Acerola (Malpighia emarginata DC)

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Acerola (Malpighia emarginata DC) Phenolic Profiling, Antioxidant Capacity, Antimicrobial Property, Toxicological Screening, and Color Stability
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1 online resource (174 p.)
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english
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Delva, Lemane
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Goodrich, Renee M
Committee Members:
Marshall, Maurice R
Gregory, Jesse F
Gu, Liwei
Roberts, Thomas Grady

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Subjects / Keywords:
acerola -- anti-microbial-properties -- antioxidant-capacity -- color-stability -- phenolic-compounds
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
This study aimed to characterize acerola fruit based on phenolic profiling, antioxidant and antimicrobial properties, toxicological evaluation, and color stability. Three specific objectives were pursued: separate, identify, and quantify the phenolic compounds; perform the antioxidant, antimicrobial, and toxicological evaluations of the phenolic extracts; study the color stability of acerola anthocyanin extracts in the presence of ascorbic acid (AA). Acerola fruits were grouped by levels of maturity. Phenolic compounds were fractionated by SPE into anthocyanins and non-anthocyanins, separated and identified by HPLC-DAD-MS2. The antioxidant capacity (AOC) was assayed by ORAC and DPPH. The antimicrobial property was determined by the disk diffusion method, and the toxicological screening was assessed by the Ames mutagenicity test. Color stability was examined by monitoring the anthocyanins degradation in acerola anthocyanin-containing AA extracts over time. The effect of AA on anthocyanins and color loss was also studied in AA-free açai extracts to which AA was added at levels that are similar to AA content of the acerola anthocyanin extracts. Pure anthocyanin model systems composed of free cyanidin and cyanidin-3-rhamnoside with added amounts of AA were assessed. Two anthocyanins, cyanidin-3-rhamnoside and pelargonidin-3-rhamnoside and various types of non-anthocyanin phenolics including caffeic, chlorogenic, ferulic and p-coumaric acid derivatives and some catechin derivatives were identified in acerola fruit. Total antioxidant capacity expressed by ORAC were higher in immature fruits (43.5 mmol TEkg-1) when compared with fruits at intermediate (36.5 mmole TEkg-1) and complete (36.2 mmol TEkg-1) stages of maturity. The phenolic fractions contributed 7.1-36.5 % while AA accounted for 18-39 % of total AOC. The flavonoid fractions of the fruit displayed antimicrobial potential against S. aureus. The results suggest that the phenolic fractions did not contribute to mutagenicity and are possibly suitable for use as food supplements. The detrimental effect of AA on anthocyanins and color was obvious in all the systems regardless of the storage conditions, resulting in increased L*, decreased a* and C* values. Acerola may be promoted as a healthy foodstuff based on its high antioxidant potential. Future studies to stabilize the color of acerola anthocyanin extracts should be oriented toward the stability of both anthocyanin and AA.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Lemane Delva.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Goodrich, Renee M.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

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lcc - LD1780 2012
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UFE0044982:00001


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1 ACEROLA ( MALPIGHIA EMARGINATA DC ) : PHENOLIC PROFILING, ANTIOXIDANT CAPACITY, ANTIMICROBIAL PROPERTY TOXICOLOGICAL SCREENING AND COLOR STABILITY By LEMANE DELVA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVE RSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Lemne Delva

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3 To GOD for keeping me healthy throughout this study cycle and for my entire life To the memory o f my beloved half sister Pierre Marie Michelle who passed away while I was in the process of completing this study ; May she rest in peace

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4 ACKNOWLEDGMENTS I would like to gratefully and sincerely thank Dr. Rene Goodrich Schneider, my Academic Superv isor and Chair of my research Committee for her guidance, constructive criticism, patience, technical support and most importantly her friendship during my journey as a graduate student at the University of Florida. Her mentorship was very important in giv ing me a well proportioned experience that is consistent with my long term career objectives. She incited me to not only grow as a Food Scientist but also as an instructor and a critical and independent thinker. I would like to express my sincere st thanks to all my committee members namely: Dr. Liwei Gu, for being very genero us in making his lab equipment available for an important portion of my research Dr. Grady Roberts for his contribution in helping me to improve my teaching skills and for his advice i n the choice of course work toward my Minor in Agricultural Education and Communication Dr. Ma urice Marshall for making his lab equipment available for me for the phenolic profiling p o rt ion of my research and Dr. Jesse Gregory III for his always construc tive criticism and counsel I would like to thank the Fulbright Laspau Scholarship Program for its precious 2 year financial support in the form of a full scholarship which puts me on the path to concretize this achievement. It is for me a great honor to be part of the very prestigious Fulbright family. Md e cine Vtrinaire (FAMV) for supporting me and my family financially during my studies in the United States.

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5 I would like to thank the Department of Food S cience and Human Nutrition especially the staff members for their hard work, their patience and their help. Tim, and Olivia; I really appreciated all the teaching and coaching. Additionally, I am grateful for the support from my close friend Joubert Fayette, and my long time friends and study partners: Valee Kalani Uma Anguswamy and Noufoh Djeri I would like to acknowledge the friendship and moral supports from my labmates: Y ael Spector Devin Lewis Chinedu Ikpechukwu Gayathrie Balakrishnan and my countrymate Annie S arah Gossin Also, I thank my parents, Clem ne (my hero) and Manius for their unwave ri ng belief in me and for allowing me to be as ambitious as I wanted. It wa s under their watchful eye that I gained so much drive and an ability to tackle challenges head on. Last but not least, I would like to thank my wife Nicole. Her support, her patience and never ending love were unquestionably the solid rock upon which the past seven years of my life have been constructed. I thank my two daughters Manisha and Adelle, both of them born in the process of getting this degree. When the stress of graduate school wanted to get the best of me, they were always there to inspire me; they are the best things that ever happen ed to my life; to me, they are and always will be an everlasting source of love, inspiration, and encouragement.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 2 LITERATURE REVIEW ................................ ................................ .......................... 21 Acerola ( Malpighia emarginata DC) ................................ ................................ ........ 21 Characteristics, Production, Harvest, Post Harvest Handling and Market Requirements ................................ ................................ ................................ ...... 21 Food and Other Uses of Acerola Fruit ................................ ................................ .... 24 Physico Chemical Properties and Nutritional Value of Acerola Fruit ...................... 25 Protein, Fat and Carbohydrate ................................ ................................ ................ 25 Vitamins and Minerals ................................ ................................ ...................... 26 pH, Acidity, Soluble Solids, and Organic Acids ................................ ................ 27 Phytochemicals in Acerola ................................ ................................ ...................... 28 Phenolic Compounds in Acerola ................................ ................................ ............. 29 Flavonoids ................................ ................................ ................................ ........ 31 Anthocyanins ................................ ................................ ................................ .... 33 Phenolic Acids ................................ ................................ ................................ .. 40 Carotenoids ................................ ................................ ................................ ............ 41 Occurrence of Anthocyanins in Acerola Fruit ................................ .......................... 42 Anthocyanin, Ascorbic Acid and Color Stability of Acerola ................................ ..... 44 Acerola Components and Potential Health Benefits ................................ ............... 45 Dietary Phenolic Compounds and Contribution to Human Health .......................... 55 Toxicological Safety of Acerola Phenolic Compounds ................................ ............ 55 3 IDENTIFICATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS IN ACEROLA FRUITS AND JUICES ................................ ................................ .......... 62 Overview ................................ ................................ ................................ ................. 62 Materials and Methods ................................ ................................ ............................ 64 Chemicals ................................ ................................ ................................ ......... 64 Fruits Harvesting, and Separation into Different Maturity Stages ..................... 64 Separation of the Fruits into Edible and Non Edible Portions ........................... 65

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7 Extraction of the Phenolic Compounds ................................ ............................ 66 Fresh fruits ................................ ................................ ................................ 66 Freeze dried samples ................................ ................................ ................ 66 Fractionation of the Crude Aqueous Phenolic Extract into Anthocyanin and Non Anthocyanin ................................ ................................ ........................... 67 Solid Phase Extraction of the Non anthocyanin Phenolics ............................... 68 HPLC Analysis of the Phenolic Compounds ................................ ..................... 68 Anthocyanins ................................ ................................ ............................. 68 Acid ic and neutral phenolic fractions ................................ .......................... 69 Results and Discussions ................................ ................................ ......................... 70 Validation of the Categorization of the Fruits by Stage of Matur ity ................... 70 Anthocyanins Identification and Quantification ................................ ................. 71 Non Anthocyanin Compounds ................................ ................................ .......... 73 Method development ................................ ................................ ................. 73 Validation ................................ ................................ ................................ ... 74 Identification by HPLC ESI MS 3 ................................ ................................ 75 Hydroxycinnamic acid derivatives ................................ .............................. 75 Flavan 3 ols ................................ ................................ ............................... 76 Flavonols ................................ ................................ ................................ .... 77 Ellagic acid and stilbene ................................ ................................ ............ 77 Summary ................................ ................................ ................................ ................ 78 4 ANTIOXIDANT ACTIVITY, ANTIMICROBIAL PROPERTIES, AND TOXIC OLOGICAL SCREENING OF PHENOLIC EXTRACTS FROM ACEROLA (MALPIGHIA EMARGINATA DC) FRUIT ................................ ................................ 84 Overview ................................ ................................ ................................ ................. 84 Materials and Methods ................................ ................................ ............................ 89 Chemicals and Biological Media ................................ ................................ ....... 89 Bacterial Strains ................................ ................................ ............................... 89 Extraction a nd Fractionation of the Phenolic Compounds ................................ 90 Total Phenolics Analysis ................................ ................................ ................... 90 Antioxidant Capacity Assays ................................ ................................ ............ 90 Oxygen Radical Absorbance Capacity (ORAC) ................................ ............... 91 DPPH (2 Diphenyl 1 picrylhydrazyl) Assay ................................ .................. 91 Determination of Ascorbic Acid in the Extract ................................ ................... 91 Antimicrobial Activity ................................ ................................ ........................ 92 Sample Preparation for Antimicrobial Test ................................ ....................... 92 The Disk Diffusion Test ................................ ................................ .................... 92 Interpretation of the Results ................................ ................................ ............. 93 Ames Mu tagenicity Test ................................ ................................ ................... 94 Statistical Analysis ................................ ................................ ............................ 95 Results and Discussion ................................ ................................ ........................... 96 Total Phenolics, Total Antioxidant Capacity and Vitamin C Content ................ 96 Contribution of Phenolic Compounds and AA to the Antioxidant Capacity of Acerola Fruit ................................ ................................ ................................ .. 98 Antimicrobial Properties ................................ ................................ .................. 100

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8 Ames Mutagenicity Assay ................................ ................................ .............. 101 Summary ................................ ................................ ................................ .............. 102 5 EFFECT OF DIFFERENT ASCORBIC ACID CONCENTRATIONS ON THE COLOR STABILITY OF ANTHOCYANIN EXTRACTS FROM ACEROLA ( MALPIGHIA EMARGINATA DC) FRUITS ................................ ........................... 114 Ove rview ................................ ................................ ................................ ............... 114 Materials and Methods ................................ ................................ .......................... 116 Acerola Fruit and Aai Puree ................................ ................................ .......... 116 Preparation of the Anthocyanin Extracts ................................ ........................ 117 Development of the Model Systems ................................ ............................... 117 Stability and Visual Color Attributes of t he Anthocyanin Extracts ................... 118 Determination of Ascorbic Acid ................................ ................................ ...... 119 Kinetics Calculations ................................ ................................ ...................... 120 Results and Discussion ................................ ................................ ......................... 120 Effect of Ascorbic Acid on the Stability of the Anthocyanin Extracts ............... 120 Eff ect of Ascorbic Acid on the Stability of the Pure Anthocyanin Solution ...... 122 Effect of Light ................................ ................................ ................................ 122 Color Stability of the Different System ................................ ............................ 123 Degradation of Ascorbic Acid over Time ................................ ........................ 125 Some Discussion on the Type of Reaction that May Take Place between Anthoc yanin and Ascorbic Acid ................................ ................................ ... 126 Summary ................................ ................................ ................................ .............. 127 6 CONCLUSIONS ................................ ................................ ................................ ... 142 APPENDIX A HPLC DAD CHROMATOGRAMS OF THE NON ANTHOCYANIN PHENOLIC COMPOUNDS DETECTED ACEROLA FRUIT ................................ .................... 145 B STATISTICAL ANALYSIS OF THE COLOR AND SOFTNESS OF THE DATA COLLECTED AT THE THREE STAGES OF M ATURITY ................................ ..... 147 C STATISTICAL ANALYSIS OF THE TOTAL ANTIOXIDANT AND VITAMIC DATA COLLECTED FOR THE FRUITS GROWN IN DAVIE, FLORIDA .............. 152 LIST OF REFERENCES ................................ ................................ ............................. 157 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 174

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9 LIST OF TABLES Table page 2 1 Nutrit ional value of acerola fruit ................................ ................................ .......... 60 2 2 Phytonutrient content of acerola fruit ................................ ................................ .. 61 3 1 Color characteristic and the hardness of acero la fruit at different stages of maturity ................................ ................................ ................................ ............... 79 3 2 Solvent gradient for reversed phase HPLC analysis of the neutral and acidic fractions of the phenolic compounds ................................ ................................ .. 80 3 3 Identification of anthocyanin using HPLC ESI/MS/MS ................................ ....... 81 3 4 Interday precision ................................ ................................ ............................... 82 3 5 Reten tion times and mass spectrometric data of non anthocyanin phenolic compounds in fruit determined by HPLC ESI MS 2 all stages of maturity included ................................ ................................ ................................ .............. 83 4 1 Total phenolic index, total antioxidant value and vitamin C content of acerola sample ................................ ................................ ................................ .............. 104 4 2 Contribution of phenolic fractions and AA in the total antioxidant value expressed by ORAC ................................ ................................ ......................... 105 4 3 Antimicrobial effect of anthocyanin fractions from acerola fruit; sample amount 500 g (n=2) ................................ ................................ ........................ 10 6 4 4 Antimicrobial effect of flavonoids fractions from acerola fruit; sample amount 500 g (n=2) ................................ ................................ ................................ ..... 107 4 5 Antimicrobial effect of phenolic acid fractions from acerola fruit; sample amount 500 g (n=2) ................................ ................................ ........................ 108 4 6 Mutagenic dose response of acerola anthocyanin fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) ................................ ................................ ............................. 109 4 7 Mutagenic dos e response of acerola flavonols fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) ................................ ................................ ............................. 110 4 8 Mutagenic dose response of acerola phe nolic acid fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) ................................ ................................ ............................. 112

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10 5 1 Degradation rate constant and the half life for anthocyanin in different systems citrate phosphate buffer pH 2.5 ................................ .......................... 128 5 2 Changes in color parameters (a and b ) for initial and final storage time ......... 129 5 3 Ascorbic acid degradation in acerola and AA fortified aai ............................... 130 B 1 SAS software code used for the statistical analysis of peel color (L a b ) and softness (H) parameters using the Duncan multiple range test ........................ 147 B 2 SAS software output used for the statistical analysis of peel color (L a b ) and softness (H) parameters using the Duncan multiple range test ........................ 148 C 1 SAS software code used for the statistical analysis of parameters (TPI, ORAC, DPPH, ORAC, vit. C) using the Duncan multiple range test ................. 152 C 2 SAS software output used for the statistical analysis of parameters (TPI, ORAC, DPPH and Vit.C) using the Duncan multiple range test ....................... 153

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11 LIST OF FIGURES Figure page 2 1 Acerola at various stages of maturity. A: immature stage (green); B: intermediate stage (orange or orange red); C: mature stage (red). .................... 58 2 2 Structure of phenolic compounds in acerola fruit: A, anthocyanins; B, flanonols; C, chlorogenic acid; D, phenolic acids. ................................ ............... 59 5 1 First order plot for some selected anthocyanin s extracts during storage under light at 20 o C: A: Ace VE light; B: Ace DA light; C: Aai+48AAlight; D: Aai+97 light. ................................ ................................ ................................ .... 131 5 2 Degradation curves of anthocyanin from acerola fruit and aai spik ed with ascorbic acid at different level and stored under light (A) and in darkness (B) at 20 o C; in citrate buffer pH 2.5.. ................................ ................................ ..... 132 5 3 Degradation curves of anthocyanin from pure cyanidin and cy anidin O rhamnoside spiked with ascorbic acid and store in darkness in citrate buffer pH 2.5.. ................................ ................................ ................................ ............. 133 5 4 Behavior of the different systems stored in the presence or in the absence of light, Pane l A: Ace VE and Ace DA, Panel B: Aai+48mgAA and Aai+97mgAA, Panel C: Aai+144mgAA and Aai+288mgAA. ........................ 134 5 5 Evolution of the lightness (L*) value for acerola extract and the aai systems enr iched with ascorbic acid of anthocyanin extracts in ph osphate citrate buffer, pH 2.5 .. ................................ ................................ ................................ 135 5 6 Changes in the color parameters a*/a 0 value for the anthocyanin extracts from acerola and the a ai in phosphate buffer solutions at pH 2.5. .................. 136 5 7 Changes in the color parameters b*/b* 0 value for the anthocyanin extracts from acerola and the aai in phosphate buffer solutions at pH 2. 5. .................. 137 5 8 Evolution of the hue value for acerola and aai systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5. ....... 138 5 9 Evolution of the chroma (C*) value for acerola and aai systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5. 139 5 10 systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5. ................................ ................................ ........................ 140

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12 5 11 Spectroph otometric profile of selected samples in the pure anthocyanin solutions with added ascorbic acid at pH 2.5.. ................................ .................. 141 A 1 HPLC DDA chromatogram for partially purified acerola anthocyanin extracts A ce DA (A), Ace VE (B), and frozen single strength juice (FSSJ) (C) acerola juice at 520 nm. ................................ ................................ ................................ 145 A 2 Sample chromatogram of the acidic fraction of phenolic compounds detected in edible portion of acerola fruit, detection wavelength: 320 nm ....................... 146 A 3 Sample chromatogram of the neutral fraction of phenolic compounds detected in edible portio n of acerola fruit, detection wavelength: 280 nm ........ 146

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13 LIST OF ABBREVIATION S ATCC American Type Culture Collection AGE Advanced Glycation End CVD Cardiovascular Disease DAD Diode Array Detector DNA Deoxyribonucleic Acid DPPH Diphenyl Picrylhydrazyl ESI Electrospray Ionization FAO Food and Agriculture Organization FDA Food and Drug Administration HPLC High Performance Liquid Chromatography LDL Low Density Lipoprotein MDR Multi Drug Resistance MHA Mueller Hinton Agar MS Mass Spe ctrometry NMR Nuclear Magnetic Resonance ORAC Oxygen Radical Absorbance Capacity PDA Photodiode Array SAS Statistical Analysis System SPE Solid Phase Extraction TSA Tryptic Soy Agar TSB Tryptic Soy Broth

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ACEROLA ( MALPIGHIA EMARGINATA DC ) : PHENOLIC PROFILING, ANTIOXIDANT CAPACITY, ANTIMICROBIAL PROPERTY, TOXICOLOGICAL SCREEN ING, AND COLOR STABILITY By Lem ne Delva December 2012 Chair: Rene Goodrich Schneider Major: Food Science and Human Nutrition This study aimed to characterize acerola fruit based on phenolic profiling, antioxidant and antimicrobial properties, toxico logical evaluation, and color stability. Three specific objectives were pursued: s eparate identify and quantify the phenolic compounds ; perform the antioxidant, antimicrobial, and toxicological evaluations of the phenolic extracts ; study the color stabil ity of acerola anthocyanin extracts in the presence of ascorbic acid (AA). Acerola fruits were grouped by levels of maturity. Phenolic compounds were fractionated by SPE into anthocyanins and non anthocyanin s separated and identified by HPLC DAD MS 2 The antioxidant capacity (AOC) was assayed by ORAC and DPPH. The antimicrobial property was determined by the disk diffusion method and the t oxicological screening was assessed by the Ames mutagenicity test. Color stability was examined by monitoring the ant hocyanin s degradation in acerola anthocyanin containing AA extracts over time. The effect of AA on anthocyanin s and color loss was also studied in AA free aai extracts to which AA was added at levels that are similar to AA content of the acerola anthocyan in extracts. Pure anthocyanin model systems

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15 composed of free cyanidin and cyanidin 3 rhamnoside with added amounts of AA were assessed. Two anthocyanins, cyanidin 3 rhamnoside and pelargonidin 3 rhamnoside and various types of non anthocyanin phenolics in cluding caffeic, chlorogenic, ferulic and p coumaric acid derivatives and some catechin derivatives were identified in acerola fruit. Total antioxidant capacity expressed by ORAC were higher in immature fruits (43.5 mmol TEkg 1 ) when compared with fruits a t intermediate (36.5 mmole TEkg 1 ) and complete (36.2 mmol TEkg 1 ) stages of maturity. T he phenolic fractions contributed 7.1 36.5 % while AA accounted for 18 39 % of total AOC The flavonoid fraction s of the fruit displayed antimicrobial potential against S. aureus The results suggest that the phenolic fractions did not contribute to mutagenicity and are possibly suitable for use as food supplements. The detrimental effect of AA on anthocyanins and color was obvious in all the systems regardless of the st orage conditions, resulting in increased L*, decreased a* and C* values. Acerola may be promoted as a healthy foodstuff based on its high antioxidant potential. Future studies to stabilize the color of acerola anthocyanin extracts should be oriented toward the stability of both anthocyanin and AA.

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16 CHAPTER 1 INTRODUCTION In the last few decades there has been a growing trend in the consumption of tropical fruits and according to the Food and Agriculture Organization (FAO), the world production and the trad e of tropical fruits is expected to expand over the next decade (FAO 2003) The less developed countries produce 98 % of the total production of tropical fruit, while the industrialized countries and major markets are responsible for 80 % of the world import trade. While the major tropical fruits (mango, pineapple, papaya, and avocado) are the dominant tropical fruits produced worldwide, the market share of va, passion fruit, acerola) have been expanding rapidly in recent years (FAO 2003) Tropical fruits are appreciated by the consumer because of their diversity of aroma, flavor, and their nutritional value, both real a nd perceived. And among the minor tropical fruits, acerola appears potentially attractive because of its very high vitamin C content, its appealing red color at complete maturity, and its biological activity. Acerola is grown in most of the Caribbean count ries, in Central America and in Brazil, and is a tropical shrub ( Matta and others 2004 ). At complete maturity it has a red color, a unique aroma, and is extremely rich in ascorbic acid. Acerola is also grown (although not at a large scale) in Florida and i n Hawaii. Acerola cultivation is straightforward in these locations, and the production is relatively cost effective (i.e. not requiring many production inputs), making it a perfect fit for low income farmers. The high levels of ascorbic acid make acerola sources of this vitamin Depending on different factors such as cultivar, level of maturation, and climate (Matta and others 2004) the ascorbic acid levels range from

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17 1000 4500 mg/100 g of fruit on a fresh weight basis. Owing to its carotenoid content (371 1881 mg/100g) (De Rosso and Mercadante 2005) and the presence of phenolic compounds such as anthocyanins, and non anthocyanin phenolics, acerola is category together with other fruits such as Maqui berry, Indian gooseberry, guarana, seabuckthorn and the like. Given the health promoting potential of carotenoids and p henolic compounds, acerola has become very popular among people that are health conscious (Hanamura and others 2006 ). Recent experiments with various acerola preparations have suggested diverse biological activities including anticarcinogenic activity agai nst lung cancer (Nagamine and others 2002) inhibitory action against nitric oxide production (Wakabayashi and others 2003) tumor specific cytotoxic activity, and multidr ug resistance reversal activity (Motohashi and others 2004) antihyperglycemic (Hanamura and others 2006), prevention of dyslipidemia and its complications (Barbalho and others 2011), and antigenotoxicity (Nunes and others 2011). The attractive red color of acerola fruit is due to anthocyanin pigments Vendramini and Trugo (2004) reported total anthocyanins of 37 mg/100g acerola skin, making acerola potentially usable as a food colorant. In addition to its colorant properties anthocyanins have been proven to demonstrate anti inflammatory effects, protection against radiation, and inhibition of low density lipoprotein (LDL) oxidation (Wan g and others 1997 ; Seeram and Nair 2002) The biological properties of anthocyanins depend on its structural scheme such as degree of glycosylation and the amount of hydroxyl groups attached to the B ring (Kong and others 2003) Therefore it is very important to determine the structure of the anthocyanins in acerola fruit. The anthocyanin

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18 composition of acerola fruit has already been reported but the results are inconsistent. I n addition, the anthocyanin contents reported by previous experimenters are also discrepant probably because those experiments have been conducted on different and sometimes unknown varieties. Vendramini and Tr ugo (2004) reported malvidin 3,5 diglucoside, cyanidin 3 glucoside, and pelargonidin as major anthocyanin s in an unidentified acerola variety. Hanamura and others (2005 ) and De Britto and others (2007) identified c yanidin 3 O rhamnoside and pelargonidin 3 O rhamnoside while report ing no free anthocyanidin in acerola fruit. The research of De Brito and others (2007) was conducted on two varieties: Acerola II47/1 and anot her variety called De Rosso and others (2008) reported two anthocyanin hexoside s : cyanidin 3 rhamnoside and pelargonidin 3 rhamnoside, and t wo free anthocyanidin s : cyanidin and pelargonidin It is worth mentioning that in addition to variety and method differences between the information available, acerola is indiscriminately referred t o as Malpighia punicifolia L Malpighia glabra L or Malpighia emarginata DC ; however, these names are synonymous and the most common one for all acerola clones, genotypes and varieties is Malpighia emarginata DC (De Rosso and others 2008). Acerola also co ntains non anthocyanin phenolic compounds such as phenolic acids and flavonoids. However these groups of phenolic compounds are poorly investigated. Very few reports exist in this area and most of them are not peer reviewed. Compounds like chlorogenic, fer ulic, and caffeic acids were reported as main phenolic acids in acerola fruit ( Ve n dramini and Trugo 2004; Righetto and Netto 2005), however

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19 the identification of compounds was made by solely comparing their retention time and their spectral information. Th e identification of phenolic compounds based on those parameters may not be accurate because compounds with similar or closely related chemical structures may have similar spectral characteristics. Therefore, it is necessary to use more powerful techniques to elucidate the structure of phenolic compounds in acerola fruit One of the problems the acerola growers are facing is the high perishability of this fruit at complete maturity. According to Vendramini and Trug o (2000) the fruits last only three days at room temperature. This perishability is thought to be caused by the climacteric nature of the fruits (Sean Carrington and King 2002) Shortly after harvest (3 4 days) the fruit loses its attractive red color and turns to a dull yellowish color that is often seen by the consumer as index of poor quality, therefore limiting the market potential of the fruit. It is believed that the color instability of the fruit is due to interaction between two important elements in the chemical composition of the fruit: ascorbic acid and anthocyanins. It has been hypothesized that ascorbic acid degrades anthocyanin in model systems but the mechanisms proposed by previous experiment ers are inconsistent. We believe that it is important to investigate the potential role of ascorbic acid in the degradation of anthocyanin in acerola which will probably help to propose ways to stabilize the color or to even develop acerola based products with better color. The overall aim of this study was to characterize acerola fruit based on phenolic profiling, antioxidant and antimicrobial properties, toxicological evaluation, and color stability. Three specific objectives were set: (1) Identify and qu antify anthocyanin s and non anthocyanin phenolic compounds, (2) Perform the antioxidant, antimicrobial, and

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20 toxicological evaluation of phenolic extracts from acerola fruit and, (3) Study the color stability of acerola fruit by monitoring anthocyanin ascor bic acid interactions in the acerola anthocyanin extracts and provide recommendations for preserving the color of fresh and processed acerola products.

