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Changes in Polyphenolics and Resultant Antioxidant Capacity in 'Tommy Atkins' Mangos (Mangifera indica L.) by Selected P...

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CHANGES IN POLYPHENOLICS AND RE SULTANT ANTIOXIDANT CAPACITY IN TOMMY ATKINS MANGOS ( Mangifera indica L.) BY SELECTED POSTHARVEST TREATMENTS By YOUNGMOK KIM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Youngmok Kim

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This thesis is dedicated to the people I love : my parents, my teachers, and my friends.

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ACKNOWLEDGMENTS First of all, I would like to express my heartfelt thanks to my supervisory committee chair, Dr. Stephen Talcott who is an amazing mentor and teacher. His warm personality, professional guidance, and profound knowledge encouraged me to complete this work. Continuing as a docto rate student with him is a gl orious opportunity and I feel extremely fortunate to have this opportunity. I would also like to expre ss my gratitude to my supervisory committee members, Dr. Jeffrey K. Brecht and Dr. Susan Percival for their thoughtful advice, consideration, and support. I am grateful for all their help and encouragement in completing this work. I thank you my beloved parents from th e bottom of my heart for supporting me intellectually, psychologically a nd physically throughout my life. I will keep trying to be a son they can be proud of. I also want to thank my special fellow laboratory members Dr. Joonhee Lee, Chris, David, Flor, Jorge, Kristi ne, Lanier, and Lisbeth. I really appreciate their advice, help, and time. I want to give my special thanks to Dr. Lee for her valuable advice and instruction. I am grateful to Sunghee for her encouragement, patience, understanding, and love. I love you so much. Finally, I would like to thank my count ry, South Korea, for allowing me to complete my masters degree here at Univer sity of Florida by fi nancially supporting me for two years. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION.......................................................................................................1 2 LITERATURE REVIEW............................................................................................4 2-1 Mango Market......................................................................................................4 2-2 Mango Cultivars...................................................................................................7 2-3 Mango Ripening...................................................................................................8 2-4 Mango Postharvest Handling.............................................................................10 2-4-1 Hot Water Immersi on Treatment (HWT)................................................10 2-4-2 Controlled Atmosphe re (CA) Storage.....................................................12 2-5 Mango Polyphenolics.........................................................................................14 2-6 Polyphenolics as Antioxidants...........................................................................17 3 PHYTOCHEMICAL CHANGES AND RESULTANT ANTIOXIDANT CAPACITY IN MANGOS DURING 4 DAY STORAGE AFFECTED BY VARYING LENGTHS OF HOT WATER IMMERSION TREATMENT..............19 3-1 Introduction........................................................................................................19 3-2 Materials and Methods.......................................................................................20 3-2-1 Fruit Prepara tion and Treatment..............................................................20 3-2-2 Identification and Quantificati on of Individual Polyphenolics................22 3-2-3 Quantification of Total Soluble Polyphenolics........................................22 3-2-4 Quantification of Antioxidant Capacity...................................................23 3-2-5 Statistical Analysis Method.....................................................................24 3-3 Results and Discussion.......................................................................................24 3-3-1 Individual Polyphenolic Concentration...................................................24 3-3-2 Total Soluble Polyphenolics (TSP)..........................................................29 3-3-3 Antioxidant Capacity...............................................................................30 v

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3-4 Conclusion.........................................................................................................31 4 TO EVALUATE POLYPHENOLICS, ANTIOXIDANT CAPACITY, AND PHYSIOLOGICAL CHANGES IN MANGOS ( Mangifera Indica L .) FOLLOWING CONTROLLED ATMOSPH ERE STORAGE COMBINED WITH HOT WATER IMMERSION TREATMENT...............................................33 4-1 Introduction........................................................................................................33 4-2 Materials and Methods.......................................................................................34 4-2-1 Fruit Prepara tion and Treatment..............................................................34 4-2-2 Phytochemical Changes and Antioxidant Capacity.................................35 4-2-3 Evaluation of Flesh Color........................................................................36 4-2-4 Quantification of Titratable Acidity (TA)................................................36 4-2-5 Quantification of Soluble Solids Content (SSC).....................................37 4-2-6 Statistical Analysis Method.....................................................................37 4-3 Result and Discussion........................................................................................37 4-3-1 External Fruit Quality..............................................................................37 4-3-2 Individual Polyphenolic Concentration...................................................38 4-3-3 Total Soluble Phenolics (TSP).................................................................46 4-3-4 Antioxidant Capacity...............................................................................48 4-3-5 Fruit Flesh Color......................................................................................51 4-3-6 Titratable Acidity (TA)............................................................................56 4-3-7 Soluble Solids Content (SSC)..................................................................58 4-4 Conclusion.........................................................................................................60 5 SUMMARY AND CONCLUSION.............................................................................62 LIST OF REFERENCES..................................................................................................64 BIOGRAPHICAL SKETCH............................................................................................73 vi

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LIST OF TABLES Table page 2-1. Main mango producing countries in 2004 (www.fao.org)..........................................5 2-2. Global imports of mangos (MT) (Sauco 2004)...........................................................6 2-3. Producing countries, selected cultivars, and main marketing season (Nakasone and Paull, 1998).........................................................................................................8 2-4. Determined dip time based on origin, sh ape, and weight of fruit. (USDA-APHIS, 2002)........................................................................................................................11 3-1. HWT with four different lengths of time (0, 70, 90, and 110 min) at 46 C with different ripeness stages (day 0 and day 4) at 23 C................................................21 3-2. Gallic acid ( g/g) and average of four hydrolyzable tannins ( g/g GAE) identified and quantified using HPLC as a result of different duration of HWT (0, 70, 90, 110 min) at two ripeness stag es (Day 0 and Day 4)................................................28 3-3. Total soluble phenolics ( g/g GAE) and total antioxidant capacity ( M TE/g) of mango treated with hot water with vary ing length of treatment times (0, 70, 90, and 110 min) at two different ripeni ng stages (Day 0 and Day 4)..........................32 4-1. Mango CA storage with 1 control (air) and 2 different air compositions (CA2 and CA3) (CA1=Air, CA2=3% O2, CA3=3% O2 + 10% CO2) at 10oC for 2 weeks. One half of the mangos were treated w ith hot water and the other half was treated with 25 C water...........................................................................................35 vii

