values were higher for most genotypes in 2013 when compared to 2012 (Fig. 2.8 and Table A.8). The difference of SS/TA means was 11.6, which was significant (P<0.0001) (Table 2.3). The difference in SS/TA means among weeks was minimal, ranging from 60.5 for week 0, 61.0 for week 1, 60.5 for week 2, and 57.2 for week 3, with only week 3 being significantly different from the others (P=0.0075). At initial date of harvest in 2012, Ison had lowest SS/TA (35.25), 153

170 Fig Soluble solids (%) of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Soluble solids (%) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

171 Fig Soluble solids to titratable acidity ratio of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). SS/TA AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

172 while AM 03 at initial date of harvest and after 2 weeks of storage in 2012 had the highest (112.1 and 112.6, respectively) (Fig. 2.8 and Table A.8). The variable response of genotypes for years, with the example of AM 03 highlighted and the variable response of genotype for weeks, with the example of AM 26 highlighted, offers insight why the interaction was significant. Berry Color The ANOVA F test indicated a significant three way interaction of year by week of storage by genotype for all of the berry color attributes measured of Chroma (P<0.0001), hue angle (P=0.0035), and L* (P=0.0001) (Table 2.4). The variability in genotypes and lack of variability among weeks and years offers some insight why the interaction for all berry color attributes was significant. L*. Differing from the other color attributes of Chroma and hue angle, the dependent variable L* had significant differences among weeks of storage and year (P<0.0001). For L* the difference in year means, averaged across genotype and week was 4.3, with 2013 having higher values overall (Fig. 2.9). The difference in L* means among weeks ranged from 42.2 for week 0, 34.4 for week 1, 34.6 for week 2, and 33.5 for week 3, with only week 0 being significantly different from the other week means (P=0.0046). After 2 weeks of storage, AM 01 in 2013 had the highest L* value (105.5), while AM 28 at week 0 in 2013 had the lowest (22.9) (Fig. 2.9 and Table A.9). When looking at the major trends in the data, one can see that generally the bronze genotypes (AM 01, AM 03, AM 15, AM 26, Fry, Ison, Summit, and Tara ) had higher L* values compared to the black genotypes (Fig. 2.11). Additionally, one can see that often, but especially in 2013, L* values were higher at initial date of harvest, and then remained fairly constant during storage (Fig. 2.9 and Table A.9). 156

173 Table 2.4. F test significance from ANOVA for Chroma, hue angle, and L* values during 3 weeks of storage at 2 C. Highest order interactions are italicized and shaded (2012 and 2013). Degrees of freedom Chroma Hue angle L* Year < < Week < Year*week < < Genotype 16 < < < Year*genotype 16 < < < Week*genotype 48 < < Year*week*genotype 48 < Fig L* values of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). L* AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

174 Hue angle. When looking at the major trends in the data it appeared that hue angle was greater in 2013 compared to 2012 (Fig. 2.10). The overall mean difference in hue angle was 37.4, which was significantly higher in 2013 (P<0.0001)(Table 2.4). Additionally, it appeared that the bronze genotypes (AM 01, AM 03, AM 15, AM 26, Fry, Summit and Tara ) generally had lower hue angles compared to the black genotypes (Fig. 2.10). Overall, no significant differences were found in hue angle during storage (P=0.0807) (Table 2.3). At date of harvest and throughout storage, Supreme and Nesbitt at date of harvest in 2012 had the largest hue angles (359.4, 359.8, 359.5, 359.0, and 359.7, respectively), while Summit at date of harvest in 2012 had the smallest hue angle (47.8 ) (Fig and Table A.9). Chroma. For the dependent variable of Chroma, no significant differences in means were found among years, when averaged across weeks of storage and genotypes (P=0.3190). Differences among weeks were minimal, ranging from 8.2 for week 0, 7.4 for week 1, 7.3 for week 2, and 7.1 for week 3, with only week 0 values being significantly different (P=0.0046). At week 0 in 2013, Southern Jewel had the highest Chroma value (23.1), while AM 27 after 1 week of storage in 2013 had the lowest (Fig and Table A.9). When looking at the major trends in the data, it appears the bronze genotypes (AM 01, AM 03, AM 15, AM 26, Fry, Summit and Tara ) generally had higher Chroma values, with the exception of Southern Jewel at week 0 in 2013, (Fig and Table A.9). Nutraceutical Content The ANOVA F test indicated a significant two way interaction of year by genotype for the berry nutraceuticals total anthocyanins (P<0.0001), total flavonols (P<0.0001), resveratrol (P<0.0001), and ORAC (P<0.0001) (Table 2.5). Additionally, there were main 158

175 Fig Hue angle of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Hue angle AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

176 Fig Chroma of muscadine genotypes stored at 2 C for 3 weeks. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Chroma AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Week

177 Table 2.5. F test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and resveratrol concentrations during. Highestorder interactions are italicized and shaded (2012 and 2013). Total anthocyanins (mg/100g) Total ellagitannins (mg/100g) Total flavonols (mg/100g) Total phenolics (mg/100g) Resveratrol (mg/100g) ORAC (µmol Trolox equivalents/g) Year < < < < Genotype < < < < < Year*genotype < < < <

178 effects of genotype and year for total ellagitannins (P<0.0001) and total phenolics (P<0.0001) (Table 2.5). The variability among genotypes and year offers some insight why the interactions were significant. Total Anthocyanins. When examining the major trends in the data it appeared that total anthocyanins were generally higher in 2012, compared to 2013 (Fig. 2.12). Averaged across genotypes, the significant difference in mean year total anthocyanins was mg/100 g (P<0.0001) (Table 2.5). Total anthocyanins were not detected in any of the bronze genotypes (AM 01, AM 03, AM 15, AM 26, Fry, Ison, Summit, and Tara ), and concentrations varied among the black genotypes (Fig and Table A.10). The black genotypes AM 27 and AM 04 in 2012 had the highest levels of total anthocyanins (122.0 mg/100g and mg/100g, respectively) (Fig and Table A.10). Total Ellagitannins. Averaged across years, the genotype AM 03 had the highest concentrations of total ellagitannins (12.5 mg/100 g), while AM 01, Tara, and Supreme had the lowest (3.1, 3.5, and 3.6 mg/100 g, respectively) (Tables 2.6 and A.10). Among years, significantly different total ellagitannin concentrations were found, 5.6 mg/100 g in 2012 and 8.3 mg/100 g in 2013 (Table 2.7). Generally, genotypes performed similarly among years, with 2013 having greater overall concentrations, offering some insight why the main effects were significant (Table A.10). Total Flavonols. The dependent variable total flavonols was significantly higher in 2012, compared to 2013, when averaged across genotypes (P<0.0001). The mean difference for total flavonol concentration was 7.4 mg/100 g among years. AM 03 and Summit in 2012 had the highest total flavonol content (70.6 and 63.1 mg/100 g, respectively), while Supreme and AM 28 in 2012 had the lowest (7.3 and 8.9 mg/100 g, 162

179 Fig Total anthocyanin concentrations of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Total anthocyanins (mg/100 g) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Year Table 2.6 Main effect of muscadine genotype means on total ellagitannin concentrations of the berry during three weeks of storage, averaged across years (2012 and 2013). Genotype Total ellagitannins (mg/100 g) AM d z AM abcd AM a AM abcd AM abcd AM cd AM cd AM abcd AM bcd Delicious 10.2 abc Fry 8.5 abcd Ison 9.6 abcd Nesbitt 4.9 bcd Southern Jewel 5.9 abcd Summit 10.8 ab Supreme 3.6 cd Tara 3.5 d z Means followed by the same letter are not significantly different at α= 0.05, separated by Tukey s HSD. 163

180 Table 2.7. Main effect of year means on total ellagitannin concentrations of the muscadine, averaged across genotypes (2012 and 2013). Year Total ellagitannins (mg/100 g) p value <

181 respectively) (Fig and Table A.10). When examining the major trends of the data, it appears that generally the bronze genotypes (AM 01, AM 03, AM 15, AM 26, Fry, Summit, and Tara ) had higher levels of flavonols compared to the black genotypes (Fig. 2.13). Total Phenolics. Total phenolics were highest in Ison (670.8 mg/100 g), while AM 28 and AM 18 had the lowest levels (335.7 and mg/100 g, respectively), averaged across years (Table 2.8). Averaged across genotypes, the difference in total phenolics among year means was 61.1 mg/100 g. Total phenolic concentrations were significantly higher in 2012 (548.7 mg/100 g), compared to 2013 (487.6 mg/100 g) (P<0.0001) (Tables 2.9 and 2.5). Generally, genotypes performed similarly among years, with 2012 having greater overall concentrations, offering some insight why the main effects were significant (Table A.10). Resveratrol. There was no significant difference in year means for the dependent variable resveratrol concentration (P=0.2010); however, when considering the major trends in the data it appeared that genotypes often performed differently among years. The genotype AM 27 in 2012 had the highest resveratrol levels (16.7 mg/100 g), while AM 28 and Southern Jewel in 2013 had the lowest (2.9 and 3.2 mg/100g) (Fig and Table A.10). With the exceptions of AM 01, AM 27, Ison, Summit, and Supreme, the genotypes generally performed similarly among years (Fig and Table A.10). Additionally, it appeared that bronze and black genotypes performed similarly (Fig. 2.14). ORAC. When considering the major trends in the data, it appears that ORAC was generally greater in 2013 compared to 2012 (Fig. 2.15). The mean difference in years was 7.4 µmol Trolox equivalents/g, which was significant (P<0.0001). The cultivar Ison in 2012 and 2013 and Southern Jewel in 2013 had the highest ORAC values (110.6, 115.5, and 165

182 Fig Total flavonol concentrations of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). 166

183 Table 2.8. Main effect of genotype on total phenolic concentrations of the muscadine berries, averaged across years (2012 and 2013). Genotype Total phenolics (mg/100 g) AM abc z AM abcde AM ab AM abcd AM abc AM f AM def AM abcd AM f Delicious abc Fry abcd Ison a Nesbitt bcdef Southern Jewel abc Summit abcdef Supreme ef Tara cdef z Means followed by the same letter are not significantly different at α= 0.05, separated by Tukey s HSD. Table 2.9. F test main effect significance for year on total phenolic concentrations averaged across muscadine genotypes (2012 and 2013). Year Total phenolics (mg/100 g) p value

184 Fig Resveratrol concentrations of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013). Year Resveratrol (mg/100 g) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara Genotype Fig Oxygen radical absorbance capacity (ORAC) of muscadine genotypes. Each standard error bar is constructed using 1 standard error from the mean (2012 and 2013) Year ORAC (µmol TE/g) AM 01 AM 02 AM 03 AM 04 AM 15 AM 18 AM 26 AM 27 AM 28 Genotype Delicious Fry Ison Nesbitt Southern Jewel Summit Supreme Tara 168

185 111.3 µmol Trolox equivalents/g, respectively), while AM 26 in 2012 had the lowest (34.2 µmol Trolox equivalents/g) (Fig and Table A.10). Averaged across years, ORAC ranged from µmol Trolox equivalents/g ( Ison ) to 45.8 µmol Trolox equivalents/g (AM 26). With the exceptions of AM 01 and AM 15, individual genotypes performed significantly different each year (Fig. 2.15), offering some insight why the interaction was significant. Black verses Bronze Storage Performance The variable response of genotypes among years for the dependent postharvest storage variables of force, weight loss, percent unmarketable, and volume change led to interest in evaluating the performance of genotypes averaged across muscadine genotype skin color, either black or bronze. Significant differences among skin colors for percent unmarketable (P=0.0012), percent volume change (P=0.0002), and force to penetrate the berry skin (P=0.0180), were found, while there was no significant difference for percent weight loss (P=0.4343). Averaged across year and week, the mean difference in percent unmarketable was 3.7%, with bronze genotypes having significantly higher percent of unmarketable berries. It was found that the black genotypes increased in volume during storage ( 1.3%), while bronze genotypes decreased in volume (3.5%), with significant differences in the means (Fig. 2.16). Overall, the black genotypes were found to have greater firmness (7.4 N), than the bronze genotypes (6.9 N) when averaged across year and week of storage (Fig. 2.16). When examining the major trends in the data, it can be determined that black genotypes have generally better postharvest storage performance compared to bronze genotypes (Fig. 2.16). Correlations 169