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21 CHAPTER 2 LITERATURE REVIEW Acerola ( Malpighia emarginata DC) Acerola ( Malpighia emarginata DC. Syn. Ma lpighia punicifolia L) is a plant originating in Central America that has spread to South America including Brazil, and the Caribbean due to its good adaptation to soil and climate. This shrub is grown in tropical and subtropical areas from the southern e nd of Texas, through Mexico and Central America to northern South America and throughout the Caribbean especially in Barbados, Trinidad & Tobago, Haiti and Puerto Rico. The tree has also been introduced widely into tropical regions of Asia and Africa. The perennial tree bears a red fruit known by the common names Barbados cherry, West Indian cherry especially in the English speaking Caribbean countries or simply cherry in Haiti. However, the name acerola, as it is called in Puerto Rico is becoming more and more popular and is the name that will be used throughout this dissertation. Characteristics, Production, Harvest, Post Harvest Handling and Market Requirements Acerola trees may reach an average height of 3 5 m with a short slender trunk that is 0.5 1 m high, and 7 10 cm in diameter. The fruit is small (1 4 cm diameter) and weighs 2 15 g. The thin skin is green during the first development stage but turns orange to orange red at the intermediate stage of maturity and become bright red at complete maturit y. Figure 2 1 portrays the three color stages of acerola fruit: green immature, semi ripe (partially mature), and fully ripe (mature). The flesh is usually of a reddish yellow hue, although some varieties with deep red skins have also a dark red pulp.

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22 Rega rdless of the size of the fruit, the three winged seeds are large in comparison to the flesh, but due to their light and pithy nature, they represent only 20 % of the weight (Miller and others 1961). The tree produces fruits 3 4 times a year and each plant produces 20 30 kg fruit per year (Mezadri and others 2006) There has been commercial cultivation of acerola in some regions of the America s but it is only in the last three decades that Brazil began to exploit it products is Brazil which commercializes it in the forms of juice, marmalades, frozen concentrates, jam, and liquors. Other plantations of commercial importance are in F lorida and Hawaii. The color of the peel has been used traditionally as an index of ripeness and therefore, as the main criterion used to determine the harvest date of the fruit. Another method of assessing the ripeness of acerola includes the measurement of sugar/acid ratio in the fruit. However the use of peel color as an index of maturity represents a more practical option especially in field situations where laboratory analyses may not be available. The harvest date depends on the intended use of the f ruit. In the case of freezing or processing into pulp or juice, fruits must be red in color but firm enough to tolerate handling. The fruit quality is high at this stage of maturation, that is, sugar content is high and acidity is low. Fruits may be picked at the beginning of maturation for use in the production of powdered products such as pharmaceuticals or concentrates for food enrichment, where the vitamin C content is the most important characteristic. The acerola fruits have high metabolic activity af ter harvest and the achievement of the ripening occurs rapidly (3 4 weeks after flowering), causing it to be

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23 too perishable for the fresh market (Alves and others 1999) Consequently, the fruits must be frozen or proce ssed quickly The mature fruit lasts only 2 3 days at room temperature (Vendramini and Trugo 2000) However, the shelf life of the mature acerola fruit maybe improved to over three days at room temperature when wr apped in PVC film. In addition, storage at 8 o C and 85 90 % relative humidity with PVC wrapping increased shelf life to a week (Alves and others 1995) The CO 2 behavior displayed by the fruit especially at the intermed iate and complete stage of ripeness suggested a climacteric behavior. According to Carrington and King ( 2002) the fruit has a very high respiratory rate (900 m L CO 2 kg 1 h 1 ) but with a low rate of peak ethylen e production (3 L C 2 H 4 kg 1 h 1 ); the high respiration rate is thought to be in part responsible for the perishable nature of the fruit. One of the problems faced by acerola producers is the great sensitivity of the mature fruits during picking, packing, p rocessing and/or distribution. The skin of the mature fruit is thin and fragile, and therefore can be easily damaged by even a very small impact. If the skin is damaged, the pulp of the fruit deteriorates rapidly. To alleviate the problem of the delicate s kin, people who have experience in handling acerola fruits for international trade suggest that the harvest be made by hand. This will also help to exclude flowers and immature fruits that are present simultaneously with the mature fruits on the acerola tr ee. However, the main drawback of hand picking is that it raises the cost of labor ( Alves and others 1999) Post harvest stability of the fruit is also affected by solar radiation; exposure of the fruit to solar radiat ion for more than 4 hours after harvest leads to substantial loss of vitamin C (Alves and others 1999) Consequently, it is suggested that the harvest of the fruit be

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24 made in the early hours of the morning, before the temperature increases to level s that can be detrimental to the mature fruits. The standard requirements for acerola fruit intended for international trade are not well established Brazilian producers experienced in international trade suggest that buyers require fruit with at least 7 o Brix for Europe, or 7.5 o Brix for Japan and about 1000 mg vitamin C per 100 g of fruits in Europe and the United States (Alves and others 1999) Japan is the most important market for ac erola products, followed by the United States and Europe. In Germany, France and Hungary, fruit is used primarily for juice while in the United States it is used by the pharmaceutical industry. Food and O ther Uses of Acerola Fruit Due to the relatively sma ll size of the fruit and its relatively large seeds, the consumption of the fruit in the raw stage has a limited fresh market The fruits have been incorporated into commercial fruit juices and energy drinks and are an increasingly attractive additive due to its current interest in developing products with health related properties. Fruits are used to enhance the vitamin C content of other fruits poor in this nutrient like apples, bananas, passion fruit, and pears. A p roduct formulated with 65 % green cocon ut water, 15 % pineapple, and 20 % acerola pulp presented the characteristics for a new commercial product ( D a Silva Pereira and others 2009) In some cases the fruit maybe cooked, strained to remove seeds and the resulting sauce or puree is utilized as topping on cake, pudding, ice cream or sliced bananas, or used in other culinary products. In a recent study an acerola ice cream has been developed and was proven to be suitable for the delivery of vi tamin C and bifidobacterium strains, while maintaining excellent viability and acceptable sensory characteristics (Favaro Trindade and others 2006)

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25 Acerola juice, sweeten ed or unsweetened due to its high ascorbic acid content maybe used to prevent browning of fruits such as banana slice s fruit salad, and will at the same time improve the ascorbic acid content of the product (Miller and others 1961) The fruits may be made into syrup or, with the addition of pectin, excellent jelly, jam, and other preserves. Cooking causes the bright red color to change to brownish red. The pasteurization process during the canning of the juice changes the color to orange red or yell ow, and packing in tin cans brings on further color deterioration. It was found that enamel lined cans lead to a better preservation of color. Wine made from acerola in the State of Hawaii was found to retain 60 % ascorbic acid (Monton 1987). Due to its ve ry high vitamin C content green acerola fruit has been extracted for use in dietary supplements. However, the high cost associated with the cultivation of the fruit seems to limit the expansion of this market. At immature stages the fruit may also be used as a source of pectin in confections or as an enriched source of dietary fiber ( Rufino and others 2010; Schreckinger and others 2010 ). Physico Chemical Properties and Nutritional Value of Acerola Fruit Protei n, Fat and Carbohydrate The physico chemical properties of acerola fruit and its nutritional value depend on several factors including: environmental conditions, growing location, cultural practices, the stage of maturation, and processing and storage (Mezadri and others 2006) One hundred gram of acerola fruit contains approximat ely 90.6 92.4 g of water, 0.21 1.20 g of protein, 0.23 0.80 g of fat, and 3.57 7.80 g of carbohydrate (Table 2 1). The main sugars in matur e acerola fruit are fructose and glucose, with small amounts of sucrose ( Mezadri and others 2006 ) The fruits from wild varieties of acerola such as those grown in the Caribbean islands are considerably tart, probably because of their low sugar and

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26 their h igh ascorbic acid contents. Recent genetic improvements have led to the development of new cultivars with higher sugar and lower ascorbic acid values such as The sweeter acerola v arieties, although contain ing a much lower vitamin C tend to be more popular in the fresh markets and juicing operations. The mature fruit contains approximately 8.98 % total carbohydrates. Vitamins and M inerals Acerola fruit is one of the most significan t sources of vitamin C obtained from plant material, and this vitamin plays a significant role in both the nutrition and the chemistry of this fruit. Reports from various sources indicated that the vitamin C content of acerola can range from 695 4827 mg/10 0 g of fruit (Table 2 1). The vitamin C content is affected by the ripening process and by the region in which the fruit is grown. Itoo and others (1990) reported reduction in the vitamin C content of acerola fruit grown in three different geographic regio ns (Nago, Naze, and Ibusuki) in Japan. From immature stage to full maturity, the vitamin C content decreased from 3.20 g/100g 1.83 g/100g, 2.78 g/100g 1.75 g/100g, and 2.15 g/100g 1.45 g/100g respectively for regions Nago, Naze, and Ibusuki. Similarly, a r eduction of about 50 % vitamin C has been observed by Vendramini and Trugo ( 2000) as the fruit ripened. This decrease in the vitamin C content has been ascribed to ascorbic acid oxidase enzyme (Butt 1980) The activity of this enzyme seems to be more intense in the mature fruits compared to the immature ones. Other authors however attributed the decrease in vitamin C content to biochemical oxidation. This hypothesis has been ver ified when the compound 3 hydroxy 2 pirone, an oxidative breakdown product of ascorbic acid was detected only in the aroma profile of mature acerola fruits (Vendramini and Trugo 2000)

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27 Given the high concentratio n of vitamin C in the green (immature) acerola fruits, it is used by some nutraceutical companies as a source of vitamin C in dietary supplements. However, the high cost associated with the cultivation of the fruit seems to limit the expansion of this mark et. Nevertheless, the utilization of the immature acerola is preferred when there is a need to develop products with high vitamin C content and when the flavor or the aroma characteristics of the fruit are not of interest. Besides maturity, post harvest ha ndling and storage conditions can substantially impact vitamin C and the shelf life of acerola fruit. The information available in the field of acerola processing, although scarce, suggests that the vitamin C content begins to decrease about 4 hours after the harvest (Alves and others 1995) One possible way to reduce post harvest loss of vitamin C is frozen storage at 18 o C which not only reduced vitamin C loss but also preserved some of the sensory qualities of the harvested material (Maciel and others 1999) Besides vitamin C, carotene (0.4 1 mg/100g), vitamins B6 (8.7 0 mg/100g), B 2 (0.07 mg/100g), and B1 (0.02 mg/100) and niacin (0.34 mg/100g) were reported in acerola fruit, but at levels below that recommended by the USRDA ( Miller and others 1961; Johnson 2003 ) Major macro minerals in acerola fruits include phosphorus (17.1 mg/100g), calciu m (11.7 mg/100g) and iron (0.22 mg/100g) (Table 2 1). The micro minerals content of this fruit, such as zinc, selenium and copper are not well studied. pH, Acidity Soluble Solid s, and Organic Acids Acerola is a very acidic fruit, and as for other componen ts of the fruit, the pH also varies with the stage of maturity. The pH value fluctuates from 3.60 3.70, the acidity

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28 expressed in gram s malic acid equivalent per 100 g fruit ranges from 1.04 1.87, and the total soluble solid varies from 7.70 9.20 g (Table 2 1). Although acerola is commonly called a cherry it s odor and flavor are more like that of tart apples than cherries. The organic acids in acerola in order of predominance are malic (0.25 0.38 g/100g), citric (0.01 0.03 g/100g), and tartaric acids (0.002 0.01 g/100g). Malic acid represents 32 % of the total acids in mature acerola fruits and 12 % in immature fruits (Righetto and Netto 2005) Another experiment reported that malic acid accounted for up to 20 % of t he acidity found in acerola fruit (Asenjo 1980) In general, the organic acids perform important functions in the metabolic process of fruits. They are directly involved in growth, maturation, and senescence; they al so influence the growth of microorganisms in fruit juices, and therefore affect the shelf life of the product; they may also participate in the synthesis of phenolic compounds and are important in the development of the characteristic flavor of the fruit (Ulrich 1970) Phytochemicals in Acerola The term phytochemical refers to bioactive non nutrient plant compounds in fruits, vegetables, grains, and other plant foods that have been associated with reducing the risk of major chronic diseases. In the literature, more than 5000 phytochemicals have been reported in fruits, vegetables, and grains, but large numbers remain unknown and need to be identified before the health benefits of phytochemicals in whole foods can be fu lly understood (Liu 2003) Convincing evidence implies that the benefits of phytochemicals in fruits, vegetables and whole grains may be even greater than is currently thought because the oxidative stress generated by free radicals is involved in the cause of a wide range of chronic diseases (Ames and Gold 1991) Because phytochemicals differ largely in composition and ratio from fruits to vegetables to grains,

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29 and often have compl imentary mechanisms to one another, it is thought that one should consume a wide variety of these plant based foods (Liu and Felice 2007) The phytochemicals include phenolic compounds, carotenoids, alkaloids, nitrogen containing compounds, and organosulfur containing compounds. The area of phytochemicals in acerola fruit is poorly documented; the most studied so far are the phenolic compounds and the carotenoids. Therefore, more emphasis will be put on phenolic compoun ds and the carotenoids in this literature review. Phenolic Compounds in Acerola Phenolic compounds in foods originate from one of the main classes of secondary metabolites in plants. They are particularly important for plant metabolism and have also becom e important for humans due to their health characteristics, particularly related to their antioxidant power. Phenolic compounds are also important because of their contribution to the sensory quality of fruits, which may be changed during the technological processes used in the production of juice and other derived products (Fernandez and others 1992) Phenolic compounds in foods may be categorized into simple phenols, phenolic acids (hydroxybenzoic and hydroxy cinnamic acid derivatives), flavonoids, stibenes, lignans, and tannins (Shahidi and Ho 2007) In foods, phenolics may occur in esterified, free or insoluble bound forms. The phenolic compounds identified in acerola f ruits are depicted in Fig ure 2 2. Due to lack of published data, only flavonoids and phenolic acids in acerola are discussed in this literature review. Phenolic compounds in foods form a large group of secondary plant metabolites which vary in chemical str ucture and reactivity. All plant phenolic compounds have one characteristic in common, an aromatic ring carrying one or more hydroxyl groups. The chemical structure may vary greatly from simple phenols to highly complex polymerized

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30 compounds like tannins. Several thousand of these natural compounds have been identified in plants, with a large diversity in their structural features (Harborne and Williams 2000) which contrast them from one another. The vast majority of dietary phenolic compounds, often defined as polyphenols, originate from plant foods (Scalbert and Williamson 2000) Their occurrence in animal tissues and non plant materials is almost entirely due to ingestion of plant sources (Shahidi and Naczk 1995) In plants, phenolic compounds exert interesting physiological attributes, such as protecting against ultraviolet radiation, pathogens and predators, imparting color and flav or, and helping growth and reproduction (Bravo 1998 ; Harborne and Williams 2000 ; Heim and others 2002) Phenolic compounds can be classi fied according to the structural characteristics of their carbon frame. The main classes of natural polyphenols include phenolic acids and derivatives, flavonoids, lignans, and stilbenes coumarins, tannins and lignans (Shahidi and Naczk 2003) Phenolic compounds occur mostly as conjugates with sugars, glucuronic or galacturonic acids, and sometimes with other phenols that are attached to hydroxyl groups or, less often, aromatic carbon atoms. The most common sugar moi ety is glucose while residues such as galactose, rhamnose, xylose or arabinose residues, are also often found (Bravo 1998) The structural differences of phenolic compounds lead in a wide variety of phytochemicals inge sted by human s In this section, the more abundant classes of dietary phenolic compounds will be discussed; with a particular focus on those that occur in acerola fruit.

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31 Flavonoids In late 1930s, the Szent Gyorgyi group observed that some flavonoids enhanc ed the biological activity of ascorbic and could cure scorbutic pigs; and recommend that the flavonoids be considered as P vitamin (Rusznyak and Szent Gyrgyi 1936) However, even though humans or animals never show the ability to synthesize flavonoids, this class of phenolic compounds has never been proven to show the usual properties of a true vitamin; therefore their classification as a vitamin has never been validated (Kuhnau 1976) Even so, the health promoting effect of flavonoids is unanimously recognized. Lampe ( 1999) believes that the health benefits of flavonoids may be enough to justify a semi essential status for these groups of phenolic compounds. Flavonoids are the most abundant class of phenolic compound in the diet (Harborne and Williams 2000) They are present in edible fruits, leafy vegetables, roots, tubers, spices, legumes, tea, c offee, chocolate, and red wine. Flavonoids are commonly classified into seven groups: flavones, flavanones, flavonols, isoflavones, flavanols and anthocyanidins (Liu and Felice 2007) They are frequently found in natur e as conjugates in glycosylated or esterified forms but can occur as aglycones, especially from the consequence s of food processing. Many different glycosides can be found in nature as more than 80 different sugars have been discovered bound to flavonoids (Hollman and Arts 2000) All fla v onoids are characterized by the flavan nucleus, a structure composed of two benzene rings (A and B) connected by an oxygen containing pyran ring (C) (Kuhnau 1976) The flavonoids are grouped into the subclasses of flavones, isoflavones, flavanols, flavonols, flavanones, anthocyanins, and proanthocyanidins, and this categorization is based on the degree of oxidation of the C ring, the hydroxylat ion

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32 pattern of the nucleus, and the substituent at carbon 3 (Scalbert and Williamson 2000) Flavonols like quercetin are ubiquitous in edible plants, isoflavones are found strictly in other foodstuffs flavonols fl avanols, and anthocyanins are abundant in the human diet, while flavones and isoflavones are less common (Scalbert and Williamson 2000) Flavonoids are excellent antioxidant and their antioxidant capacity depends o f their structure. Rice Evans and others ( 1997) studied the structural requisite for effective antioxidant action of flavonoids and phenolic acids. Due to their reduction potential, polyphenols can protect endogen ous antioxidants. This property is similar to that exhibited by ascorbic acid which exerts a vitamin E recycling ability. For example phenolic acids can effectively remove free radicals in model systems ( Laranj inha and others 1994 ; Chen and Ho 1997 ), delay lipid oxidation, spare vitamin E, and regenerate tocopherol from its tocopheroxyl radical in human LDL, erythrocyte membrane hosts, and monocytic cells ( Laranjinha and others 1995 ; Nardini and others 1995 ; Nardini and others 1998 ; Laranjinha and Cadenas 1999 ; Liao and Yin 2000 ). It was proven in a rat model system that caffeic acid spared vitamin E and increased the resistance of LDL towards oxidative stress (Nardini and others 1997) Lotito and Frei ( 2004) demonstrated in vitro and in vivo that flavonoids and phenolic acids from apples delay the oxidation of tocopherol in blood plasma. In the last few years, efforts have been made to study flavonoids in acerola; flavonols such as quercetin and kaempferol have been i solated in acerola (Vendramini and Trugo 2004 ; Hanamura and others 2006) however no quantitative data is currently available. Acerola anthocyanins, however, have been re latively well studied. This

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33 subgroup of flavonoids is very important because they are responsible for the attractive red color of the acerola fruits at complete maturity. Anthocyanin s Anthocyanins include a group of natural pigments that are responsible f or a wide range of color in the plant world including blue, purple, violet, and magenta. Chemically, anthocyanins are polyhydroxy and polymethoxy derivatives of 2 phenylbenzopyr y lium also known as flavylium salt. For quite a long time the safety of synthet ic pigments has been a subject of concern which fosters an increased interest in the use of naturally occurring coloring compounds such as anthocyanin (Francis 1984). However, the stability of anthocyanin in food is also a major problem. Anthocyanins demon strate greatest stability in acidic media, but are generally unstable and degrade by one of several possible mechanisms to form colorless and insoluble brown pigments (Jackman and others 1984). The changes in anthocyanins usually occur during processing an d storage; therefore, thorough knowledge of the factors that affect the stability of anthocyanins and their degradation mechanism is important if these pigments are to be utilized in the manufacture of food products. A number of factors influence the stabi lity of the anthocyanin pigment including pH, temperature, sugars, metal ions, co pigments, and ascorbic acid. Another important factor that may influence the stability of anthocyanin is an internal factor, also called the structural effect. Given the obje ctives of this study, the structural effect of pH an d Vitamin C will be discussed. The anthocyanins are structurally characterized by C 6 C 3 C 6 carbon skeleton. All their biosynthetic origin is similar to other naturally occurring flavonoids; they differ fr om these compounds by showing strong absorption in the visible range of the spectrum. In

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34 addition to the various external factors, the stability of anthocyanins is a function of their inherent molecular structure. All the naturally occurring anthocyanins a re glucosides of mainly six anthocyanin aglycone also called anthocyanidins, these being polyhydroxy and polymethoxy derivatives of flavylium salts. The aglycone moiety is highly reactive and this reactivity is responsible for diverse structural modificati ons that anthocyanin s undergo under acidic conditions (Timberlake and Bridle 1967; Brouillard 1982). Ano ther aspect of the anthocyanin structure that may influence its stability is the position of attachment of the sugars, acyl, and methoxy groups on the p igment molecules (Timberlake 1980). Because anthocyanidins are in general unstable and less soluble in aqueous media than anthocyanins, it is assumed that glycosylation confers stability and solubility to the anthocyanin molecule (Harbone 1979). Jurd (1972 ) showed that loss of the glycosyl moiety at the 3 position is accompanied by rapid decomposition of the aglycone in model systems, with irreversible loss of color. If a second site in the anthocyanin molecule is glycosylated, it is often located at positi on five (Brouillard 1982). Each glycosidic substitution is associated with a characteristic bathochromic shift (shift to a longer wavelength) (Harbone 1958). According to Brouillard (1982), a free r the formation of a quinoidal (anhydrobase) structure. This structure is derived from the flavylium cation form of the anthocyanin, by loss of acidic hydroxyl hydrogen, generally above pH 3. The quinoidal structure of anthocyanins is primarily responsible for the pigmentation of flower and fruit tissues. The flavylium cation, from which the quinoidal structure is derived, is relatively stable under acidic pH (1 3). This cation has been described as a heterocyclic carboxonium cation with its positive charge delocalized over the entire structure giving

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35 rise to six contributing resonance forms. According to Bendz and others (1967), the highest partial positive charges occur at the 2 and 4 positions of the flavylium cation. The stability of the cation is highly dependent upon nucleophilic attack at either position, by such compounds as water and sulfite ions (Bendz and others 1967). In addition, increases in pH. Under acidic conditions, the color of anthocyanins is determined largely by the degree of hydroxylation in the B ring of the aglycone, the greater the substitution the bluer the color (Asen 1977). Thus glycosides of delphinidin are bluer than those of cyanidin, which t hemselves are bluer than those of pelargonidin (Jackman and others 1984). pH has a marked effect of the color of anthocyanin solutions. Anthocyanins behave somewhat like pH indicators as a result of their amphoteric nature. Below pH 3, anthocyanin solution s display their most intense red coloration. When the pH of such solutions is raised, their red color normally fades to the point where they appear colorless in the pH range of 4 to 5. Further increases in pH of anthocyanin solutions give rise to purple an d blue, and these, upon storage or heat treatment, have been observed to change in pigmentation from blue to yellow (Jackman and others 1984). According to prior studies the observed changes in pigmentation with variations in pH may be attributed to the eq uilibrium reaction scheme presented in E quation 2 1 (Jurd 1963). AH + H + +H + A B flavyli um cation quinoidal anhydrobase carbinol pseudobase (2 1)

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36 In this scheme, under acidic conditions there is equilibrium between the flavylium cation (AH + ) and the carbinol pseudobase (B) form of the anthocyanin, with the supposed existence of a transient species, the quinoidal anhydrobase (A) structure obtained by the deprotonation of the flavylium cation. Pigment solutions above pH 7, held for an extended period or at elevated temperatures, were presumed to gradually change in pigmentation from blue to yellow as an indirect result of the formation of a chalcone (C) structure via ring fission of the anhydrobase (Hrazdina and Franzese 1974) The existence of the chalcone structure was postulated by Markakis and others (1957) in studies on the thermal degradation of anthocyanin. The chalcone has been described as a colorless compound; however, its ionized form is associated with a pale yellow color (Brouillard 1982). Timberlake (1980) and Brouillard (1982) su ggested that the distribution of the four different anthocyanin structures at a particular pH, under equilibrium conditions, may lead to some interesting conclusions. Research by Brouillard and Delaporte (1978) has shown that in very acid media (pH 0.5), t he red flavylium cation exists as the only species at equilibrium. With an increase in solution pH, both the concentration of this species and the pigmentation of the solution decrease as the cation hydrate to the colorless carbinol base. The formation of colorless chalcone and blue quinoidal anhydrobase begins at a pH slightly below that corresponding to the pK characteristic of the equilibrium between the flavylium cation and carbinol base and continue s to increase with increasing pH at the expense of the red flavylium cation. In the pH range of 4 to 5, the concentrations of

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37 the two colored anthocyanin forms tend to be very small, their total color effect contributing little to the pigmentation of the solution. The possible involvement of ascorbic acid in anthocyanin instability has been pointed out in 1940 by Battie and in the late 1940s by Pederson and others (1947). The two authors were among the first to observe the concurrent disappearance of ascorbic acid and anthocyanin in stored fruit juices and sug gested possible interaction between the two compounds. Oxygen and metal ions are essential requirements in the decolorization of anthocyanin by ascorbic acid (Sondheimer 1953; Timberlake1960; king and others 1980). It is reported that cranberry juice pigme nts degraded more rapidly when the greatest amount of ascorbic acid and oxygen were present. Under ascorbic acid conditions, the addition of transition metal for example copper ions stimulates the destruction of ascorbic acid and anthocyanin in their mutua l presence. Under these conditions, hydrogen peroxide (H 2 O 2 ) produced by copper catalyzed breakdown of ascorbic acid is believed to be the cause of the degradation (Timberlake 1960). Copper ions were found to accelerate and flavonols such as quercetin and quercitrin to slow down the degradation of both cranberry anthocyanin and ascorbic acid in model systems when they are present simultaneously (Shrikande and Francis 1974). However, in the model system containing no ascorbic acid, no anthocyanin degradation was noticed suggesting that ascorbic acid plays a role in anthocyanins break down. Recently, in a more contradictory result, Garzn and Worlstad (2002) found that fortification of strawberry juice with ascorbic acid at certain level s increase the half lif e of the pigment. In contrast, Rababah and others (2005) found that the addition of 1 %

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38 ascorbic acid increase s the lightness and decrease s the redness and yellowness color values of fresh strawberry, peach, apple slices and puree from them. The mechanism of degradation of anthocyanin by ascorbic acid has been investigated. However, the results are subjected to debate and up to now a resolution has yet to be found. When ascorbic acid is oxidized in the presence of copper, hydrogen peroxide (H 2 O 2 ) is produc ed; and since H 2 O 2 is as an anthocyanin bleacher, it is believed that the ascorbic acid induced anthocyanin degradation is mediated by H 2 O 2 (Markakis and others 1957). Jurd and others (1972) speculated that ascorbic acid degrades anthocyanins by a mechanis m involving direct condensation of the ascorbic acid to the position 4 on the flavylium structure. However, the authors did not provide experimental diketone dimedone condenses very readily with flavylium salts, and assuming that ascorbic acid has similar structure, the authors speculated that a similar condensation react ion may justify the observed effect of this substance. The direct condensation hypothesis is discarded by other experimenters. According to L acobucci and Sweeny ( 1983) color bleaching of anthocyanin by ascorbic acid occurs by cleavage of the pyrilium ring of the anthocyanin molecule. Garcia Viguera and Bridle (1999) studied the color stability of anthocyanin and flavylium salt with ascorbic acid. They found that ascorbic acid had a destructive effect on the flav ylium salts even when the position 4 is substituted. They concluded that the degradation is more likely to occur via a free radical mechanism proposed by L acobucci and Sweeny ( 1983) rather than by direct condensa tion as proposed by Jurd and others (1972). Garcia Viguera (1999) made some observations that let them to question the

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39 direct condensation hypothesis: (1) the loss of color happened slowly rather than instantaneously; red color does not return immediately upon acidification, (2) no new UV could be an indication of formation of new compounds, (3) the degradation effect of ascorbic acid was seen of the flavylium salt even when the position 4 was substituted. Another hypothesis was tentatively explained by Meschter and others (1953). According to this hypothesis, dehydroascorbic acid which is a product of ascorbic acid oxidation can also decolorize anthocyanins, but no mechanism has been proposed. In acidic solution, malvidin 3 5 diglucoside was oxidized faster than its acylated counterpart (Hrazdina and Franzese 1974) an effect attributed to reduced activity of the C 2 position and/or steric hindrance. The influence of ascorbic acid is slightly greater for a monoglucoside solution than a diglucoside solution .The presence of anthocyanins and the nature of these compounds influence the rate of degradation of ascorbic acid at pH 2.35 (Garcia Vig uera and Briddle 1999). Anthocyanins provide some level of protection toward ascorbic acid. Garcia Viguera and Briddle (1999) reported total loss of ascorbic acid after 15 days in the presence of malvidin 3 glucoside, while 5 % remains after 17 days in th e presence of the diglucoside. At low pH, the predominant anthocyanin form is the flavylium cation, known to be more active as a free radical scavenger (Garcia Viguera and Briddle 1999). Decrease in chroma value means loss of red color, lower contribution of the a value to the red color observed. The type of flavylium showed no significant influence on the rate loss of ascorbic acid (less than 3 % AA left after 10 days for 4 phenyl and 0 % left for 4 H and 4 CH 3 (Garcia Viguera and Bridle 1999).