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LIST OF FIGURES Figure page 2-1. Structure of gallotannin ( -1,2,3,4,6-pentagall oyl-O-D-glucose) (Zalewska et al. 1995)........................................................................................................................16 2-2. A depside bond which is formed between the phenolic group of the upper and the acid group of the lower gallic acid units (Mueller-Harvey 2001)...........................16 3-1. Changes in average gall ic acid concentration ( g/g GAE) for two ripeness stages (day 0 and day 4) affected by HWT with varying length of treatment times (0, 70, 90, and 110 min). Average values and standard error bars of triplicate samples for all treatments are represented..............................................................25 3-2. Changes in total hydrolyzable tannin concentration ( g/g) for two ripeness stages (day 0 and day 4) as a result of diffe rent lengths of HWT (0, 70, 90, 110 min). Average values and standard error bars of triplicate samples for all treatments are represented.........................................................................................................26 3-3. HPLC chromatogram of polyphenolics, gallic acid and four hydrolyzable tannins (HT), present in mango flesh at Day 0 and Day 4. Peaks were tentatively identified based on retention time and spect ral similarities against an authentic standard of gallic acid..............................................................................................28 3-4. Changes in TSP (Total Solu ble Phenolic) concentration ( g/g GAE) for two different ripeness stages (day 0 and day 4) effected by four different durations of HWT (0, 70, 90, and 110 min). Average va lues and standard error bars of triplicate samples for all treatments are represented...............................................29 3-5. Changes in total antioxidant capacity ( M TE/g) for two different ripeness stages day 0 and day 4) affected by four di fferent lengths of HWT (0, 70, 90, and 110 min). Average values and standard erro r bars of triplicate samples for all treatments are represented.......................................................................................31 4-1. Changes in average gallic acid c oncentration (mg/L) for CA1, CA2 and CA3 during ripening for fruit samples at gr een, mid, and full ripeness. The ripeness stages were green (on the day of harves t), mid (after 2 week CA storage), and full (the time fruit starte d deterioration) ripe...........................................................40 viii

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4-2. CA storage induced effects in average gallic acid concentrat ion (mg/L) with or without HWT. Control was expres sed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2)............................................................................................................42 4-3. Changes in average total hydrolyzable tannin concentration (mg/L GAE) for CA1, CA2, and CA3 during mango ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA stor age), and full (the time fruit started deterioration) ripe....................................................................................................43 4-4. CA storage induced change in aver age HT concentration (mg/L GAE) in mango with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2)............................................................................................44 4-5. Gallotannin biosynthesis via meta-d epside bond by esterification. Depending on the number of gallic acid to glucose, gallotannin have 5 different structure such as -glucogallin, 1-6-Digalloylglucose, 1,2,6-Trigalloylglucose, 1,2,3,6Tetragalloylglucose, and 1,2,3,4,6-Pentag alloylglucose. (Grundhfer et al., 2001)........................................................................................................................45 4-6. Change in average total soluble phenolics (TSP) ( g/g GAE) in mango for CA1, CA2 and CA3 during ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA stor age), and full (the time fruit started deterioration) ripe....................................................................................................47 4-7. Changes in average total soluble phenolics ( g/g GAE) as a result of CA storage (Air, O2, and O2+CO2) with or without HWT. C ontrol was expressed as CA1 (21% air + 97% N2) and two CA storages we re shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2)........................................................48 4-8. Change in average antioxidant capacity (Trolox Equivalent M TE/g) for CA1, CA2 and CA3 determined by ORAC (O xygen Radical Absorbance Capacity) assay during fruit ripening. Each stage re presents green (on the day of harvest), mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe.49 4-9. Antioxidant capacity (Trolox equivalent M TE/g) effected by CA storage with or without HWT. It is measured by ORAC (Oxygen Radical Absorbance Capacity) assay........................................................................................................................50 4-10. The CIELAB color space system. L* represents lightness and a*, and b* show the strength of own colors (a: red to green and b: yellow to blue) (Lpez and Gmez, 2004)..........................................................................................................52 4-11. Average rate of reducing lightness (L *) for HWT during fruit ripening. Each stage represents mid (after 2 week CA st orage) and full (the time fruit started deterioration) ripe....................................................................................................52 ix

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4-12. Reduction rate of average hue angle (o) for HWT depends on CA storage air composition during ripening. Each stage represents mid (after 2 week CA storage) and full (the time fruit started de terioration) ripe. Hu e angle was defined by the coordination a* and b*.................................................................................53 4-13. Reduction in average chroma for HW T depends on CA storage air composition during ripening. Each stage represents mi d (after 2 week CA storage) and full (the time fruit started deteriorati on) ripe. Chroma was defined by the coordination a* and b*............................................................................................54 4-14. Changes of average hue angle (o) affected by CA storage with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2)....55 4-15. Changes of average chroma affected by CA storage with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2)....56 4-16. Change of average titratable acid ity (%) for CA1, CA2 and CA3 during mango ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe..............................57 4-17. Change of titratable acidity (%) in mango affected by CA storage with or without HWT. Titratable acidi ty was measured twice at 10 C (mid ripe) and 25 C (full ripe)........................................................................................................58 4-18. Effect of the fruit ripening on average soluble solids content ( Brix) for all CA storage and HWT in mangos. Each stage repr esents green (on the day of harvest), mid (after 2 week CA storage) and full (the time fruit started deterioration) ripe..59 4-19. Average soluble solids content affected by CA storage with or without HWT. Soluble solids content was measured twice at 10 C (mid ripe) and 25 C (full ripe).........................................................................................................................6 0 x