186 Fig Storage performance of black and bronze muscadine genotypes, averaged across year (2012 and 2013) stored for 3 weeks at 2 C. Each standard error bar is constructed using 1 standard error from the mean. Force (N) Unmarketable (%) Volume change (%) Color Black Bronze 2.0 % Weight Loss

187 Though some strong correlations did occur, the dependent variables measured were generally minimally correlated (Table A.11). Percent unmarketable berries was negatively correlated with force (r= 0.74) (Table A.11). TA was negatively correlated with SS/TA (r= 0.89) (Table A.11). L* value was positively correlated with soluble solids (r=0.71) and Chroma (r=0.72), and negatively correlated to hue (r= 0.73) (Table A.11). Chroma was strongly negatively correlated with hue (r= 0.93) and total anthocyanins (r= 0.87), while hue angle was positively correlated with total anthocyanins (r=0.75) and negatively correlated with total flavonols (r= 0.73) (Table A.11). Soluble solids were positively correlated with total flavonols (r=0.73) and L* (r=0.71) (Table A.11). Discussion Berry Storage Attributes The average percent unmarketable berries and percent weight loss were generally similar in both years of the study; however, genotypes generally performed differently each year. Both percent weight loss and percent unmarketable increased during storage and varied among genotypes, and these results were consistent with other studies (Ballinger and Nesbitt, 1982a; James et al., 1997; James et al., 1999; Lutz, 1938; Silva et al., 1994; Takeda et al., 1983). For example, James et al. (1997) found that after 2 weeks of storage, Summit had 72% unmarketable berries and 4.9% weight loss, while Fry had 6.2% unmarketable berries and 4.3% weight loss. The postharvest berry diseases present were identified as black rot (Guignardia [Phyllosticta] bidwellii [ampelicida] Ellis.), myrothecium leaf spot (Myrothecium sp./spp.), and botrytis fruit rot, (Botrytis sp./spp.). This is similar to reports by Lane (1978), Smit et al. (1971) and Takeda et al. (1983). Generally, berry diseases were not a major cause of 171

188 unmarketability until 3 weeks of storage. The primary factors involved in unmarketable berries were browning (especially in bronze genotypes), leakage from torn or wet stem scars, and shriveling, which is consistent with similar work reported by Perkins Veazie et al. (2012). Occasionally an unknown species of fruit fly (Drosophila sp.) was present, but only in berries with wet or torn stem scars and did not contribute to unmarketable berries. Unmarketable Berries. A major cause of unmarketability in the bronze genotypes was skin browning during storage likely caused by chilling injury (Fig. 2.17). The abiotic disorder of chilling injury is common in many horticultural crops, including bananas (Musa paradisiaca L.), citrus (Citrus spp.), sweet potatoes (Ipomoea batatas L.), and tomatoes (Solanum lycopersicum L.) (Wang, 1990). Chilling injury can increase susceptibility to decay through providing a favorable medium for the growth of pathogens (Wang, 1990). The primary symptom of chilling injury identified in my study was brown discoloration of the skin, pulp, and vascular strands of berries, as reported previously by Himelrick (2003) and Wang ( 1990). Chilling injury has been reported in muscadines stored at or below 1.7 C (Himelrick, 2003; Smittle 1990), but is uncommon in muscadines stored at 2 C. It was surprising to find possible symptoms of chilling injury on the muscadine berries in my study, as they were stored above the previously reported threshold of 1.7 C (Himelrick, 2003; Smittle 1990). This illustrates chilling injury susceptibility is likely genotype specific and creates the potential for opportunities of improvement through tolerance to chilling injury selection in the University of Arkansas muscadine breeding program. Other V. vinifera table grapes were successfully stored at 1 C without showing symptoms of chilling injury (Burg, 2004). 172

189 Fig Muscadine genotype AM 01 at date of harvest (left) and after 3 weeks of storage (right), demonstrating the browning that occurred, likely caused by chilling injury (2013). 173

190 Weight Loss. Leakage and shriveling are common problems in muscadines during storage and are managed with elimination prior to storage of berries with wet stem scars and by maintaining high RH during storage (Perkins Veazie et al., 2012; Smit et al., 1971). Leakage and shriveling did occur and was most prevalent after 3 weeks of storage (Fig. 2.18). It has been shown that use of plastic film packaging of other fruits (Citrus limon L. and Capsicum annuum L.) prevented water loss, resulting in less leakage and shriveling (Ben Yehoshua et al., 1983). Conversely, it has been found that the use of polyethylene bags resulted in less weight loss of muscadines during storage, but did not prevent leakage and shriveling; due to the juice retention in the bags (Walker et al., 2001). It has also been found that edible coatings prevent weight loss during storage of fresh highbush blueberries (Vaccinium corymbosum L.), but this is unknown on muscadines (Duan et al., 2011). Other table grapes (V. vinifera) experienced less leakage and shriveling during storage, as the berries were retained on the pedicel, and thus did not have stem scar damage potential during marketing unlike muscadines where berries are harvested singularly. It was uncommon that a single genotype performed well in both years of storage. After 3 weeks of storage, the genotypes with the least percent weight loss in 2013 were AM 28, Nesbitt, and Southern Jewel, while in 2012 AM 03, AM 27, Delicious and Tara had the least (Fig. 2.1 and Table A.7). The genotypes with the greatest percent weight loss after 3 weeks of storage in 2013 were AM 01, AM 03, and Fry, while in 2012 AM 04, AM 18, and Nesbitt had the greatest weight loss (Fig. 2.1 and Table A.7). In 2013, the genotypes with the least percent unmarketable after 3 weeks of storage were AM 03, AM 04, AM 26, and AM 28, while in 2012 AM 03, Southern Jewel, Summit, and Supreme had the least percent unmarketable (Fig. 2.2 and Table A.7). The genotypes in 2013 with the highest 174

191 Fig Muscadine genotype AM 04 after 3 weeks of storage, demonstrating shriveling that occurred, likely caused by berry leakage. (2012) 175

192 percent unmarketable were AM 01, Fry, and Tara, while in 2012 the genotypes with the highest percent unmarketable were AM 04, AM 26, and Fry (Fig. 2.2 and Table A.7). This strongly shows the influence of environmental factors (rainfall and temperature) on storage performance of muscadines (Table A.1). Additionally, this illustrates the importance of testing multiple years for postharvest performance in a muscadine storage protocol. Similar to our findings, Ballinger and Nesbitt (1982b) found Nesbitt to have acceptable postharvest storage quality, while James et al. (1999) found Summit to have the greatest percent decay and weight loss. I found Summit to have intermediate quality during storage, which is similar to the findings of James et al. (1997). Force. Force to penetrate muscadine skin has been shown to be a useful characteristic to assess berry firmness and texture as well as berry maturity (Conner, 2013); however, use of force to determine storability of muscadine grapes has previously shown results with no clear trend (Silva et al., 1994; Walker et al., 2001). It has been shown that muscadines require a force up to 13.9 N to penetrate the skin at date of harvest, which is nearly twice that of V. vinifera cultivars (Conner, 2013). Similarly, I found that muscadine grapes require up 13.2 N to penetrate the skin at date of harvest (Fig. 2.3 and Table A.7). Similar to the findings of Conner (2013), I determined that Nesbitt was among the most firm cultivars. It was found that the berries stored in 2013 were generally firmer than the berries stored in 2012, further illustrating the significance of environmental influences (temperature and rainfall) on storage quality (Figs. 2.1, 2.2, and 2.3, and Table A.7). Percent unmarketable berries was negatively correlated with force (r= 0.74), potentially illustrating that berries requiring greater force to penetrate the berry skin store better as they were firmer (Table A.11) and likely one of the more important relationships among 176

193 variables measured to assist in evaluating a genotype s postharvest storage potential. Overall, I found that berry firmness decreased during storage, but was occasionally lowest after 2 weeks of storage; similar results were reported by James et al. (1999), and this could possibly be due water loss during storage resulting in an increase of firmness at week 3 (Fig. 2.3 and Table A.7). It was found that the genotypes requiring the most force to penetrate the skin at date of harvest also required the most force to penetrate the skin after 3 weeks of storage (especially in 2013), and indicating force to be a good indicator of potential storage performance (Fig. 2.3 and Table A.7). Volume Change. Volume change of muscadine berries during storage is widely unstudied. Unexpectedly, no relationship between storage time and volume change was found. Conversely, it has been found that a decrease in size occurred in mangoes (Mangifera indica L.) and citrus during storage due to water loss (Jha et al., 2006; McCornack, 1975). The berries stored in 2012 generally increased in volume during storage while those stored in 2013 generally decreased (Fig. 2.4 and Table A.11). This may have been due to the substantial differences in growing seasons, as 2012 was extremely hot and dry, while 2013 had more moderate temperatures and more rainfall particularly near harvest (Table A.1). The lack of correlations found between volume change and the other independent variables was potentially due to the extreme variation of berry size within each genotype (Table A.11), limiting volume change as a useful variable in determining storability. Berry Composition Muscadines have been shown to increase in soluble solids and decrease in TA during ripening (Carroll and Marcy, 1982; Flora and Lane, 1979; Johnson and Carroll, 1973; 177

194 Lanier and Morris, 1979; Peynard and Riberau Gayon, 1971; Walker et al., 2001). Unlike many other fruits, muscadines are nonclimacteric, and do not continue to accumulate sugars after harvest. ph. The overall ph measured in 2013 was higher than in 2012 and ranged from 3.2 ( Ison ) to 3.8 (AM 04) in 2012 and from 3.4 (AM 15) to 4.0 (AM 02) in 2013 (Fig. 2.5 and Table A.8), this is contradictory to Jackson (1986), who found that high ph was often associated with warmer temperatures during the growing season. I found that ph of muscadines remained constant during storage (Fig. 2.5), which was consistent with several other studies (James et al., 1997; James et al., 1999; Silva et al., 1994; Takeda et al., 1983; Walker et al., 2001). Additionally, stable ph during storage was found in kiwifruit (Actinidia deliciosa L.), mango, pineapple (Ananas comosus L.), strawberry (Fragaria x ananassa Weston.), and watermelon (Citrullus lanatus Thunb.), while ph was shown to decrease in muskmelon (Cucumis melo L.) (Gil et al., 2006). The ph reported for some V. vinifera cultivars was higher than I found in muscadines in 2012 and 2013 (Mascarenhas et al., 2012). Titratable Acidity. It was found that percent TA stayed relatively constant during storage (Fig 1.7 and Table A.3); this was consistent with the results of other studies (James et al., 1997; James et al., 1999; Takeda et al., 1983; Walker et al., 2001) and is contradictory to Silva et al., (1994), who reported that TA increased during storage, and Lutz (1938), who reported that TA decreased during storage. Percent TA has also been shown to remain constant during storage of muskmelon, kiwifruit, mango, pineapple, strawberry, and watermelon (Gil et al., 2006). There was a strong negative correlation between TA and SS/TA (r = ) (Table A.11), and a relationship was expected as TA is used in the 178