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40 Phenolic A cids Phenolic acids are divided into three subclasses the hydroxycinnamic acids and their derivatives which are the most important subclass, the benzoic acid derivatives, and the hydrolysable tannins. These compounds differ in patterns of hydroxylation and methylation of their aromatic rings. Fig ure 2 2 shows the chemical structures of some of these compounds. Some common examples of hydroxycinnamic acids are p coumaric, ferulic, and caffeic acids. Of these hydroxycinnamic acids, caffeic acid is thought to be the most abundant in fruits and vegetables (Shahidi and Naczk 1995) and also the human diet (Clifford 2000) Hydroxycinnamic acids are generally present in the bound form and rarely present in free form. Bound forms of hydroxycinnamic acids are esters of hydroxyacids like quinic, shikimic, and tartaric acids as well as their sugar derivatives. The quantitatively most important conjugate of caffeic acid is its ester with qu inic acid, 5 caffeoylquinic acid also known as chlorogenic acid. Hydroxycinnamic acids are generally present in the bound form and rarely present in free form. The presence of chlorogenic acid in many foods of plant origin including apples, apricots, black berries (Herrmann 1973) and carrots (Babic and Amiot 1993) has been reported. Especially high concentrations of phenolic acid are found in coffee, apples, citrus fruits and juic es, and the bran of cereal grains. Excessive coffee drinkers may achieve a daily consumption of phenolic acids in excess of 1 g (Clifford 2000) The intake of caffeic acid alone was reported to be up to 983 mg per day in a southern German population, but also as low as 5 mg per day in some individuals. Hydroxybenzoic acids are commonly present in bound form. They are components of complex structures such as lign a ns and hydrolysable tannins. Hydroxybenzoic acids ar e also found in the forms of organic acids and derivatives of sugar (Schuster and

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41 Herrmann 1985) The content of hydroxybenzoic acids in foods of plant origin is generally low, except for blackberry, raspberry, blac kcurrant, red currant, and strawberry, as well as vegetables such as onions and horseradishes in which the content of hydroxyl benzoic acid partially protocatechuic acid, p hydroxybenzoic acid, and gallic acids may be very high. In acerola fruit, p coumar ic and ferulic acids were identified as two major phenolic acids (Vendramini and Trugo 2004) The same authors identified chromatographic peaks corresponding to chlorogenic and caffeic acids. In addition, benz oic acid derivatives like gallic, and syringic acids have also been reported in acerola (Righetto and others 2005) Carotenoids Carotenoids are yellow, orange, and red pigments present in many fruits and vegetables. More than 600 carotenoids have been identified in nature and approximately 20 are present in quantifiable amount in human serum (Cooper and others 1999) carotene, cryptoxanthin, lycopene, lutein, and zeaxanthin (Bendich 1989 ) only lycopene has not been identified in acerola fruit. De Rosso and Mercadante ( 2005) studied the carotenoids composition of tw carotene (265 1669 g/100g), lutein (37.6 10 1 cryptoxanthin (16.3 56.5 carotene (7.8 0 59.3 g/100g) as major carotenoids in both acerola genotypes. Other types of dietary carotenoids o f less quantitative importance including: diepoxy cryptoxanthin, 5,6 epoxy cryptoxanthin, 5,8 epoxy diepoxy carotene, 5,8 epoxy carotene were also reported. In another experiment, Lima and

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42 others ( 2005) reported the total carotenoid contents in acerola fruit cultivated in Brazil at different stages of maturity and different weather conditions. The results sh owed that the levels of carotenoids were very low in green fruit and then greatly increased as the fruit matured (Table 2 2 ); changes that were thought to reflect degradation of chlorophylls with a concomitant rise in carotenoids (Alves and others 1995 ) In addition, a higher level of carotenoids was reported for mature fruits harvested in the rainy season compared to those harvested in the dry season (Table 2 2 ). These data show that the carotenoids content vary accor ding to environmental conditions such as harvest season and stage of maturity. Occurrence of A nthocyanins in A cerola F ruit Acerola fruit has a very attractive red color at full maturity due the presence of anthocyanin pigments. Recently, the anthocyanins i n acerola have been characterized and quantified. However, there are some discrepancies in the results. Anthocyanins from two different acerola cultivars (Waldy and Olivier) have been extracted using 0.5 % HCL in methanol and identify by liquid chromatogra phy, mass spectrometry (LC MS). With 76 78 % of the total anthocyanin, cyanidin 3 rhamnoside represented the major anthocyanin in acerola fruit followed by pelargonidin 3 rhamnoside (13 16 %), cyanidin (6 8 %), and pelargonidin (2 3 %) ( D e Rosso and others 2008) While those results seem to be more or less consistent with the results obtained by Hanamura and others ( 2005) who identified cyanidin 3 O rhamnoside and pelargoni din 3 O rhamoside in acerola by NMR, they were less in agreement with another study in which the authors used different extraction and analytical techniques. Vendramini and Trugo ( 2004) identified three type s of anthocyanins in acerola by means of chromatographic and spectral data, finding only malvidin 3, 5 diglucoside, cyanidin 3 glucoside, and free

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43 pelargonidin. Recently in our lab we identified only two anthocyanins, cyanidin 3 rhamnoside and pelargonidi n 3 rhamnoside in acerola fruit from the variety Florida Sweet grown in Florida; cyanidin 3 rhamnoside being the most abundant form (Delva and Goodrich 2010) In contrast to the results obtained by Hanamura and others ( 2005) or Vendramini and Trugo ( 2004) no anthocyanin aglycons (anthocyanidins) were identified unde r our experimental conditions. The kind of anthocyanin present in ace rola fruit is variety dependent; some varieties may contain free anthocyanin aglycon while other varieties may not. Therefore it is important to mention the type of variety used in anthocyanin related stud ies of acerola fruit. With regards to the total ant hocyanin, amounts of 6.5 7.6 mg/100g and 7.9 8.4 mg/100g acerola pulp for the varieties Waldy and Olivier respectively have been reported ( D e Rosso and others 2008) Total anthocyanin in the pulp of the two varietie s mentioned above can be considered low when compared to fruits known as good sources of anthocyanin such as red raspberry (cultivar Jewel) (197.2 mg/100g), blackberry (cul tivar Chester Thornless) (153 mg/100g), and mulberry (14. 7 272 mg/100 g) (Wang and Lin 2000 ; Liu and others 2004) Conversely, acerola skin is much more concentrated in anthocyanin; the amount of 37.5 mg/100g of ripe acerola skin has been reported (Vendramini and Trugo 2004) This anthocyanin content is higher than that found in red cabbage (25 mg/100g), plum (2 25 mg/100 g), strawberries (15 35 mg/100g) and banana bracts (32 mg/100g FW) (Timberlake 1988 ; P azmino Duran and others 2001) Therefore, acerola skin, which is considered as a byproduct of the acerola pulp production, may be used as a commercial source of natural pigment.

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44 A nthocyanin, Ascorbic Acid and Color Stability of Acerola Attractive color is a very important sensory attribute for the consumer of fruits and processed food products derived from them. Unfortunately the appealing red color of fresh acerola fruit at comple te maturity is not maintained through processing and storage. Anthocyanin containing products during processing and storage are prone to color change resulting from the effect of anthocyanin degradation and brown pigment formation. When color changes occur extensively, visually unacceptable products result. Under normal processing conditions, a dull, brownish, pigment often appears which is often perceived by the consumer as indicative of poor quality and impl ies the spoilage of the fruit. Therefore, the in stability of the color has a negative impact on the market potential of the fruit. Acerola anthocyanin is very unstable and its degradation is responsible for the loss in red color of the frozen acerola pulp and processed juice. Although losses occur throu ghout the entire processing system, the main problem occurs during the commercial storage of these products. Because acerola anthocyanins appear to be more unstable than anthocyanins found in other fruits, the instability is thought to be due to structural differences in the individual anthocyanins. It has been reported that the color of some foodstuffs containing anthocyanins that are rich in cyanidin or delphinidin aglycones is less stable than foods containing anthocyanins that are rich in petunidin or m alvidin aglycones. Using a different hypothesis, it has been demonstrated in two separate model systems that the presence of elevated concentrations of ascorbic acid may be the main cause of the low stability of acerola anthocyanins, which occurs mainly du e to the direct condensation of ascorbic acid on carbon 4 of the anthocyanin, resulting

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45 in losses of both compounds (De Rosso and Mercadante 2007) The mechanism by which ascorbic acid may degrade anthocyanin is st ill unclear with further research needed for clarification of the degradation mechanism. Acerola Components and Potential Health Benefits The consumption of fruits and vegetables is considered to be beneficial to health. Many recent clinical studies suppor t the fact that increased consumption of fruits and vegetables is beneficial for the prevention of cancer (Steinmetz and Potter 1991 ; Block and others 1992 ; Margetts and others 1994; Steinmetz and Potter 1996 ) and cardiovascular disease (Ness and Powles 1997) An increase in consumption of fruits and vegetables to 400 g or five portions a day has been promoted by national and intern ational bodies inferring that such a change would reduce the incidence of both cancer and cardiovascular disease (James and others 1988) Due to the worldwide increase in obesity, the United States Department of He alth and Human services suggests increasing the consumption of fruits and vegetables as an effective strategy for weight management. According to Tohill (2007), fruits and vegetables have high water and dietary fiber contents and their consumption contribu te to satiety and reduce energy intake. Nutrients such as dietary fiber, vitamins, and bioactive phytonutrients such as phenolic compounds, dietary carotenoids, and lignans are held responsible for the anti cancer activity and other health related benefits provided by fruits and vegetables ( Van't Veer and others 2000) Acerola fruit is an outstanding source of vitamin C, a good source of phenolic antioxidant especially anthocyanins and phenolic acids, dietary carot biological activity is due to the high antioxidant power of the polyphenols, vitamin C and potential carotenoids

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46 Vitamin C is a very significant water soluble antioxidant in biological fluids ( Frei and others 1989 ; Frei and others 1990) It readily scavenges reactive oxygen and nitrogen species, such as superoxide and hydroperoxyl radicals, singlet oxygen, ozone, peroxynitrite, nitrogen dioxide, nitroxide radicals, and hydrochlorous acid (Halliwell 1996) therefore efficiently sparing other substrates from oxidative damage. Acute deficiency of vitamin C contributes to scurvy, expressed by blood vessel fra gility, connective tissue damage, fatigue, and ultimately death (Li and Schellhorn 2007) Some observational studies report a negative correlation between dietary intake of vitamin C, taken alone or in combination with ot her antioxidant vitamins, and the risk of cardiovascular complications ( Taniyama and Griendling 2003 Salonen and others 2000 ; Salonen and others 2003), although this correlation was not seen in randomized controll e d trials ( Willett and Stampfer 2001; Stanner and others 2004 ) Ascorbic acid has the ability to regenerate vitamin E by rapidly reacting with tocopherol radical and reducin g it back to its original form (Nagoaka and others 2007). In addition, it was found in cigarette smokers that the pace of the blood vitamin E oxidation cause by increased oxidative stress is significantly reduced by vitamin C supplementation ( Bruno and others 2005 ), suggesting a vitamin E recycling function of vitamin C and potential cooperative relationship between the two vitamins. The transport mechanism of vitamin C imposes a physiological restriction on its bioa vailability that is attainable by oral consumption (Padayatty and others 2004) Recent investigations suggest that ascorbic acid from natural sources is more readily absorbed by the human body than the synthetica lly produced vitamin C. In a double blind experiment, it was found that vitamin C in acerola powder is 1.63 times more bio

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47 available in the human body than vitamin C synthetically produced (Tang 1995) Studies showed th at infants consuming apple juice supplemented with acerola juice exhibited above average or average growth and development for their age and weight (Johnson 2003) Vitamin C levels in the blood were above average for all infants after the acerola juice was introduced in the diet. No allergic reaction was observed during this study, suggesting that acerola juice combined with apple is a good alternative to orange juice as a source of vitamin C in the diet. In a con troversial study it was shown that administration of a high dose of ascorbic acid improved the survival of patient s with last stage cancer ( Cameron and Campbell 1974 ; Cameron an d Pauling 1976 ). Their results led to the suggestion of using megadoses of vitamin C to combat degenerative diseases, including cancer and CVD. Vitamin C may protect against cancer through several mechanisms in addition to inhibition of DNA oxidation. One potential mechanism is chemoprotection against mutagenic compounds such as nitrosamines ( Tannenbaum and others 1991 ; Hecht 1997 ). Although there still is doubt on the res ult of this study, acerola fruit with its very high ascorbic acid content represents an appealing option for the use of mega dose ascorbic acid in cancer therapy. Vitamin C also acts as a coantioxidant by regenerati tocopherol (vitamin E) tocopheroxyl radical produced via scavenging of lipid soluble radicals ( Bowry and others 1995 ; Packer and Fuchs 1997 ). This is an important fu nction because in tocopherol can act as a prooxidant in the absence of coantioxidants such as vitamin C ( Bowry and others 1995 ; Neuzil and oth ers 1997 ).

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48 Numerous studies in humans have investigated the effects of the oxidazability of LDL by vitamin C supplementation in combination with vitamin E or carotene or both ( Frei and McCall 2000 ). Studies have bee n carried out in smokers (Steinberg and Chait 1998; Nyyssnen and others 1994 ), non smokers ( Jialal and Grundy 1993 ) and persons with hypercholesterolemia or cardiovascular disease ( Rifici and Khachadurian 1993 ; Gilligan and others 1994) In all cases, a significant reduction in LDL oxidizability was observed. The potential health promoting effect of ascorbic acid is due to the fact that this vitamin is a powerful antioxidant. Research conducted on the antioxidant potential of acerola fruit and acerola based drinks always mention the potential contribution of ascorbic acid although this contribution has never been quantified to the very b est of our knowledge. The antioxidant activity of acerola fruit and acerola juices has been investigated. Low density lipoprotein (LDL) has been incubated with acerola extract containing 25 and 51 M equivalents of ascorbic acid in the presence and absence of 0.1 M genistein/daidzein equivalents of soy extract or 0.2 M coumestrol/apigenin equivalents of alfalfa extract. It was found that the inhibition of copper mediated LDL oxidation by soy extract was significantly enhanced in the presence of 25 M acer ola extract, and an even higher inhibition was reported with 51 M acerola extract (Hwang and others 2001) In the same experiment, it was also demonstrated that low density lipoprotein radical (LDL ) formation is decrea sed in cells pretreated with soy, alfalfa, and acerola extracts. Pretreatments with soy, alfalfa, and acerola extracts noticeably reduced the extent to which 100 g/mL of LDL was converted to LDL after 24 h. The protective

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49 effect of these extracts is thou ght to be related to the presence of flavonoids in soy and alfalfa extracts and to the presence of ascorbic acid in acerola extract, which may act synergistically as antioxidants. More recently the antioxidant activity of juice extracts from mature and imm ature acerola fruit and of a concentrated juice from immature acerola fruit was evaluated with the objective of assessing the potential of immature and mature acerola used in food formulation s It was demonstrated that all acerola juices tested exhibited a ntioxidant activity in a lipid model system consisting of methyl linoleate. The oxidation curves of the methyl linoleate in the presence of acerola extracts (fractionated or not with a solid phase extraction (SPE) column) showed an increase in the rate of the oxidation after 48 h, which was an indication of the start of the propagation of the oxidation process. The antioxidant power of samples collected after 48 h indicated the hi ghest antioxidant activity (72. 1 %) for the immature concentrated juice extrac t followed by mature acerola and immature acerola juice extract at 68.5 % and 52.7 % respectively. Alpha tocophenol which was used as a standard antioxidant exhibited an antioxidant capacity of 86.2 % (Righetto and ot hers 2005) The fact that the mature acerola extract which contained smaller amount s of ascorbic acid and total phenolics exhibited higher antioxidant capacity than the immature acerola juice extract allowed the authors to speculate that phenolic compound s and/or other constituents with antioxidant activity rather than the total phenolics or vitamin C content were responsible for this activity. Since ferulic acid was detected in mature fruit, the higher antioxidant activity may be related to the presence o f ferulic acid. In fact, due to its ability to prevent the autooxidation of oils, ferulic acid has been largely utilized as a food preservative (Trombino and others 2004)

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50 Dietary flavonoids and other plant phenolic s have been found to have strong anti oxidant capacity, and antimicrobial and anti inflammatory effects (Huang and others 1992) They are also known for their health promoting effect by reducing the risk of cardiovasc ular diseases (CVD) and cancer (Temple 2000) Caffeic acid is thought to be the most abundant phenolic acid in the diet. Especially high concentrations are found in coffee, apples, and the bran of cereal grains. Exc essive coffee drinkers may achieve a daily consumption of phenolic acids in excess of 1 g (Clifford 2000) The intake of caffeic acid alone was reported to be up to 983 mg per day in a Southern German population, but also as low as 5 mg per day in some individuals (Frank 2004) Phenolic acids have been reported to efficiently scavenge free radical s in various model systems. In a rat model, caffeic acid spared vitamin E and enha nced the resistance of LDL towards oxidative stress (Nardini and others 1997) It has been proven that phenolic acids in apple delayed the oxidation of ascorbic acid in blood plasma (Lotito and Frei 2004) ; but no increase resistance to oxidation of endogenous antioxidants was found in blood plasma collected from volunteers up to 4 hours after the consumption of five apples. The evaluation of acerola fruit as a source of phenoli c antioxidant has been conducted. Also biological activity of a phenolic extract from acerola fruit has been studied. Total phenolic compounds have been quantified in 12 different acerola genotypes harvested during the dry and rainy seasons at three ripeni ng stages in Brazil. The phenolic concentration decreased as the fruits ripened. For the 12 different genotypes evaluated, it was reported that mature fruits harvested in the dry season showed higher

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51 total phenolic contents than those harvested in the rain y season (Lima and others 2005) For one genotype, 1703 mg/100g and 930 mg/100g catechin equivalent (fresh weight) were reported for mature acerola harvested in the dry and rainy seasons, respectively A possible explanation is that the rainfall may dilute the cellular juice, and therefore, decrease the total phenolic level (Lima and others 2005) In the same year however, Righetto an d others ( 2005) quantified the total phenolics in acerola and found that the mature acerola juice had 1.35 mg of catechin/g juice (57 mg of catechin/g dry material), and the immature acerola juice exhibited a level of 3.8 mg/g juice (24.5 mg of catechin/g of dry matter). Other authors reported similar results for berries and other fruits (K hk nen and others 1999; K hk nen and others 2001 ). Hanamura and others ( 2005) isolated three polyphenols from acerola fruit: cyanidin 3 O rhamnoside and pelargonidin 3 O rhamnoside as anthocyanins, and quercetin 3 O rhamnoside (quercetin) as flavonol. These acerola polyphenols were found to have radical glucosidase and advance glycation end (AGE) production, wh ich are both closely related to diabetes mellitus and its complications. Recently, Hanamura and others ( 2006) reported that crude acerola polyphenol inhibits glucose uptake in Caco 2 cells in a dose dependent fashion by adding acerola with an IC 50 value of nearly 0.2 mg/ L In addition, crude acerola polyphenols significantly suppress the glucose and maltose plasma levels after administering both glucose and maltose to ICR mice suggesting that crude acerola polyphenols had a preventive effect on hyperglycemia in the postprandial state by a mechanism that includes either the suppression of the intestinal glucose transport or the glucosidase (Hanamura and others 2006)

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52 In a relatively recent study, Motohashi and others ( 2004) investigated acerola extracts for cytotoxic, antibacterial, and antifungal activities and demonstrated that acerola extracts contained various biocidal subst ances. Cytotoxic activity against normal and tumor cells, antibacterial activity, radical quenching effect, multi drug resistance (MDR) reversal activity and other activities were enriched by fractionation with organic solvent extraction, and silica gel or reversed phase column chromatography. Among the biological effect tested, the reversal multidrug resistance in tumor cells was the most impressive The authors found that some acerola fractions such as hexane acetone fraction, acetone fraction, both from fresh acerola and a hexane acetone fraction from dried acerola powder showed highest tumor specific cytotoxic activity. In addition, those fractions of acerola inhibited the P glycoprotein (PgP) function in the MDR cancer cell more effectively than did ver apamil (a drug used in cell biology as an inhibitor of drug efflux pump proteins such as P glucoprotein) alone, thus improving the efficacy as a cancer chemotherapeutic treatment Based on these results and the overall biological activity, the authors stat ed that acerola extract is not only a good candidate as a new type of MDR reversal agent but may also be applied in the future for cancer therapy. Multidrug resistance is one of the major obstables to long term successful cancer chemotherapy The use of MD R reversal (MDRR) agents is a promising approach to overcome undesired MDR. In another experiment, Nagamine and others ( 2002) reported that acerola extract at 70 mg/kg body weight and 700 mg/kg body weight reduced t he number of 4 methylnitrosamino 1 3 pyridyl 1 butanone (NNK) initiated cells at the initiation stage in mice. NNK is a potent carcinogen formed from nicotine during tobacco processing and cigarette smoking (Hecht 199 7) Because it has

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53 been shown in an epidemiological study that the risk of tobacco induced lung cancer is lower in persons receiving high intake of vitamin C, the authors speculated that vitamin C might contribute to a part of acerola extract dependent in hibition of the initiation. However, it was also observed that the inhibition reached a plateau at the lowest level of acerola extract (70 mg/kg body weight), suggesting that other factors might play significant role s in the suppression of the initiation. In fact, as presented earlier, acerola may be a good source of carotenoids especially when it is cultivated in rainy season and harvested in the mature state (De Rosso and Mercadante 2005) Therefore, as suggested i n the publication of Nagamine and others ( 2002) carotenoids in the acerola extract may also participate in the inhibition process against NNK induced lung tumorigenesis through the suppression of the initiation sta ge. Besides ascorbic acid and dietary phenolics, carotenoids represent a significant group of phytochemicals in acerola fruit. Godoy and Rodriguez Amaya ( 1994) reported that acerola has a higher vitamin A value (64 R etinol Equivalents, RE /100g ) compared to some other fruits cultivated in Brazil such as nectarine (47 RE/100g) or peach cultivar (Cv Diamante) (58 RE/100g). In another experiment, Lima and others ( 2005) reported t he total carotenoid contents in 12 different acerola genotypes cultivated at the active Germplasm Bank in the Federal Rural University of Pernambuco, Brazil at different stages of maturity and different weather conditions. For a given genotype, the levels of carotenoids were very low in green fruit and then greatly increased as the fruit matured; changes that were thought to reflect degradation of chlorophylls with a concomitant rise in carotenoids (Alves and others 199 5) Within different genotypes, the carotenoid content was higher in some genotypes compared to others. In addition, a higher level of

