GUAVA (Psidium guajava L.) FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND OVERALL QUALITY AS INFLUENCED BY POSTHARVEST TREATMENTS By FLOR DE MARIA NUNEZ RUEDA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Flor de Maria Nunez Rueda DEDICATION To the Lord Jesus, for giving me the strength and motivation to keep pursuing my dreams. To Sabina, my grandmother, backbone of my family and shimmering light in our lives. To my nephew Diego, with lots of love from your always spoiling aunt. ACKNOWLEDGMENTS I wish to extend my special thanks to my major advisor, Dr. Stephen Talcott, for his support, advice, friendship and for being a leading example of motivation and hard work to us. I could not have asked for more. I thank my advising committee, Dr. Susan Percival and Dr. Donald Huber, for their invaluable assistance and time for my project. I thank my lab partners and friends, Kim, Kristine, Jorge, Lanier, Chris, Lisbeth, and Stacy, for their never-ending assistance in my project and for making the work in the lab such an enjoyable and nurturing experience. Special thanks go to Dr. Joonhee Lee and soon Dr. to be David del Pozo, for investing their valuable time, sharing their knowledge and providing guidance, as well as a friendly shoulder to lean on. Graduate school would definitely not have been as much fun without David’s friendship and craziness. I would have not reached where I am without the unconditional love, support, and advice of my mother Nora, the biggest example of strength I have seen in a person. I love her so much. I thank my sister and best friend Tania, for her advice and big sis support during all these years. Big thanks go to my brother Jesus and my sister-in-law Yalile, for their warm support and example of perseverance and love, as well as to my little brother Guille, for having patience with me. Special thanks go to my family in Honduras, Nunez, Rueda, and Maier, for all their support and best wishes through the distance. Last but not least, big thanks go to Gary, for sharing his love, patience, jokes and for just adding much happiness to my graduate school days. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 2.1 Guava Market and Industrial Applications.............................................................4 2.2 Guava Fruit.............................................................................................................5 2.2.1 Origin............................................................................................................5 2.2.2 Morphology..................................................................................................5 2.2.3 Postharvest Physiology.................................................................................6 2.3 Guava Phytochemicals............................................................................................8 2.3.1 Phytochemicals.............................................................................................8 2.3.3 Dietary Fiber.................................................................................................9 2.3.4 Carotenoids and Lycopene.........................................................................10 2.3.5 Guava Polyphenolics..................................................................................11 2.4 Postharvest Treatments.........................................................................................13 2.4.1 Guava Postharvest Handling and Storage..................................................13 2.4.2 Quarantine Heat Treatments.......................................................................13 2.4.3 Shelf-life Extension Treatments.................................................................15 2.5 1-Methylcyclopropene..........................................................................................15 2.5.1 1-Methylcyclopropene................................................................................15 2.5.2 1-MCP Application Conditions..................................................................16 2.5.3 1-MCP on Climacteric Fruits.....................................................................17 2.5.4 Guava and 1-MCP......................................................................................18 2.6 Polyphenolics........................................................................................................18 2.6.1 Polyphenolics.............................................................................................18 2.6.2 Polyphenolic Classification........................................................................19 2.6.3 Polyphenolics as Antioxidants...................................................................23 v 3 EFFECTS OF HOT WATER IMMERSION TREATMENT ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY.............25 3.1 Introduction...........................................................................................................25 3.2 Materials and Methods.........................................................................................26 3.2.1 Materials and Processing............................................................................26 3.2.1.1 Fruit preparation and HW treatment................................................26 3.2.1.2 Guava fruit processing.....................................................................27 3.2.2 Chemical Analysis......................................................................................27 3.2.2.1 Moisture content determination.......................................................27 3.2.2.2 Quantification of total soluble phenolics.........................................28 3.2.2.3 Analysis of ascorbic acid by HPLC.................................................28 