195 calculation of SS/TA. Titratable acidity was strongly affected by year and genotype, with 2012 generally having higher TA than 2013 (Fig. 2.6), which can be explained by the warmer growing season in Jackson (1986) reported that warmer temperatures during the growing season resulted in higher acidity. In my study, the cultivar Supreme generally had the highest percent TA and AM 01 and Southern Jewel had the lowest (Fig. 1.7 and Table A.3). TA was found to be lower than that of V. vinfera, reported to have ranged from 0.6 to 1.5% (Mascarenhas et al., 2012). Soluble Solids. Probably due to the extremely hot and dry conditions, the percent soluble solids was uncharacteristically higher in 2012 than in 2013 (Fig. 2.7). Overall soluble solids remained constant during storage (Fig. 2.7and Table A.8); this is consistent with most other studies (James et al., 1997; James et al., 1999; Takeda et al., 1983), but was contradictory to Silva et al. (1994) and Walker et al. (2001), who reported decreased soluble solids during storage, and Lutz (1938), who reported increased solids during storage. Percent soluble solids has also been shown to remain constant during storage of muskmelon, kiwifruit, mango, pineapple, strawberry, and watermelon (Gil et al., 2006). Soluble solids in my study was found to be similar to that reported for V. vinifera (Mascarenhas et al., 2012). There were positive correlations for both L* (r=0.71) and total flavonols (r=0.73) with soluble solids (Table A.11). A possible explanation for these correlations could be that higher soluble solids resulted in lighter berries (higher L*), and increased flavonol concentrations, as soluble solids has been shown to be an indicator of muscadine berry ripeness (Carroll and Marcy, 1982; Flora and Lane, 1979; Johnson and Carroll, 1973; Lanier and Morris, 1979; Peynard and Riberau Gayun, 1971; Walker et al., 2001). There have been strong influences of both year and genotype on soluble solids 179

196 reported, for example Threlfall et al. (2007) found that Supreme had the lowest soluble solids of all genotypes evaluated, while I identified Supreme as being midrange in soluble solids (Fig. 2.7 and Table A.8). However, berry maturity at harvest could have played a major role in these differences. Soluble Solids to Titratable Acidity Ratio. The SS/TA was strongly influenced by year and genotype (Fig. 2.8 and Table A.8). The overall average SS/TA measured in 2013 was higher than in 2012 (Fig. 2.8 and Table A.8). The berries in 2012 had higher soluble solids and TA, compared to the berries harvested in 2013 (Figs. 2.6 and 2.7), resulting in the differences in SS/TA among years. It was found that SS/TA remained relatively constant during storage, which is contradictory to Lutz (1938), who reported an increase of SS/TA during storage time. The SS/TA reported here was higher than reported for some V. vinfiera cultivars (Mascarenhas et al., 2012). Berry Color The affect of storage on the berry skin color attributes of L* value, Chroma, and hue angle of fresh market muscadine grape berries is widely unstudied, while it is well studied in juice and wine. The United States Department of Agriculture (USDA) currently has no standards in place to grade muscadine berries for the color attributes of Chroma, hue, and L*. The standards for skin color of muscadines state the berries should be well colored to be considered marketable; for black and red cultivars 75% of the surface of the berry must have characteristic color for the variety, while no color requirement exist for bronze genotypes except that Carlos, Fry or similar cultivars can show any amount of blush or bronze color on the berry (USDA, 2006). Additionally the USDA states that black cultivar colors can include reddish purple, purple, and black; red cultivar colors include light pink, 180

197 pink, red, dark red, and purple; and bronze cultivar colors include light green, straw, amber, and bronze with allowance for an amount of blush or pink color that may also be characteristic for certain cultivars (USDA, 2006). L* value. Generally, L* values remained relatively constant during storage; however, the genotypes in AM 04 in 2012, and AM 03, AM 15, AM 18, Delicious, and Southern Jewel in 2013 had L* values decreased after the date of harvest, but remained relatively stable during storage (Fig. 2.9 and Table A.9). For week 0, the average L* value for the bronze genotypes was 55.4 and 35.4 for the black genotypes. The average L* values for the black genotypes was 26.5 for week 1, 26.4 for week 2, and 26.5 for week 3, while the average L* value for the bronze genotypes was 45.7 for week 1, 46.2 for week 2, and 43.4 for week 3, with no significant differences in any of the means; this illustrates the berries darkened after date of harvest, but remained relatively unchanged during storage. A decrease in L* value would represent a darkening of the berries during storage, as L* measures lightness from completely opaque (0) to completely transparent (100) (Walker et al. 2001). Silva et al. (1994) found that L* values increased during storage, though the differences were not discernable by panelists. Conversely, I found that generally, L* values remained stable or slightly decreased during storage (Fig. 2.9 and Table A.9). Similarly, Hernandez Herrero and Frutos (2014) found that the L* values of model juice of grape, plum, and strawberry stayed relatively constant during storage. Possibly due the more favorable growing season (Table A.1), the L* values were higher in 2013, than in 2012 (Fig. 2.9 and Tables A.1 and A.9). Overall, L* values were strongly affected by genotype and year, while less affected by week of storage. 181

198 Hue Angle. I found the hue angles were on average for the black and 89.5 for the bronze genotypes. Hue angle ranged from 69.9 ( Summit ) to ( Supreme ), when averaged across year and week of storage. Conversely, Conner and MacLean (2013) found hue values that ranged from 1.5 to 91.8, Threlfall et al. (2007) found values ranging from 53.4 to 98.6, and Walker et al. (2001) found values that ranged from 76.5 to I found no clear relationship between week of storage and hue angle, with values remaining stable during storage (Fig. 2.10). Similarly, Hernandez Herrero and Frutos (2014) found that the hue angles of model juice of grape, plum, and strawberry stayed relatively constant during storage. There was a negative correlation between hue angle and L* value (r= ) (Table A.11), showing that as L* increased (berries became lighter), hue angle decreased. Overall, hue values were greater in 2013 than in 2012 (Fig and Table A.9), which might be due to the milder growing season in 2013, likely resulting in less berry sunburn (Table A.1). The average hue angle for the black genotypes was 266.3, which falls between blue and green coloration value for hue, while the average hue angle was 89.5 for bronze genotypes, which is approximately yellow for hue. Chroma. I found that Chroma was influenced by storage time, year, and genotype. Walker et al. (2001) found that Chroma of the bronze cultivar Fry ranged from 12.1 to 14.2 based on maturity level, this is comparable to the findings of my study, with Chroma for Fry ranging from 13.1 to Conner and MacLean (2013) found Chroma values ranging from 2.4 to 22.8 and Threlfall et al. (2007) found Chroma values ranging from 8.0 to 52.8 on four black cultivars ( Black Beauty, Ison, Nesbitt, and Supreme ) and 2 bronze cultivars ( Granny Val and Summit ), with the bronze genotypes generally having lower Chroma values, both of which are consistent with my findings. I found Chroma generally 182

199 had no clear pattern during storage (Fig and Table A.9), similarly Hernandez Herrero and Frutos (2014) found that the Chroma values of model juices of grape (V. vinifera), plum (Prunus spp.), and strawberry stayed relatively constant during storage. Chroma values were generally highest for bronze genotypes, with the exception of the black Southern Jewel at initial date of harvest in 2013 (Fig and Table A.9), which was unusually high. These results are similar to the findings of Conner and Maclean (2012). There was a strong negative correlation between Chroma and hue angle (r= 0.93) (Table A.11). Overall, Chroma values were higher in 2012 than in 2013 (Fig 2.11 and Table A.9), which could possibly be explained by the milder growing season in 2013 (Table A.1). I found that averaged across year and storage, Chroma for the bronze genotypes was 12.9, while the black genotypes had an average Chroma of 3.7, showing on average the bronze genotypes were less grey than the black genotypes. Nutraceutical Content Total Anthocyanins. Total anthocyanin concentrations found were similar to those previously reported (Ballinger et al., 1973; Brown, 1940; Conner and MacLean 2013; Lee et al., 2005; Lee and Talcott, 2004; Goldy et al., 1987; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Sandhu and Gu 2010; Striegler et al., 2005; Stringer et al., 2009; Threlfall et al., 2007). As expected, anthocyanins were not detected in any of the bronze genotypes in either year of the study (Fig and Table A.10). A negative correlation with total anthocyanins and Chroma (r= ) and a positive correlation with hue angle (r=0.7507) was found (Table A.11), showing that lower Chroma values and greater hue angles were related to higher total anthocyanins, which is not surprising as bronze genotypes generally had higher Chroma values and lower hue angles and no anthocyanins (Figs. 2.9, 2.10, and 183

200 2.12 and Tables A.9 and A.10). I found greater total anthocyanin concentrations than those reported for V. vinifera grapes (Hernandez Herrero and Frutos, 2014). Total anthocyanins found were greater than or comparable to those reported for blackberry (Rubus sp. L.), highbush blueberry, red raspberry (Rubus ideaus. L.), and strawberry (Cordenunsi et al., 2002; Ehlenfeldt and Prior et al., 2001; Maatta Riihinen et al., 2004; Siriwoharn et al., 2004). Anthocyanin concentrations were generally higher in 2012 than in 2013 (Fig. 2.12). The differences in total anthocyanins among years may have been due to higher temperature and greater sun exposure, and therefore, greater color development in the 2012 growing season (Table A.1). The cultivar Nesbitt was identified as having among the highest levels of anthocyanins by Lee and Talcott (2004), while I found Nesbitt to have some of the lowest levels among black genotypes. I identified Supreme as having among the lowest levels of total anthocyanins, which was also found by Threlfall et al. (2007) and Striegler et al. (2005). It is unclear then why my levels agree with some and not other studies, but this could be due to differences in environment, maturity, cultural management, or methodology of measurement (Awad et al., 2001). Total Ellagitannin. Concentrations of total ellagitannins were found to be lower than those reported for muscadines by Marshall et al. (2012) and Lee and Talcott (2004), but similar to those reported by Boyle and Hsu (1990), Lee et al. (2005), Pastrana Bonilla et al. (2003), Stringer et al. (2009), and Talcott and Lee (2002). Ellagitannin concentrations were found to be higher than those reported for strawberry (Cordenunsi et al., 2002), but lower than those reported in blackberry and raspberry (Maatta Riihinen et al., 2004; Siriwoharn et al., 2004). Additionally, ellagitannins were absent in other V. vinifera and V. lubrusca grapes (Marshall et al., 2012). Total ellagitannin concentrations were very genotype 184

201 specific, and no clear relationship was identified between color (bronze verses black) and ellagitannins (Table 2.6). Overall, total ellagitannin concentrations were greater in 2013 compared to 2012 (Table 2.7), which is similar to the findings of Tharayil et al. (2011), who found that warmer and drier growing conditions were associated with lower levels of ellagitannins in the leaves of Red Maple (Acer rubrum L.) compared to wetter growing conditions. Total Flavonols. Total flavonol concentrations found were lower than those reported by Marshall et al. (2012) and Talcott and Lee (2002). Overall, total flavonols were higher in 2013 compared to 2012 (Fig and Table A.11). Generally, results showed that genotypes differed among years, with the exceptions of AM 15 and Summit, which had among the highest total flavonol concentrations both years of my study (Fig. 2.13). The bronze genotypes were found to be generally higher in total flavonols than the darker genotypes (Fig. 2.13), this potentially due to the presence of the flavonol myricetin in the bronze genotypes (Marshall et al., 2012). A positive correlation with total flavonols and soluble solids (r=0.73) and a negative correlation with hue angle and total flavonols (r= 0.73) occurred (Table A.11). These correlations possibly illustrate that riper berries have higher flavonol concentrations, as soluble solids has been shown to be an indicator of muscadine berry ripeness (Carroll and Marcy, 1982; Flora and Lane, 1979; Johnson and Carroll, 1973; Lanier and Morris, 1979; Peynard and Riberau Gayon, 1971; Walker et al., 2001), and berries with lower hue angles had higher total flavonols, which is supported by the data as the bronze genotypes generally had higher total flavonol levels and lower hue angles (Figs and 2.13). I found total flavonols at higher levels than those found in strawberry, but lower than those found in blackcurrant, chokeberry (Aronia mitschurinii 185