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHANGES IN POLYPHENOLICS AND RE SULTANT ANTIOXIDANT CAPACITY IN TOMMY ATKINS MANGOS ( Mangifera indica L.) BY SELECTED POSTHARVEST TREATMENTS By Youngmok Kim August 2005 Chair: Stephen T. Talcott Major Department: Food Science and Human Nutrition Mango ( Mangifera indica L.) is one of the most important tropical fruits worldwide and is gaining popularity in the US. The demand of mangos far exceeds domestic supply thus extending markets for fr uit import, whereby a hot water treatment (HWT) is required against invasive pests. Additionally, controlle d atmosphere (CA) storage is sometimes used to preserve quali ty and extend shelf-life of mango fruit during transportation and storage. Post harvest studies with fresh mangos have primarily focused on quality characteristics and limited data ex ist for fruit phytochemicals and antioxidant content and their resultant ch anges during postharvest hand ling and ripening. Therefore, these studies were conducted to investigat e phytochemical and antioxidant changes in fresh mangos as influenced by the stage of fr uit ripeness for the a pplication of CA in combination with a HWT. The first objective was to quantify phytoche mical changes and resultant antioxidant capacity in mangos during 4 days storage by varying lengths of hot water immersion xi

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treatment before storage. In this study, unripe mangos (Tommy Atkins) were first subjected to three different HW T (0, 70, 90, and 100 min all at 46 C) and then stored at 25 C for 4 days. Phytochemical and antioxidant content was monitored after each HWT and throughout storage. Hot water treatment at 46C for 70 min increased total hydrolyzable tannin (30%) but did not induce significant changes in gallic acid, total soluble phenolics and antioxidant content. Hot water treatments of >70 min did not affect fruit antioxidant content but resulted in decreas es of gallic acid (40%), total hydrolyzable tannins (35%) and total solubl e phenolics (25%). No remarkable phytochemical changes associated with ripening were dete cted during the 4 days storage. The second objective was to identify and quantify pol yphenolics and resultant antioxidant capacity in mango following contro lled atmosphere (CA) storage combined with HWT. In this study, unripe mangos were held under three CA conditions with or without a HWT (46C for 75 min). Controlled atmosphere treatments included CA1 (21% O2 + 79% N2), CA2 (3% O2 + 97% N2,) and CA3 (3% O2 + 10% CO2 + 87% N2) for 2 weeks at 10 C. Half of the fruit was immediatel y collected for analysis after CA storage while remaining fruits were held at 25C until fully ripe. Analyses for this study included individual and total phenolics by HP LC, total soluble phenolics (Folins assay), antioxidant capacity (ORAC assay), soluble so lid content (SSC), titratable acidity (TA), and flesh color. Hot water treatment at 46C for 75 min before CA storage did not change the phytochemical and antioxidant content of mangos. Fruit in CA1 and CA2 contained equivalent phytochemical and antioxidant le vels while CA3 fruit contained significantly higher amounts of gallic acid (12%), hydrolyzab le tannins (15%), to tal soluble phenolics 18%) and antioxidant content (13 %) compared to CA1 (P < 0.05). xii

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CHAPTER 1 INTRODUCTION Mango ( Magnifera Indica .L) is a popular tropical fruit all over the world due to its luscious color, characteristic taste, and excellent nutritional value. Since mango imports to the US have grown due to greatly incr eased demand and mangos have come down in price, the mango market has shown a rapid growth. For example, between 1996 and 2004, there was 40% increase in mango imports to the US (Saco, 2004). Even though several cultivars such as Tommy Atkins, Keitt and Ke nt are commercially cultivated in Florida and Hawaii, the production covers less than 1% of domestic consumption (2,800 MT) in 2004 (FAO, 2004). Now, more than 99% of mangos (278,422 MT) are imported mainly from Mexico (about 70% in 2000) followed by Brazil, Ecuador, Peru, Haiti and Guatemala (Saco, 2004). Mango is a valuable source of phenolic com pounds that have bitter and astringent taste that improve the characteristic taste of foods and develop food quality such as color and flavor (Grundhofer et al., 2001; Haard and Chism, 1996). Phenolic compounds are the most widely distributed secondary metabol ites in all higher plant, which have a phenol with one or more hydroxy substitutions (Hagerman et al., 1998; Robbins, 2003). Several studies reported phe nolic compounds found in mang o such as quercetin, iso querectin, kaempferol, mangiferin, gallic acid, m-digallic acid, m-trigallic acid, ellagic acid, and hydrolyzable tannin such as gallota nnin (El-Ansari et al., 1969; El-Sissi and Saleh, 1965). Especially, gallic acid and hydrolyzable tannins are known as major phenolic compounds found in mango. 1

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2 Oxidation is a fundamental process for th e cells in the body. However, there is a critical side effect of oxi dation, generation of free radi cals and other reactive oxygen species (ROS) such as peroxyl radical (ROO ), hydroxyl radical (HO superoxide ion (O2 ) and singlet oxygen (1O2) (Lpez et al., 2003). Free radicals involving oxygen are considered ROS and most ROS are classified as free radi cals (Karakaya et al., 2001). Since free radicals have unpaired electron(s) and continuously find another electron to be stable, important cellular components in th e body such as DNA and cell membrane could be damaged because cellular respiration is inhibited (Antolovich et al., 2001). However, antioxidant compounds mainly found in fruits and vegetables prot ect the body from free radicals and other ROS. Polyphenolic compounds abundantly found in mango not only provide various functionalities in foods but also act as a strong antioxidant in the body. Phenolic compounds have a strong antioxidant capacity in quenching and/or neutralizing free radicals by donating hydrogen to reactive free radicals (Karakaya et al., 2001; Fernndez-Pachn et al., 2004). Since mango is a climacteric fruit, which implies a highly perishable nature, several postharvest treatments have been used to k eep the quality and exte nd shelf-life. Mangos from other countries can be infested by fruit f lies such as Tephripid fruit flies, so all the imported mangos must go through a thermal qua rantine treatment such as a hot water treatment (HWT) to eliminate invasive pests before importing to the US (Jacobi et al., 2001). Controlled Atmosphere (CA) storage is us ed for fruits and vegetables to prolong their shelf-life and preserve them from quality deterioration (Sanchez-Mata et al., 2003). Since most of mangos consumed in the US (99%) are imported and transportation usually takes a few days to a few weeks, applica tion of CA storage must be considered.