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54 carotenoids was reported for mature fruits harvested in the rainy season compared to those harvested in the dry season. carotene) content of acerola can be used to promote the consumption of acerola fruit and acerola based drinks and other p roducts as healthy foodstuffs. In fact, carotenoids have been studied widely and proven to show diverse beneficial effect on human health. carotene is believed to have antioxidant activity. It has been shown to exhibit radical trapping behavior only at partial pressures of oxygen significantly less than in normal air (Burton and Ingold 1984) carotene appears to potentially act against angiogenesis in vivo (using male C57BL/6 mice as well as B16F 10 cells) and in vitro (using rat aortic ring assay, human umbilical vein endothelial cell proliferation, migration, and tube formation) ( Guruvayoorappan and others 2007) Angiogenesis is the formation of new blood vessels out of the preexisting vascular network and involves a sequence of events that are importan t for some pathological processes including the growth of tumor and metastasis. There is a growing body of evidence, including, in vivo, in vitro and epidemiological studies supporting the claim that lutein and zeaxanthin contribute to health and delay age related macular degeneration of the eyes and, to a lesser extent, cancer and heart diseases (Snodderly 1995; Rong and others 2007) Another epidemiological study conducted in the Pacific Isla nd indicated that people with higher carotene, and lutein had the lowest risk of lung cancer (Le Marchand and others 1993) Lutein showed anti tumor promoting activity in a two stage carci cryptoxanthin showed anti tumor promoting activity in two stage carcinogenesis in skin of IRC mice (Nishino and others 2002) Bishay ee and others ( 2000) showed that carotenoids can inhibit the initiation

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55 stage of the tumorigenic process in rat liver carcinogenesis initiated by a single injection of diethylnitrosamine (200 mg/kg) followed by promotion with phenobarbital (0.05 %) in a b asal diet. Dietary Phenolic Compounds and Contribution to Human Health Phenolic compounds in foods form a large group of secondary plant metabolites which vary in chemical structure and reactivity. All plant phenolic compounds have one characteristic in c ommon, an aromatic ring carrying one or more hydroxyl groups. The chemical structure may vary greatly from simple phenols to highly complex polymerized compounds like tannins. Several thousand of these natural compounds have been identified in plants, with a large diversity in their structural features (Harborne and Williams 2000) which contrast them from one another. The vast majority of dietary phenolic compounds, often defined as polyphenols, originate from plan t foods (Scalbert and Williamson 2000) Their occurrence in animal tissues and non plant materials is due to ingestion of plant foods (Shahidi and Naczk 1995) In plants, phen olic compounds exert essential physiological functions, such as protecting against ultraviolet radiation, pathogens and predators, contributing to their color and flavor, and facilitating growth and reproduction (Bravo 1998 ; Harborne and Williams 2000 ; Heim and others 2002) Toxicological Safety of Acerola Phenolic Compounds s that impart benefits to human health. Those functional properties include action against virus, hyperglycemia, and hypersensitivity. As it was described in previous sections of this chapter, acerola fruit contains previously isolated and identified polyp henols such as anthocyanins (cyanidin 3 rhamnoside, pelargonidin 3 rhamnoside) and flavonols such

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56 as quercetin (quercetin 3 O rhamnoside) (Hanamura and others 2005). The same authors demonstrated that those polyphenols exert radical scavenging power and p otential anti hyperglycemic activity particularly with regards to diabetes mellitus. A new type of polyphenolic constituent isolated in green acerola fruit and arbitrarily named display radical scavenging capacity (Kawaguchi and o thers 2007). Th e identification of this new type of phenolic compound may contribute to the enhance d health benefits of acerola polyphenols. It may also raise concern about the safety of acerola phenolic extracts. Phenolic extracts from other sources have been evaluated for their biological properties and their toxicological safety. Yamane and others (1996) showed that epigallocatechin gallate (EGCG) and green tea extracts inhibited chemical carcinogenesis of the gastrointestinal tract in rodent. In the sam e research, green tea extract was shown to be non toxic and its clinical use showed no harmful effect. It was also reported that procyanidins from grapes were not mutagenic in Salmonella mutagenesis assay system (Yu and Swamitan 1987). It was also reported that aplephenon, a commercial apple polyphenolic extract display s little mutagenicity at high concentration 2500 g/plate and chromosomal aberration test s and micronucleus tests exhibited no significant mutagenicity (Shoji and others 2004). However, rep orts on the toxicology of acerola polyphenols both in vivo in vitro and in animal studies are very scarce to the best of our knowledge. Hanamura and Aoki (2008) evaluated the toxicological safety of acerola polyphenols in rats and they suggested that the minimum lethal dose maybe higher than 2000 mg/kg. In addition, the authors reported no abnormal clinical signs regarding the administration of acerola polyphenols in the experimental subjects. While this research provide some promising

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57 results with regard to the safety of acerola polyphenols, it is clear that more research need s to be conducted in this area.

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58 Figure 2 1. Acerola at various stages of maturity. A: immature stage (green); B: intermediate stage (orange or orange red); C: mature stage (red).

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59 Figure 2 2. Structure of phenolic compounds in acerola fruit: A, anthocyanins; B, flanonols; C, chlorogenic acid; D, phenolic acids. D Antho cyanins R1 R2 R3 R4 Malvidin 3,5 diglycoside OCH3 OCH3 glucose glucose Cyanidin 3 glucoside OH Glucose glucose glucose Pelargonidin H H H H Cyanidin 3 rhamnoside OH H rhamnose H Pelargonidin 3 rhamnoside H H rhamnose H Flavonols R1 R2 Quercetin OH H Kaempferol H H Chlorogenic acid Phenolic acids R1 R2 R3 Caffeic acid H OH OH p coumaric acid H OH H Ferulic acid H OH OCH3 A B C

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60 Table 2 1. Nutritional value of acerola fruit Nutrient Content for 100 g acerola fruit A uthors Water 90.6 92.4 g Mezadri and others (2006) Protein 0.21 0.80 g 0.90 1.20 g Mezadri and others (2006) Vendramini and Trugo (2000) Fat 0.23 0.80 g Mezadri and others (2006) Carbohydrate 3.57 7.80 g 4.30 4.40 g Mezadri and others (2006) Vendrami ni and Trugo (2006) Fructose 0.25 0.38 g Glucose 2.14 3.33 g Righetto and others (2005) Sucrose 0.02 g 970 1900 mg 1074 2164 mg 695 4827 mg Righetto and others (2005) Vendramini and Trugo (2000) (Righetto and o thers 2005 ) Mezadri and others (2006 ) Vitamin C Vitamin B6 Vitamin B2 Vitamin B1 8.70 mg 0.07 mg 0.02 mg Mezadri and others (2006) Phosphorus 17.1 mg Mezadri and others (2006) Calcium 11.7 mg Iron 0.22 mg Ash 0.40 g Mezadri and othe rs (2006) Dietary fiber 3.00 g Mezadri and others (2006) Soluble solid 7.70 9.20 g Vendramini and Trugo (2000) Acidity 1.04 1.87 g Mezadri and others (2006) pH 3.60 3.70 Vendramini and Trugo (2000) Malic acid 0.25 0.38 g Righetto and others ( 2005) Citric acid 0.01 0.03g Tartaric 0.002 0.01 g *Acidity expressed in gram malic acid equivalent

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61 Table 2 2. Phytonutrient content of acerola fruit Phytonutrients Content for 100 g acerola fruit Authors Anthocyanins 3.79 59.74 mg 6.5 8.4 mg 37.5 mg Meza dri and others (2006) De Rosso and others (2008) Vendramini and Trugo (2004) Total carotenoids 32 323 a g 100 352 b g 75 419 c g 147 589 d g 940 3100 e g 1410 4060 f g 371 1881 g Lima and others (2005) De Rosso and Mercadante (2005) caroten e 265.5 1669 g 536.55 g De Rosso and Mercadante (2005) Mezadri and others (2006) trans carotene 340 g Godoy and Rodriguez Amaya(1994) carotene 7.8 59.3 g De Rosso and Mercadante (2005) Lima and others (2005) Lutein 37.6 100.7 g 99.21 g De Ro sso and Mercadante (2005) Mezadri and others (2006) Cryptoxanthin 16.3 56.5 g 417.46 g De Rosso and Mercadante (2005) Mezadri and others (2006) trans cryptoxanthin 40 g Godoy and Rodriguez Amaya (1994) Violaxanthin 395.3 g Mezadri and others (2006) Vitamin A value 46.2 283 RE* De Rosso and Mercadante (2005) Godoy and Rodriguez Amaya (1994) RE: Retinol equivalent. a, b: green acerola fruit cultivated in dry and rainy season respectively. c, d: half mature acerola fruit cultivated in dry and rainy seasons respectively. e, f: mature acerola fruit cultivated in dry and rainy seasons respectively.

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62 CHAPTER 3 IDENTIFICATION AND QUANTIFICATION OF PHENOLIC COMPOUND S IN ACEROLA FRUITS AND J UICES Overview Acerola is a tropical shrub grown in the Americas that produces a deep red cherry like fruit called differently (acerola, Barbados cherry, West Indian, cherry etc. ) depending on the region This fruit is particularly known for its very high vitamin C content and has become very attractive especially among people that are health conscious (Hanamura and others 2006) Recent investigations gave indication of some interesting biological activity of acerola fruit extract such as anticarcinogenic effect against lung cancer (Nagamine and others 2002) inhibition of nitric oxide prod uction (Wakabayashi and others 2003) antimicrobial properties, and tumor specific cytotoxic effect (Motohashi and others 2004) These effects are thought to be attributable to the presence of phytonutrients other th an vitamin C such as carotenoids and phenolic compounds (Hanamura and others 2006) Phenolic compounds are very important groups of secondary metabolites in plants. They play a significant role in the nutritional and sensory characteristics of different fruits and vegetables. Over the years, fruits and vegetables containing phenolic compounds have received considerable attention due to their potential biological and health promoting effects (Ahmed and Beigh 2009; Cart ea and others 2011) Anthocyanins are brightly colored polyphenolic pigments responsible for the red color of acerola fruit. The visual impact of anthocyanins associated with their potential health benefits make then potentially attractive as natural food colorants. The beneficial health related effect linked with a nthocyanin intake may include: a reduced risk of heart disease (Sumner and others 2005) protection against obesity and low blood glucose

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63 (Jayaprakasam and others 2006) enhancement of memory (Andres Lacueva and others 2005) and the protection against fetal brain tissue (Loren and others 2005) Anthocyanins are known as good antioxidants which may explain the health advantage they deliver ( D e Brito and others 2007) Kong and others ( 2003) describ ed the protection efficiency of anthocyanins as a function of the chemical structure of the molecule, such as degree of glycosylation, and number of hydroxyl group s in the B ring. Therefore, the determination of anthocyanins structure in foods and food pro ducts is a topic of interest. Recently, anthocyanins from acerola fruits have been reported; however the results are very inconsistent. De Rosso and others ( 2008) identified two anthocyanin hexosides: cyanidin 3 rha mnoside and pelargonidin 3 rhamnoside, and two free anthocyanidin s : cyanidin and pelargonidin by HPLC PDA MS/MS. Hanamura and others ( 2005) identified cyanidin 3 O rhamnoside and pelargonidin 3 O rhamoside in ac erola by NMR, but reported no free anthocyanidins. Vendramini and Trugo ( 2004) identified malvidin 3, 5 diglucoside, cyanidin 3 glucoside, and pelargonidin by means of chromatography and spectral data. In addi tion to those conflicting results, the identification of anthocyanins in the acerola variety used in this experiment (the variety Florida sweet) has never been reported to the best of our knowledge. Moreover, quantitative analysis of anthocyanins based on concentration basis is lacking. The quantification analysis performed by De Rosso and others (2008) was based on peak area percent of the identified compounds. Another gap of knowledge is that the non anthocyanin phenolics profile in both edible and non ed ible portions of acerola fruit and in acerola juice is not well understood. Vendramini and Trugo ( 2004) identified p coumaric and ferulic acids as two major phenolic acids in acerola. The same authors

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64 identifi ed chromatographic peaks corresponding to chlorogenic and caffeic acids. Furthermore, benzoi c acid derivatives like gallic, and syringic acids have also been reported in acerola ( Righeto and others 2005; El Malak and others 2010) The objective of this study was to identify and quantify phenolic compounds in acerola fruit. Materials and Methods Chemicals % purity), pelargonidin chloride, ferulic (99 % purity), p % purity), caffeic, gallic, and chlorogenic acids, Saint Louis, MO were purchased azobis (2 amidinopropane) dihydrochloride (AAPH) 6 hydroxy 2 5 7 8 tetr amethylchroman 2 carboxylic acid (Trolox) were purchased from Wacko Chemicals, Bellwood, RD, USA. L (+) ascorbic acid (99 % purity) was obtained from Acros Organic, NJ, USA. All other reagents were purchased from Fisher (Fair Lawn, NJ). Methanol, ethyl ace tate, and acetonitrile were of HPLC grade; all other solvents were of analytical grade. Fruits Harvesting, and Separation into Different Maturity Stages in Davie, South Flori da a nd by several growers in Vero Beach, Central Florida. The fruits were packed in Ziploc bags and transported the same day to the pilot plant of the Food Science and Human Nutrition Department at the University of Florida. The fruits were grouped into th ree different stages of maturity based on the visual color of the peel. The green, orange red, and deep red fruits were selected as the initial, intermediate, and complete stages of maturity respectively. The visual categorization was instrumentally and st atis tically validated using a machine vision method described in Y agiz and others (2009). The machine vision system was composed of a light box

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65 and a Nikon D200 digital color camera (Nikon Corp., Tokyo, Japan) coupled with a computer having a fire wire con nection. The camera settings were the f ollowing: 36 mm compensation and direct sunlight white balance. A computer program was created to collect images and to obtain color results based on lightness (L ), redness (a ) an d yellowness (b ) values of the fruits. The fruits were placed in the light box and the digital camera captured a picture of the fruits for each treatment. The machine vision system was calibrated with a standard red plate (L = 51.1 a = 50. 0, b = 24. 0 ) from Labsphere (North Sutton, NH, USA). Average L a b calculated using a color analysis program. Firmness analysis was performed as an additional means of validating the grouping of the fruits by stages of matu rity. The firmness analysis was performed using a TA.XT Plus texture analyzer (Texture Technologies Corp., Scarsdale, NY). The experiment al condition was as follows: compression to 3 mm using a 35 mm diameter cylinder Perspex probe at a test speed of 2 mm/ sec. The results were obtained in terms of kilogram force (kgf). The lower the force applied means the softer the fruit. The categorization of the fruits by stage of maturity was statistically validated by performing a one way analysis of variance with th e Duncan pair wise comparison test of the mean L a b and softness values. The statistical analysis was performed using the SAS (statistical analysis system, SAS Institute Inc. Cary, NC, USA ). Separation of the Fruits into Edible and Non Edible Porti ons The frui ts were separated into edible portions (skin+pulp) and non edible portion (seed). In addition to the fruits, frozen single strength acerola juice was purchased from ITI Tropicals, Lawrenceville, NJ. The fruit portions and the juice were kept at 20 C until

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66 needed. The edible and non edible portions of the fruits were packed in new Ziploc bags in 100 g increment and freeze dried; the frozen single strength juice was also freeze dried. The freeze dried material was finely ground using a Waring Bl ender (model 51BL31, Torrington, CT, USA) and stored at 20 o C prior to extraction. Extraction of the Phenolic Compounds Fresh fruits The extraction and fractionation of the phenolic compounds were achieved according to the method described in Kim and Lee (2002) with necessary modifications. Briefly, 100 g edible portion of fruit was mixed with 100 mL of methanol in a beaker. The content was transferred into a chilled blender and immediately macerated at high speed for 3 min. The ground material was crushed and returned to the original beaker with 50 ml of 80 % aqueous methanol placed in an ice bath for 20 min and sonicated every 5 min at low temperature. After 20 min the mixture was passed through two strainers with different pore sizes. The residues were re extracted with 100 mL of absolute methanol. The liquid extract was filtered through a Whatman no. 2 filter paper. The filtrates were combined and transferred to a 1000 mL round bottom evaporating flask with 40 mL of 80 % aqueous methanol. The methanol w as evaporate d in a rotary evaporator under vacuum at 30 o C to a volume of 20 25 mL. The concentrated extract was dissolved to a volume of 100 m L with deionized water and stored at 20 o C until fractionation. Freeze dried samples Ground freeze dried powder from 100 g of fruits was mixed with 100 m L of 80 % aqueous methanol in a 500 mL Erlen me yer flask which was immersed into an ultrasonic bath and sonicated for 20 min at room temperature and periodic shaking; the temperature of the ultrasonic bath was kept l ow throughout the extraction process The

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67 mixture was strained and filtered through a Whatman no. 2 filter paper by vacuum suction using a chilled Buchner Funnel The filter cakes were re extracted with 100 m L of 80 % aqueous methanol and the filtrates wer e combined and transferred into a 1000 m L round bottom evaporating flask with 50 m L of 80 % aqueous methanol and evaporated at 40 o C using a rotary evaporator until a volume of 10 30 m L was reached. The concentrate was solubilized into a 100 m L volume with deionized water, flushed with nitrogen and stored at 20 o C. Fractionation of the Crude Aqueous Phenolic Extract into Anthocyanin and Non Anthocyanin Twenty (20) certified C 18 Sep Pak cartridges were precondition by sequentially passing 6 m L of ethyl ace tate, 6 mL of absolute methanol and 6 m L of 0.01 N HCl into each cartridge. The aqueous phenolic extract was filtered through a 0.45 Millipore filter and 5 mL was loaded onto each cartridge. The cartridges were washed with 6 mL of 0.01 N aqueous HCl to rem ove sugars, acids, vitamin C and other water soluble compounds. The cartridges were allowed to dry for 10 min under vacuum. Each cartridge was rinsed with 10 mL of ethyl acetate to elute the non anthocyanin phenolic compounds and the eluates were combined in a 200 mL round bottom flask. The adsorbed anthocyanins from each cartridge were eluted with 3 mL of acidified methanol and the eluates were combined in a 50 mL round bottom flask. The ethyl acetate from the non anthocyanin fraction was removed under red uced pressure at 20 o C using a rotary evaporator and the methanol in the anthocyanin fraction was removed under the same condition but at 40 o C. Each fraction was dissolved in 15 mL deionized water, flushed with nitrogen and stored at low temperature for l ater use.

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68 Solid Phase Extraction of the Non anthocyanin Phenolics Three (3) SPE cartridges were preconditioned for the fractionation of the non anthocyanin phenolics into neutral fraction by sequentially passing 2 mL of absolute methanol and 2 mL of deion ized water. The aqueous phenolic extract was filtered through a 0.45 m PVDF filter and the pH was adjusted to 7.0 with concentrated NaOH. The extract was passed through the preconditioned cartridge to absorb the neutral phenolic compounds and acidic fract ions. For neutral phenolic compounds, the cartridges were preconditioned; for the acidic fraction 2 mL of 0.01N HCl was used instead of deionized water. The aqueous phenolic extract was adjusted to pH 7 .0 with concentrated sodium hydroxide and passed thro ugh the cartridges preconditioned for neutral. Three (3) more cartridges were preconditioned for the acidic fractions by sequentially passing 2 mL of methanol and 2 mL of 0.01N HCl. The pH of the effluent portion from the neutral cartridges was adjusted to pH 2.0 using 1N HCl and passed through the acidic cartridges to absorb the acidic phenolic compounds. Both the acidic and the neutral fractions were eluted with 5 mL of absolute methanol. The solvent was removed at 30 o C using a rotary evaporator. The fra ctions were solubilized with 5 mL of deionized water, sonicated to remove dissolved oxygen and stored at very low te mperature for HPLC analysis. HPLC Analysis of the Phenolic Compounds Anthocyanins Chromatographic analyses were performed on an Agilent 1200 series HPLC system (Agilent, Palo Alto, CA) coupled with an autosampler/injector and diode array detector (DAD). A Zorbax Stablebond Analytical SB C 18 column (4.6 mm x 250 mm, 5 n was

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69 performed using mobile phase A ( 1 % formic acid aqueous solution) and mobile phase B (100 % methanol). UV vis spectra were scanned from 220 to 600 nm on a diode array detector with detection wavelength of 520 nm. The flow rate was 1 mL/min, and the f ollowing convex gradient was used: 5 to 20 % B from 0 to 2 min, 20 to 30 % B from 2 to 5 min, 30 to 45 % B from 5 to 10 min, 45 to 55 % B from 10 to 15 min, 55 to 70 % B from 15 to 30 min, isocratic (70 % B) from 30 to 32 min, followed by re equilibration of the column for 3 min for the next run. Electrospray mass spectrometry was performed with a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). The anthocyanins are positively charged therefore, the mass spectrometer was operated in positi ve ionization mode. Other experimental conditions for the mass spectrometer were as follows: nebulizer, 45 psi; dry gas, 1 1.0 L/min; dry temperature, 350 o C; ion trap, scan from m/z 100 to 2200; smart parameter settin g (SPS), compound stability, 50 %; trap drive level, 60 %. The mass spectrometer was operated in auto MS/MS mode to capture and fragments the most abundant ion in full scan mass spectra. The identification of the anthocyanin was based on mass spectral information, chromatography of pure standar ds when available, and UV vis spectra of the diode array detector. Individual anthocyanin contents was calculated using standard calibration curves, cyanindin 3 rhamoside was calculated as cyanindin equivalent while pelargonidin 3 rhamnoside was expressed as pelargonidin equivalent. Acidic and neutral phenolic fractions The acidic and the neutral fractions were diluted 1:1 (v/v) with methanol and filtered through a 0.45 m filter. Chromatographic analyses were performed on an Agilent 1200 series HPLC system (Agilent, Palo Alto, CA) coupled with an

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70 autosampler/injector and diode array detector (DAD). A Zorbax Stablebond Analytical SB C 18 used for separation. The separations were performed by gradient elution of increasing concentration of acetonitrile in acidified water at a flow rate of 0.2 mL/min. The starting elue nt (A) and the gradient former (B) consisted of water and acetonitrile, respectively, both containing 1 .0 % (v/v) formic acid, and the elution was performed by a multisegment gradient, according to the program described in Nicoletti and others ( 2008) and summarized in Table 3 2 The DAD detector was set at 280 nm for the neutral phenolic compounds or 320 nm for acidic phenolic compounds. Results and Discussions Validation of the Categorization of the Fruits by Stag e of Maturity To validate the grouping of the fruits by stage of maturity, the mean values of certain color quality parameters mainly the lightness (L ) yellowness (b ), and redness (a ) and the softness were statistically compared using a Duncan multiple range test performed by the Statistical Analysis Software (SAS). The SAS program used and the SAS output are presented in Appendix B (Tables B 1 and B 2). Table 3 1 shows that the L values increased from the immature to the intermediate stage of the fruit ; a decrease in the L was seen from the intermediate to the complete stage of development of the fruit. The a values increased from the immature to the fully mature stage; while the yellowness (b ) decreased as the fruit ripened. The softness of the frui t followed the following trend: softness fully mature fruit > softness fully intermediate>softness immature. Statistical analysis showed significant difference (p<0.05) between the parameters within a given stage of maturity; therefore validating

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71 the visua l categorization of the fruit by stage of maturity made after harvest. The observed changes in the parameters (increase L decrease a*, and decrease b*, and decrease softness) translate the complex biochemical transformation that takes place during the ri pening process (Vendramini and Trugo 2000). Anthocyanins Identification and Quantification The anthocyanin analysis was performed only on the edible portions of the fully mature (red) fruits. Two chromatographic peaks were detected in the partially purifie d anthocyanin fraction s from fruits grown in Davie ( Ace DA), Vero Beach ( Ace VE) and the frozen single strength acerola juice ( F SSAJ ) by HPLC DAD MS 2 The chromatograms of the identified anthocyanins are shown in Figure A 1 ( Appendix A ) and the characteri stics of the peaks are given in Table 3 3 The peaks obtained from different samples showed similar mass spectral characteristics. The molecular ion of peak 1, respectively from Ace DA, Ace VE, and the FSSAJ was found at a mass to charge ratio (m/z) 433 an d product ions at m/z 287. The presence of these ions suggests that the aglycon cyanindin was glycosylated with a deoxyhexose due to the lost of 146 u (433 287), this peak was tentatively identified as cyanidin 3 rhamnoside. The Molecular ions of peak 2 wa s found at m/z 417, and fragment ions at 271 (M 146 + ) corresponding to aglycone pelargonidin with the loss of one molecule of hexose. Therefore, peak 2 was tentatively identified as pelargonidin 3 rhamnoside. The results show that acerola fruits grown in D avie, those grown in Vero Beach and the juice which was processed from an unknown variety have similar anthocyanin profile. Cyanidin 3 rhamnoside and pelargonidin 3 rhamnoside are the only anthocyanins identified in the samples. The identification of cyan idin 3 rhamnoside and pelargonidin 3 rhamoside is in agreement with the information reported in the literature (Hanamura and others 2005 ;

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72 De Brito 2007, De Rosso and others 2008). However, malvidin 3 5 di glucoside, malvidin 3 glucoside, free cyanidin and free pelargonidin reported in the literature (Vend ramini and Trugo 2004; De Rosso and others 2005) were not identified under the conditions that this experiment was conducted. Each individual anthocyanin detected was quantified using a standard calibratio n curve from 0 50 g/ mL of pure standard compound; the results are presented in Table 3 3 Due to unavailability of cyanidin 3 rhamnoside and pelargonidin 3 rhamnoside standards, the individual anthocyanin contents were expressed as cyanidin o r pelargonidi n equivalents. The results show that the concentration of cyanidin 3 rhamoside in Ace DA sample is 2.67 mg/100g FW and nearly is twice as high as the concentration of pelargonidin 3 rhamnoside, 1.34 mg/100 mg FW in the same sample. In contrast, the concent ration of cyanidin 3 rhamnoside in the Ace VE sample (8.47 mg/100g) is just a little bit higher than the concentration of pela rgonidin 3 rhamnoside (6.52 mg/ 100g). The results also show that the fruits collected in Vero Beach exhibited a higher individual anthocyanin content than those collected in Davie. The cyanidin 3 rhamnosid e content in Vero Beach sample on a fresh weight basis was 8.47 mg/100g, about three times the cyanidin 3 rhamnoside content in the samples grown in Davie. In addition, pelargonidin 3 rhamnoside content in the Vero Beach sample was more than four times as high as the content found in samples collected in Davie. While the individual anthocyanin content in acerola fruit appears to be low when compared to other fruits, there is no doub t that these pigments are responsible for the appealing red color of acerola fruit.