202 A.K.Skvortsov & Maitul), cranberry (Vaccinium oxycoccos L.), and red raspberry (Cordenunsi et al., 2002; Hakkinen et al., 1999; Maatta Riihinen et al., 2004). Total Phenolics. Total phenolic concentrations found were similar to those previously reported for muscadines (Lee et al., 2005; Lee and Talcott, 2004; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Striegler et al., 2005; Stringer et al., 2009; Talcott and Lee, 2002; Threlfall et al., 2007). Similar phenolic concentrations were reported in blackberry (Siriwohorn et al., 2004), while lower phenolic concentrations were found in highbush blueberry and strawberry (Cordenunsi et al., 2002; Ehlenfeldt and Prior et al., 2001). Total phenolic concentrations were significantly higher in 2012, compared to 2013 (Table 2.9), likely due to the added stress on the vines from hot and dry growing conditions, and the plants responding with increased phenolic production. I found Summit to have among the highest levels of total phenolics, which was similar to the findings of Threlfall et al. (2007). Additionally, I found the cultivar Supreme to have the overall lowest total phenolics (average value across years) among the genotypes measured, while Striegler et al. (2005), found Supreme to have among the highest total phenolic level of 6072 mg/kg (607.2 mg/100 g) in their study. The lower level found in my study for Supreme could be due to environment, maturity, or cultural management. Resveratrol. Trans resveratrol concentrations found were similar to those previously reported in muscadines (Ector et al., 1996; Magee et al., 2002; Marshall et al., 2012; Pastrana Bonilla et al., 2003; Stringer et al., 2009). As I did, Marshall et al. (2012) found resveratrol in nearly every genotype evaluated. There was no significant difference among year means of resveratrol concentrations, though variation did occur among genotypes each year (Fig. 2.15). Highlighted by the large error bars associated with the 186

203 genotypes AM 01, Ison, Nesbitt, Summit and Supreme, resveratrol concentrations varied within individual genotypes as well, due to the variation among replications within some genotypes (Fig. 2.14). No clear relationship between berry color and resveratrol concentrations were found, conversely Ector et al. (1996) found resveratrol to be greater in black genotypes. Magee et al. (2002) found the bronze Summit to have among the highest levels of resveratrol in a group of both black and bronze genotypes, which is similar to the findings of my study. Trans resveratrol concentrations were found to be equivalent to those in V. vinifera (Vincenzi et al., 2013), which was unexpected. Ector et al. (1996) found that resveratrol concentrations were higher than those reported for V. vinvifera grapes. The different resveratrol concentrations found in my study could be due to environment, maturity, or cultural management, as resveratrol is produced in response to environmental factors during the growing season (Marshall et al., 2012). ORAC. Oxygen radical absorbance capacity is widely accepted as being a good estimation of antioxidant capacity of fruits, although its significance is often questioned, as it does not accurately represent the bioactivity of the antioxidants in the human body. The ORAC values I found were similar to those previously reported by Sandhu and Gu (2010) and Talcott and Lee (2002), but were considerably higher than those reported by Lee et al. (2005), Striegler et al. (2005), and Threlfall et al. (2007). I found ORAC values to be greater than or comparable to those found in apple (Malus domestica Borkh.), blackberry, highbush blueberry, plum, orange, red table grapes, strawberry, and white table grapes (Ehlenfeldt and Prior et al., 2001; Siriwoharn et al., 2004; Wang et al., 1996; Wu et al., 2004). ORAC levels were found to be higher in 2013 compared to 2012, which could possibly be due to the extremely hot and dry growing season in 2012 (Table A.1), although it was difficult to 187

204 identify this clearly among the genotypes (Fig 2.15). The cultivar Ison had the highest ORAC values both years of the study, while Supreme and Tara had among the lowest both years (Fig and Table A.10). Conversely, Threlfall et al. (2007) reported Nesbitt having among the lowest ORAC levels, while Striegler et al. (2005) identified Supreme as having among the highest. It is important to note the vast variation in ORAC levels among genotypes (Fig. 2.15), which illustrates ORAC as having potential as a character to be selected for in muscadine breeding programs. Black Verses Bronze Storage Performance Throughout this experiment, the bronze genotypes had higher levels of unmarketability compared to the black genotypes, which was often due to browning and discoloration that occurred during storage, though individual causes of unmarketable berries were not recorded. It was found that overall the black genotypes had better storage performance than the bronze genotypes (Fig. 2.16). As expected, significant differences were seen between bronze and black genotypes for percent unmarketable, but surprisingly differences were also identified in percent volume change and force to penetrate the berry skin. However, there were no differences found in percent weight loss between berry color (Fig. 2.16). In addition to browning, the bronze genotypes often showed greater signs of decay. This could potentially have caused the differences found in force and volume change, though no data were collected specifically on the effects of decay on these variables. It is hypothesized that the black genotypes could have also had symptoms of browning or discoloration during storage, but these symptoms were not visible due to the darkness of the berry skin. Storage Protocol 188

205 A major component of this study was to determine the important parameters of storage performance of muscadine genotypes, and in so doing to develop a storage protocol for the University of Arkansas muscadine breeding program. Overall, both percent unmarketable and percent weight loss increased during storage, showing importance as storage parameters. Force to penetrate the berry skin generally decreased during storage, also showing potential as an important postharvest storage parameter, particularly since some genotypes had significantly less loss in force during storage. Percent change in berry volume showed no clear pattern during storage, probably due to the variation in individual berries within each genotype limiting the usefulness of berry volume as a storage measurement. Composition parameters TA, ph, soluble solids, and SS/TA remained relatively constant during storage, therefore are not important postharvest storage measurements to routinely measure in evaluating storage potential. Though no clear correlations were identified in this study, it has been shown that soluble solids can be useful in determining maturity, which has been shown to be related with storage performance (Ballinger and McClure, 1983; Carroll and Marcy, 1982). The berry color measurements, Chroma and hue angle, generally showed no clear pattern during storage, while L* showed a sharp decrease after date of harvest and then remained relatively constant during storage. Therefore, it is potentially valuable to determine L* value at date of harvest and again after storage is complete to evaluate color change during storage. The retention of nutraceutical content (total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol) and antioxidant capacity (ORAC) was not evaluated in this study. 189

206 Among the sources of variation in my study, genotype was the most common source with differences among most dependent variable means. This is a major finding, in that in differentiating the potential value of breeding selections, particularly for postharvest storage potential, adequate variation for a characteristic or trait is needed. It appears there is substantial variation among genotypes in the program for most variables to select for those with improved or superior values. Further, the differences among years for many dependent variables indicated the importance of multi year evaluations of breeding selections for storage potential. Since this study was conducted early in the muscadine breeding program, the findings reported here, including the most critical variables to measure, should lead to improved precision in identifying and releasing improved cultivars for fresh market production with enhanced postharvest potential. Literature cited Awad, M.A., P.S. Wagenmakers, and A. de Jager Effects of light on flavonoid and chlorogenic acid levels in the skin of Jonogold apples. Scientia Hort. 88: Ballinger, W.E., E.P. Maness, and W.B. Nesbitt Anthocyanins of black grapes of 10 clones of Vitis rotundifolia, Michx. J. Food Sci. 38: Ballinger, W.E. and W. F. McClure The effect of ripeness on storage quality of Carlos muscadine grapes. Scientia Hort. 18: Ballinger, W.E. and W.B. Nesbitt. 1982a. Postharvest decay of muscadine grapes (Carlos) in relation to storage temperature, time, and stem condition. Amer J. Enol. Viticult. 33: Ballinger, W.E. and W.B. Nesbitt. 1982b. Quality of muscadine grapes after storage with sulfur dioxide generators. J. Amer. Soc. Hort. Sci. 107: Banini, A.E., L.C. Boyd, J.C. Allen, H.G. Allen, and D.L. Sauls Muscadine grape products intake, diet and blood constituents of non diabetic and type 2 diabetic subjects. Nutrition 22:

207 Ben Yehoshua, S., B. Shapiro, Z.E. Chen, and S. Lurie Mode of action of plastic film in extending life of lemon and bell pepper fruit by alleviation of water stress. Plant Physiol. 73: Boyle, J.A., and L. Hsu Identification and quantitation of ellagic acid in muscadine grape juice. Amer. J. Enol. Viticult. 41: Bralley, E.E., D.K. Hartle, P. Greenspan, and J.L. Hargrove Topical anti inflammatory activities of Vitis rotundifolia (muscadine grape) extracts in the tetradecanoylphorbol acetate model of ear inflammation. J. Medicinal Food 10: Brown, W.L The anthocyanin pigment of the Hunt muscadine grape. J. Amer. Chem. Soc. 62: Burg, S.P Postharvest physiology and hypobaric storage of fresh produce. 1 st ed. CABI Publishing, Wallingford, Oxfordshire, U.K. Carroll, D.E. and J.E. Marcy Chemical and physical changes during maturation of muscadine grapes (Vitis rotundifolia). Amer. J. Enol. Viticul. 33: Cho, M.J., L.R. Howard, R.L. Prior, and J.R. Clark Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry, and red grape genotypes determined by highperformance liquid chromatography/mass spectrometry. J. Sci. Food Agri. 84: Cho, M.J., L.R. Howard, R.L. Prior, and J.R. Clark Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high performance liquid chromatography/mass spectrometry. J. Sci. Food. Agr. 85: Conner, P.J A century of muscadine Grape (Vitis rotundifolia Michx.) breeding at the University of Georgia. Acta Hort. 827: Conner, P.J Instrumental texture analysis of muscadine grape germplasm. HortScience 48: Conner, P.J., and D. Maclean Evaluation of muscadine grape genotypes for storage ability. HortScience 47:S386. (abstr.). Conner, P.J., and D. MacLean Fruit anthocyanin profile and berry color of muscadine grape cultivars and Muscadinia germplasm. HortScience 48: Cordenunsi, B.R., J.R. Oliveira do Nascimento, M.I. Genovese, and F.M. Lajolo Influence of cultivar on quality parameters and chemical composition of strawberry fruits grown in Brazil. J. Agr. Food. Chem. 50:

208 Duan, J., R. Wu, B.C. Strik, and Y. Zhao Effect of edible coatings on the quality of fresh blueberries (Duke and Elliot) under commercial storage conditions. Postharvest Biol. Technol. 59: Ector, B.J Compositional and nutritional characteristics, p In: F.M. Basiouny and D.G. Himelrick, (eds.). Muscadine Grapes. ASHS Press, Alexandria, VA. Ector, B.J., J.B. Magee, C.P. Hegwood, and M.J. Coign Resveratrol concentration in muscadine berries, juice, pomace, purees, seeds, and wines. Amer. J. Enol. Viticult. 1: Ehlenfeldt, M.K., and R.L. Prior Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbush blueberry. J. Agr. Food Chem. 49: Flora, L.F. and R.P. Lane Effects of ripeness and harvest date on several physical and composition factors of Cowart muscadine grapes. Amer. J. Enol. Viticult. 30: Gil, M.I., E. Aguayo, and A.A. Kader Quality changes and nutrient retention in freshcut versus whole fruits during storage. J. Agr. Food. Chem. 54: God, J.M., P. Tate, and L.L. Larcom Anticancer effects of four varieties of muscadine grape. J. Medicinal Food 10: Goldy, R.G., W. E. Ballinger, E. P. Maness, and W. H. Swallow Anthocyanin content of fruit, stem, tendril, leaf, and leaf petiole in muscadine grape. J. Amer. Soc. Hort. Sci. 112: Greenspan, P., A. Ghaffar, J.L. Hargrove, D.K. Hartle, E.P. Mayer, J.D. Bauer, S.H. Pollock, and J.D. Gangemi Anti inflammatory properties of the muscadine grape (Vitis rotundifolia). J. Agr. Food Chem. 53: Hakkinen, S.H., S.O. Karenlampi, L.M. Heinonen, H.M. Mykkanen, and A.R. Torronen Content of edible flavonols quercetin, myricetin, and kaempferol in 25 edible berries. J. Agr. Food. Chem. 47: Hager, T.J., L.R. Howard, R. Liyanage, J.O. Lay, and R.L. Prior Ellagitannin composition of blackberry as determined by HPLC ESI MS and MALD TOF MS. J. Agr. Food. Chem. 56: Hernandez Herrero, J.A., and M.J. Frutos Colour and antioxidant capacity stability in grape, strawberry, and plum peel model juices at different phs and temperatures. Food. Chem. Himelrick, D.G Handling, storage, and postharvest physiology of muscadine grapes. Small Fruits Rev. 2:(4):