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3 Limited study has shown the relationship between postharvest treatments and polyphenolics in mango, but mostly physiological changes such as color, firmness, shelflife and ripening rate. Thus, two studies were designed to investig ate how postharvest treatments affect polyphenolics and antioxi dant capacity in mango. The first study was conducted to figure out the effect of HWT on mango polyphenolics and resultant antioxidant capacity when mangos were subjec ted to HWT with three different treatment times. The second study was conducted to find out phytochemical changes and resultant antioxidant capacity by CA storage of mango combined with HWT. Controlled atmosphere storage having two different ga s compositions was applied to the mangos with a control for 2 weeks. The hypothesis was hot water immersion treatment with varying lengths of time and CA storage with different ga s composition combined with HWT will induce phytochemical and antio xidant capacity changes in mango. The specific objectives in th is study were as follows: 1. To quantify phytochemical changes and re sultant antioxidant capacity in mangos during 4 days storage by varying lengths of hot water imme rsion treatment (0, 70, 90 and 110 min) applied before storage. 2. To identify and quantify polyphenolics and re sultant antioxidant capacity in mangos following different CA treatments combined with or without hot water immersion treatment.

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CHAPTER 2 LITERATURE REVIEW 2-1. Mango Market Mango ( Magnifera indica L) is widely considered as the king of tropical fruits due to its world-wide popularity, production, an d acreage. Additionally, it is a valuable and economically important tropi cal fruit throughout the world due to its vibrant colors, characteristic taste, and nutri tional composition. Fresh fruit im ports to the US and Europe have increased due to increased consumer demands for fresh and processed mango products. In turn, mangos have become an af fordable tropical fru it that has found favor with consumers in a variety of foods and beverages. Mango has been cultivated for about 4,000 years and its production and consumption has gradually increased as its popularity grows. Originating over 4,000 years ago in India and Burma, its cultivati on has spread to Malaysia, Eastern Asia, and Eastern Africa (Mitra and Baldwin, 1997). A ccording to history records in the US, mangos began to be cultivated in Cape Sa ble, Florida in 1833 (Crane and Campbell, 1994; CRFG, 2001). Now, at least 87 count ries are known to grow over 26,286,255 MT in 2004 throughout the world (Food and Agricultural Organization [FAO], 2004; Sauco, 2004). Mango production is highest in Indi a, the leading mango producing country at 41% of the worlds production (10,800,000 MT ), followed by China, Thailand, Mexico, Pakistan, Indonesia, the Philippines, Nigeria, and Brazil (FAO, 2004) (Table 2-1). Other mango producing countries such as Australia, Peru, Venezu ela, Haiti, and Dominican Republic also produce and export mangos (DA-AMAS., 2003; Jacobi et al., 2001; 4

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5 Mossler et al., 2002; Sauco, 2004) (Table 2-1) Asia produces 76.9% of the total world production followed by the Americas (13.8%) and Africa (9%) (Sauc o, 2004). Mexico is the leading mango exporter c ountry at 41% of the world market (102,500 MT), followed by Philippines (7.8%) and Pakistan (7.6%) (Sauco 2004). The worlds largest mango importing country is the US (Table2-2) and the mango market in th e US has steadily grown in response to increasing demand. Mexi co is also the lead ing mango exporter to the US market followed by Brazil, Ecuador Peru, Haiti and Guatemala (Sauco, 2004). Even though the history of mango production and consumption in the US is relatively short compared to other countries such as Eu rope and China, now th e US is the largest mango importing country due to great demand in the domestic mango market. Table 2-1. Main mango producing c ountries in 2004 (www.fao.org) Rank Country Production (MT) 1 India 10,800,000 2 China 3,400,000 3 Thailand 1,750,000 4 Mexico 1,503,010 5 Pakistan 1,072,000 6 Philippines 890,000 7 Brazil 845,000 8 Indonesia 800,000 9 Nigeria 730,000 10 Vietnam 337,000 11 Egypt 327,000 12 Haiti 261,000 13 Bangladesh 243,000 14 Cuba 235,000 15 Madagascar 210,000 16 Democratic Republic of the Congo 200,000 17 Sudan 195,000 18 United Republic of Tanzania 195,000 19 Guatemala 187,000 20 Dominican Republic 180,000

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6 Table 2-2. Global imports of mangos (MT) (Sauco 2004) Country 1998 1999 2000 USA 197,000 219,000 235,000 Europe 115,000 170,000 172,000 Hong Kong 47,000 33,000 33,000 UAE 39,000 38,000 38,000 Malaysia 21,000 25,000 25,000 Saudi Arabia 14,000 14,000 14,000 Singapore 11,000 14,000 15,000 Japan 9,000 9,000 10,000 Since mangos are grown in tropical climate, they are also cultivated in the southern states in the US. However, Fl orida is the only state in the United States where agricultural statistics are reported for mangos even t hough mangos are also cultivated to various extents in Hawaii, California, Texas, and Puerto Rico (Mossler and Nesheim, 2002). More than 80% of Florida mangos are cult ivated in Miami Dade County with the remaining acreage in Lee, Palm Beach, and scattered other counties where adequate conditions for growth exist (Mossler et al., 2002). Recently, new mango trees are being planted in Merritt Island in Florid a (Mossler and Nesheim, 2002). Before Hurricane Andrew in 1992, mango production in Florida was over 10,000 MT from 1172 ha (Crane and Balerdi, 1997). However, the production was reduced to 1,250 MT from 648 ha. Recovery from damage was poor and slow because many remaining trees continued dying, damaged bark was uncovered from excessive sunlight and roots of trees got damaged (Crane and Balerdi, 1997). As a result of poor recovery, Florida production is still le ss than 1% of total consumption in the US (HAP, 2002; Sauco, 2004).