3.2.2.4 Quantification of antioxidant capacity.............................................29 3.2.2.5 Analysis of lycopene by HPLC........................................................29 3.2.2.6 Quantification of non-lycopene carotenoids....................................30 3.2.2.7 Analysis of polyphenolics by HPLC................................................30 3.2.3 Quality Analysis.........................................................................................31 3.2.4 Statistical Analysis.....................................................................................31 3.3 Results and Discussion.........................................................................................32 3.3.1 Chemical Analysis......................................................................................32 3.3.1.1 Moisture content...............................................................................32 3.3.1.2 Total soluble phenolics.....................................................................33 3.3.1.3 Ascorbic acid....................................................................................35 3.3.1.4 Antioxidant capacity........................................................................36 3.3.1.5 Lycopene and yellow carotenoids....................................................38 3.3.1.6 Polyphenolics by HPLC...................................................................41 3.3.2. Quality Analysis........................................................................................49 3.3.2.1 pH and soluble solids.......................................................................49 3.3.2.2 Overall fruit quality..........................................................................49 3.3.3 Stage III Fruit.............................................................................................50 3.4 Conclusions...........................................................................................................51 4 EFFECTS OF 1-METHYLCYCLOPROPENE ON GUAVA FRUIT PHYTOCHEMICALS, ANTIOXIDANT PROPERTIES AND QUALITY.............53 4.1 Introduction...........................................................................................................53 4.2 Materials and Methods.........................................................................................54 4.2.1 Materials and Processing............................................................................54 4.2.1.1 Fruit preparation and 1-MCP treatment...........................................54 4.2.1.2 Guava fruit processing.....................................................................56 4.2.2 Quality Analysis.........................................................................................56 4.2.2.1 Aesthetic fruit quality assessment during storage............................56 4.2.2.2 Firmness determination during storage............................................56 4.2.2.3 Titratable acidity, soluble solids and pH..........................................56 4.2.3 Chemical Analysis......................................................................................57 4.2.4 Statistical Analysis.....................................................................................57 4.3 Results and Discussion.........................................................................................57 vi 4.3.1 Quality Analysis.........................................................................................57 4.3.1.1 Aesthetic fruit quality during storage...............................................57 4.3.1.2 Firmness during storage...................................................................60 4.3.1.3 Titratable acidity, soluble solids, and pH during storage.................62 4.3.2 Chemical Analysis......................................................................................64 4.3.2.1 Moisture content...............................................................................64 4.3.2.2 Total soluble phenolics.....................................................................65 4.3.2.3 Antioxidant capacity........................................................................66 4.3.2.3 Ascorbic acid....................................................................................67 4.3.2.4 Lycopene..........................................................................................68 4.3.2.5 Polyphenolics by HPLC...................................................................70 4.3.3 1-MCP Treatment to Boxed Guavas..........................................................73 4.4 Conclusions...........................................................................................................76 5 SUMMARY AND CONCLUSIONS.........................................................................78 LIST OF REFERENCES...................................................................................................80 BIOGRAPHICAL SKETCH.............................................................................................89 vii LIST OF TABLES Table page 3-1. Gradient elution running program for HPLC analysis of polyphenolics.................31 3-2. Tentative identification of guava polyphenolics at 280 nm by HPLC based on retention time, spectral properties, and comparison to authentic standards.............43 3-3. Guava gallic acid (GA), gallic acid derivatives, and an ellagic acid derivative as affected by a hot water quarantine treatment and ripening stage.............................45 3-4. Guava procyanidins, other characteristic unknown compounds, and total polyphenolics by HPLC as affected by a hot water quarantine treatment and ripening