209 Huang, Z., R.D. Pace, P. Williams, and B. Wang Identification of anthocyanins in muscadine grapes with HPLC ESI MS. Food Sci. Tech. 42: Jackson D.I Factors affecting soluble solids, acid, ph, and color in grapes. Amer. J. Enol. Viticult. 37: James, J., O. Lamikanra, G. Dixon, S. Leong, J.R. Morris, G. Main, and J. Silva Shelf life study of muscadine grapes for the fresh fruit market. Proc. Fla. State Hort. Soc. 110: James, J., O. Lamikanra, J.R. Morris, G. Main, T. Walker, and J. Silva Interstate shipment and storage of fresh muscadine grapes. J. Food Quality. 22: Jha, S.N., A.R.P Kingsly, and S. Chopra Physical and mechanical properties of mango during growth and storage for determination of maturity. J. Food. Eng. 72: Johnson, L.A. and D.E. Carroll Organic acid and sugar contents of Scuppernong grapes during ripening. J. Food. Sci. 38: Lane, R.P Effect of vineyard fungicide treatments on the shelf life of muscadine grapes. Georgia Agr. Res. 19: Lane, R.P., and L.F. Flora Some factors influencing storage of muscadine grapes. HortScience 15:273 (abstr.). Lanier, M.R. and J.R. Morris Evaluation of density separation for defining fruit maturities and maturation rates of once over harvested muscadine grapes. J. Amer. Soc. Hort. Sci. 104: Lee, J H. and S.T. Talcott Fruit maturity and juice extraction influences ellagic acid derivatives and other antioxidant polyphenolics in muscadine grapes. J. Agr. Food Chem. 52: Lee, J H., J.V. Johnson, and S.T. Talcott Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC ESI MS. J. Agr. Food Chem. 53: Lutz, J.M Factors influencing the quality of American grapes in storage. U. S. Dept. Agr. Tech. Bul. 606:1 27. Maatta Riihinen, K. R., A. Kamal Eldin, and A.R. Torronen Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (Family Rosaceae). J. Agr. Food Chem. 52: MacLean, D., P.J. Conner, J. Paulk, and L. Grant Postharvest control of decay organisms. The Southern Region Small Fruit Consortium. Prog. Rpt

210 Magee, J.B., B.J. Smith, and A. Rimando Resveratrol content of muscadine berries is affected by disease control spray program. HortScience 37: Marshall, D.A., S.J. Stringer, and J.D. Spiers Stilbene, ellagic acid, flavonol, and phenolic content of muscadine grape (Vitis rotundifolia Michx.) cultivars. Pharmaceutical Crops 3: Mascarenhas, R.d.J., S.d.M. Silva, M.A.C.d. Lima, R.M.N. Mendonca, and H.J. Holschuh Characterization of maturity and quality of Brazilian apirenic grapes in the Sao Francisco river Valley. Cie ncia e Tecnologia de Alimentos. 32: McCornack, A.A Postharvest weight loss of Florida citrus fruits. Proc. Fla. State Hort Soc. 88: Morris, J.R Handling and marketing of muscadine grapes. FruitSouth, 4(2): Morris, J.R., O.L. Oswald, G.L. Main, J.N. Moore, and J.R. Clark Storage of new seedless grape cultivar with sulfur dioxide generators. Amer. J. Enol. Viticult. 43: Musingo, M.N., S.F. O'Keefe, O. Lamikanra, C.A. Sims, and R.P. Bates Changes in ellagic acid and other phenols in muscadine grape (Vitis rotundifolia) juices and wines during storage. Amer. J. Enol. Viticult. 52: Pastrana Bonilla, E., C.C. Akoh, S. Sellappan, and G. Krewer Phenolic content and antioxidant capacity of muscadine grapes. J. Agr. Food Chem. 51: Perkins Veazie, P., S. Spayd, B. Cline, and C. Fisk Handling and marketing guide for fresh market muscadine grapes. SFRC E03: Peynard, E. and P. Ribercau Gayun The grape, p In: A.D. Hulme (ed.). The bio chemistry of fruits and Their Products. Vol. 2. Academic Press, Waltham, MA. Prior, R.L., H. Hoang, L. Gu, X. Wu, M. Bacchiocca, L. Howard, M. Hampschwoodill, D. Haung, B. Ou, and R. Jacob Assays for hydrophilic and lipophilic antioxidant capacity [oxygen radical absorbance capacity (ORACF1)] of plasma and other biological and food samples. J. Agr. Food Chem. 51: Prior, RL, S.A. Lazarus, G. Cao, H. Muccitelli, and J.F. Hammerstone Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium Spp) using highperformance liquid chromatography/mass spectrometry. J Agr. Food Chem. 49: Sandhu, A.K., and L.W. Gu 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 MSn. J. Agr. Food Chem. 58:

211 Silva, J.L., E. Marroquin, C.P. Hegwood, G.R. Silva, and J.O. Garner Jr Quality changes in muscadines for table grapes during refrigerated storage in various packaging systems. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 17: Siriwoharn, T., R.E. Wrolstad, C.E. Finn, and C.B. Pereira Influence of cultivar, maturity, and sampling on blackberry (Rubus L. hybrids) anthocyanins, polyphenolics, and antioxidant properties. J. Agr. Food Chem. 52: Slinkard, K. and V.L. Singleton Total phenol analysis: automation and comparison with manual methods. Amer. J. Enol. Viticult. 28: Smit, C.J.B., H.L. Cancel, and T.O.M. Nakayama Refrigerated storage of muscadine grapes. Amer. J. Enol. Viticult. 22: Smittle, D.A Requirements for commercial CA storage of muscadine grapes. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 13: Striegler, R.K., P.M. Carter, J.R. Morris, J.R. Clark, R.T. Threlfall, and L.R. Howard Yield, quality, and nutraceutical potential of selected muscadine cultivars grown in southwestern Arkansas. HortTechnology 15: Stringer, S.J., D.A. Marshall, and P. Perkins Veazie Nutraceutical compound concentrations of muscadine (Vitis rotundifolia Michx.) grape cultivars and breeding lines. Acta. Hort. 841: Takeda, F., M. Starnes Saunders, C.F. Savoy, and T.T. Hatton Storageability of muscadines for use as fresh fruit. Proc. Viticult. Sci. Symp. Fla. A and M. Univ. 3: Takeda, F., M. Starnes Saunders, J.A. Saunders, and T.T. Hatton Effects of prestorage treatment and storage temperature on incidence of decay and chemical composition in muscadine grape. Proc. Fla. State. Hort. Soc. 95: Talcott, S.T., and J H. Lee Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. J. Agr. Food Chem. 50: Tharayil, N., V. Suseela, D.J. Triebwasser, C.M. Preston, P.D. Gerard, and J.S. Dukes Changes in the structural composition and reactivity of Acer rubrum leaf litter tannins exposed to warming and altered precipitation: climatic stress induced tannins are more reactive. New Phytol. 191: Threlfall, R.T., J.R. Morris, J.F. Meullenet, and R.K. Striegler Sensory characteristics, composition, and nutraceutical content of juice from Vitis rotundifolia (muscadine) cultivars. Amer. J. Enol. Viticult. 58:

212 Threlfall, R.T., J.R. Morris, L.R. Howard, C.R. Brownmiller, and T.L. Walker Pressing effects on yield, quality, and nutraceutical content of juice, seeds, and skins from Black Beauty and Sunbelt grapes. J. Food Sci. 70: U.S. Department of Agriculture United States standard for grades of muscadine (Vitis rotundifolia) grapes. U.S. Dept. Agr. Washington, D.C. Vincenzi, S., D. Tomasi, F. Gaiotti, L. Lovat, S. Giacosa, F. Torchio, S. Rio Segade, and L. Rolle Comparative study of the resveratrol content of twenty one Italian red grape varieties. South African J. Enol. Viticult. 34: Walker, T.L., J.R. Morris, R.T. Threlfall, G.L. Main, O. Lamikanra, and S. Leong Density separation, storage, shelf life, and sensory evaluation of 'Fry' muscadine grapes. HortScience 36: Wang, C.Y Chilling injury of horticultural crops. 1 st ed. CRC Press, Boca Raton, FL. Wang, H., G. Cao, and R.L. Prior Total antioxidant capacity of fruits. J. Agr. Food Chem. 44: Wu, X., G.R. Beecher, J.M. Holden, D.B. Haytowitz, S.E. Gebhardt, and R.L. Prior Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agr. Food Chem. 52: Yi, W., C.C. Akoh, and J. Fischer Study of anticancer activities of muscadine grape phenolics in vitro. J. Agr. Food Chem. 53:

213 Appendix A: Meteorological data, interaction means, and correlations. Table A.1. Average monthly maximum and minimum temperatures and total rainfall recorded at the Fruit Research Station; Clarksville, AR (lat N and long W) (2012 and 2013). Year Month Maximum temperature ( C) Minimum temperature ( C) Precipitation (mm) 2012 January February March April May June July August September October November December January February March April May June July August September October November December

214 Table A.7. Interaction means of the postharvest attributes of percent weight loss, percent unmarketable, force to penetrate berry skin, and percent volume change for year, genotype, and week of storage at 2 3 C for 0 3 weeks. Genotype Year Week Weight loss (%) Force (N) Volume change (%) Unmarketable (%) AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM

215 Genotype Year Week Weight loss (%) Force (N) Volume change (%) Unmarketable (%) AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM Delicious Delicious Delicious Delicious

216 Genotype Year Week Weight loss (%) Force (N) Volume change (%) Unmarketable (%) Delicious Delicious Delicious Delicious Fry Fry Fry Fry Fry Fry Fry Fry Ison Ison Ison Ison Ison Ison Ison Ison Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Summit Summit Summit Summit

217 Genotype Year Week Weight loss (%) Force (N) Volume change (%) Unmarketable (%) Summit Summit Summit Summit Supreme Supreme Supreme Supreme Supreme Supreme Supreme Supreme Tara Tara Tara Tara Tara Tara Tara Tara

218 Table A.8. Interaction means of the composition attributes of percent titratable acidity, percent soluble solids, ph, and soluble solids to titratable acidity for year, genotype, and week of storage at 2 C for 0 3 weeks. Genotype Year Week Titratable acidity (%) ph Soluble solids (%) SS/TA AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM

219 Genotype Year Week Titratable acidity (%) ph Soluble solids (%) SS/TA AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM Delicious Delicious Delicious Delicious Delicious