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7 2-2. Mango Cultivars Among over 1,000 different mango cultivars throughout the world, about 800 mango cultivars have been named (Pandey, 1986). Since each mango growing country possesses different climate, geological featur e, harvest time, and marketing season, each country generally has its own major cultiv ars for commercial use (Nakasone and Paul, 1998; Sauco, 2004) (Table 2-3). For example, Australian production is dominated >95% by a single variety Kensington (also known as Kensington pride) and Tommy Atkins and Keitt are the major varieties grown in Florida (Crane and Campbell, 1994; Jacobi et al., 1998). Since many cultivars have their own characteristics, each mango is cultivated according to their regional and climatic conditions in their respective countries. Mango may be divided into two types, which are Indian and Indo-Chinese. Since two types have quite different features, this is a good indicator to classify mangos. For example, Indo-Chinese type mangos have polyembryonic seeds that have multiple embryos while Indian type mangos have monoembryonic seeds (Crane and Campbell, 1997). Indo-Chinese type mango has better resistance to anth racnose caused by Colletotrichum gloeosporioides Penz which is the most important fruit disease in Florida (Crane et al., 1997; Gamagaea et al., 2004; Vivekanant han et al., 2004). Since Tommy Atkins mango is an Indo-Chinese type, it is relatively resistan t to anthracnose. Even though it has been reported that a bout eighty mango culti vars are found in Florida, only a few varietie s are commercially cultivated. 'Tommy Atkins', 'Keitt', 'Van Dyke', 'Palmer', 'Kent', 'Haden', 'Sensation', and 'Parvin' are commercially grown varieties in Florida (Crane and Campbell, 1994; Sauc o, 2004). Especially, 'Tommy Atkins' and 'Keitt' accounts for >50% of the total production in Florida (Campbell, 1992; Morton, 1987). 'Tommy Atkins' is oval in shape (8 to 15 cm in diameter) and its size ranges from

PAGE 20

8 medium to large at 450 to 750g with a thick, toug h adherent skin that is yellow to orange in color (Gilman and Watson, 1993). Even t hough Tommy Atkins has relatively poor eating quality (rated fair to good) and has fibrous pulp, it is the most widely distributed and commercially variety produced in Florid a due to great resistance to anthracnose, tolerance to handling damage and endurance to hot water immersion treatment (Campbell and Campbell, 1993). Table 2-3. Producing countries, selected cultiv ars, and main marketing season (Nakasone and Paull, 1998) Country Selected Cultivar Marketing on Seas USA-Florida Keitt, Irwin, Tommy Atkins, Kent, Van Dyke, Palmer Jul-Aug Australia Kensington Pride, Keitt, Kent, Palmer, Irwin Oct-Mar India Alphonso, Banganpalli, Dashehari, Bangalora, L Mulgoa, Neelum, Pairi angra, Apr-Jul Brazil Haden, Tommy Atkins, Kent, Keitt, Palmer, Bourbon, Espada, Itamarco, Caco, Rosa, Car lota Oct-Feb Indonesia Arumanis, Dodol, Gedong, Golek, Cengkir Sep-Jan Israel Keitt, Tommy Atkins, Kent, Maya, Haden Jul-Aug Malaysia ) Harumanis, Golek, Maha 65, MA 200 (Malgoa Jun-Aug Mexico Haden, Manila, Esmeralda, Kent, Keitt, Tommy Atk Jan Dyke, Palmer ins, Apr-Oct The Philippines Carabao, Pico, Julie Jun-Sep South Africa Peach, Zill, Fascell, Sensation, Tommy Atkins, Keitt Nov-Jan Spain Tommy Atkins, Keitt, Lippens, Osteen Jul-Aug Taiwan Irwin, Yellow No.1, Haden Jul-Oct Thailand Nan Dok Mai, Rad, Tongdum, Okrong Mar-May ipening Ripening is a com any physiological changes but many chemical changes due t st fruits continue to respire. Mang 2-3. Mango R plex process that invol ves not only m o the fact th at postharve o is a climacteric fruit, which means that its respiration rate ri ses as fruit ripening proceeds in response to ethylene (Alexande r and Grierson, 2002; Silva et al., 2003).

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9 Physiological changes occur as a result of chem ical alterations in climacteric fruits durin ripening such as a change in pulp and skin color (loss of chlorophy lls and synthesis o carotenoids), loss of weight and volume (loss of moisture), decline of firmness (softening caused by pectin breakdown), altered taste (loss of acidity and in creased soluble solids due to conversion of starch to sugar) and s ynthesis of more ethylene (a ripening hormone) (Jacobi et al., 1998; Kader, 1997; Lizada, 1991; Modi and Reddy, 1967). Since mangos are harvested when they are still green as a climacteric fruit, they are expected to withstand postharv est handling, and then complete their ripenin g f g (riper stages gener uit was e of ally preferred by consumers) at the fres h market, determining exact ripening indices are important for market consideration. Chemi cal parameters may include soluble solids contents, acidity, starch content, and phenolic constituents while physical parameters may include shape, size, surfa ce color, and shoulder growth (Mitra and Baldwin, 1997). The most definitive indicator of a mangos maturity is to determine shoulder growth (from green to mid-ripe) and color development (alm ost all yellow color pulp) (Holmes et al., 1990; Jacobi et al., 1998). Even if mangos ar e of the same variety and harvested on the same day, they could be different from each other, for example, their shape, color development, and weight. This is becaus e the amount of sunli ght, rain, and other environmental factors are different, depending on location on a tree even though fr grown on the same tree. In conclusion, accurate standards to distinguish the degre maturity as well as the degree of ripeness ar e important for mangos to adjust time for the fruit market.

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10 2-4. Mango Postharvest Handling 2-4-1. Hot Water Immers ion Treatment (HWT) Thermal quarantine treatment is legally required for fresh mangos to be accepted by several importing countries such as the United States and Japan because imported mangos are highly susceptible to infection by frui t flies in the form of adults, larvae, or eggs. In the US, mangos from other countries must go through a thermal quarantine treatment to eliminate invasive pests (e.g. Ceratitis capitata (Mediterranean fruit fly) and Anastrepha spp., Anastrepha ludens (Mexican fruit fly)) before importing to the US (APHIS, 2002). Several methods for quarantin e treatments are available for fruits including irradiation, vapor heat, forced hot -air, and hot water immersion treatments (APHIS, 2002). However, only hot water immers ion treatment (HWT) is approved in the US to disinfect mango; not only is HWT the mo st effective treatment, but it has several direct advantages (Jacobi et al., 2001). Advantages include ease of handling and strict control of treatment environments, which a llows for exact temperature control during treatment, and less cost as an additional benefit of HWT (about 90% less than vapor heat treatment) (Sharp and Hallman, 1994; Fallik, 2004). Required HWT conditions such as treatment time, temperature, and water purity are rigidly and specifically developed and controlled by USDA-APHIS (Animal and Plant Health Inspection Service) because fru it fly and/or its larvae and eggs may remain under a variety of storage conditions. The targ et disinfestation probability in eliminating pests in fruit is over 99.9968% (probit 9) in many countries (Jacobi et al., 2000). Since the profit value is so high, every condition for thermal quarantine must be controlled strictly. Legally required time and temperature for HWT in the US is 70, 90, or 110 min immersion at 46C, so the guidelines publishe d by USDA-APHIS sets these times based