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73 Non Anthocyanin Compounds Method development The effect of different parameters affecting the efficacy of the extraction and the performance of the diode array detection ( DAD) of the polyphenols was considered during this experiment. The extraction and isolation of the polyphenols in acerola fruit were accomplished according to a protocol developed by Kim and Lee ( 2002) with minor but n ecessary adjustments. Two types of extraction were performed, a dry extraction where freeze dried powdered acerola fruits where used and a wet extraction in which frozen fruits were extracted directly. In the case of the dry material, aqueous methanol comb ined with sonication was used for the extraction. The cavitation generated by ultrasound when used in aqueous methanol extraction helps to enhance mass transfer rate and favors higher production yield with less extraction time and solvent usage (Kim and Lee 2002) The disadvantage of using the freeze dried extraction is that freeze drying is a very expensive process where a freeze drier is not always available. The sample preparation in which the polyphenols in the fres h material are extracted using absolute methanol and homogenization is simple although time consuming. Following the extraction, the crude polyphenolic extracts were fractionated into anthocyanin and non anthocyanin fractions. In our preliminary experiment s HPLC separation of the non anthocyanin phenolic compounds lead to very crowded chromatograms due to too many interfering peaks such as sugars, organic acid and most importantly ascorbic acid in the case of acerola fruit. Therefore, the non anthocyanin ph enolic compounds were further fractionated into neutral and acidic fractions based on the fact that polyphenolic acids are completely ionized at pH 7.0 and un ionized at pH 2.0 Kim and Lee ( 2002)

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74 After the extraction and the purification steps the samples were analyzed by HPLC. The separation was performed using an Agilent HPLC system composed of a DAD detector, and the separation was carried out on a Zorbax Stablebond An alytical SB C 18 column (4.6 mm x 250 mm ) operati ng under a gradient of elution mode composed of water containing 1 % formic acid (solvent A) and acetonitrile containing 1 % formic acid (solvent B). One percent formic acid was added in the mobile phase in order to keep carboxyl and hydroxyl groups of the analytes in their protonated form and help to minimize peak broadening (Nicoletti and others 2008) Validation The validation of a chromatographic method is usually judged based on the precision, linearity, and ac curacy ; only the precision of this method was measured. The identification of the phenolic compounds in acerola fruits was accomplished on the basis of their retention time and visible spectra collected with the DAD detector. The identification of each pea k was tentatively confirmed by ESI MS n detection. The parameters set for the ESI MS n are described in the materials and methods section, almost all phenolic compounds produced mass spectra with the base peak corresponding to the so called molecular ion. Th e precision of the method was evaluated in terms of interday repeatability of the retention time for most of the standard phenolic compounds used in this study. The standard phenolic compounds were analyzed in sextuplicate over six consecutive days. The re sults are expressed as mean values, standard deviation and relative standard deviation of the retention times Table 3 4 show very small interday variability in the retention times of the standard phenolic compounds analyzed

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75 Identification by HPLC ESI MS 3 Two samples of chromatograms of the acidic and neutral fractions of the phenolic compounds in the edible portion of the acerola fruit are shown in Appendix A ( Figure A 2 and Figure A 3 ) Because the phenolic compounds at the different maturity stages are not qualitatively different, only chromatograms of samples from the full stage of maturity are presented. The chromatograms were captured at 320 nm and 280 nm for acidi c and neutral fractions respectively. However, most of the acidic phenolic compounds co uld also be detected at 280 nm. The acidic fraction is mostly composed of phenolic acids which are derivatives of hydroxycinnamic or hydroxybenzoic acids while flavonols and to a lesser extent flavan 3 ols are the predominant groups of phenolic compounds i dentified in the neutral fractions. The identification of the phenolic compounds in the different samples was based on mass spectral data and chromatography of pure standards (when available), and the published literature data. All samples combined, a tota l of twelve (12) phenolic compounds were tentatively identified. Among the twelve compounds, 4 of them are reported for the first time that include two flavan 3 ols epi catechin and another epicatechin derivative identified only in the edible portions at c omplete maturity, free ellagic acid and another ellagic acid derivative identified only in the non edible portions (seed) of the mature fruit, and another phenolic compound in the subclass of stilbene: resveratrol hexoside, only identified in the edible po rtion of the fruit at full maturity. Hydroxycinnamic acid derivatives Compound 22 from Table 3 5 and Figure A 2 (Appendix A) had a mass to charge ratio (m/z) 377 [M+Cl ] ion indicating that a chloride based adduct was fragmented to give rise to a base pea k at m/z 341 [M H ] This compound was further fragmented to

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76 give another ion at m/z 179 probably after the removal of a molecule of hexose. This compound was tentatively identified as a derivative of caffeic acid. Other hydroxycinnamic acid derivatives id entified include derivatives of clorogenic, ferulic and p coumaric acids (Table 3 5). Caffeic acid is known as one of the most abundant phenolic acid s in fruits and vegetables, and in the human diet (Shahidi and Nacz k 1995; Clifford 2000) Chlorogenic acid is one of the most abundant conjugates of caffeic acid and it has been reported in fruits such as apple, apricots, blackberries (Herrmann 1973) and in c arrots (Babic and Amiot 1993). p c oumaric acid and ferulic aci d were reported as main phenolic acids in acerola fruit (Vendramini and Trugo 200 4 ). The authors also identified chromatographic peaks corresponding to chlorogenic acids. In c ontrast benzoic acid der ivatives like gallic, and syringic acids reported in the literature (Righetto and others 2005, El Malak and others 2010) were not found under the conditions of this experiment. Flavan 3 ols Compound 16 (Table 3 5) and Figure A 3 (Appendix A) shows m/z at 289 [M H] Although we did not see the characteristic ba se peak usually seen at m/z 245 at MS 2 however at MS 3 a minor ion was observed at m/z 205 probably from the cleavage of ring A from the flavan 3 ol molecule, this compound was tentatively identified as epicatechin. Compound 46 with m/z 453 [M H] was frag mented and shows one product ion at m/z 289 probably from the cleavage of the epicatechin molecule from the mother ion; therefore this compound was tentatively identified as an epicatechin derivative. Epicatechin and its derivatives were not previously rep orted in acerola fruit and our identification is tentative with further experiments needed to confirm the presence of epicatechin and its derivative in mature acerola fruit.

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77 Flavonols Flavonols are a very important subclass of flavonoid s in acerola fruit. In this research, the flavonols identified are all glycosides: quercetin 3 O rhamnoside ( compound 55, Table 3 5 ), kaemferol 3 rhamnoside ( compound, 54 Table 3 5 ) and another kaempferol derivative ( compound 41, Table 3 5 ). Compound 41 showed a parent ion at m/z 593 [M H] which was fragmented to give a base peak at m/z 285 corresponding to the loss of an hexose molecule. Further degradation of this ion showed another product at m/z 211. This compound was identified as a kaempferol derivative. Compound 55 s howed a parent at m/z 463[M H] the dissociation of this ion lead to a fragment ion at m/z 301 (MS 2 ) which in turn was degraded in to fragments observed at m/z 277, 271, 179 typical of quercetin fragmentation observed in the literature. Compound 55 was the refore identified as quercetin 3 O rh amnoside In addition, compound 54 was also identified as kaempferol derivative on the basis of mass data and the chromatography of pure standard. The flavonols identified in this experiment are in line with literatur e data. The presence of quercetin and kaempferol in acerola fruit has been mentioned in at least two previous experiments. Vendrami and Trugo (2004) showed by means of chromatography and spectral data two chromatographic peaks having similar characteristic s with quercetin and kaempferol. The existence of a quercetin hexoside (quercet in 3 O rhamnoside) was detected and identified in acerola fruit by nuclear magnetic resonance (NMR) (Hanamura and others 2005). Ellagic acid and s tilbene Free ellagic acid (compound 26 Table 3 5) as well as a potential derivative of ellagic acid (compou nd 33, Table 3 5) was tentatively identified in the non edible portion

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78 (seed) of the mature fruit. D ue to lack of a standard compound we could not confirm the identification of this compound. Free ella gic acid as we ll as other derivatives of ella gic acid h as been reported in seeds of muscadine grape (Sandhu and Gu 2010). A compound having similar spectral characteristics with resveratrol hexoside was observed in the edible portion of the fruits at complete maturity, but as for the ellagic acid and its der ivative, the identification was not confirmed. Summary Our results show that all stages of maturity for the acerola variety Florida Sweet contained two types of anthocyanins: cyanidin 3 rha m noside and pelargonidin 3 rhamnoside and various groups of non an thocyanin phenolic compounds including hydroxycinnamic acid derivatives, flavonols, flavan 3 ols, ellagic acid and stilbene. While the identification of some of the non anthocyanin phenolic s was not confirmed, the chromatograms and the mass spectral data g enerated can be used as fingerprint for future research in this area. B ecause anthocyanins and non anthocyanin phenolic compounds display interesting health promoting effects, the i r presence in the acerola fruit may contribute to the expansion of this frui t to market s of exotic tropical fruit.

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79 Table 3 1. Color characteristic and the hardness of acerola fruit at different stages of maturity Maturity stage L a b Hardness (kgf) Immature (green) 52.09 a 8. 30 c 40.66 a 6.03 a Intermediate (Orange) 52.39 a 19 .49 b 38.33 b 3.2 0 b Mature (red) 43.80 b 38. 60 a 31.33 c 2.21 c L : lightness (0 indicate black, 100 indicates white); a : redness or greenness (positive values indicate red, negative values indicate green); b : yellowness or blueness (positive values indicate yellow, negative values indicate blue). Values in a column followed by different letters are significantly different (P 0.05) according to the Duncan test.

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80 Table 3 2. Solvent gradient for reversed phase HPLC analysis of the neutral and acidic fractions of the phenolic compounds a Time (min) Solvent A (%) Solvent (%) 0 95 5 3 95 5 15 91 9 27 86.5 13.5 32 86.5 13.5 42 81.5 18.5 44 81.5 18.5 51 77.5 22.5 55 70 30 56 60 40 57 95 5 a Solvent A, 1 : 9 9 % (v/v) formic acid/water; solvent B, 1 : 9 9 % (v/v) formic acid/acetonitrile

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81 Table 3 3. Identification of anthocyanin using HPLC ESI/MS/MS Sample Peak t R max (nm) [M + ] (m /z) [MS/MS] (m/z) Compound Content* Ace DA 1 12. 6 280, 520 433 287 Cyanidin 3 rhamnoside 2.67 2 13.5 270, 508 417 271 Pelargonidin 3 rhamnoside 1.34 Ace VE 1 12.6 280, 520 433 287 Cyanidin 3 rhamnoside 8.47 2 13.6 270, 506 417 271 Pelargonidin 3 rham noside 6.52 FSAJ 1 11.9 280, 520 433 287 Cyanidin 3 rhamnoside 3.49 2 13.0 270, 506 417 271 Pelargonidin 3 rhamnoside NQ Anthocyanin content in mg/100g FW Ace DA: Sample from the cultivar Florida Sweet grown in Davie Ace VE: Sample from the cultivar Florida Sweet grown in Vero Beach FSSAJ: Frozen single strength acerola juice NQ: Not quantified

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82 Table 3 4. Interday precision Repeatability (n=6) Retention time (min) Peak no Analyte Average SD RSD % 1 Gallic 8.31 0.03 0.31 2 Protocatechuic acid 15.77 0.09 0.60 3 Chlorogenic acid 28.48 0.21 0.74 4 Caffeic acid 31.19 0.15 0.49 5 Syringic acid 35.92 0.05 0.13 6 Ferulic acid 44.41 0.33 0.75 7 p coumaric 51.62 0.24 0.46 8 Sinapic 54.43 0.25 0.47

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83 Table 3 5. Retention times and m ass s pectr ometric d ata of non anthocyanin phenolic compounds in fruit determined by HPLC ESI MS 2 all stages of maturity included Cpd No. t R (min) Mwt MS 1 (m/z) MS 2 (m/z) MS 3 (m/z) Identification (tentative) Acidic Fraction Hydroxycinnamic acid derivatives a 22 3 1.9 342 377[M+Cl ] f 341 279, 149 179 Caffeic acid derivative 20 28.8 345 344 [M H] f 181 163 Chlorogenic acid derivative 31 44.2 356 355[M H] 193 337, 265, 209 Ferulic acid hexoside 36 51.7 522 521[M H] f 503 415 459, 415, 307, 265, 221 p coumaric acid hexoside derivative Neutral Fraction Flavan 3 ols b 48 29.1 454 453[M H] f 289, 433 f 127 271 Epicatechin derivative 16 16.8 290 289[M H] f 271, f 209, 113 253, 197, f 203 113 (+) -Epicatechin Flavonols c 55 41 464 463[M H] f 301, 273 271, 255, f 179 Quercetin 3 O rhamnoside 54 38.5 432 431[M H] f 285 255, 229, 195, 174 Kaemferol 3 rhamnoside 41 21.7 594 593[M H] 534, 533, 288, f 285 211 Kaemferol derivatives Conjugate of Ellagic acid d 26 36.0 302 301[M H] 284, f 257 Ellargic acid 33 47.3 434 4 33[M H] f 301 177, 133 396, 340, 265, 219, 177, f 133 Ellagic acid derivative Stilbenes e 56 41.9 390 389[M H] f 371 297, 221, 177, 133 327, 283, 221, 197, f 133 Resveratrol hexoside a, c identified in edible portion of fruits at both immature and mature stages, b,e identified in edible portion only at full maturity, d identified only in non edible portion (seed) of mature fruits. f most intense product ion s

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84 CHAPTER 4 ANTIOXIDANT ACTIVITY ANTIMICROBIAL PROP ERTIES, AND TOXICOLO GICAL SCREENING OF PHENOLI C EXT RACTS FROM ACEROLA ( MALPIGHIA EMARGINATA DC) FRUIT Overview In recent years, the relationship between food and good health has become a very important issue. Many c ommon foods are now considered addition to f ulfilling the basic nutritional needs should be able to provide additional physiological benefits, such as preventing or delaying the occurrence of chronic diseases in human (Kaur and Kapoor 2001). Research in the field of food science and nutrition has bee n focusing on the development of food products with higher nutritional values, and the evaluation of foods for their health promoting potential (Nicoli and others 1999). Recently, phytochemicals in fruits and vegetables have attracted a great deal of atten tion mainly owing to their role in the prevention of degenerative diseases caused by oxidative stress. Oxidative stress has been defined as a disturbance in the equilibrium status of pro oxidant/antioxidant systems in intact cells resulting in oxidative damage to nucleic ac ids, lipids, proteins and carbohydrates (Thomas 1994). Oxidative stress releases free oxygen radicals in the body, and is involved in a number of disorders including heart disease, cataracts, cancers, rheumatism, ageing and many other auto immune diseases (Kaur and Kapoor 2001). Phytochemicals act as antioxidant compounds and are very effective free radical scavengers. Epidemiological evidence has shown correlation of dietary patterns with the prevention of non transmissible chronic diseases such as cancer and cardiovascular disease. Many clinical studies have consistently demonstrated positive correlations between the consumption of fruits and vegetables and the reduction rate of heart

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85 disease mortality, certain forms of cancer and other types of degenerat ive disorders (Steinmetz and Potter 1991; Steinmetz and Potter 1996; Block and others 1992; Margetts and others 1994; Ness and Powell 1997) This is due to the fact that fruits and vegetables contain different class of phytochemical compounds such as vitam in C, vitamin E, dietary fiber, dietary phenolic compounds and dietary carotenoids. For all the reasons indicated above there is an increased interest in the evaluation of the antioxidant activity in fruits and vegetables, and there is a plethora of public ation in this area (Wada and Ou 2002; Kolayli and others 2003; Chinnici and others 2004; Silva and other 2004; Roesler and others 2006). The consumption of exotic tropical fruits has increased on both domestic and international markets due to increase reco gnition of its nutritional and health promoting effects. Acerola is a shrub grown in tropical and subtropical areas from the southern end of Texas, through Mexico and Central America to northern South America and throughout the Caribbean. It has also been introduced widely into tropical areas of Asia especially in the Island of Okinawa in Japan, and in Africa. The tree bears a soft red fruit that can be consumed fresh or processed for use as an ingredient in a variety of foods including commercial fruit ju ices, and energy drinks. In Germany, France, and Hungary, the fruit is used primarily for juice while in the United States it is utilized by the supplement and pharmaceutical industries as a rich source of vitamin C. Acerola is therefore an exotic fruit th at has excellent agro industrial potential and represents an appealing economic products rich in nutrients for maintaining health and preventing degenerative diseases (Alve s and others 2008). Recent research showed that in addition to vitamin C, acerola

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86 fruit may be a good source of phytochemicals such as anthocyanins (De Rosso and others 2008; Delva and Goodrich 2010), non anthocyanin phenolic compounds (Vendramini and Trug o 2004; Hanamura and others 2005), and dietary carotenoids (De Rosso and Mercadante 2005; Lima and others 2005). The presence of those compounds is indicative of potential high antioxidant capacity. Using a linoleic acid model system it was shown that acer ola juice exhibited high antioxidant capacity (Righetto and others 2005). It was also demonstrated that acerola extracts have the ability to enhance the antioxidant capacity of soy and alfalfa extracts in a variety of low density lipoprotein oxidation syst em s (Hwang and others 2001). However, not only published data in this area is scarce, but the majority of research reported does no t mention the variety of acerola used; given that the antioxidant capacity is dependent on the variety, it becomes difficult to compare results across laboratories. Food spoilage and food poisoning by microbes represent a serious problem that has not yet been satisfactorily controlled in spite of the powerful preservation techniques available. The antibiotic resistance by patho genic organisms to conventional drugs has preferences for foods that are prepared without preservatives of chemical origin have driven the search for natural surrogates providing s ufficiently long shelf life of foods and a high level of safety with regards to foodborne microorganisms. Previous studies have indicated that medicinal plants are one of the best resources for the isolation and development of new bioactive compounds (Moha n and others 2008). In addition, plant derived preparations have drawn the attention of people worldwide because of their fewer side effects and lesser toxicity in comparison to synthetic drugs (Jain and others

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87 2011). There are a wide range of antimicrobia l compounds occurring naturally and that play a significant role in the defense of different kinds of living organisms (Rauha and others 2000). The phenolic compounds represent a large group of secondary metabolites that are widespread in superior plants. Attention has been paid to their antimicrobial activity, but no impressive proof of their efficacy has been found (Rauha and others 2000). Scientific information about the antimicrobial property of acerola fruit is very scarce. A publication by Motohashi a nd others (2004) reported that hexane and ethyl acetate extracts from acerola fruit showed relatively high antibacterial activity particularly against Gram positive bacteria such as Staphyloccocus epidermidis (ATCC 1228). However, since the experiment was not conducted on purified extracts, the nature of the active compounds is not well known. To evaluate the ability of acerola fruit phenolic extracts to be used as a dietary supplement, it is important to perform its toxicological evaluat ion. The Food and Drug Administration (FDA) recommended a list of toxicological tests to the food industry. Tests that are relying on genetic toxicity such as the bacterial reverse mutation test are among the tests recommended to evaluate all chemicals for their toxicologic al safety. The Ames mutagenicity assay employs different strains of Salmonella typhimurium which were mutated for different sensitivity towards different types of DNA damaging chemical mutagens. In contrast to common Salmonella the abi lity to synthesize biotin from histidine is lacking in these mutants. Because biotin is required for their growth and development the mutant Salmonella strains lose their capacity to grow in an environment where biotin is a limiting factor. However, the gr owth capacity can b e restored in case of reverse mutation which may be caused by exposure to mutagenic

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88 compounds (Maron and Ames 1983). In the Ames test, a glucose minimal agar plate with top agar having a very small amount of histidine is used in order to produce an environ ment deficient in histidin e and biotin. Acerola fruit at incomplete, intermediate and complete stage of maturity contain phenolic compounds. Acerola phenolic extracts have demonstrated interesting biological activity. However, very limited information is a vailable on the toxicological evaluation of the acerola phenolic extracts. This lack of information on the toxicology of the acerola phenolic extracts limits their use as a food supplement. Acerola phenolic extract contain both simple and polyphenol, and p otentially some non identified compounds. It is important to perform the toxicological evaluation of acerola phenolic extracts in order to assess if they are safe to be used as dietary supplement. In a tier approach suggested for general screening, two st rains of S almonella Typhimurium (TA 98 and TA 100) are recommended to be employed in the initial step. Results are usually presented as mean of revertant colonies per plate standard deviation. The mutagenicity was determined by a metho d described in Mort elmans and Zeiger (2000). In this method, the mutagenicity is determined by setting up fold increase as a cut off point. In general, when there is a 2 3 fold increase in the number of colonies from the negative control, the extract is considered mutagenic (Mortelmans and Zeiger 2000). This study had three major objectives. (1). To evaluate the total antioxidant value of acerola fruit, and, given the importance of ascorbic acid and phenolic compounds in this fruit, we assess their contribution in the antioxi dant activity of the fruit. (2). To investigate the antimicrobial potential of phenolic fractions from acerola fruit. To achieve

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89 this goal, three different microbial species were used. Among the species investigated, Staphylococcus aureus is one of the mos t common gram positive bacteria causing food poisoning (Rauha and others 2000). Escherichia coli which are well understood indicator organism s was chosen as a representative of Gram negative bacteria Pseudomonas putida also a gram negative organism was used as a representative of spoilage microorganism s (3). To perform preliminary screening of acerola phenolic fractions (anthocyanins, flavonols and phenolic, and phenolic acids) for mutagenicity based on the Ames mutagenicity test. Materials and Methods Chemicals and Biological Media Folin Ciocalteu phenol reagent was purchased from MP Biochemicals, LLC. 1,1 diphenyl 2 picrylhydrazil (DPPH), Fluorescein (free acid), and 6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid (Trolox) were from Sigma Aldric h. 2 azobis (2 amidinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals. Glucose was obtained from Difco (Houston, TX, USA). Tryptic soy agar (TSA) was acquired from Bacto (Bexton, Dickinson and company, Sparks, MD, USA). Agar power was provided by Fisher Scientific. Donomycine, top agar supplemented with 0.6 % L histidine and D biotin, Vogel Bonner E salts (VB salts 50x) Dubelco sodium phosphate buffer, 0.1 mM, pH 7 .0 was obtained from S igma (Saint Louis, MO, USA). Bacterial Strains To perform the Ames mutagenicity test, cultures from two strains of Salmonella Typhimurium TA 98, and TA 100 were employed. For the antimicrobial activity assay by the disc diffusion method, cultures of E coli (ATCC 25922), Staphylococcus aureus

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90 (ATCC 2924 7), and Pseudomonas putida (ATCC 12633) were used as test organisms. They were all purchased from the American Type Culture Collection (VA, U.S.A.). Extraction and Fractionation of the Phenolic Compounds The extraction and fractionation of the phenolic com pounds were accomplished according to the method by Kim and Lee (2002) as described in C hapter 3 (see materials and methods section of C hapter 3). Total Phenolics Analysis The crude water extracts was diluted to proper strength for analysis. The total phen olic content was determined using the Folin & Ciocalteu assay (Singleton and Rossi 1965) The extracts were mixed with diluted Folin Ciocalteu reagent and 15 % sodium carbonate. Absorption at 765 nm was measured in a microplate reader (S PECTRA max 190 Molec ular Devices, Sunnyvale, CA ) after incubation for 30 min at room temperature. The results were expressed as milligrams gallic acid equivalents per kilogram of fresh weight (mg of GAE/kg) for the fruits or milligram of gallic acid equivalent per liter (mg of GAE/L) for the juice, using a standard curve generated with 100 600 mg of gallic acid per liter. Antioxidant Capacity Assays One of the objectives of this research was to determine the contribution of phenolic compounds to the total antioxidant activity of the acerola fruit. Total antioxidant capacity was determined on the acerola phenolic extracts (crude water extract). After the fractionation of the phenolic compounds, the antioxidant capacity of each fraction was determined by the ORAC and DPPH assays

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91 Oxygen Radical Absorbance Capacity (ORAC) The ORAC assay was conducted according to a modified method of Sandhu and Gu (2010). The assay was conducted on a Spectra XMS Gemini plate reader (Molecular Devices, Sunnyva added to the designated wells of a 96 well black plate. This was followed by the addition o C for 10 min before f the free radical AAPH. The fluorescence was monitored using 485 nm excitation and 530 nm emission at 1 min intervals for 40 min. Trolox was used to generate a standard curve. The antioxidant capacity of the samples was expressed as mmol Trolox equivalent (TE) per kg (mmol of TE/kg) on a fresh weight basis DPPH (2 Diphenyl 1 picrylhydrazyl) Assay The DPPH assay was conducted according to Sandhu and Gu (2010) Fifty L samples was mix ed with 950 L DPPH solution. The mixture was incubated for 60 minutes in the dark. Fifty (50) L Trolox solutions were added to 950 L DPPH solution to generate a standard curve. Fifty (50) L MeOH was mixed with 950 L DPPH working solution and used as a control. After the incubation, 200 L of mixture was pipette d into a 96 well plate and the plate was read in a spectrophotometer at 515 nm. The result was expressed as mmol Trolox equivalent per kilogram fresh weight (mmol TE/kg). Determination of Ascorbic Acid in the Extract The AA analysis was conducted according to the method described in Lee and Coates (1999) with necessary modifications. Briefly, the acerola crude extracts and single strength juice were diluted to proper strength using potassium phosphate monobasic (KH 2 PO 4 ) solution (pH 2.4).The diluted extract was fil tered through a 0.45

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92 m Nylon filter and analyzed by HPLC, The ascorbic acid content in the extracts was determined using a standard calibration curve with concentration of 0 60 g/mL ascorbic acid. The ascorbic acid content was expressed in mg ascorbic ac id per 100 g sample on a fresh weight basis. The HPLC analysis was conducted on a Dionex model P680 liquid chromatograph equipped with a Dionex model AS 100 automated sample injector, and a Dionex model 100 photodiode array (PDA) detector set at 254 nm. A Dionex model Acclaim C 18 column (4.6 mm x 250 mm, 5 m ) operated at ambient temperature was used. A 0.2 M potassium phosphate monobasic (KH 2 PO 4 ) (Merck) in deionized water solution was used as the mobile phase with a flow rate of 1.0 mL/min. The pH of th e mobile phase was adjusted to 2.4 with phosphoric acid (H 3 PO 4 ). Antimicrobial Activity Sample Preparation for Antimicrobial Test Concentrated stock of freeze dried acerola phenolic extracts were reconstituted with sterile deionized water to achieve dilut ed concentrations of 0.25, 2.5, and 25 mg/mL. During preliminary experiments, only selected samples from the concentration of 25 mg/mL showed some types of antimicrobial activity. Therefore, the full scale antimicrobial assay was carried out only on that c oncentration. The Disk Diffusion Test The antimicrobial activity of the acerola phenolic ex tracts was conducted using the Kerby Bauer disk diffusion susceptibility protocol (Hudzicki 2009). One day prior to the inoculum preparation, the microorganisms were sub cultured. Using a sterile inoculating loop, five well defined colonies were touched and suspended in 6 mL of sterile tryptic soy broth (TSB) and incubated until a cell density of 1x10 8 cfu/mL was achieved. The