220 Genotype Year Week Titratable acidity (%) ph Soluble solids (%) SS/TA Delicious Delicious Delicious Fry Fry Fry Fry Fry Fry Fry Fry Ison Ison Ison Ison Ison Ison Ison Ison Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Summit Summit Summit Summit Summit

221 Genotype Year Week Titratable acidity (%) ph Soluble solids (%) SS/TA Summit Summit Summit Supreme Supreme Supreme Supreme Supreme Supreme Supreme Supreme Tara Tara Tara Tara Tara Tara Tara Tara

222 Table A.9. Interaction means of the berry color attributes of Chroma, Hue angle, and L* values for year, genotype, and week of storage at 2 C for 0 3 weeks. Genotype Year Week L* Chroma Hue angle AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM

223 Genotype Year Week L* Chroma Hue angle AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM Delicious Delicious Delicious Delicious Delicious Delicious Delicious Delicious Fry Fry

224 Genotype Year Week L* Chroma Hue angle Fry Fry Fry Fry Fry Fry Ison Ison Ison Ison Ison Ison Ison Ison Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Nesbitt Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Southern Jewel Summit Summit Summit Summit Summit Summit Summit Summit Supreme Supreme Supreme Supreme

225 Genotype Year Week L* Chroma Hue angle Supreme Supreme Supreme Supreme Tara Tara Tara Tara Tara Tara Tara Tara

226 Table A.10. Interaction means of the berry nutraceutical concentrations of total phenolics, total anthocyanins, total ellagitannins, total flavonols, and resveratrol, and the antioxidant capacity (ORAC) for year and genotypes. Genotype Year Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmol TE/g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM AM Delicious Delicious Fry Fry Ison Ison Nesbitt Nesbitt S. Jewel S. Jewel Summit Summit Supreme Supreme Tara Tara

227 Table A.11. Study 2 multivariate correlation coefficients among muscadine berry storage quality, composition, color, and nutraceutical content for 2012 and *Significant at P=0.05. WL z UM y TA w ph SS v L* Chr u Hue t For s VC x Anth q Ella p ORAC o Fla n WL z UM y 0.00 TA w ph SS v L* * Chr u Hue t * * For s * VC x Anth q * Ella p ORAC o Fla n * * TP m * 0.50 Res l z WL=weight loss of berries (%) y UM=unmarketable berries (%) x VC=volume change (%) w TA=titratable acidity (%) measures as tartaric acid v SS=soluble solids (%) u Chr=Chroma t Hue=hue angle s For=force to penetrate berry skin (%) q Anth=total anthocyanins (mg/100 g) p Ella=total ellagitannins (mg/100 g) o ORAC=oxygen radial absorbance capacity (µmol Trolox equivalent/ g) n Fla=total flavonols (mg/100 g) m TP=total phenolics (mg/100 g) l Res=trans-resveratrol (mg/100 g) 211

228 Chapter 3 THE EFFECT OF STORAGE TIME ON NUTRACEUTICAL CONTENT OF SUPREME MUSCADINE BERRY SEGMENTS Abstract A common goal of muscadine (Vitis rotundifolia Michx.) breeding programs is increased storability, as a major limiting factor in muscadine commercialization is deterioration during storage. Genotypes with improved skin and flesh texture have shown some promise for increased storability. Muscadines have been shown to have high levels of nutraceuticals in the berry skin and seeds; however, studies on the retention of these nutraceuticals are limited. The effect of storage time on total anthocyanins, total ellagitannins, total flavonols, total phenolics, trans resveratrol, and the antioxidant capacity measurement of oxygen radical absorbance capacity (ORAC) were evaluated on whole berries, as well as berry flesh (skin and pulp) and seeds from three vines of Supreme in 2012 and Muscadine berries were harvested and stored at 2 C and 85 95% relative humidity. Every 7 d for 6 weeks in 2012, and only at harvest in 2013 berries nutraceutical measurements were taken. In 2012 total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and trans resveratrol were significantly affected by week of storage, vine, and berry segment, though varying differences occurred. The nutraceutical measurements at harvest indicated total anthocyanins, ORAC, total flavonols, total phenolics, and trans resveratrol were significantly affected by year, vine, and berry segment, while total ellagitannins were only affected by berry segment. It was found that total anthocyanins were greatest in the flesh and whole berries, and not detected in the seeds. Total ellagitannins, total flavonols, and trans resveratrol were found to be greatest 212

229 in the seeds. Total phenolics and ORAC in the whole berries had the greatest concentrations. Differences in nutraceutical concentrations among vines were generally minimal; however, vine 1 was found to generally have the highest total flavonol concentration. Nutraceutical concentrations varied among weeks of storage, but no clear pattern of change was evident during storage. Total anthocyanins, ORAC, and total phenolics were greater in 2012, while trans resveratrol was greater in Overall, it was determined that nutraceutical and antioxidant capacity varied by berry segment and year of the study, but were generally less affected by vine and storage time. 213

230 Introduction Muscadine grapes (Vitis rotundifolia Michx.) are indigenous to the southeastern United States. Muscadines have been under cultivation for over 400 years, originally in the North Carolina Colony followed soon after by surrounding colonies and states (Conner, 2009). Muscadines are the most important Vitis species cultivated in the southeastern U.S. (Marshall and Stringer, 2014). This native grape is presently grown in small vineyards and home plantings, ranging from North Carolina and Florida to eastern Oklahoma and Texas. Arkansas has approximately 230 ha of muscadines in production, making up 10% of total US production. The recent recognition that the berries are important sources of beneficial antioxidants has increased consumer demand (Perkins Veazie et al., 2012). A major limiting factor in muscadine grape commercialization is deterioration during storage. One solution for extending market seasons and preventing market saturation for fresh muscadines could be the release of new cultivars with improved postharvest storability. In related studies it was found that genotypes with improved texture and increased firmness, e.g. Supreme, have the potential to remain marketable following storage time of up to 3 weeks (Studies 1 and 2). Despite that, the retention of nutraceuticals in freshmarket muscadines in storage is widely unstudied (Marshall and Stringer, 2014). Nutraceuticals are compounds found in foods that have physiological benefits. Among fruits, muscadines contain some of the highest levels of nutraceuticals; additionally, several of the compounds present are unique to muscadines (Marshall et al. 2012). Brown (1940) first researched the anthocyanin pigments of muscadines, and found that the anthocyanins present in muscadines are unique in that they are 3,5 diglucosides, this differs from V. vinifera L. and V. aestivalis Michx., which contain 3 monoglucosides. 214

231 Individual anthocyanins present in muscadines have been identified as cyanidin (Cy), delphinidin (Dp), malvidin (Mv), peonidin (Pn), and petunidin (Pt) (Ballinger et al., 1973; Flora, 1978, Goldy et al., 1986). The presence of anthocyanins have been shown to protect blood vessels, and play a role in cancer prevention, though anthocyanin absorption in the blood appears to be low in humans (Prior, 2004). Although commonly found in other fruits, muscadines have been shown to contain ellagitannins, which is unique within Vitis (Marshall et al., 2012). Ellagic acid in muscadines is expressed as free ellagic acid, ellagic acid glycosides, and ellagitannins (Marshall et al., 2012; Talcott and Lee, 2002). Ellagic acid and its derivatives have been widely studied due to their antiproliferative properties through their ability to directly inhibit DNA binding of certain carcinogens, and their chemoprotective effect in cellular models by reducing oxidative stress (Talcott and Lee, 2002, Lesca, Patrana Bonilla, 2003, Mertens Mertens Talcott, et al., 2003, Stoner and Morse, 1997, Khanduja et al., 1999). Talcott and Lee (2002) identified that potential for increased marketability of muscadines exists, due to the possible health benefits associated with the presence of ellagitannins. In addition to ellagitannins, the presence of the flavonol myricetin in bronze muscadines is also unique (Marshall et al., 2012). It has been shown that flavonols protect against the initiation of cancer formation through protection against DNA mutations (Hollman, and Katan, 1999; Williamson, and Manach, 2005). Certain flavonol compounds have also been shown to prevent heart disease through relaxation of blood vessel walls and increased production of enzymes that dissolve blood clots (Abou Agag, et al., 2001; Rendig, et al., 2001). Sandhu and Gu (2010) identified the flavonols present in muscadines as glycosides of quercetin, kaempferol, and myricetin. They also identified the flavonols 215

232 myricetin hexoside, kaempferol hexoside, quercetin glucoside, and kaempferol rutinoside for the first time in muscadines. Phenolics are secondary metabolites that are ubiquitous in the kingdom Plantea, and are involved in plant response to abiotic and biotic stresses (Marshal et al., 2012). In addition to protecting the parent plant, often phenolics exhibit significant pharmaceutical benefits (Marshall et al., 2012). Measurements of total phenolics to provide an overall assessment of the content and chemical activity of compounds present, and aids in determining the antioxidant capacity of fruits and vegetables (Lee and Talcott, 2004; Thiapong et al., 2006). Muscadine grapes have been identified as having high levels of total phenolics (Marshall et al., 2012; Pastrana Bonilla et al., 2003; Striegler et al., 2005; Stringer et al., 2009; Threlfall et al., 2007), and Threlfall et al. (2007) found that total phenolic concentrations are not related to berry skin color, and ranged in concentrations among bronze and black genotypes. Stilbenes are synthesized in grape leaves in response to both biotic and abiotic induction treatments, and the capacity to produce stilbenes is correlated with the resistance of grape leaves to fungal infection (Creasy and Coffee, 1988; Marshall et al., 2012). Resveratrol (trans 3,5,4 trihydroxystilbene) is a phytoalexin, or stilbene, produced as a response to fungal infection, stress including injury, and UV irradiation (Jeandet et al., 1995; Jeandet et al., 1991; Marshall et al., 2012; Magee et al., 2002; Threlfall et al., 1999). Resveratrol has two forms, cis and a trans resveratrol; this study will only focus on the trans isomer. Resveratrol has long been confirmed in both red and white V. vinifera grape skins and pulp, but not in seeds. Only within the last 20 years has resveratrol been measured in muscadines. The fleshy parts of both black and bronze muscadine berries have 216

233 higher concentrations of resveratrol than reported for V. vinifera and V. labrusca (Ector et al., 1996). Resveratrol can potentially act as a chemopreventative agent and afford protection against cardiovascular and coronary heart disease (Hudson et at., 2007; Jang et al., 1997; Lu and Sorreno, 1999; Magee et al., 2002). Resveratrol has lipid lowering action, inhibition of human low density lipoprotein oxidation and thus may delay atherosclerosis onset, inhibition of platelet aggregation in the blood, reduce cholesterol levels, and has been shown to have anticarcinogenic activity in all stages of prostate and breast cancer (Ector et al., 1996; Ector, 2001; Hudson et at., 2007; Jang et al., 1997; Lu and Sorreno, 1999; Magee et al., 2002). Measurements of peroxyl radical (free radical) scavenging activity using oxygen radical absorbance capacity (ORAC) assay is a common index that provides an overall assessment of the content and chemical activity of compounds present, and aids in determining the antioxidant capacity of fruits and vegetables, although its significance is often questioned, as it does not accurately represent the bioactivity of the antioxidants in the human body (Lee and Talcott, 2004; Prior et al, 2003). Free radicals may contribute to the cause of a number of diseases including cancer and atherosclerosis (Wang et al., 1996). Studies have been conducted to determine the ORAC values of muscadines (Sandhu and Gu, 2010; Striegler et al., 2005; Talcott and Lee, 2002; Threlfall et al., 2007). It has been determined that ORAC is generally higher in fully ripe muscadines, and levels vary among genotypes and berry segments (Sandhu and Gu, 2010; Striegler et al., 2005; Talcott and Lee, 2002; Threlfall et al., 2005). Studies have been conducted to understand the concentration of nutraceutical compounds in the different segments (juice, skins, pulp, and seeds) of muscadine berries 217