stage...........................................................................................................45 3-5. Quality parameters, soluble solids and pH, in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 °C) and ripening stage (Stage I and Stage II). Data are expressed as fresh weight basis (mean ± standard error), n = 4..........................................................................................................................49 3-6. Phytochemical content and quality parameters in Stage III guavas as affected by a hot water quarantine treatment at 46 °C (mean ± standard error). n = 4...............51 4-1. Changes in skin coloration in non-treated (control) and 1-MCP-treated guavas during 1-MCP application and storage at 15 °C......................................................59 viii LIST OF FIGURES Figure page 2-1. Chemical structure of lycopene, a 40-C open hydrocarbon chain...........................10 2-2. Chemical structures of a condensed tannin (A) and hydrolysable tannin (B). A is a typical condensed tannin composed of catechin and epicatechin; B is a polygalloyl glucose composed of a glucose core esterified with gallic acid residues.....................................................................................................................21 2-3. Chemical structures of flavonoids apigenin and myricetin, which have previously reported in guava. They are composed of three pyrane rings.................22 3-1. Moisture content (%) of ripe guavas as affected by a hot water immersion treatment (0,15, 30, and 60 min at 46 °C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n =4..............................................33 3-2. Total soluble phenolics (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 °C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4................................34 3-3. Ascorbic acid content (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 °C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4................................36 3-4. Antioxidant capacity (μmol Trolox Equivalents/g DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 °C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4.........37 3-5. Lycopene content (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46 °C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4.............................................39 3-6. Non-lycopene carotenoids (mg/kg DW) in guava as affected by a hot water immersion treatment (0, 15, 30, and 60 min at 46°C) and ripening stage (Stage I and II). Error bars represent standard error of the mean, n = 4................................40 3-7. HPLC chromatogram of polyphenolic compounds found in guava juice-A) gallic acid, B) gallic acid derivatives, C) unknown-characteristic guava polyphenolics, D) procyanidins, and E) ellagic acid derivative. Identification (280 nm) was done by comparison to authentic standards and spectral properties........................42 ix 4-1. Firmness (kg) of guavas treated with 1-MCP (1000 nL/L ,10°C, 24 h) during storage at 15 °C. Error bars represent the standard error of the mean, n = 5...........61 4-2. Titratable acidity (% citric acid) of guavas treated with 1-MCP during storage at 15 °C. Error bars represent the standard error of the mean, n = 5............................63 4-3. Effect of a 1-MCP treatment (1000 nL/L ,10°C, 24 h) on guava pH during storage at 15 °C. Error bars represent the standard error of the mean, n = 5...........63 4-4. Effect of 1-MCP treatment (1000 nL/L at10°C, 24 h) on guava soluble solids (°Brix) during storage at 15 °C. Error bars represent the standard error of the mean, n = 5...............................................................................................................64 4-5. Effect of 1-MCP (1000 nL/L, 10°C, 24 h) on total soluble phenolics (mg/kg DW) in guava. Error bars represent the standard error of the mean, n = 23............66 4-6. Effect of 1-MCP treatment (1000 nL/L, 10°C, 24 h) on guava antioxidant capacity (μM Trolox equivalents/g DW). Error bars represent the standard error of the mean, n = 23...................................................................................................66 4-7. Guava ascorbic acid content (mg/kg DW) as affected by 1-MCP (1000 nL/L, 10°C, 24 h). Error bars represent the standard error of the mean, n = 23................68 4-8. Effect of 1-MCP (1000 nL/L ,10°C, 24 h) on guava lycopene content (mg/kg DW). Error bars represent the standard error of the mean, n = 23...........................69 4-9. Guava procyanidin content (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard error of the mean, n = 23......................................................71 4-10. Guava ellagic acid derivative content (A) and characteristic polyphenolics content (B) (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard error of the mean, n = 23...........................................................................72 4-11. Guava gallic acid content (mg/kg GAE) as affected by 1-MCP. Error bars represent the standard error of the mean, n = 23......................................................73 4-12. Firmness (kg) of boxed guavas treated with 1-MCP (1000 nL/L ,10°C, 24 h) during storage at 25 °C. Error bars represent the standard error of the mean, n=3..74 4-13. Titratable acidity (% citric acid) of boxed guavas treated with 1-MCP during storage at 15 °C. Error bars represent the standard error of the mean, n = 3...........75 4-14. Effect of 1-MCP treatment (1000 nL/L at10°C, 24 h) on boxed guavas soluble solids (°Brix) during storage at 15 °C. Error bars represent the standard error of the mean, n = 3.........................................................................................................76 x