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93 density was monitored according to the Mc Farland standard (absorbance measured with a spectrophotometer was approximately 0.1 at 625 nm). The suspension was used within 15 min of preparation. A sterile swab was dipped into the inoculum tube; the swab was rotated against the side of the tube to re move excess of liquid. The dry surface of the Mueller Hinton agar (MHA) plate was inoculated by streaking the swab three times over the entire surface of the agar. Twenty (20) L of acerola phenolic extract was impregnated onto sterile 6 mm diameter blank paper discs (final amount of 500 g of phenolic compounds). The discs were allowed to rest in an aseptic hood until complete dryness and placed in triplicate onto the surface of the pre treated agar plates. Prepared antibiotic disks of penicillin and ampic illin were used as positive control while discs impregnated with reagent alcohol were used as negative control. The plates were inverted and incubated at 37 o C for 24 hours for E. coli and S. aureus and at room temperature for 24 hours for P. putida Foll owing the incubation, the inhibition zone sites were measured to the nearest millimeter usin g a ruler, and recorded. The experiment was then repeated at least two times. Interpretation of the Results When a known concentration of an antimicrobial compound is applied on a disc and the disc is placed on the surface of an MHA plate, there is immediate movement of water from the agar to the disc. And subsequently, the antimicrobial compound begins to diffuse into the surrounding agar. The rate of the diffusion of the antimicrobial compound through the agar is dependent on the diffusion and the solubility properties of the antimicrobial compound in the agar and the molecular weight of the compound (Bauer and others 1996) If the agar plate has been inoculated wit h a suspension of the testing microorganism before the disc has been placed on its surface the growth of the

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94 micro organism and the diffusion of the antimicrobial compounds take place simultaneously The growth occurs in the presence of antimicrobial comp ounds when the bacteria reach a critical mass and can overpower the inhibitory effect of the antimicrobial compound ( Hudzicki 2009 ) The diameter of the inhibition zone is a function of concentration, potency and diffusion coefficient. One of the methods u sed to interprete the result is described in Rauha and others (2000) In this method, the inhibition zone (i.z.) of the sample is compared to that of the negative controls (antibiotics). Briefly when i.z. sample < i.z. reagent alcohol + 1 mm, the samples i s considered to exhibit no antimicrobial activity, when i.z. sample is 1 3 mm > i.z. reagent alcohol, the sample is considered to have a slight antimicrobial activity, samples having i.z. between 3 4 mm > i.z. reagent alcohol, the sample is consid ered havi ng moderate antimicrobial properties, when i.z. samples is 4 10 mm > i.z. reagent alcohol, the sample is said to have clear antimicrobial property; finally when sample i.z. > i.z. reagent alcohol + 10 mm, the sample is considered to have strong antimicrobi al property. Ames Mutagenicity Test The Ames mutagenicity test was conducted according to the procedure available in Mortelmans and Zeiger (2000) with necessary modifications. In summary, glucose minimal agar plates were prepared by aseptically adding 50 mL of sterile glucose solution, 20 mL sterile VB salt solution and 930 mL of sterile agar at 65 o C; the mixture was then mixed with a magnetic stirrer. Twenty five (25) mL of the agar medium was poured into each of the 100 x 15 mm petri dishes; all the mani pulations were conducted aseptically. The bacterial cultures were grown on TSA and five (5) well defined colonies were selected and inoculated in 25 mL of TSB broth in the flask. The flasks were placed

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95 in a shaking water bath at 37 o C until a desired cultu re density of 1 2x10 9 colony forming unit s (cfu)/mL was reached after nearly 16 hours. The density of the culture was monitored spectrophometrically at 660 nm and the absorbance at the desired density was between 1.2 1.4. Two (2) mL of sterile agar contain ing 0.6 % histidine and biotin were transferred to aseptic glass tubes and kept in water bath at 48 o C until needed. The Ames test was conducted on the three phenolic fractions of acerola fruit (anthocyanins, flavonols and phenolic acids) at three differe nt concentrations: 0.25, 2.5, and 25 mg/mL. The test was performed by pipetting aseptically 0.5 mL of 0.1 mM sodium phosphate buffer pH 7.4, 20 L of acerola phenolic fraction (corresponding to amounts of 5, 50, and 500 g phenolic compounds per plate), 0. 1 mL of overnight salmonella culture into top agar and vortexed. The mixture was quickly poured and evenly distributed onto the surface of the GM agar mixture. Following the solidification of the surface of the agar, the plates were inverted and incubated at 37 o C for 48 hours. For all the samples, sterile deionized water was chosen as negative control, daunomycin at a concentration of 60 g/mL was used as a positive control for assay with TA 98, and sodium azide at a concentration of 60 g/mL was chosen a s positive control for assay with TA 100; all experiments were conducted at least in duplicate. After an incubation time of 48 hours, the number of colonies were counted and recorded, and the background of each sample dish was also compared to the negativ e control in the absence of the background. Statistical Analysis The effect of maturity on the total phenolic index, antioxidant capacity, and vitamin C content was studied by performing a one way analysis of variance with the Duncan multiple range test co mparing the mean values within each type of extraction (fresh or

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96 freeze dried extraction). The statistical analysis was performed using SAS ( S tatistical Analysis S ystem, SAS Institute Inc. Cary, NC ). The SAS program codes used and the SAS output are prese nted in Appendix C (Tables C 1 and C 2). Total phenolics, DPPH, ORAC, and v itamin C values are expressed as means plus or minus the standard deviation. The mutagenic dose response of acerola phenolic fractions to the S. typhimurium strains are also express ed as means plus or minus the standard deviation. Results and Discussion Total Phenolics, Total Antioxidant Capacity and Vitamin C Content As shown in Table 4 1, all the acerola samples analyzed, no matter the treatments (stage of maturity, conditions of e xtraction, edible or non edible portion of the fruits) show higher total phenolic values than other tropical fruits such as mango (16.4 mgkg 1 ), pineapple (13.4 mgkg 1 ) (Gorinstein and others 1999); and other food products like virgin olive oil (3000 mgkg 1 ) (Gallina Toschi and others 2005), and honey (3500 mg kg 1 ) (Gheldof and others 2002). For the fruits grown in Davie, the edible portions from the immature fruits show higher total phenolic values than the edible fractions obtained from the mature fruits (Table 4 1). The total phenolic value decreases from the immature stage to the intermediate stage and increased again as the fruits reach ed the full maturity stage. The same trend is also observed for the fruits cultivated in Vero Beach. Righetto and othe rs (2005) reported a decrease in total phenolic content of acerola juice from the immature to the mature stages. Extracts obtained from the freeze dried powder showed a higher total phenolic index. This is because the powdered plant material maximizes poly phenolic extraction due to its high surface contact area with solvent and easy destruction of biological cell walls (Kim and Lee 2002) The total phenolics were also determined in the non edible fraction of the fruits; only seeds from mature fruits

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97 were co nsidered and only the freeze dried extraction was performed. Overall, the total phenolic content of the seeds is higher than the phenolic content of the edible portion of the fruits. On a fresh weight basis, the seed from the fruits grown in Vero Beach sho wed a higher total phenolics value (18155 mg GAE kg 1 ) than all the other edible samples. It is also important to mention that acerola juice purchased from a supplier shows higher total phenolics value even higher than some of the edible portions of the fr uit. The antioxidant capacity was performed by ORAC and DPPH assays, the results are gathered in Table 4 1. For the edible portion of the fruits and regardless of the condition of extraction, the ORAC values vary from 79 43.5 mM TE kg 1 62 36 mM TE kg 1 53 36 mM TE kg 1 respectively for immature intermediate and mature stage of ripeness ORAC values of acerola samples were higher than values reported in the literature for cauliflower (17.7 mM), strawberry (15.4 mM) and spinach (12.6 mM) (Cao and others1 996). The DPPH values vary from 251 95 mM TE kg 1 142 54.4 mM TE kg 1 53 36 mM TE kg 1 respectively for immature intermediate and mature stage of ripeness these values are higher than the DPPH values reported for wine, green tea infusion, and pomegranate (Fogliano and others 1999; Prior and Cao 2000; Gil and others 2000). The results also show that regardless of the extraction technique applied and the stage of maturity, fruits grown in Vero Beach show higher antioxidant capacity (expressed by ORAC or DPP H) than those grown in Davie. As it was observed for the total phenolic index, freeze dried samples show higher DPPH scavenging capacity than fresh extraction. The DPPH value of the samples decreased as the fruit goes from the immature to complete maturity At complete maturity, the seeds from the fruits grown in Vero Beach show a much higher DPPH scavenging capacity nearly 148 (mmol kg 1 FW)

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98 than other samples. The ORAC value follows a similar trend; it is decreasing as the fruits ripen. At complete maturi ty, the non edible fractions from the fruits grown in Vero Beach exhibited much higher ORAC value (85 mmol TE kg 1 FW) than the other samples analyzed. Table 4 1 shows AA content of the different samples analyzed; the AA analysis was carried out only in t he edible fractions of the fruits. The vitamin C content ranged from 1161 1744 g/100g, 970 1049 g/100g, and 405 987 g/100g respectively for immature intermediate and mature stages These values are slightly lower than those reported by Vendramini and Tru go ( 2000 ) but higher that those reported by Mezadri and others ( 2008). These results are however much higher than the AA contents reported for other fruits or fruit juices such as orange juice (0.516 g L 1 ), grapefruit juice (0.274 g L 1 ) or lemon juice ( 0.327 g L 1 ) (Ashoor and others 1984). The fruits grown in Vero Beach exhibited lower vitamin C content than those grown in Davie. For a given growing location, the AA content decreases as the fruit ripens. As for the fruits grown in Davie, the AA content decreases from 1744 mg/kg FW at the immature stage (green), to 1049 mg/kg FW at the intermediate stage, and reached 987 mg/kg FW (nearly 43 % decrease) at complete maturity. Vendramini and Trugo (2000) reported a 50 % reduction of AA from the green to the red fruits and explained the loss of AA by biochemical oxidation. Contribution of Phenolic Compounds and AA to the Antioxidant Capacity of Acerola Fruit One of the main objectives of this study was to investigate the contribution of different phenolic fra ctions, and AA to the antioxidant capacity of acerola fruit. The procedure includes the determination of the total antioxidant of the crude extract before

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99 fractionation, the determination of ascorbic acid, and the fractionation of the crude extract into an thocyanin s (F1), flavonoids (F2), and phenolic acid s (F3) as described in earlier sections. Following the fractionation of the phenolic compounds, the antioxidant capacity of each fraction was determined and the contribution of each fraction to the total a ntioxidant capacity was calculated. The contribution of AA to the total antioxidant capacity of the samples was also accomplished by performing the antioxidant activity of AA solutions having similar concentrations to the analyzed samples. Table 4 2 shows that the antioxidant capacity of the phenolic fractions are in the following order: anthocyanin s
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100 a good source of (De Rosso and Mercadante 2005; Lima and others 2005) and also a novel class of flavonoid compound s named aceronidin recently isolated in acerola fruit and whose DPPH sca tocopherol (Kawaguchi and others 2007). Antimicrobial Properties The antimicrobial properties of the phenolic extract were evaluated by the disc diffusion method. Tables 4 3, 4 4, and 4 5 show the inhi bition by the phenolic fractions. The entire activities correspondent to sample amount of 500 g. The phenolic extracts in the concentration tested showed limited antimicrobial activity against the bacterial strains tested. The anthocyanin fractions (F1) ( Table 4 3) did not demonstrate antimicrobial activity In contrast, two extracts from the flavonoids fraction (F2) show moderate or clear antimicrobial properties. For instance, freeze dried edible portion of mature fruits from Davie showed moderate antimi crobial activity while freeze dried edible portions of immature and freeze dried non edible portion of the fruit from Davie both showed clear antimicrobial activity against the strain of S. aureus used in this experiment (Table 4 4). For the phenolic acid fraction (F3), only the non edible portion of the fruits shows some moderate activity against S. aureus (Table 4 4). Overall the flavonoids show more activity especially against S. aureus than the other phenolic fractions. The main flavonoid in acerola fru it has been identified as quercetin 3 O rhamnoside (Hanamura and others 2005); and various quercetin glucosides have been identified as the active antimicrobial compounds in plant extracts especially against S aureus (Rhamaswamy and others 1972; Khanna and others 1980; Rauha and others 2000). It is also important to point out that all the fractions (F1, F2, and F3), did not demonstrate activity against E. coli and P. putida strains tested in this experiment. T his

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101 is not different from the results reporte d by Motohashi and others ( 2004) who reported good antioxidant properties of acerola extracts against bacteria such as: S. epidermidis (ATCC 12228), but almost no activity against Gram negative bacteria such as E. coli (ATCC 25925) and P. aeruginosa (ATCC 27853). Overall, under the conditions that this experiment was conducted, the flavonoids fraction of the fruits shows relatively good antioxidant activity against S. aureus It is important to mention that the antimicrobial test was performed using a 6 mm disc that has a very limited loading capacity. After the application of 25 30 L liquid sample corresponding to 500 g of active compound, the disc apparently reached its limit The investigation of the antimicrobial properties of acerola phenolic extracts using other method s is therefore necessary in order to generate more information on the antimicrobial potential of acerola phenolic extracts. Ames Mutagenicity Assay Acerola phenolic extracts were screened for mutagenicity. Three (3) types of phenolic fr actions were studied: anthocyanin s phenolic acids and the flavonols fractions. The Ames mutagenicity test was conducted on the two mutant strains of Salmonella typhimurium TA 98 and TA 100. The results, expressed in terms of the number of revertant coloni es grown on the glucose minimum agar with histidin e limited top agar is shown in Table 4 6, Table 4 7, and Table 4 8 respectively for anthocyanin, flavonol and phenolic acid fractions. Each phenolic fraction was tested at 3 three different concentrations: 5, 50, and 500 g per plate. For the three types of phenolic fractions tested, regardless of the conditions: type of extraction, stage of maturity, the number of revertant colonies at the tested concentrations was lower than that of the negative control. O nly a few samples present a number of revertant colonies higher

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102 than that of the negative control, one example of such samples is the anthocyanin fraction from the fresh edible portion of acerola fruit at intermediate stage of maturity on the S. typhimuriu m strain T100. More importantly, in all the samples, the number of revertant colonies were not at least two fold higher than the negative control, suggesting that the phenolic fractions in acerola fruit, regardless of their nature did not contribute to mut agenicity Because the number of revertant colonies was not at least 2 fold higher than that of the negative control, it can be concluded that the phenolic compound fractions are not mutagenic. The toxicological evaluation of acerola phenolic extract has ne ver been conducted using the Ames mutagenic test. The concentration (5, 50. 500 g per plate) corresponds to the chronic, subchronic and the acute levels used by Hanamura and Aoki (2008). The results reported are similar to those reported by Hanamura and A oki (2008) who showed no toxic effect for acerola extract a concentration as high as 2000 ppm in rats. Summary Acerola fruits exhibit high total phenolics value with significant antioxidant capacity expressed by both the ORAC and DPPH methods. Ascorbic ac id accounted for higher contribution in the overall antioxidant capacity of the fruit, but still much lower than expected considering the high ascorbic acid content of the fruit. Other antioxidant compounds such as carotenoids and newly isolated flavonoid s may also contribute in the overall antioxidant capacity of the fruit. Overall this study demonstrates the antioxidant potential of the fruit, but more research needs to be conducted in order to better understand the contribution of compounds other than AA and phenolic compounds in the antioxidant capacity of the fruit. The results also show the

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103 antimicrobial potential of the flavonoids fraction of the fruit particularly against S. aureus. However, further research using different assays is needed for a tho rough assessment of the antimicrobial potential of the acerola phenolic extracts. The results show that no matter the method of extraction (freeze dried or fresh) and the stage of maturity (green, red, or intermediate) the phenolic fractions did not contri bute to mutagenicity.

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104 Table 4 1. Total phenolic index, total antioxidant value and vitamin C content of acerola sample Sample TPI ORAC ** DPPH *** Vit. C **** Fruits grown in Davie Fresh extraction ED G 9403 a 43.5 a 0.02 95.0 a 0.30 1744 a ED O 7944 c 36.5 b 0.10 54.7 b 0.15 1049 b ED R 8340 b 36.2 b 0.05 40.7 c 0.35 987 c Freeze dried extraction ED G 16285 a 48.0 a 0.04 136 .0 a 1161 a ED O 12915 b 39.7 b 0.07 106 .0 b 970 b ED R 18155 c 40.0 b 0.02 74 .0 c 405 c NED 18155 85.0 0.50 1 47 .0 NQ FSSJ 12562 85.0 0.02 30 .0 921 Fruits grown in Vero Beach Fresh extraction ED G 11317 a 79.0 a 0.30 251 .0 a 1238 a ED O 10191 b 62.0 b 142 .0 b 894 b ED R 10283 c 53.0 c 0.04 101 .0 c 470 c ED G: edible portion green; ED O: edible portion orange red; ED R: edible portion red; NED: non edible portion (seed); FSSJ: frozen single strength juice. TPI: total phenolic index in mg GAE/kg; ** ORAC value in mmol TE/kg, *** DPPH value expressed in mmol TE/kg; **** Vitamin C content in mg per 100g. NQ: not quantified. Within each type of extraction, values in a column followed by different letter s are All results are expressed on fresh weight basis.

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105 Table 4 2. Contribution of phenolic fractions and AA in the total antioxidant value expressed by ORAC Sample F1 F2 F3 Sum (F1+F2+F3) ORAC AA Tot. ORAC a sample % Contribution P henolics % Contribution AA Fruit grown in Davie Fresh extraction ED G N/A 4.20 1.18 5.38 17. 1 43.5 12.4 39.2 ED O 1.64 2.17 0.44 4.25 12.8 36.5 11.6 35.0 ED R 1.77 2.63 0.94 5.34 13. 8 36.2 7.10 38. 1 Freeze dried extraction ED G N/A 8.90 1.60 10.5 12. 2 48.1 21.8 25.3 ED O 6.05 6.88 1.58 14.5 13.6 39.7 36.5 34. 3 ED R 6.03 6.28 2.30 14.6 14.9 40.0 36.5 37. 4 NED 3.37 6.11 1.05 10.5 N/A 85.0 12.3 NQ FSSJ 4.19 4.90 0.09 9.20 17.9 85.2 10.8 21.1 Fruit grown in Vero Beach Fresh extraction ED G N/A 5.38 0.52 5.90 14. 3 79.3 7.44 18.1 ED O 2.26 6.03 0.54 8.83 14.2 61.2 14.4 23.2 ED R 1.63 2.76 0.65 5.04 12.2 53.0 9.50 23.0 F1: anthocyanin, F2: flavonols, F3: phenolic acids. ED G: edible portion green; ED O: edible portion orange red; ED R: edible portion red; NED: non edible portion (seed); FSSJ: frozen single strength juice. a total ORAC value in mmol TE/kg on a fresh weight basis. NQ: not quantified.

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106 Table 4 3. Antimicrobial effect of anthocyanin fractions from acerola fruit; sample amount 500 g (n=2) Anthocyanin fraction Staphylococcus aureus 29247 E coli 25922 Pseudomonas putida ATCC 12633 Fruit grown in Davie Fresh Extraction ED R ED O Freeze dried extraction E D R ED O Non edible freeze dried NED Fruit grown in Vero Beach Fresh extraction ED R ED O FSSJ Reference/activity Ampicillin + Ampicillin +++ Penicillin : No antimicrobial activity, inhibition zone (i.z) of sample < i.z. reagent alcohol plus 1 mm; +: moderate antimicrobial activity, i.z. of sample 3 4 mm > i.z. reagent alcohol; ++: clear antimicrobial activity, i.z of sample 4 10 mm > i.z. reagent alcohol; +++: strong antimicrobial activity, i.z of sample > i.z. of distilled water plus 10 mm. ED G: edible portion green; ED O: edible portion orange red; ED R: edible portion red; NED: non edible portion (seed); FSSJ: frozen single strength juice.

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107 Table 4 4. Antimicrobial effect of flavonoids fractions from acerola fruit; sample amount 500 g (n=2) Flavonoid fractions Staphylococcus aureus 29247 E. coli 25922 Pseudomonas putida ATCC 12633 Fruits grown in Davie Fresh extraction ED R ED O ED G Freeze dried extraction ED R + ED O ED G ++ Non edible freeze dried NED R (seed) ++ Fruits grown in Vero Beach Fresh extraction ED R ED O ED G FSSJ Reference/activity Ampicillin + Ampicillin +++ Penicillin : No antimicrobial activity inhibition zone (i.z) of sample < i.z. reagent alcohol plus 1 mm; +: moderate antimicrobial activity, i.z. of sample 3 4 mm > i.z. reagent alcohol; ++: clear antimicrobial activity, i.z of sample 4 10 mm > i.z. reagent alcohol; +++: strong antimicrobial activity, i.z of sample > i.z. of distilled water plus10 mm. ED G: edible portion green; ED O: edible portion orange red; ED R: edible portion red; NED: non edible portion (seed); FSSJ: frozen single strength juice.

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108 Table 4 5. Antimicrobial effect of phe nolic acid fractions from acerola fruit; sample amount 500 g (n=2) phenolic acid fractions Staphylococcus aureus 29247 E. coli 25922 Pseudomonas putida ATCC 12633 Fruits grown on Davie Fresh extraction ED R ED O ED G Freeze dried e xtraction ED R ED O ED G Non edible freeze dried NED + Fruit grown in Vero Beach Fresh extraction ED R ED O ED G FSSJ Reference/activity Ampicillin + Ampicillin +++ Penicillin : No antimic robial activity, inhibition zone (i.z) of sample < i.z. reagent alcohol plus 1 mm; +: moderate antimicrobial activity, i.z. of sample 3 4 mm > i.z. reagent alcohol; ++: clear antimicrobial activity, i.z of sample 4 10 mm > i.z. reagent alcohol; +++: strong antimicrobial activity, i.z of sample > i.z. of distilled water plus10 mm. ED G: edible portion green; ED O: edible portion orange red; ED R: edible portion red; NED: non edible portion (seed); FSSJ: frozen single strength juice.

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109 Table 4 6. Mutagenic do se response of acerola anthocyanin fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) Number of colonies (CFU/Plate TA 98 TA 100 Dose level (g/plate) Fruits grown in Davie Fresh extrac tion Sterile DI water (NC) 35.0 8. 0 0 15.0 4.0 0 Daunomycine (60 g/plate) 400 20.0 Sodium azide (PC) 998 125 5 g ED R 26.0 3.0 0 10.0 4.5 0 5 g ED O 21.5 5.1 0 12.0 2.0 0 25 g ED R 15.0 5.0 0 2.7 0 3.0 0 25 g ED O 11.0 2.7 0 4 .7 0 2.5 0 50 g ED R 5.0 0 0.0 0 8.0 0 3.0 0 50 g ED O 7.5 0 2.0 0 6.0 0 1.0 0 500 g ED R 2.0 0 1.2 0 0.0 0 0.0 0 500 g ED O 3.1 0 0.0 0 0.0 0 0.0 0 Freeze dried extraction 5 g ED R 21.0 7.0 0 16.4 4.0 0 5 g ED O 17.0 3.0 0 12.2 6.0 0 2 5 g ED R 17.0 7.0 0 11.0 3.0 0 25 g ED O 12.4 5.0 0 8.4 0 2.0 0 50 g ED R 11.0 7.0 0 2.0 0 0.0 0 50 g ED O 13.1 4.0 0 19.0 3.0 500 g ED R 6.7 0 2.0 0 4.5 0 1.5 0 500 g ED O 9.5 0 5.0 0 7.0 0 0.0 0 Non edible freeze dried 5 g NED 27.0 8.2 0 16.0 4.0 0 50 g NED 12.5 3.0 0 8.2 0 1.5 0 500 g NED 4.0 0 0.0 0 4.0 0 2.5 0 Fruit grown in Vero Beach Fresh extraction 5 g ED R 17.0 6.5 0 11.5 7.1 0 5 g ED O 13.0 2.5 0 21.0 9.0 0 5 g FSSJ 15.5 0.0 0 10.0 0.0 0 50 g ED R 7 .0 0 2.0 0 9.1 0 5.0 0 50 g ED O 10.3 6.5 0 5.7 0 2.0 0 50 g FSSJ 13.5 2.5 0 11.0 1.0 0 500 g ED R 5.0 0 3.5 0 0.0 0 0.0 0 500 g ED O 8.3 0 1.0 0 7.0 0 1.0 0 500 g FSSJ 10.2 2.0 0 6.5 0 4.5 0 Values are presented as mean SD of at least two experiments. Two fold or more of number of revertant colonies is an indicator of mutagenicity; NC: negative control; PC: positive control.