PAGE 23

11 on cultivar, weight, size, shap e, and country of origin (U SDA-APHIS 2002) (Table 2-4). However, hot water treatment may damage the quality of mangos and cause more internal and external injury than vapor treatment if the treatment temperature is out of optimum range (Jacobi and Wong, 1992). Table 2-4. Determined dip time based on origin, shape, and weight of fruit. (USDAAPHIS, 2002) Origin of fruit Fruit shape Fruit weight (grams) Dip time (minutes) Up to 400 65 Flat, elongated varieties1 400 to 570 75 Up to 500 75 500-700 90 Puerto Rico, U.S. Virgin Islands, or West Indies Rounded varieties2 701-900 110 Up to 375 65 Flat, elongated varieties1 400 to 570 75 Up to 500 75 500 to 700 90 Mexico or Central America (North of and including Costa Rica) Rounded varieties2 701 to 900 110 Up to 375 65 Flat, elongated varieties1 375 to 570 75 Up to 425 75 Panama, South America or West Indies islands of Aruba, Bonaire, Curacao, Margarita, Tortuga, or Trinidad and Tobago Rounded varieties2 425 to 650 90 *1Cultivars: Frances, Carrot, Zill, Ataulfo, Carabao , Irwin, and Manila. *2Cultivars: Tommy Atkins, Kent, Heyden, and Keitt. An organized process for HWT was also set as a standard by USDA-APHIS. According to the Hot Water Treatment Sche dule (APHIS, 2002), mangos must be sorted by origin, weight and shape before HWT. Water quality for washing, dipping, or showering the fruit should be considered before treatment and the water must be chlorinated (50-200 ppm). During the treatment, the fruit must be submerged at least 4 inches (10.16 cm) below the wate r level at the target temperature. In addition, the water must be constantly circulated and the temp erature of the water should be kept at 46oC 0.3 C throughout the treatment. After HWT, ma ngos should stand for at least 30 minutes

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12 prior to refrigeration to ensure that all fru it fly larvae would be completely killed during this time. Recommended temperatures for refrigeration are 55-57 F (12.8-13.9 C) at 85 to 90% RH. This storage condition will delay softening and extend storage life to 2 to 3 weeks. 2-4-2. Controlled Atmosphere (CA) Storage During transport from an exporting country to an importing country by trucks and/or ships, fruits can easily perish unle ss they are held under proper storage conditions because transport usually takes from a few days to a few weeks (usually 2-3 weeks). Since the main mango exporter for the US mark et is Mexico followed by Brazil, Ecuador, Peru, Haiti and Guatemala, shipping mangos ma y vary according to the targeted countries. In order to protect quality, delay ripening, reduce ethylene production and respiration rate, prevent fruit disease, and extend shelf-lif e during shipping, CA storage is sometimes employed (Abd. Shukor et al., 2000; Rattanapa none and Watada, 2000). Domestic mango production satisfies a small portion (about 1%) of total mango consumption while the mango market is getting larger in the US. Since about 99% of mangos are imported, CA storage should easily be applie d to the US mango market as a means to extend shelf-life and keep the quality during transportation and storage. CA storage is a common method used to preserve fruits and vegetables from quality deterioration and to extend shelf-life. CA storage, an artifi cially increased CO2 and decreased O2 level environment at reduced temp eratures, decreases ethylene (ripening hormone) production, reduces sensitivity to ethylene and lessens respiration rate. Thus, it can prolong shelf-life of fruits, postpone chlo rophyll degradation, and keep the texture of fruit (Mitra and Baldwin, 1997; Kader et al., 1989; Kader, 2002). Since fruits are still

PAGE 25

13 alive even after harvest by c ontinuing to respire (consuming O2 and producing CO2), CA storage conditions are effective to inhibit respiration. CA storage also has additional benefits such as maintaining titratable ac idity, preserving fruit firmness, and reducing pitting (Wang and Vestrheim, 2002; Chen et al., 1981; Sanchez et al ., 2003). Therefore, CA storage can help maintain fruit quality deterioration and it also gives additional advantages to fruits such as extent po stharvest shelf-life and delayed ripening. The optimum O2 and CO2 composition and temperature in a CA chamber varies with the cultivar and grow ing region of fruits. In ge neral, optimum atmosphere composition for mature-green mangos is 3 to 5% of O2 and 5 to 8% of CO2 at 12 to 13 C for up to 3 weeks and for tree-ripe mangos is 3 to 5% of O2 and 5 to 8% of CO2 at 8 C or 5% O2 and 10% CO2 at 5 C for 3 weeks (Bender et al., 2000). These recommended conditions vary due to natural variability of fruit and dynamic response to storage conditions (Saltveit, 2003). Too low O2 and too high CO2 levels in CA storage can induce anaerobic respiration and fr uit injury (Kader et al., 1989). Anaerobic respiration can cause shortening of shelf-life, increasing susceptibility to decay, producing off-flavors, and leading to physiological di sorders (Brecht, 1980; Kader et al., 1989). Furthermore, the symptoms of CO2 injury (too high CO2) are development of abnormal and grayish color, prevention of normal aroma developmen t, production of off-fl avors, and generation of grey spots. The symptom of O2 injury (too low O2) is irreversible inhibition of ripening (chlorophyll decline) (Bender et al., 2000.; Mitra and Baldwin, 1997). Keeping the atmosphere concentration in the optimum ra nge in the CA chamber is important to prevent anaerobic respiration and fruit inju ry. Thus, atmosphere composition has to be monitored and adjusted to mainta in optimum gas concentration.