234 (Lee and Talcott, 2004; Marshall et al., 2012; Sandhu and Gu, 2010; Shi et al., 2003). Ellagic acid has been identified in higher concentrations in the skins of the berry when compared to the pulp or juice (Lee and Talcott, 2004). Anthocyanin concentrations were found to be generally higher in the berry skins and juice than in the pulp or seed (Lee and Talcott, 2004; Marshall et al., 2012; Threlfall et al., 2005). Total phenolics were higher in the seeds than in the skin or pulp of muscadines (Lee and Talcott, 2004; Marshall et al., 2012; Shi, et al., 2003; Takeda et al., 1983; Threlfall et al., 2005). ORAC has been measured in muscadine seeds, skins, pulp, and juice (Lee and Talcott, 2004; Threlfall et al., 2005). Lee and Talcott (2004) found ORAC values to be highest in the skin, which is often exceptionally thick in muscadines. However, the effect of storage time on the nutraceutical levels of individual muscadine berry segments is widely unstudied. Since the implementation of a muscadine breeding program at the University of Arkansas in 2005, selections have been made based on improved texture, and dry stem scar, potentially resulting in improved postharvest storability. Nutraceutical levels in muscadines can vary among genotypes (Marshall et al., 2012), and no information has been collected on the nutraceutical content of the University of Arkansas breeding selections; however, nutraceutical concentrations have merit as possible variables for selection. (J.R. Clark, personal communication). The cultivar Supreme was used as it has shown to have improved texture and the potential for maintaining high quality extended postharvest storage. The objective of this study was to determine the effect of storage time on nutraceutical concentrations and antioxidant capacity of Supreme muscadine berry flesh (skin and pulp), seeds, and whole berries. It was hypothesized that nutraceutical content 218

235 and antioxidant capacity of muscadine grapes would vary during storage. Materials and Methods Grapes and Vineyard Vines of Supreme muscadines used for the study were grown at the University of Arkansas Fruit Research Station, Clarksville, AR (lat N and long W). One vine used in this study was 7 years old, while the other 2 vines were three years old. The vines were grown in Linker fine sandy loam, in USD hardiness zone 7a, where average annual minimum temperatures reach 15 to 17.7 C. Vine spacing was 6.1 m apart and rows were spaced 3.0 m apart. A single wire trellis was used, and vines were trained to a bilateral cordon. The vines were dormant pruned annually in February using spur pruning with spurs retained of two to four buds in length. Weeds were controlled with pre and postemergence herbicides as needed and vines did not have any stress from weed competition. Vines were irrigated by drip irrigation as needed, beginning in early June (prior months received adequate rainfall) and continuing through the harvest period. Vines received N fertilization in March of each year at a rate of approximately 70 kg/ha. No insecticides, fungicides or other pest control compounds were applied to the vines. The vines used in the study had full crops produced each year, and no crop reduction due to winter injury or other limitations occurred. Thus, the vines produced berries under representative conditions. Daily maximum and minimum temperatures along with rainfall were recorded at the research location to characterize the environment the vines were subjected to and potential differences among years (Table A.1). Harvest and Transport 219

236 The muscadine berries were once over, hand harvested at the Fruit Research Station. Berries were harvested late in the afternoon and transported to University of Arkansas Institute of Food Science and Engineering, Fayetteville, AR., in an air conditioned car on the same day. Harvest date/maturity was based on soluble solids of 18 22% in 2012 and 15 18% in 2013 (due to differences in summer temperature and precipitation), ease of release from the pedicel, and berry color. Composition Analysis Composition measurements were taken only at harvest. Titratable acidity and ph were measured by an 877 Titrino Plus (Metrohm AG, Herisau Switzerland) with an automated titrimeter and electrode standardized to ph 2.0, 4.0, 7.0, and 10.0 buffers. Titratable acidity was determined using 6 g of juice diluted with 50 ml of deionized, degassed water by titration of 0.1 N sodium hydroxide (NaOH) to an endpoint of ph 8.2, and results were expressed as percent tartaric acid. Soluble solids were measured using a Bausch and Lomb Inc. Abbe Mark II refractometer (Rochester, NY). Soluble solids, TA, and ph were measured from the juice of the whole berries, strained through cheesecloth to remove any solids. Postharvest Storage Berries were then hand sorted to remove any split, shriveled, or decayed fruit before packaging. Only sound berries, showing no signs of unmarketability, were stored. The berries were packaged into hinged standard vented clamshells (18.4 cm x 12.1 cm x 8.9 cm) (H116, FormTex Plastics Corporation, Houston, TX) and stored in plastic harvest lugs in cold storage at 2 C with 85 89% relative humidity (RH). From the harvested berries, three vented clamshell containers were filled to approximately 500 g. Every 7 d for 6 220

237 weeks, six randomly selected berries from each replication were removed, placed in plastic bags and stored at 20 C until analysis. Of the six berries, three were kept as whole berries, and three were divided into flesh (skin and pulp) and seeds for nutraceutical analysis. Nutraceutical Analysis For nutraceutical analysis, the frozen berries were thawed in 30 C water for 30 s. The berries were then cut open and the seeds were removed from the flesh for analysis. The berries and berry segments were homogenized three times each for 1 min in alternating washes of 80 ml (whole berries and flesh) or 25 ml (seeds) of extraction solution containing methanol/water/formic acid (MWF) (60:37:3 v/v/v) and acetone/water/acetic (70:29.5:0.5 v/v/v) to the smallest particle size using a Euro Turrax T18 Tissuemizer (Tekmar Dohrman Corp, Mason, OH). Homogenates were then centrifuged for 5 min at 10,000 rpm and filtered through Miracloth (CalBiochem, LaJolla, CA). The samples were taken to a final volume of 250 ml (whole berries and flesh) or 100 ml (seeds) with extraction solvent and stored at 70 C until further analysis. Prior to high performance liquid chromatography (HPLC) analysis, the samples were filtered through 0.45 μm filters (Whatman PLC, Maidstone, UK). Nutraceutical concentrations and antioxidant capacity evaluations were based on modified methods determined by Cho et al. (2004), Cho et al. (2005), Hager et al. (2008), and Prior et al. (2003). Total Phenolics. Total phenolics were measured using the Folin Ciocalteu assay (Slinkard and Singleton 1977) on a diode array spectrophotometer (8452A; Hewlett Packard, Palo Alto, CA), with a gallic acid standard and a consistent standard curve based on sequential dilutions. Samples were prepared with 1 ml 0.2N Folins reagent, 0.8 ml Na2CO3 (75 g/l) and 0.2 ml of extracted sample with a reaction time of 2 h. Absorbance 221

238 was measured at 760 nm, and results were expressed as gallic acid equivalents (GAE), on a per weight basis. Anthocyanin, Ellagitannin, and Flavonol Analysis. For anthocyanin, ellagitannin, and flavonol analysis, subsamples (5 ml) of supernatant were evaporated to dryness using a SpeedVac concentrator (ThermoSavant, Holbrook, NY) with no radiant heat and suspended in 1 ml of aqueous 3% formic acid solution. Samples (1 ml) were analyzed using a Waters HPLC system equipped with a model 600 pump, a model 717 Plus autosampler, and a model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Symmetry C18 column (Waters Corp, Milford, MA) with a 3.9 mm 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid and methanol from 2% to 60% for 60 min at 1 ml min 1. Prior to each injection, the system was equilibrated for 20 min at the initial gradient. Detection wavelength was 510 nm for anthocyanins. Individual anthocyanin diglycosides were quantified as Dp, Cy, Pt, Pn, Pg, and Mv glycoside equivalents. Total anthocyanins were calculated as the sum of individual glycosides and their derivatives. For total flavonol and ellagitannin analysis, samples (5 ml) of supernatant were evaporated to dryness using a SpeedVac concentrator with no radiant heat and suspended in 1 ml of aqueous 50% methanol solution. The samples were analyzed using a Waters HPLC system (Waters Corp, Milford, MA) equipped with a model 600 pump, model 717 plus autosampler and model 996 photodiode array detector. Separation was carried out using a 4.6 mm 250 mm Aqua C18 column (Phenomenex, Torrance, CA) with a 3.0 mm 4.0 mm ODS C18 guard column (Phenomenex). The mobile phase was a gradient of 20 g kg 1 acetic acid (A) and 5 g kg 1 acetic acid in water and acetonitrile (50:50 v/v, B) 222

239 from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min. Prior to each injection, the system was equilibrated for 20 min at the initial gradient. A detection wavelength of 360 nm was used for flavonols and 280 nm for ellagitannins at a flow rate of 1 ml min 1. Flavonols and ellagitannins were expressed as mg rutin equivalents kg 1 fresh weight. Analytical Standard and HPLC/MS. For flavonol and ellagitannin confirmation, a representative bronze and black genotype were analyzed using mass spectrometry (MS). For HPLC/MS analysis the HPLC apparatus was interfaced to a Burker Esquire (Burker Corporation, Billerica, MA) LC/MS ion trap mass spectrometer. Mass spectral data were collected with the Bruker software, which also controlled the instrument and collected the signal at 360 or 510 nm. Typical conditions for mass spectral analysis in negative ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 ml min 1 and a temperature of 300 C. Data were collected in full scan mode over a mass range of m/z at 1.0 s per cycle. Characteristic ions were used for peak assignment. Resveratrol (3,4,5 Trihydroxy trans stilbene, 5 [(1E) 2 (4 Hydroxyphenyl)ethenyl] 1,3 benzenediol) concentrations were confirmed using an analytical standard (ID: ; Sigma Aldrich Co. LLC, St. Louis, MO). Oxygen Radical Absorbance Capacity. The ORAC of muscadine extracts was measured using the method of Prior et al. (2003) modified for use with a FLUOstar Optima microplate reader (BMG Labtechnologies, Durham, NC) using fluorescein as a fluorescent probe. Muscadine extracts were diluted 1600 fold with phosphate buffer (75 mm, ph 7) prior to ORAC analysis. The assay was carried out in clear 48 well Falcon plates (VWR, St. Louis, 223

240 MO), each well having a final volume of 590 μl. Initially, 40 μl of diluted sample, Trolox equivelants (TE) standards (6.25, 12.5, 25, 50 μm) and blank solution of phosphate buffer were added to each well. The FLUOstar Optima instrument equipped with two automated injectors was programmed to add 400 μl of fluorescein (0.108 μm) followed by 150 μl of 2,2 azobis(2 amidino propane) dihydrochloride (AAPH) (31.6 mm) to each well. Fluorescence readings (excitation 485 nm, emission 520 nm) were recorded after the addition of fluorescein and AAPH and every 192 s for 112 min to reach 95% loss of fluorescence. Results were based upon differences in areas under the fluorescein decay curve between the blank, samples, and standards, and expressed relative to the initial reading. The standard curve was obtained by plotting the four concentrations of TE against the net area under the curve of each standard. Final ORAC values were calculated using the regression equation between TE concentration and the net area under the curve and expressed as μmol TE equivalents kg 1 fresh weight. ORAC was expressed on a per weight basis. Experimental Design The data were analyzed separately for 2012 and at date of harvest for 2012 and 2013, due to the lack of differences in nutraceutical levels for storage time in In 2012 the experimental design was a split split plot, with the first split being storage (weeks 0, 1, 2, 3, 4, 5, and 6) and the second split being berry segment (flesh [skin and pulp], seed, and whole berry). When the date of harvest data were combined for both years the experimental design was a split split plot, with the first split being year (2012 and 2013) and the second split being berry segment, with three replications consisting of the harvest of each vine. 224