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110 Table 4 7. Mutagenic dose response of acerola flavonols fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) Number of colonies (CFU/Plate ) TA 98 TA 100 Dose level (g/plate) Fruits grown in Davie Fresh extraction Without metabolic activation Sterile deionized water (NC) 35.0 6 .00 10.0 Dauno mycine (60 g/plate; PC) 1500 Sodium azide (60 g/plate; PC) 128 15.0 5 g ED R 31.5 6.0 0 18.5 2.0 0 5 g ED O 28.5 7.0 0 16.5 5.0 0 5 g ED G 27.0 1.0 0 16.0 1.0 0 25 g ED R 24.5 4.5 0 12.5 4.5 0 25 g ED O 18.7 1.0 0 12.7 1.0 0 2 5 g ED G 20.0 8.0 0 12.0 2.0 0 50 g ED R 15.4 2.0 0 9.5 0 1.0 0 50 g ED O 15.7 7.5 0 11.0 5.0 0 50 g ED G 17.0 4.5 0 7.0 0 4.5 0 500 g ED R 11.5 2.5 0 4.7 0 2.0 0 500 g ED O 8.0 0 1.0 0 3.0 0 1.0 0 500 g ED G 8.3 0 2.4 0 2.7 0 2.0 0 Fr eeze dried extraction 5 g ED R 29.5 4.0 0 18.5 6.0 0 5 g ED O 30.5 7.0 0 19.5 4.6 0 5 g ED G 37.0 2.0 0 19.0 1.0 0 25 g ED R 25.0 11.0 14.5 5.4 0 25 g ED O 27.0 2.5 0 13.3 3.0 0 25 g ED G 23.0 10.0 14.0 5.0 0 50 g ED R 14.4 2.0 0 11.5 2.0 0 50 g ED O 16.7 5.7 0 13.0 2.0 0 50 g ED G 17.0 1.5 0 7.0 0 4.5 0 500 g ED R 9.5 0 3.0 0 5.7 0 2.0 0 500 g ED O 13.0 0.0 0 5.6 0 0.0 0 500 g ED G 10.3 0.0 0 3.7 0 1.0 0 Non edible freeze dried 5 g NED 25.0 0.0 0 18.5 6.0 0 50 g NED 16.8 2.5 0 19.5 4.6 0 500 NED 11.7 4.0 0 19.5 4.6 0

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111 Table 4 7. Continued. Number of colonies (CFU/Plate ) TA 98 TA 100 Fruit grown in Vero Beach Fresh extr action 5 g ED R 24.5 3.0 0 15.0 4.0 0 5 g ED O 27.5 6.0 0 16. 5 3.6 0 5 g ED G 30.0 3.0 0 14.0 2.0 0 5 g FSSJ 23.2 5.1 0 13.1 0 1.0 0 50 g ED R 20.0 6.0 0 8.3 0 2.0 0 50 g ED O 14.0 2.0 0 9.10 2.0 0 50 g ED G 19.7 5.0 0 8.5 0 2.0 0 50 g FSSJ 17.0 5.0 0 13.0 2.0 0 500 g ED R 10.7 0.0 0 7.0 0 2.5 0 500 g ED O 12.5 5.0 0 4.7 0 1.0 0 500 g ED G 10.0 0.0 0 3.6 0 0.0 0 500 g FSSJ 9.7 0 0.0 0 2.9 0 2.0 0 Values are presented as mean SD of at least two experiments. Two fold or more of number of revertant colonies is an indicator of mutageni city; NC: negative control; PC: positive control

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112 Table 4 8. Mutagenic dose response of acerola phenolic acid fraction to S. Typhimurium (TA98 and TA100) as represented by mean number of revertant colonies (CFU/plate) (n=2) Number of colonies (CFU/Plate TA 98 TA 100 Dose level (g/plate) Fruits grown in Davie Fresh extraction Without metabolic activation Sterile deionized water (NC) 28.0 6.0 0 10.0 2.0 0 Daunomycine (60g/plate; PC) 300 15.0 Sodium azide (60 g/plate; PC) 1150 5 g ED R 30.0 0.0 0 12.4 0.0 0 5 g ED O 27.7 7.1 0 11.1 2.5 0 5 g ED G 27.0 5.0 0 10.0 0.0 0 25 g ED R 17.5 2.6 0 6.7 0 2.1 0 25 g ED O 16.2 1.0 0 6.0 0 2.0 0 25 g ED G 18.7 2.6 0 6.0 0 2.0 0 50 g ED R 12.7 1.0 0 5.0 0 1.0 0 50 g ED O 1 4.4 0.0 0 4.5 0 0.0 0 50 g ED G 13.6 2.5 0 4.7 0 3.2 0 500 g ED R 2.5 0 1.7 0 2.0 0 1.6 0 500 g ED O 3.7 0 0.0 0 2.7 0 2.3 0 500 g ED G 4.7 0 4.5 0 2.0 0 1.0 0 Freeze dried extraction 5 g ED R 35.0 1.4 0 15.0 1.0 0 5 g ED O 40.5 3.2 0 1 7.5 2.1 0 5 g ED G 37.1 2.2 0 16.4 4.2 0 25 g ED R 21.4 3.5 0 11.5 5.3 0 25 g ED O 24.7 4.0 0 9.7 0 0.0 0 25 g ED G 26.8 1.0 0 10.6 0.0 0 50 g ED R 19.1 1.2 0 7.4 0 0.0 0 50 g ED O 18.2 4.0 0 6.8 0 1.0 0 50 g ED G 16.7 2.0 0 7.0 0 3.2 0 500 g ED R 7.8 0 1.0 0 2.7 0 0.0 0 500 g ED O 8.9 0 0.0 0 2.0 0 0.0 0 500 g ED G 7.2 0 0.0 0 3.0 0 1.0 0 Non edible freeze dried 5 g NED 32.7 1.0 0 18.2 2.1 0 50 g NED 25.0 4.5 0 10.8 2.3 0 500 g NED 15.2 0.0 0 5.0 0 3.1 0

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113 Ta ble 4 8. Continued. Number of colonies (CFU/Plate TA 98 TA 100 Fruit grown in Vero Beach Fresh extr action 5 g ED R 20.0 5.1 0 14.8 4.8 0 5 g ED O 24.4 0.0 0 12.7 1.7 0 5 g ED G 24.2 2.0 0 13.5 2.5 0 5 g FSSJ 19.5 4.8 0 13.7 2.1 0 50 g ED R 15.6 2.6 0 8.9 0 2.0 0 50 g ED O 15.0 0.0 0 7.9 0 0.0 0 50 g ED G 18.7 4.8 0 9.1 0 4.0 0 50 FSSJ 14.0 2.7 0 8.7 0 1.7 0 500 g ED R 7.2 0 1.2 0 2.1 0 0.0 0 500 g ED O 6.7 0 0.0 0 4.3 0 1.3 0 500 g ED G 5.0 0 1.0 0 4.1 0 0.0 0 500 FS SJ 4.6 0 0.0 0 3.8 0 1.2 0 Values are presented as mean SD of at least two experiments. Two fold or more of number of revertant colonies is an indicator of mutagenicity; NC: negative control; PC: positive control.

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114 CHAPTER 5 EFFECT OF DIFFERENT ASCORBIC ACID CONCENTRATIONS ON THE COLOR STABILITY OF ANTHOCY ANIN EXTRACTS FROM A CEROLA ( MALPIGHIA EMARGINATA DC) FRUITS Overview Acerola tree belongs to the Malpighiaceae family. This tree gives fruit that has a smooth and thin skin, a soft pulp, and an excepti onal bright red color at complete maturity. The attractive red color of acerola is due to the presence of anthocyanin pigment which we identified in the variety Florida Sweet used in this study as cyanidin 3 rhamnoside, the major kind; and pelargonidin 3 r hamnoside, the minor type. One of the problems the acerola growers are facing is the high perishability of this fruit at complete maturity. Shortly after harvest (3 4 days) the fruit losses its attractive red color and turns to a dull yellowish color that is often seen by the consumer as index of poor quality, therefore limiting the market potential of the fruit. The low stability of acerola anthocyanins is also a problem during processing and storage of the acerola juice. Preventing the degradation of anth ocyanin can therefore be beneficial to both the acerola growers, processors and ultimately the consumers of acerola juice or related products. During processing and storage, food products that contain anthocyanin are prone to color degradation occurring a s the result of the conjoined effect of anthocyanin degradation and the formation of brown pigment (Abers and Wrolstad 1979 ; Skrede and others 1983) The type of anthocyanins of fruit is dependent on the variety (Timberlake and Briddle 1982), and according to Markakis (1982) the type of anthocyanin may affect the resistance to color change. The stability of anthocyanin in foods maybe affe cted by

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115 several factors including the chemical structure of the pigment; for instance diglucosidic substitution is known to impart more stability to the molecule than monoglucosidic (Markakis 1982 ; Mazza and Miniati 1993) Other elements in the composition of the fruit may also affect the stability of anthocyanin pigment such as phenolic compounds and ascorbic acid (Timberlake and Bridle 1982) Acerola fruit when compared with other fruits is relatively low in anthocyanin pigment, but very high in ascorbic acid. It has been proven that when anthocyanin and ascorbic acid are present in the same system, depending on the conditions, one may degrade the other. It is suggested that the high vitamin C content of acerola fruit maybe the cause of the red color instability in this fruit. The mechanism of degradation of anthocyanin by ascorbic acid has been investigated. However, the results are su bjected to debate and up to now a mechanism has yet to be found. Two theories exist: (1) When ascorbic acid is oxidized in the presence of copper, hydrogen peroxide (H 2 O 2 ) is produced; and since H 2 O 2 is an anthocyanin bleacher, it is believed that the asco rbic acid induced anthocyanin degradation is mediated by H 2 O 2 (Markakis 1982) Jurd (1972) speculated that involving direct c ondensation of the ascorbic acid to the position 4 on the flavylium the objective is to study the color stability of acerola anthocyanin in the presence of ascorbic ac id while additionally further explaining the degradation kinetics of anthocyanin in the presence of ascorbic acid in a model system and the mechanism by which ascorbic acid may degrade anthocyanin.

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116 To understand the kinetic s of anthocyanin degradation, an thocyanins were extracted from acerola fruits, and the stability of those extracts were monitored over time and compared with an aai anthocyanin model system in which ascorbic acid was added to levels that match the ascorbic acid contents in acerola antho cyanin extracts; this model was proposed by De Rosso and others (2007). Materials and Methods To understand the kinetic s of anthocyanin degradation, anthocyanins were extracted from acerola fruits, and the stability of those extracts were monitored over t ime and compared with an aai anthocyanin model system in which ascorbic acid was added to levels that match the ascorbic acid contents in acerola anthocyanin extracts; this model was proposed by De Rosso and others (2007). Aai is a good model because lik e acerola, it contains monoglucosylated anthocyanins. Pure anthocyanin model systems with added ascorbic acid were also developed and the possible formation of anthocyanin breakdown products was monitored by spectrophotometry. Acerola Fruit and Aai Puree Acerola fruits from the variety Florida Sweet (FSW) were harvested from different in Vero Beach (VE), Central Florida. The fruits were manually harvested and transpor ted to the Food Science and Human Nutrition Department at the University of Florida. Upon arrival, the fruits were washed with clean water and separated into edible portion (pulp+ skin) containing the anthocyanins and non edible portion containing the seed s were discarded. The edible portions of the fruits were stored in a freezer at 20 o C until needed for analysis. Frozen aai puree was donated by ITI Tropicals (Lawrenceville, NJ). The puree was stored in a freezer at 20 o C for later use.

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117 Preparation of the Anthocyanin Extracts Edible portions of fruits, 353 g, and 362 g respectively for fruit collected in Davie (Ace DA) and Vero Beach (Ace VE) and aai puree 344 g were blended with 500 mL 0.1 % HCl in methanol and allowed to stay overnight in a refrige rator. The mixtures were strained and centrifuged ( 4000 g 4 o C, and 10 min) to obtain an extract free of sediment. The extract was then concentrated in a Buchi rotary evaporator at low temperature (~30 o C) to evaporate the methanol; the concentrated extra ct was stored at 20 o C until needed. Development of the Model Systems The anthocyanin model systems were developed in citrate phosphate buffer, pH 2.5. The buffered crude acerola extracts had AA contents of 288 mg/100 mL and 97 mg/100 mL for Ace DA, and Ace VE samples respectively. Therefore, 288 mg AA/100 mL was added to the aai extract to match the AA level in Ace DA samples. Similarly, 97 mg AA/100 mL was added to aai extract to simulate the AA content in Ace VE samples. In another separate treatment half of the AA content of each type of acerola (144 mg in the case of Ace DA or 48 mg in the case of Ace VE) was added to the aai extracts. A similar model system was used by De Rosso and Mercadante ( 2007) The crude anthocyanin extracts were diluted and scanned from 400 and 700 nm to obtain the wavelength of the maximum absorption. The wavelengths of maximum absorption were 505, and 517 nm for diluted acerola and aai solutions respectively. All absorbance readi ngs were made against the dilution buffer as a blank. Spectrophotometric measurements were conducted using a DU 730 Life Science UV Vis spectrophotometer (B e ckman Coulter ). The solutions were distributed in glass

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118 tubes in 20 mL increments and stored unde r two different conditions (light or dark at 20 o C 1). The major anthocyanin in the acerola variety used in this experiment is cyanidin 3 O rhamnoside. Therefore, a model system was developed with this particular type of anthocyanin. Cyanidin 3 O rhamno %), and free %) were purchased from Extrasynthse Genay Cedex, ( Lyon, France ) and stored at 15 o C until needed. Anthocyanin solutions having absorbance values ca 1.5 were prepared in citrate phosphate buffer (pH 2.5) with 0.1 % sodium benzoate, using cyanidin 3 O rhamnoside (1.19 mg/L) and cyanidin (1.45 mg/L) with initial absorbance value of 1.0. To a portion of each solution, ascorbic was added to give final concentration of 330 mg/ L (Garcia Viguera and Bridle 1999). The anthocyanin solutions were scanned from 400 700 nm to determine the wavelength of maximum absorption which was 520 nm for both cyanidin and cyanidin 3 O rhamnoside. Reaction mixtures (20 mL) were placed in tubes in 2 0 mL increment and stored in darkness at 20 o C. Stability and Visual Color Attributes of the Anthocyanin Extracts The stability of the anthocyanins in the different systems was monitored periodically by spectrophotometry, measuring changes in absorption a t maxi mum time absorbance value was considered as the initial absorbance. The anthocyanin retention for each time period was calculated as a percentage of the zero time time absorbance readings taken at 100 % retention (zkan 2002) Absorbance readings at 700 nm were recorded to correct for turbidity. Browning index, a reading of the changes in browning compounds was determined as follows:

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119 ABS 420 ABS 700 )/(ABS max ABS 700 ) (Reyes and Cisneros Zevallo s 2007). Changes in the color of the anthocyanin solutions were determined using the CIELAB system, using the Color Quest XE colorimeter (Hunter Lab., Reston, United States) equipped with light source D65 and observation angle of 10 o Color parameters ligh tness (L ), red ness (a ) and yellow ness (b ) were read. Other parameters such as chroma value ( C = [(a ) 2 + (b ) 2 ] 1/2 ) and hue angle ( h= arctan (b /a )) were calculated. These parameters were calculated because L a b coordinates d o not directly express hue and chroma and are difficult to translate independently (Reyes and Cisneros Zevallos 2007). Determination of Ascorbic Acid The AA analysis was conducted according to the method described in Lee and Coates (1999) with necessary mo difications. Briefly, the samples were diluted to proper strength using potassium phosphate monobasic (KH 2 PO 4 ) solution (pH 2.4).The diluted samples was filtered through a 0.45 m Nylon filter and analyzed by HPLC, the ascorbic acid content was determined using a standard calibration curve with concentration of 0 60 g/mL ascorbic acid. The HPLC analysis was conducted on a Dio n ex model P680 liquid chromatograph equipped with a Dio n ex model AS 100 automated sample injector, and a Diomex model 100 photodiode array (PDA) detector set at 254 nm. A Dio n ex model Acclaim (4.6 mm x 250 mm 5 m ) C 18 column operated at ambient temperature was used. A 0.2 M potassium phosphate monobasic (KH 2 PO 4 ) (Merck) in deionized water solution was used as the mobile phase with a flow rate of 1.0 mL/min. The pH of the mobile phase was adjusted to 2.4 with phosphoric acid (H 3 PO 4 ).

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120 Kinetics Calculations The degradation kinetics for anthocyanins assuming first order could be modeled using the E quation 5 1 log [A] t = 2.303 kt + lo g [A] 0 ( 5 1) But since the disappearance of the anthocyanin over time was monitored by UV visible spectrophotometry, the concentration was replaced by the absorbance (A) in the E quation 5 1 We assumed that both anthocyanin and potential anthocyanin degradation product absorb at the monitored wavelength, therefore the final absorbance (absorbance read at the final storage time) is non zero. Under these conditions, the E quation 5 2 presented in Billo (2001) was used ln|A t A inf | = kt + ln|A i A inf | ( 5 2) Where A i is the initial absorbance reading and A inf is the absorbance value when the reaction is a ssumed complete. The first order behavior was verified by the straight line fit of the data shown in Fig ure 5 1 Results and Discussion Effect of Ascorbic Acid on the Stability of the Anthocyanin Extracts Among all the samples studied in this experiment, t he degradation of anthocyanins followed a first order kinetics. Aai extracts showed a greater stability than did the acerola samples. Aai samples in which 288 mg AA was added showed greater stability than Ace VE samples (containing lower AA contents), bu t similar stability with the Ace DA samples (containing higher AA acid contents). It is important to not e that for both the dark and light samples near the end of storage, the residual anthocyanins in the Ace DA sample became unexpectedly higher than the r esidual anthocyanin in Aai+144 mg AA, Ace VE and A ai +288 mg AA (Fig ure 5 2). This is due to the development of more

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121 brown pigments in Ace DA samples as a result of higher AA content than the other samples. Under light, the addition of 48, 97, 144, or 288 mg AA to the aai anthocyanin solution lead to a 3.7, 3.07, 5.2, or 6.48 fold increase in the degradation rate constant respectively when compared with non enriched aai solution (Table 5 1) Under dark, the same trend was also observed, the degradation r ate constant increased with increasing level of AA fortification. Fortification levels of 48, 97, 144, or 288 mg AA lead to 3.34, 3.78, 4.19, and 5.21 fold increase in the degradation rate. The degradation rate constant for anthocyanin from Ace DA is 1.27 times as high as that of Ace VE under the presence of light. Under light, the degradation rate constant of the anthocyanin solutions from Ace DA is nearly similar to the degradation rate constant of aai sample enriched with 288 mg AA. Higher stability of a ai anthocyanins can be ascribed to the presence of much higher total flavonoids which may protect anthocyanin through intermolecular copigmentation (Mazza and Brouillard 1990) The protective effect of flavonoids lik e querc e tin and quercitrin against the deleterious effect of AA on cranberry anthocyanin has been reported (Shrikhande and Francis 1974) Regardless of the system considered, addition of AA decreased the half life of anthocyanins. Lower half lives are reported for samples enriched with higher amount s of AA. Under light, the addition of 288 mg AA decreased the half life from 104 hours (no AA added) to as low as 16 hours. Under light or in darkness, anthocyanin extra ct from Ace VE showed a higher stability than Ace DA. For example in the presence of light, the half life of the anthocyanin solutions from Ace VE was 26.9 hours while the half life of anthocyanin of extract from Ace DA was nearly 16 hours. This is due to the fact that Ace VE sample contains less ascorbic acid than Ace DA samples.

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122 Effect of Ascorbic Acid on the Stability of the Pure Anthocyanin Solution The rate of decrease of the anthocyanin is faster in samples containing ascorbic acid especially at the start (the first 5 hours) of the experiment. The influence of ascorbic acid is greater for the cyanidin aglycone than its corresponding monoglucoside (cyanidin 3 O rhamnoside). After storage for nearly 36 hours, the percentage loss of cyanidin with AA (39 % remain) or without added ascorbic acid (62 % remain) is much higher than that of cyanidin 3 O rhamnoside with AA (65 % remain ) or without AA (89 % remain) ( Fi g ure 5 3). The greater stability of cyanidin 3 O rhamnoside over cyanidin corroborates the fact that glucosidic substitution confers more stability to the anthocyanin molecule (Markakis 1982; Mazza and Miniati 1993). Also as another proof of lower stability, a higher brow n ing index was observed in the pure cyanidin system (data not shown). Effect of Light In the acerola model systems, the anthocyanin degradation was expectedly faster for samples stored in the presence of light than those stored in darkness (Fig ure 5 4). This tendency was similar for both acerola fruits grown in Davie, in which the as corbic acid content is higher, and acerola fruits grown in Vero Beach Florida. Overall, the deleterious effect of light appears to be more intense on acerola anthocyanin, this tendency however was reversed with increasing aai fortification with ascorbic a cid. No matter the system considered, Ace VE, Ace DA, Aai+48 mgAA, Aai+97mgAA, Aai+144mgAA, or Aai+288mgAA, storage in darkness lead to a higher percentage of anthocyanin remain ing near the end of storage (Fig ure 5 4, panels A, B, and C). This is not s urprising since light is one of the factors usually involved in anthocyanin degradation (Markakis 1982).

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123 Color Stability of the Different System Noticeable changes were observed in L a b hue and chroma values for all the extracts, confirming the degradat ion of visual parameters in the anthocyanin extracts over time. The changes however were more obvious in acerola anthocyanin extracts containing higher level s of ascorbic acid and in aai extracts enriched with higher level s of ascorbic acid. The lightness (L ) is the intensity of the luminosity transmitted by the solution. The linear behavior of LnL over time as shown in Fig ure 5 5 proved that the anthocyanin degradation in all the systems followed a first order pattern. The increase of L values under li ght or in darkness is related to the formation of translucent extracts due to color fading (Reyes and Cisneros Zevallos 2007). The redness (a ) (Fig ure 5 6) and the yellowness (b ) (Fig ure 5 7) values in all the samples decreased. Redness (a ) value decrea sed at a much faster pace in Ace DA samples than the Ace VE samples. Ace DA samples stored under light or under dark at 20 o C showed more than 96 % decrease in a value after 120 hours storage time. The same tendency was also observed for aai samples enri ched with ascorbic acid (Table 5 2). The color parameters a and b decrease quicker with the addition of ascorbic acid in the aai model systems. For a given storage condition (under light or in darkness) aai samples enriched with 97 mg AA exhibited high er percent decrease of a and b value than aai sample enriched with 48 mg AA (Table 5 2). It is also important to point out that initial color parameters a and b of the aai samples appeared to be degraded at a slower pace compared to a and b from th e acerola anthocyanin extracts. This could be related to the higher total flavonoids content in aai samples which may confer some level of protection of anthocyanins against the potential harmful effect of ascorbic

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124 acid. T he fact that a and b decrease f aster in the Ace DA in comparison with Ace VE anthocyanin extracts maybe due to the higher ascorbic acid content in the former. In the CIELAB color space, the hue parameter (h) is the angle made by the parameters b (yellow) and a (red). It usually defines specific colors which include yellow, red, blue, green or any combination of these colors (Gonnet 1998). During storage, in the presence of light or under dark, the hue angle in all the samples increased over the 120 hour period of time (Fig ure 5 8). The m ost spectacular increase was observed in the Ace DA samples while only minor increase was observed in Ace VE samples and in aai solutions enriched with AA (48 mg AA/100 m L or 97 mg AA/100 m L ). The difference in the behavior of h values in all the model sy stems during storage under different conditions (light, dark) indicat es that the color was changing from orange red to yellow during storage of Ace DA samples whereas the tonality in the Ace VE samples and the AA enriched aai systems remained red. The inc rease of h value has been previously reported in strawberry syrup fortified with AA (Skrede and others 1992); an d in aqueous extracts of purple and red flesh potatoes where the increase in the hue angle was associated to the formation of yellow chalcone sp ecies (Reyes and Cisneros Zevallos 2007) .The chroma (C ) is another parameter that is usually used to describe color intensity. In all the samples, the C values also decreased over the course of storage (Fig ure 5 9), confirming that intensive degradation of anthocyanin occurred. Decrease in chroma is often associated to degradation of monomeric anthocyanin (Reyes and Cisneros Zevallos 2007).The detrimental effect of AA enrichment on the color was obvious in all the systems regardless of the storage condit ions, resulting in the increased L decreased a and C values over time.

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125 Another parameter used in the assessment of color is the overall color difference ). Fig ure 5 10 shows that the overall color difference for the model systems showed Ace Ace Aai + 48mg AA. Th were, 26.3, and 28.4 for Ace Ve sample stored under light and dark at 20 o C respectively, and the values for the Ace Da system were 32.5, and were 22.8, 23.8 and 29.2, 31.6 for aai +48 mg AA and aai+97 mg AA under light and dark storage respectively. These results show that storage under light produce more color difference than sample stored in darkness. Degradation of Ascorbic Acid over Time The change in ascorbic acid content either in the acerola anthocyanin solution where this compound is naturally present, or in the aai systems where the ascorbic acid was added was monitored by high performance liquid chromatography HPLC. The ascorbic acid was measured before and at the end of th e storage period. Table 5 3 shows that the degradation of ascorbic acid occurred faster in the presence of light. Lower percent decrease was observed in aai samples enriched with AA when compared with the acerola samples, probably because flavonoid s in a ai confer some level of protection towards the AA. In summary anthocyanins from aai extracts show greater stability in the presence of ascorbic acid. Addition of ascorbic acid negatively influences the color parameters in aai extracts. Acerola anthocyani n extracts having highest AA content showed highest degradation rate constant and lower half life.

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126 Some Discussion on the Type of Reaction that May Take Place between Anthocyanin and Ascorbic Acid The results suggest that AA play a significant role in the instability of acerola and aai anthocyanin. Now the question is what type of reaction took place between the anthocyanin and the ascorbic acid molecule. The flavylium nucleus of the anthocyanin molecule lacks electron and is therefore very susceptible to nucleophilic attack. It has diketone dimedone to yield colorless 4 substituted adducts, and based on the similarities of the AA structure to dimedone, Jurd (1972) speculated that a similar condensa tion reaction may occur. Based on the results generated in this experiment and certain observations made especially in the pure anthocyanin model system, conclusion similar to that of Garcia Viguera and Bridle (1999) can be drawn. It is improbable that dir ect condensation between anthocyanin and AA occurred because the red color faded away rather slowly in both the acerola anthocyanin solutions, and aai extracts, and the pure anthocyanin model systems, suggesting that the reaction that took place was not s pontaneous. Timberlake and Bridle (1968) described a spontaneous condensation reaction between SO 2 (a nucleophilic agent like ascorbic acid) and anthocyanin. Another observation that argues against the direct condensation theory is that in the pure cyanidi n and cyanidin 3 O rhamnoside model systems, no changes were seen in the wavelength scans. The spectrophotometric profile of cyanidin+AA at time zero is similar to the profile obtained at the middle of the storage period; and the same trend was also observ ed in the scanning profile of cyanidin 3 O rhamnoside model system (Fig ure 5 1 1 ). Therefore, the degradation of anthocyanin through a free radical mechanism as proposed by L acobucci and Sweeny (1983) and supported by Garcia Viguera and Bridle (1999) is

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127 mor e likely. In the free radical mechanism theory, it is believed that ascorbic acid activates molecular oxygen by producing free radical that leads to the cleavage of the flavylium ring. Summary The results show that ascorbic acid plays an important role in the degradation of anthocyanin in acerola fruit. However, the degradation seems to be promoted by the degradation product s of ascorbic acid. Therefore, until the mechanism of this degradation is fully elucidated, it is important to store acerola fruit or i ts related products under conditions that favor the stability of ascorbic acid.

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128 Table 5 1. Degradation rate constant and the half life for anthocyanin in different systems citrate phosphate buffer pH 2.5 Samples k obs (h 1 ) Half life (hr) R^2 Ace VE Light 3.43 x 10 2 27.0 0.99 Ace VE Dark 6.20 x 10 2 26.0 0.98 Ace DA Light 4.35 x 10 2 16.0 0.95 Ace DA Dark 4.34 x 10 2 16.0 0.98 Aai NoAA Light 6.60 x 10 3 104 0.81 Aai NoAA Dark 8.20 x 10 3 84.0 0.84 Aai+48mgAA Light 2.45 x 10 2 28.0 0.99 Aai+48mg AA Dark 2.74 x 10 2 25.0 0.93 Aai+97mgAA Light 2.03 x 10 2 23.0 0.96 Aai+97mgAA Dark 3.10 x 10 2 22.0 0.98 Aai+144mgAA Light 3.43 x 10 2 20.0 0.96 Aai+144mgAA Dark 3.44 x 10 2 20.0 0.96 Aai+288mgAA Light 4.28 x 10 2 16.0 0.94 Aai+288mgAA Dark 4 .40 x 10 2 16.0 0.96 Ace VE Light: acerola anthocyanin s extract from fruits harvested in Vero Beach and stored under light; Ace VE Dark: acerola anthocyanin s extract from fruits harvested in Vero Beach and stored in darkness; Ace DA Light: acerola anthocy anin s extract from fruits harvested in Davie and stored under light; Ace DA Dark: acerola anthocyanin s extract from fruits harvested in Davie and stored in darkness; Aai NoAA Light: a ai anthocyanin s extract with no added ascorbic acid and stored under lig ht; Aai NoAA Dark: a ai anthocyanin s extract with no added ascorbic acid and stored in darkness; Aai+48mgAA Light :a ai anthocyanin s extract enriched with 48 mg/100 mL ascorbic acid and stored under light; Aai+48mgAA Dark : a ai anthocyanin s extract enri ched with 48 mg/100 mL ascorbic acid and stored in darkness; Aai+97mgAA Light: a ai anthocyanin s extract enriched with 97mg/100mL ascorbic acid and stored under light; Aai+97mgAA Dark: a ai anthocyanin s extract enriched with 97mg/100 mL ascorbic acid and stored in darkness; Aai+144mgAA Light: a ai anthocyanin s extract enriched with 144mg/100 mL ascorbic acid and stored under light; Aai+144mgAA Dark: a ai anthocyanin s extract enriched with 144mg/100 mL ascorbic acid and stored in darkness; Aai+288mgAA L ight : a ai anthocyanin s extract enriched with 288 mg/100 mL ascorbic acid and stored under light ; Aai+288mgAA Dark: a ai anthocyanins extract enriched with 288mg/100 mL ascorbic acid and stored in darkness.