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14 2-5. Mango Polyphenolics Mango is a source of phenolic compounds th at improve food quality, and prevent food deterioration. Those are gallic acid, gall otannin, p-OH-benzoic ac id, and ferulic acid, which are found in mango pulp and skin (Sch ieber et al., 2000). Phenolic compounds are the most widely distributed plant secondary me tabolites and are found in all higher plants (Hagerman et al., 1998). Basically, they have one common structural feature, which is a phenol (an aromatic ring possessing at le ast one hydroxyl substituent) (Robbins, 2003). Phenolics may be divided into two cate gories that include polyphenols and simple phenols based on the number of phenol subunits present, for example, polyphenolics have at least two phenol subunits a nd tannins have at least three phenol subunits. Phenolic acid is a phenol that has one carboxylic acid functio nal group and is related to color, sensory quality, and antioxidant capacity of foods (Robbins, 2003). Polyphenolics are abundantly found in fruits and vegetables and are important compounds in reducing risks of human diseases and oxidative damage to biological membrane (Kelly et al., 2002; Robbins, 2003). Thus, it is important for human to increase consumption of polyphenolics with antioxidant properties by eating fruits and vegetables (Kang and Saltveit, 2002). Moreover, polyphenolics provide various func tionalities, such as color, flavor, and astringency in foods, and polyphenolics are likel y to be involved in the plant defense mechanism as a chemical barrier, helping the plant to recover from injured surface (Grundhofer et al., 2001; Haard and Chism, 1996). Technically, phenolics that have three or more phenol subunits and have an ability to precipitate proteins are categorized as tannin and are categorized as either condensed or hydrolyzable tannins (Hag erman, 2002; Robbins, 2003). Tannins are a specific class of polyphenolics that have b een identified, but poorly characterized in

PAGE 27

15 mangos. Generally, tannins have high molecu lar weights (>1000 amu) and can be found in abundance in oak, tea, sumac, nuts, and some fruits. Several va luable functions of tannin include protein precipitation, cell wall stabilization, insecticid es, herbicides, and anti-carcinogenic agents due to their antioxidant capacity (Hartzfeld et al., 2002; Osslpov et al., 1997; Werner et al., 1999). Tannin ha s about 15-30 times better peroxy radical quenching capacity than simple phenol a nd Trolox because it has relatively many hydroxyl groups and it is highly polymerized (Hagerman et al., 1998). Condensed tannins are polymeric forms of (+)-catechin and (-)-e picatechin and/or vary from dimers to considerably larger polymeric procyanidins, due to their ability to form cyanidin or delphinidin (prodelphinidins) in the presence of acid and heat. There are two kinds of hydrolyzable tannins: gallotannin and ellagic ac id which are derivatives of gallic acid (Hagerman, 2002). Gallic acid (3,4,5-trihydroxybenzoic acid) is an abundant polyphenolic compound found in mango with gallotannins and it is deri ved from quinic acid via the shikimic acid pathway (also known as the phenylpropanoid path way). Gallotannin is the simplest form of hydrolyzable tannin. As a polygalloyl ester of glucose, gallotannin has five hydroxyl groups attached to the core sugar (glucose ) (Hagerman, 2002) (Fig 2-1). Gallotannin is divided into two parts, glucose and gallic acid, upon hydrolysis with strong acid and is extended by adding galloyl esters via meta-d epside bonding to glucose (Hagerman, 2002; Niemetz et al., 1999) (Fig 2-3).

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16 Figure 2-1. Structure of gallotannin ( -1,2,3,4,6-pentagalloyl-O-D-g lucose) (Zalewska et al. 1995) Figure 2-2. A depside bond which is formed be tween the phenolic group of the upper and the acid group of the lower gall ic acid units (Mueller-Harvey 2001) Many polyphenolics in mango were prev iously identified. Quercetin and kaempferol were the first polyphenolics to be tentatively identified in mango using 2dimensional paper chromatography (ElAnsari et al., 1969). Subse quently, El-Ansari et al. (1969) reported the presence of ellagic acid, gallic acid (GA), m -digallic acid, m trigallic acid, isoquercetin, mangiferin, querc etin and gallotannin in mango. Gallic acid and gallotannin are known as the major polyphenolic compounds found in mango, which are important antioxidants to quench harmful free radicals (Saleh and El-Ansari,1970).

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17 2-6. Polyphenolics as Antioxidants Oxygen is necessary for human beings and other living organisms to function and the oxidative mechanism is necessary for the cells to survive in the body. However, when oxygen is transformed to free radicals and ROS (reactive oxygen species), which then act as oxidants that have a tendency to donate their oxygen to other materials, oxygen plays a harmful role in our body (Karakaya et al ., 2001). Many ROS are free radicals. Free radicals and other ROS such as peroxy radi cal, hydroxyl radical, singlet oxygen derived from normal essential metabolic process or ot her external sources can harm all kinds of biological materials such as proteins, carbohydrates, lipids, an d nucleic acid (Karakaya et al., 2001; Fernndez-Pachn et al., 2004). In the human body, ROS and free radicals can affect the formation of biological membrane s, change enzyme act ivity, induce abnormal organ metabolism, and influence the genera tion of cataracts, atherosclerosis, and degenerative diseases (Karakaya and Nehir, 1999; Leitao and Mensor, 1999). ROS and free radicals cause oxidation that induces dete rioration of food, re sulting in rancidity, changes in color, and declines in nutri tional quality, flavor, texture and safety (Antolovich et al., 2002). Fortunately, antioxidants such as ascorb ic acid, tocopherol, carotenoid, and polyphenolics can quench and neutralize free radicals (Shadidi and Naczk, 1995). Consequently, antioxidant capacity also reduces the possibility of several diseases like cancer, stroke, atherosclerosis, and cardi ovascular diseases (Karakaya et al., 2001; Shadidi and Naczk, 1995). Antioxidant capacity of phenolic compounds comes from their redox properties in acting as a reducing agen ts, hydrogen donator, metal chelator and singlet oxygen quencher (Pyo et al., 2004). Even though there are other antioxidant compounds in fruits and vegetables such as ascorbic acid, tocopherol, carotenoids, and

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18 lycopenes, phenolic compounds have stronger radical quenching capacity (Pyo et al., 2004; Toit et al., 2001). For example, fla vonoid including multiple functional phenolic groups has 2 to 5 fold stronger radical quenching capacity than ascorbic acid and tocopherol (Toit et al., 2001).