241 Experimental Analysis The data were analyzed by analysis of variance (ANOVA) using JMP (version 11.0; SAS Institute Inc., Cary, NC). Tukey s Honest Significant Difference and Student s t Test was used for mean separations (p = 0.05). Associations among all dependent variables were determined using multivariate pairwise correlation coefficients of the mean values using JMP (version 11.0; SAS Institute Inc., Cary, NC). Results Initial Attributes The initial nutraceutical concentrations and composition of Supreme muscadine berry segments for 2012 and 2013 were presented in Table 3.1. In 2012 the soluble solids for vines one, two, and three were 19.7, 18.5, and 20.2%, respectively, the ph for vines one, two, and three were 3.8, 3.6, and 3.8, respectively, and the TA for vines one, two, and three were 0.29, 0.32, and 0.25%, respectively. In 2013 the soluble solids for vines one, two, and three were 18.1, 16.9, and 17.9%, respectively, the ph solids for vines one, two, and three were 3.9, 4.2, and 3.9, respectively, and the TA solids for vines one, two, and three were 0.21, 0.15, and 0.15%, respectively Nutraceutical Analysis For the 2012 data, the ANOVA F test indicated a significant three way interaction of vine by week of storage by berry segment for total anthocyanins (P=0.0001), total ellagitannins (P=0.0309), and ORAC (P<0.0001) (Table 3.2). The ANOVA F test also indicated a significant two way interaction of vine by berry segment for total flavonols (P<0.0001), total phenolics (P=0.0001), and trans resveratrol (P<0.0001), and a significant two way interaction of week of storage by berry segment for total flavonols (P=0.0227) 225

242 Table 3.1. The initial nutraceutical concentrations of Supreme muscadine berry segments averaged across vines for 2012 and Year Segment Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) ORAC (µmol Trolox equivalents/ g) Resveratrol (mg/100 g) 2012 Whole Flesh Seed ND z Whole Flesh Seed ND znd=not detected. 226

243 and trans resveratrol (P=0.0012) (Table 3.2). Additionally, a significant interaction of week of storage by vine occurred for total phenolics (P=0.0235) and trans resveratrol (P=0.0182) (Table 3.2). The variability of all nutraceuticals during storage of berry segments from different vines offers some insight as to why the interactions were significant. Total Anthocyanins. Major trends in the data for anthocyanins showed their presence only in the flesh and whole berries, but not in the seeds (Fig. 3.1). Additionally, it was found that total anthocyanin concentrations varied among weeks, but no clear pattern occurred during storage, and no significant differences occurred for mean anthocyanins across weeks (P=0.1124). There were also no significant differences in mean anthocyanin concentrations for vines of the study (P=0.2513); however, the muscadine flesh after 3 weeks of storage and whole berries after 4 weeks of storage from vine two and flesh after 5 weeks of storage from vine one had the highest anthocyanin concentrations (71.6, 76.6, and 63.3 mg/100 g, respectively), while whole berries from vine two at harvest had the lowest (14.8 mg/100 g), excluding seeds (Fig. 3.1 and Table A.12). Total Ellagitannins. In 2012, it was found among berry segments that the seeds had the highest concentrations of total ellagitannins, while the flesh and whole berry contents were lower and similar to one another (Fig. 3.2). Though significant differences in total ellagitannin means among weeks occurred (P=0.0003), no clear pattern showed an increase or decrease during storage (Fig. 3.2). Additionally, the differences in mean ellagitannin concentrations that occurred among vines varied, but was not significant (P=0.8704) (Fig. 3.2). It was found that ellagitannin concentrations in the seed from vine one after 1 week of storage were the highest (43.9), while the flesh after 4 weeks of storage 227

244 Table 3.2. F test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and trans resveratrol concentrations of Supreme muscadine berries stored for 6 weeks at 2 C in Highest order interactions are italicized and shaded. DF z Total anthocyanins (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmol Trolox equivalents/ g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) Vine < < < Week < < Week*vine Segment 2 < < < < < < Vine*segment < < < Week*segment < < Vine*week*segment < z Degrees of freedom. 228

245 Fig Total anthocyanin concentrations of Supreme muscadine berry segments (flesh = pulp and skin) stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. Total anthocyanins (mg/100 g) Week Vine Segment Flesh Seed Whole 229

246 Fig Total ellagitannin concentrations of Supreme muscadine berry segments (flesh = pulp and skin) stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. Total ellagitannins (mg/100 g) Week Vine Segment Flesh Seed Whole 230

247 and whole berries after 5 weeks of storage from vine one had the lowest ellagitannin concentrations (0.51 and 0.59 mg/100 g, respectively) (Fig. 3.2 and Table A.12). ORAC. In examining the major trends in the data, whole berries had the highest ORAC values, but there was no clear overall increase or decrease in ORAC during storage (Fig. 3.3). With few exceptions, it was found that ORAC was generally lowest in whole berries and berry segments from vine one, compared to the other vines (Fig. 3.3). The flesh and seed of berries from vine one after 1 week of storage had the lowest ORAC values (20.6 and 22.2 µmol Trolox equivalents/g, respectively), while the whole berries from vine two at harvest had the highest (81.1 µmol Trolox equivalents/g) (Fig. 3.3 and Table A.12). Total Flavonols. In examining the major trends in the data, one can see that total flavonol concentrations were highest in the seeds, and overall the variation among vines was minimal with the exception of seeds from muscadine berries in vine one (Fig. 3.4). Additionally, it was found that though variation occurred in total flavonol concentrations among the berry segments, there was no clear increase or decrease during storage (Fig. 3.5). Averaged across week of storage the seeds of berries from vine one had the greatest total flavonol concentration (56.8 mg/100 g), while the flesh of berries from vine 1 had the lowest (8.4 mg/100 g) (Fig. 3.4). Averaged across vines, the seeds after 6 weeks of storage and at harvest had the highest total flavonol concentrations (41.6 and 40.9 mg/100 g, respectively), while the whole berries after 3 weeks of storage had the lowest (6.9 mg/100 g) (Fig. 3.5). Total Phenolics. The major trends in the data showed that though variation did occur among weeks of storage and vines, it was difficult to identify a clear pattern of increase or decrease of total phenolic concentrations during storage, or to identify a single 231

248 Fig Oxygen radical absorbance capacity (ORAC) of Supreme muscadine berry segments (flesh = pulp and skin) stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. ORAC (µmol TE/g) Week Vine Segment Flesh Seed Whole 232

249 Fig Total flavonols of Supreme muscadine berry segments (flesh = pulp and skin) from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. Fig Total flavonols of Supreme muscadine berry segments (flesh = pulp and skin) stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. 233

250 vine that generally had higher phenolics, averaged across berry segments (Fig. 3.6). Additionally, on a per weight basis, the seeds generally had the lowest total phenolics, while the whole berries generally had the highest (Fig. 3.7). Averaged across berry segments, muscadines from vines two and three after 6 weeks of storage had the highest total phenolics (312.2 and mg/100 g, respectively), while berries from vine one after 1 week of storage had the lowest (230.3 mg/100 g) (Fig. 3.6). The whole berries from vines two and three had the highest total phenolics concentrations (388.7 and mg/100 g, respectively) while seeds from vine one had the overall lowest (166.9 mg/100 g), when averaged across weeks of storage (Fig 3.7). Resveratrol. Averaged across berry segments, it was difficult to identify a vine that produced berries with consistently the highest or lowest levels of resveratrol; however, when looking at the major trends in the data it appeared that after 5 weeks of storage, resveratrol concentrations in berries from vine three were highest (Fig. 3.8). Vine one after 1 week of storage had the lowest resveratrol concentrations (2.6 mg/100 g),while vine three after 5 weeks of storage had the highest (10.3 mg/100 g), when averaged across berry segments (Fig. 3.8). Overall the seeds from vines two and three had the highest resveratrol concentrations (10.2 and 7.1 mg/100 g, respectively) when averaged across weeks of storage, while seeds from vine one had the lowest (2.3 mg/100 g, respectively) (Fig. 3.9). Furthermore, when averaged across vines, it appeared that resveratrol concentrations were highest in whole berries, flesh, and seed after 5 or 6 weeks of storage (7.2, 7.9, 9.2, 10.2, 7.6, and 8.0 mg/100 g, respectively), with the exception of seeds after 1 week of storage (8.5 mg/100 g) (Fig 3.10). 234

251 Fig Total phenolics of Supreme muscadine berries stored at 2 C for 6 weeks, averaged across berry segment in Each standard error bar is constructed using 1 standard error from the mean. Total phenolics (mg/100 g) Week Vine Fig Total phenolics of Supreme berry muscadine segments (flesh = pulp and skin) from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. Total phenolics (mg/100 g) Flesh Seed Whole Segment Vine Fig Resveratrol concentrations of Supreme muscadine berries from three different vines of Supreme stored at 2 C for 6 weeks in Values are averaged across berry segments. Each standard error bar is constructed using 1 standard error from the mean. Resveratrol (mg/100 g) Week Vine

252 Fig Resveratrol concentrations of Supreme muscadine berry segments (flesh = pulp and skin) from three different vines of Supreme in Each standard error bar is constructed using 1 standard error from the mean. Resveratrol (mg/100 g) Flesh Seed Whole Segment Vine Fig Resveratrol concentrations of Supreme muscadine berry segments (flesh = pulp and skin) stored at 2 C for 6 weeks in Each standard error bar is constructed using 1 standard error from the mean. Resveratrol (mg/100 g) Week Segment Flesh Seed Whole 236

253 Combined Nutraceutical Analysis Due to the lack of consistent differences in storage time for the dependent variables, the storage aspect of this study was discontinued after the 2012 season. For the combined 2012 and 2013 data, the ANOVA F test indicated significant three way interactions of year by vine by berry segment for total anthocyanins (P=0.0235), total flavonols (P<0.0001), total phenolics (P=0.0213), and resveratrol (P=0.0011) (Table 3.3). For the dependent variable ORAC, significant two way interactions of year by vine (P=0.0325), year by berry segment (P<0.0001), and vine by berry segment (P<0.0001) occurred (Table 3.3). The variability of nutraceuticals in berry segments from different vines among years offers some insight as to why the interactions were significant. The only significant main effect of berry segment was for total ellagitannins (P>0.0001) (Table 3.3). Total Anthocyanins. When examining the major trends in the data, it appeared that total anthocyanins were detected in the flesh and whole berries in varying concentrations, but not the muscadine seeds, and overall total anthocyanin concentrations were greater in 2012 compared to 2013 (Fig. 3.11). The difference in total anthocyanins among berry flesh and whole berries was not significant. The whole berries from vine three in 2012 had the greatest anthocyanin concentration (37.4 mg/100 g), while the berry flesh from vine one in 2013 had the lowest anthocyanins detected (9.9 mg/100 g) (Fig and Table A.13). Averaged across berry segments and vines, the overall anthocyanin concentration in 2012 was 15.6 mg/100 g, and 8.8 mg/100 g in Additionally, vine three was identified as having the greatest anthocyanins, compared to vines one and two (P<0.0001) (Fig. 3.11). Total Ellagitannins. Overall, the berry seeds were found to contain the highest concentrations of ellagitannins averaged across years and vines (Fig and Tables A

254 Table 3.3. F test significance from ANOVA for total anthocyanins, total ellagitannins, ORAC, total flavonols, total phenolics, and transresveratrol concentrations of Supreme muscadines at harvest in 2012 and Highest order interactions are italicized and shaded. DF z Total anthocyanin s (mg/100 g) Total ellagitannins (mg/100 g) ORAC (µmol Trolox equivalents /g) Total flavonols (mg/100 g) Total phenolics (mg/100 g) Resveratrol (mg/100 g) Year 1 < < Vine < Year*vine Segment 2 < < < < < Year*segment < Vine*segment < < Year*vine*segment < z Degrees of freedom. 238