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129 Table 5 2. Changes in color parameters (a and b ) for initial and final storage time Samples Initial time (T 0 ) Light 120 h Dark (120 h) a value Ace Ve 35.8 14.0 (60.9) 12.74 (64.5) Ace Da 22.8 0.82 (96.4) 0.71 (97.0) Aai+ 48mg AA 43.3 27.2 (37.3) 25.18 (41.8) Aai+ 97mg AA 42.0 19.8 (51.7) 16.51 ( 59.8) b value Ace Ve 22.6 9.56 (58.0) 11.3 (50.4) Ace Da 27.3 14.9 (45.4) 13.2 (51.6) Aai+ 48mg AA 15.8 13.9 (11.9) 14.4 (8.79) Aai+ 97mg AA 16.8 14.1 (16.1) 14.6 (13.4) Value within parentheses represent the percent decrease of the a and b values after 120 h of storage under different conditions Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie ; Aai+48mgAA : a ai anthocyanins extract enriched with 48 mg/100mL a scorbic acid; Aai+97mgAA: a ai anthocyanins extract enriched with 97mg/100mL ascorbic acid.

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130 Table 5 3. Ascorbic acid degradation in acerola and AA fortified aai Samples Acid ascorbic content (mg/100mL) T 0 T f =(120h) Ace VE Light 97 .0 22 .4 (77) Ace VE Dark 97 .0 29.7 (69) Ace DA Light 288 169 (23) Ace DA Dark 288 169 (41) Aai+48mgAA Light 48 .0 41.6 (13) Aai+48mgAA Dark 48 .0 41.0 (15) Aai+97mgAA Light 97 .0 57.0 (41) Aai+97mg AA Dark 97 .0 64.7 (33) Aai+144mgAA Light 144 79.2 (50 ) Aai+144mgAA Dark 144 87.0 (39) Aai+288mgAA Light 288 96.5 (67) Aai+288mgAA Dark 288 102 (64) *Values in parenthesis represent % decrease of AA Ace VE Light: acerola anthocyanins extract from fruits harvested in Vero Beach and stored under light; Ace VE Dark: acerola anthocyanins extract from fruits harvested in Vero Beach and stored in darkness; Ace DA Light: acerola anthocyanins extract from fruits harvested in Davie and stored under light; Ace DA Dark: acerola anthocyanins extract from fruits ha rvested in Davie and stored in darkness; Aai+48 mgAA Light :a ai anthoc yanins extract enriched with 48 mg/100mL ascorbic acid and stored under light; Aai+48 mgAA Dark: a ai anthocyanins extract enriched with 48 mg/100mL ascorbic acid and stored in darkness ; Aai+97 mgAA Light: a ai anthocyanins extract enriched with 97 mg/100mL ascorbic acid and stored under light; Aai+97 mgAA Dark: a ai anthocyanins extract enriched with 97mg/100mL ascorbic acid and stored in darkness; Aai+144mgAA Light: a ai anthocyanins e xtract enriched with 144 mg/100mL ascorbic acid and stored under light; Aai+144 mgAA Dark: a ai anthocyanins extract enriched with 144 mg/100mL ascorbic acid and stored in darkness; Aai+288 mgAA Light : a ai anthocyanins extract enriched with 288 mg/100mL asco rbic acid and stored under light; Aai+288 mgAA Dark: a ai anthocyanins extract enriched with 288 mg/100mL ascorbic acid and stored in darkness.

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131 Figure 5 1. First order plot for some selected anthocyanin s extracts during storage under light at 20 o C : A: Ace VE light; B: Ace DA light; C: Aai+48AA light; D: Aai+97 AA light A B C D

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132 Figure 5 2 Degradation curves of anthocyanin from acerola fruit and aai spiked with ascorbic acid at different level and stored under light (A) and in darkness (B) at 20 o C; in citrate buffer pH 2.5. Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai NoAA: a ai anthocyanins extract with no added ascorbic acid; Aai+48mgAA : a ai ant hocyanins extract enriched with 48 mg/100mL ascorbic acid; Aai+97mgAA: a ai anthocyanins extract enriched with 97 mg/100 mL ascorbic acid. A B

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133 Figure 5 3. Degradation curves of anthocyanin from pure cyanidin and cyanidin O rhamnoside spiked with ascorbic aci d and store in darkness in citrate buffer pH 2.5. Cyanidin+NoAA: Cyanidin with no added ascorbic acid ; Cyanidin +AA: cyanidin enriched with ascorbic acid; Cyanidin 3 R+ N oAA : cyanidin 3 rhamnoside with no added ascorbic acid; Cyanidin 3 R+AA: Cyanidin 3 rham noside enriched with ascorbic acid.

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134 Figure 5 4. Behavior of the different systems stored in the presence or in the absence of light, Panel A: Ace VE and Ace DA, Panel B: Aai+48mgAA and Aai+97mgAA, Panel C: Aai+144mgAA and Aai +288mgAA. A B C

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135 Figure 5 5. Evolution of the lightness (L*) value for acerola extract and the aai systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5 stored under light at 20 o C (A), under dark at 20 o C (B ). Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA : a ai anthocyanins extract enriched with 48 mg/100 mL ascorbic acid; Aai+97mgAA: a ai anthocyanin s extract enriched with 97 mg/100 mL ascorbic acid. Result presented as m ean plus or minus standard deviation A B

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136 Figure 5 6 Changes in the color parameters a*/a 0 value for the anthocyanin extracts from acerola and the aai in phosphate buffer soluti ons at pH 2.5 in the presence (A) or the absence (B) of light. Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA : a ai anthocyanins extract enriched wi th 48 mg/100 mL ascorbic acid; Aai+97mgAA: a ai anthocyanins extract enriched with 97mg/100 mL ascorbic acid. Result presented as m ean plus or minus standard deviation A B

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137 Figure 5 7. Changes in the color parameters b*/b 0 value for the anthocyanin extracts from acerola and the aai in phosphate buffer solutions at pH 2.5 in the presence (A) or the absence (B) of light. Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA: aai anthocyanins extract enriched with 48 mg/100 mL ascorbic acid; Aai+97mgAA: aai anthocyanins extract enriched with 97 mg/100 mL ascorbic acid. Result presented as m ean plus or minus standard deviation A B

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138 Figure 5 8. Evol ution of the hue value for acerola and aai systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5 stored under light at 20 o C (A), under dark at 20 o C (B ). Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA: aai anthocyanins extract enriched with 48 mg/100 mL ascorbic acid; Aai+97mgAA: aai anthocyanins extract enriched with 97 mg/100 mL ascorbic acid. Result pre sented as m ean plus or minus standard deviation A B

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139 Figure 5 9. Evolution of the chroma (C*) value for acerola and aai systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5 stored under light at 20 o C (A), un der dark at 20 o C (B). Ace VE: acerola anthocyanins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA: aai anthocyanins extract enriched with 48 mg/100 mL ascorbic acid; Aai+97mg AA: aai anthocyanins extract enriched with 97 mg/100 mL ascorbic acid. Result presented as m ean plus or minus standard deviation A B

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140 Figure 5 10 systems enriched with ascorbic acid of anthocyanin extracts in phosphate citrate buffer, pH 2.5 stored under light at 20 o C (A), under dark at 20 o C (B). Ace VE: acerola anthocy anins extract from fruits harvested in Vero Beach; Ace DA: acerola anthocyanins extract from fruits harvested in Davie; Aai+48mgAA: aai anthocyanins extract enriched with 48 mg/100 mL ascorbic acid; Aai+97mgAA: aai anthocyanins extract enriched with 97 mg/100 mL ascorbic acid. Result presented as m ean plus or minus standard deviation A B

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141 Figure 5 1 1 Spectrophotometric profile of selected samples in the pure anthocyanin solutions with added ascorbic acid at pH 2.5. A: cyanidin+NoAA, T 0 ; B: cy anidin+AA, halftime; C: cyanidin 3 O R+ N oAA T 0 ; D: cyanidin 3 O R+AA, half time. Cyanidin+NoAA: Cyanidin with no added ascorbic acid; Cyanidin+AA: cyaniding enriched with ascorbic acid; Cyanidin 3 R+ N oAA : cyanidin 3 rhamnoside with no added ascorbic acid; Cyanidin 3 R+AA: Cyanidin 3 rhamnoside enriched with ascorbic acid. A B C D

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142 CHAPTER 6 CONCLUSIONS In this study, the phenolic profile, the antioxidant capacity, the antimicrobial property, the toxicological screening, and the color stability of acerola fruit w ere examined geographic locations in Florida. Two types of anthocyanins: cyanidin 3 O rhamnoside and pelargonidin 3 O rhamnoside were identified The non anthocyanin phenolic compounds identified include various types of phenolic acids, and some neutral phenolic compounds such as: quercetin, quercerin 3 rhamnoside, (+ ) -epicatechin, and resveratrol. Resveratrol, and (+ ) epicatechin were reported for the first time in acerola fruit. The non edible (seed) portion of the fruits showed exceptionally high total phenolic contents. Overall phenolic extracts at the concentration considered (25 mg/mL) showed limited antimicrobial properties against the microbial strains tested However, selected flavonol extracts (especially from seeds) show antimicrobial effects against S. aureus Using an AA free aai anthocyanin model system (aai) it was indicated that AA was the main cause of anthocyanin degradation in acerola anthocyanin extracts. The mecha nism of the degradation is still not completely understood however results and observations suggest that direct condensation of AA on 4 position of flavylium cation of the anthocyanin molecule is unlikely. Although some phenolic compounds including antho cyanins and non anthocyanin phenolics were identified, chromatograms of the non anthocyanin phenolic compounds contain ed many unidentified peaks. Therefeore, the use of new analytical and more informative analytical techniques such as nuclear magnetic reso nance (NMR) could help to identify the unidentified compounds Regarding the antimicrobial testing, the

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143 results demonstrate limited antimicrobial properties at the concentration tested. The antimicrobial property was conducted on 6 mm diameter discs with l imited sample retention capacity. It would be important for future research in this area to use disc having higher retention. In addition, the antimicrobial activity of the acerola phenolic extracts was conducted only on three strains of microorganisms; it would be important to test the effect of the acerola phenolic fractions on a much larger number of bacterial strains to gather more information that would help to better assess the antimicrobial potential of acerola phenolic extracts. Regarding the effec t of ascorbic acid on the anthocyanins and color loss of the acerola anthocyanin extracts the overall conclusion is that the degradation of anthocyanin is more likely to occur through a free radical mechanism rather than by direct condensation between asco rbic acid and the anthocyanin molecule. Given the co existence of the two compounds in the same systems the stabilization of anthocyanin in acerola fruit or its derived products would be a difficult task Therefore until the mechanism is elucidated, it is important to store the anthocyanin extracts under conditions (packaging, temperature, etc.) that favor the stability of both anthocyanin and ascorbic acid. Another potential solution to improve the stability of anthocyanin in acerola extract would be the addition of polyphenolic compounds to anthocyanin solutions as copigment. This method has been reported to improve the stability of anthocyanin during storage in model and fruit juice systems (Brenes and others 2005; Talcott and others 2005). As the additi on of pure phenolic compounds is not applicable in the food industry, the general method is the use of phenolic extracts from natural sources to stabilize anthocyanins. For instance, Pozo Insfran and others (2007)

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144 reported that the addition partially purif ied rosemary and thyme phenolic extracts as copigment increase Muscadine grape juice color, antioxidant capacity, and also reduced phytochemical losses during high hydrostatic pressure processing and storage. The application of natural polyphenolic extract s to stabilize the anthocyanin in acerola extracts and juices could be an interesting area of research where question like the practical commercial levels that have no effect on the flavor and other sensory attributes of acerola extracts or juices could be addressed.

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145 APPENDIX A HPLC DAD CHROMATOGRAMS OF THE NON ANTHOCYANIN PHENOLIC COMPOUNDS DETECTED A CEROLA FRUIT Figure A 1. HPLC DDA chromatogram for partially purified acerola anthocyanin extracts Ace DA (A), Ace VE (B), and frozen single s trength juice (FSSJ) (C) acerola juice at 520 nm. Peak identification is given in Table 3 3 1 2 A B 1 2 1 2 C

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146 Figure A 2. Sample chromatogram of the acidic fraction of phenolic compounds detected in edible portion of acerola fruit, detection wavelength: 320 nm Figure A 3. Sample chromatogram of the neutral fraction of phenolic compounds detected in edible portion of acerola fruit, detection wavelength: 280 nm

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147 APPENDIX B STATISTICAL ANALYSIS OF THE COLOR AND SOF TNESS OF THE DATA COLLECTED AT THE THR EE STAGES OF MATURIT Y Table B 1. SAS software code used for the statistical analysis of peel color (L a b ) and softness (H) parameters using the Duncan multiple range test Data experiment; Input maturity $ sample L a b H @@; Datalines ; 1 1 50.09 8.30 40.60 5.99 1 2 52.09 8.31 40.59 6.03 1 3 54.09 8.31 40.66 6.03 2 1 52.39 19.24 38.00 3.19 2 2 52.45 19.48 38.66 3.20 2 3 52.6 9 19.59 38.31 3.23 3 1 43.80 38.60 31.37 2.21 3 2 43.78 38.67 31.40 2.26 3 3 43.60 39.00 31.33 2.30 ; Proc glm ; Class maturity sample; Model L = maturity sample; Means maturity/ Duncan ; Proc glm ; Class maturity sample; Model a = maturity sample; Means matu rity/ Duncan ; Proc glm ; Class maturity sample; Model b = maturity sample; Means maturity/ Duncan ; Proc glm ; Class maturity sample; Model H = maturity sample; Means maturity/ Duncan ; Run ;

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148 Table B 2. SAS software output used for the statistical analysis of pee l color (L, a, b) and softness (H) parameters using the Duncan multiple range test The GLM Procedure Dependent Variable: L Source DF Sum of Squares Mean Square F Value Pr > F Model 4 150.0703778 37.5175944 28.46 0.0034 Error 4 5.2729778 1.3182444 Corrected Total 8 155.3433556 R Square Coeff Var Root MSE L Mean 0.966056 2.322202 1.148148 49.44222 Source DF Type I SS Mean Square F Value Pr > F m aturity 2 147.2686889 73.6343444 55.86 0.0012 sample 2 2.8016889 1.4008444 1.06 0.4264 Source DF Type III SS Mean Square F Value Pr > F maturity 2 147.2686889 73.6343444 55.86 0.0012 sample 2 2.8016889 1.4008444 1.06 0.4264 The GLM Procedure Duncan's Multiple Range Test for L Alpha 0.05 Error Degrees of Freedom 4 Error Mean Square 1.318244 Means with the same letter are not significantly different. Duncan Grouping Mean N maturity A 52.5100 3 2 A A 52.0900 3 1 B 43.7267 3 3

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149 Table B 2. Continued. The GLM Proc edure Dependent Variable: a Source DF Sum of Squares Mean Square F Value Pr > F Model 4 3358.004644 839.501161 53264.1 <.0001 Error 4 0.063044 0.015761 Corrected Total 8 3358.067689 R Square Coeff Var Root MSE a Mean 0.999981 0.754971 0.125543 16 .62889 Source DF Type I SS Mean Square F Value Pr > F maturity 2 3357.912289 1678.956144 106525 <.0001 sample 2 0.092356 0.046178 2.93 0.1646 Source DF Type III SS Mean Square F Value Pr > F maturity 2 3357.912289 1678.956144 106525 <.0001 sample 2 0 .092356 0.046178 2.93 0.1646 The GLM Procedure Duncan's Multiple Range Test for a Note: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 4 Error Mean Square 0.015761 Number of Means 2 3 Critical Range .2846 .2908 Means with the same letter are not significantly different. Duncan Grouping Mean N maturity A 38.7567 3 3 B 19.4367 3 2 C 8.3067 3 1

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150 Table B 2. Continued. The GLM Procedure Dependent Variable: b Source DF Sum of Squares Mean Sq uare F Value Pr > F Model 4 139.2941778 34.8235444 952.04 <.0001 Error 4 0.1463111 0.0365778 Corrected Total 8 139.4404889 R Square Coeff Var Root MSE b Mean 0.998951 0.520149 0.191253 36.76889 Source DF Type I SS Mean Square F Value Pr > F ma turity 2 139.2170889 69.6085444 1903.03 <.0001 sample 2 0.0770889 0.0385444 1.05 0.4289 Source DF Type III SS Mean Square F Value Pr > F maturity 2 139.2170889 69.6085444 1903.03 <.0001 sample 2 0.0770889 0.0385444 1.05 0.4289 The GLM Procedure Duncan 's Multiple Range Test for b Note: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 4 Error Mean Square 0.036578 Number of Means 2 3 Critical Range .4336 .4431 Means with the same letter are not significantly different. Duncan Grouping Mean N maturity A 40.6167 3 1 B 38.3233 3 2 C 31.3667 3 3

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151 Table B 2. Continued. The GLM Procedure Dependent Variable: H Source DF Sum of Squares Mean Square F Value Pr > F Model 4 22.94106667 5.73526667 20 242.1 <.0001 Error 4 0.00113333 0.00028333 Corrected Total 8 22.94220000 R Square Coeff Var Root MSE H Mean 0.999951 0.439874 0.016833 3.826667 Source DF Type I SS Mean Square F Value Pr > F maturity 2 22.93620000 11.46810000 40475.6 <.0001 sam ple 2 0.00486667 0.00243333 8.59 0.0357 Source DF Type III SS Mean Square F Value Pr > F maturity 2 22.93620000 11.46810000 40475.6 <.0001 sample 2 0.00486667 0.00243333 8.59 0.0357 The GLM Procedure Duncan's Multiple Range Test for H Note: This test c ontrols the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 4 Error Mean Square 0.000283 Number of Means 2 3 Critical Range .03816 .03899 Means with the same letter are not significantly differ ent. Duncan Grouping Mean N maturity A 6.01667 3 1 B 3.20667 3 2 C 2.25667 3 3

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152 APPENDIX C STA TISTICAL ANALYSIS OF THE TOTAL ANTIOXIDAN T AND VITAMIC DATA COLLECTED FOR THE FR UITS GROWN IN DAVIE, FLORIDA Table C 1. SAS software code used for the statistical analysis of parameters (TPI, ORAC, DPPH ORAC, vit C ) using the Duncan multiple range test Data experiment; Input extraction maturity $ sample TPI ORAC DPPH VitC @@; Datalines ; 1 1 1 9403 43.5 90 1745 1 1 2 9406 43.5 90 1743 1 1 3 9406 43.5 105 1744 1 2 1 7944 36.5 54.77 1049 1 2 2 7945 36.5 54.77 1049 1 2 3 7947 36.5 54.77 1049 1 3 1 8342 36.2 41.75 987 1 3 2 8340 36.2 39.75 980 1 3 3 8338 36.2 39.75 994 2 1 1 16280 48.0 134.0 1162.0 2 1 2 16280 48.0 138.0 1160.0 2 1 3 16295 48.0 136.0 1161.0 2 2 1 12917 39.7 1 06.0 966.0 2 2 2 12915 39.7 106.0 972.0 2 2 3 12913 39.7 106.0 972.0 2 3 1 11942 40.0 78.0 407.0 2 3 2 11942 40.0 71.0 405.0 2 3 3 11942 40.0 73.0 403.0 ; Proc glm ; Class extraction maturity sample; Model TPI = extraction maturity sample; Means extraction maturity/ Duncan ; Proc glm ; Class extraction maturity sample; Model ORAC = extraction maturity sample; Means extraction maturity/ Duncan ; Proc glm ; Class extraction maturity sample; Model DPPH = extraction maturity sample; Means extraction maturity/ Duncan ; P roc glm ; Class extraction maturity sample; Model VitC = extraction maturity sample; Means extraction maturity/ Duncan ;

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153 Table C 2. SAS software output used for the statistical analysis of parameters ( TPI ORAC DPPH and Vit.C ) using the Duncan multiple range test The GLM Procedure Dependent Variable: TPI Source DF Sum of Squares Mean Square F Value Pr > F Model 5 145830326.9 29166065.4 43.04 <.00 01 Error 12 8132742.7 677728.6 Corrected Total 17 153963069.6 R Square Coeff Var Root MSE TPI Mean 0.947177 7.390818 823.2427 11138.72 Source DF Type I SS Mean Square F Value Pr > F extraction 1 119377001.4 119377001.4 176.14 <.0001 maturity 2 26453306.8 13226653.4 19.52 0.0002 sample 2 18.8 9.4 0.00 1.0000 Source DF Type III SS Mean Square F Value Pr > F extraction 1 119377001.4 119377001.4 176.14 <.0001 maturity 2 26453306.8 13226653.4 19.52 0.0002 sample 2 18.8 9.4 0.00 1.0000 The GLM P rocedure Duncan's Multiple Range Test for TPI Alpha 0.05 Error Degrees of Freedom 12 Error Mean Square 677728.6 Number of Means 2 3 Critical Range 1036 1084 Duncan Grouping Mean N maturity A 12845.0 6 1 B 10430.2 6 2 B 10141.0 6 3

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154 Table C 2. Continued. The GLM Proced ure Dependent Variable: ORAC Source DF Sum of Squares Mean Square F Value Pr > F Model 5 300.2150000 60.0430000 567.34 <.0001 Error 12 1.2700000 0.1058333 Corrected Total 17 301.4850000 R Square Coeff Var Root MSE ORAC Mean 0.995788 0.800296 0.32 5320 40.65000 Source DF Type I SS Mean Square F Value Pr > F extraction 1 66.1250000 66.1250000 624.80 <.0001 maturity 2 234.0900000 117.0450000 1105.94 <.0001 sample 2 0.0000000 0.0000000 0.00 1.0000 Source DF Type III SS Mean Square F Value Pr > F extraction 1 66.1250000 66.1250000 624.80 <.0001 maturity 2 234.0900000 117.0450000 1105.94 <.0001 The GLM Procedure Duncan's Multiple Range Test for ORAC Alpha 0.05 Error Degrees of Freedom 12 Error Mean Square 0.105833 Number of Means 2 3 Critical Range .4092 .4283 Duncan Grouping Mean N maturity A 45.7500 6 1 B 38.1000 6 3 B 38.1000 6 2

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155 Table C 2. Continued. The GLM Procedure Dependent Variable: DPPH Source DF Sum of Squares Mean Square F Value Pr > F Model 5 18270.22088 3654.04418 108.8 7 <.0001 Error 12 402.75 457 33.56288 Corrected Total 17 18672.97544 R Square Coeff Var Root MSE DPPH Mean 0.978431 6.867049 5.793348 84.36444 Source DF Type I SS Mean Square F Value Pr > F extraction 1 7914.49742 7914.49742 235.81 <.0001 maturity 2 10336.27901 5168.139 51 153.98 <.0001 sample 2 19.44444 9.72222 0.29 0.7536 Source DF Type III SS Mean Square F Value Pr > F extraction 1 7914.49742 7914.49742 235.81 <.0001 maturity 2 10336.27901 5168.13951 153.98 <.0001 sample 2 19.44444 9.72222 0.29 0.7536 The GLM Pro cedure Duncan's Multiple Range Test for DPPH Alpha 0.05 Error Degrees of Freedom 12 Error Mean Square 33.56288 Number of Means 2 3 Critical Range 7.287 7.628 Duncan Grouping Mean N maturity A 115.500 6 1 B 80.385 6 2 C 57.208 6 3

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156 Table C 2. Continued. The GLM Procedu re Dependent Variable: VitC Source DF Sum of Squares Mean Square F Value Pr > F Model 5 2507431.333 501486.267 23.73 <.0001 Error 12 253630.667 21135.889 Corrected Total 17 2761062.000 R Square Coeff Var Root MSE VitC Mean 0.908140 13.81082 145.3 819 1052.667 Source DF Type I SS Mean Square F Value Pr > F extraction 1 773768.000 773768.000 36.61 <.0001 maturity 2 1733647.000 866823.500 41.01 <.0001 sample 2 16.333 8.167 0.00 0.9996 Source DF Type III SS Mean Square F Value Pr > F extraction 1 773768.000 773768.000 36.61 <.0001 maturity 2 1733647.000 866823.500 41.01 <.0001 sample 2 16.333 8.167 0.00 0.9996 The GLM Procedure Duncan's Multiple Range Test for VitC Alpha 0.05 Error Degrees of Freedom 12 Error Mean Square 21135.89 Number of M eans 2 3 Critical Range 182.9 191.4 Means with the same letter are not significantly different. Duncan Grouping Mean N maturity A 1452.50 6 1 B 1009.50 6 2 C 696.00 6 3

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174 BIOGRAPHICAL SKETCH Lemne Delva received his b achelor degree in 2000 from the College of Agriculture and Veterinary Medicine of the State University of Haiti, majoring in Animal Sciences. After obtaining his b d egree he wo rked as a t eaching assistant at his home University for nearly one and a half year. In 2003, he was granted a s cholarship form the International Cooperation and Development Fund (ICDF) and went to the National Pintung University of Science and Technology i n Taiwan, where two years later he graduated with a m degree in Food Science. He returned to Haiti in 2005 and started to work as research assistant and instructor at his home University; h e kept that University position for two years. In summer 200 7, he came to the United States as a Fulbright grantee to p ursue a doctoral degree in Food Science at the Department of Food Science and Human Nutrition of the University of Florida. Under the m entorship of Dr. Ren e Goodrich Schneider, Lemne received hi s Ph.D. in Food Science and Human Nutrition with a Minor in Agricultural Education and Communication in the fall of 2012