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CHAPTER 3 PHYTOCHEMICAL CHANGES AND RESU LTANT ANTIOXIDANT CAPACITY IN MANGOS DURING 4 DAY STOGRAGE AFFECTED BY VARYING LENGTHS OF HOT WATER IMMERSION TREATMENT 3-1. Introduction Mango ( Magnifera indica L.) has been a popular and economically important tropical fruit throughout the world due to its excellent eating quality (bright color, sweet taste and luscious flavor) and nutritional composition (diverse amount of vitamins, minerals, fiber and various antioxidant compounds). Fresh fruit imports to the US, Europe, and Japan have increased due to increased consumer demand for fresh and processed mango products. In turn, mangos have become an affordable tropical fruit that has found favor with consumers in a variety of foods and beverages. In the US, although mangos are commercially produced in Florid a and Hawaii, the amount of production is not enough to meet domestic demands. Thus, mango importation in the US has gradually increased in the market. Since imported mangos can easily be a host for Ceratitis capitata (Mediterranean fruit fly) and Anastrepha spp., Anastrepha ludens (Mexican fruit fly) causing quarantine risks in the fo rm of adults, larvae, or eggs (Jacobi et al., 2001; USDAAPHIS, 2002), all mangos imported to the US must be subjected to a thermal quarantine treatment to eradicate invasive pests. Several methods for quarantine are available such as irradiation, fumigation, vapor he at, forced hot-air, and hot water immersion treatments. However, only the hot water immersion treatment is legally allowed in the US because it is the most effective quarantine met hod for mangos (Jacobi et al., 2001). 19

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20 Numerous studies have addressed physio logical changes (e.g. heat injury, heat tolerance, ripening velocity, and shelf-life) caused by hot water immersion treatment (HWT). Polyphenolic compounds such as gal lic acid and hydroly zable tannin are found in mango and are important because these phytochemicals have a strong antioxidant capacity and therefore serv e to improve food quality. However, limited information concerning phytochemical changes after HWT is available. This study concentrated on phenolic compound changes caused by HWT with varying length of treatment time and resultant antioxidant capacity. The objective of this study was to identify and quantify polyphenolic compounds and antioxidant capacit y in fresh mangos after three HWT times for two ripeness stages and its affect on fruit quality. 3-2. Materials and Methods 3-2-1. Fruit Preparation and Treatment Mangos for this study were obtained from Lyons Farms in Homestead, FL in June 2002. Forty mature-green mangos were chosen ba sed on the uniformity of color, size, and weight. Fruits were transported on the day of harvest to the University of Florida and subjected to HWT with a laboratory scale fruit heating system (Model HWH-2, Gaffney Engineering, Gainesville, FL). All mangos were stored for 2 days at 14C prior to the treatment. The mangos were randomly divided in to four groups and the first three groups were immersed into a hot water bath fo r 70 min (HW70), 90 min (HW90), or 110 min (HW110) at 46C. The fourth group remained unt reated to use as a control (HW0). Since median weight of sample mangos was 497.5g, required treatment time was 75 min in accordance with the direction of APHIS tr eatment manual for mangos (USDA-APHIS, 2002). In this study, 70 min, the nearest time to 75 min, was determined as required treatment time for a regular 20 min interv al between treatments (70, 90 and 110 min).

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21 Because treatment time usually varies within a range of 10 min according to hydrocooling rate and the median was le ss than 500g, 5 min difference might not influence on the result caused by HWT. Samples were acquired for immediate analysis after HWT while remaining fruit was held at 23oC for an additional 4 days prior to obtaining analysis samples. A total of eight een mangos were evaluated for each of the four treatments and included a control, divided into five groups with the edible flesh of three mangos pooled for analysis. The sixth gr oup was utilized to measure fruit core temperature and they were not included in the dataset. Core temperature was obtained by placing a thermocouple inside the fruit during treatment. Table 3-1. HWT with four different lengths of time (0, 70, 90, and 110 min) at 46 C with different ripeness stages (day 0 and day 4) at 23 C Treatment Period HW 0 (control) HW 70 HW 90 HW 110 C-1 H70-1 H90-1 H110-1 C-2 H70-2 H90-2 H110-2 C-3 H70-3 H90-3 H110-3 C-4 H70-4 H90-4 H110-4 C-5 H70-5 H90-5 H110-5 Day 0 & Day 4 C-6 H70-6 H90-6 H110-6 Total D0-18, D4-18 D0-18, D4-18 D0-18, D4-18 D0-18, D4-18 Mango skins were manually peeled and fl esh blended using a laboratory blender (Waring commercial, Model 31BL91) for 5 min to obtain a consistent puree and held at 20C until analysis whereby the samples were thawed for chemical analysis. A 5g puree was treated with 20l of pectinase (Pectinex Ultra SP-L from Aspergillus aculeatus SIGMA chemical Co.), then incubated at 35C for 3 hours, and centrifuged until a clear

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22 supernatant (clarified mango juice) was obt ained from which phytochemical analyses were conducted. 3-2-2. Identification and Quantifi cation of Individual Polyphenolics Individual polyphenolics were identified and quantified using HPLC as described by Talcott et al. (2000). Clarif ied juice isolates were filt ered through a 0.45 m PTFE filter (Whatman, Clifton, NJ) and then injected into a Waters 2695 Alliance chromatography system. Compounds were se parated on a Waters Spherisorb ODS 2 column using a gradient elution program. Mobiles phases consisted of Phase A (98% H2O : 2% Acetic acid) and Phase B (68% H2O : 30% Acetonitrile : 2% Acetic acid) run at 0.8 mL/min. Polyphenolics were separated usi ng a gradient elution system that kept mobile phase B 0% for 5 min and then cha nged phase B 0% to 10% in 20 min; 10% to 25% in 30 min; 25% to 50% in 40 min; 50% to 75% in 50 min; 75% to 100% in 70 min and returned to original c ondition in 2 min for the next injection. Polyphenolics were detected and quantified at 280 nm using a Waters 996 photodiode array (PDA) detector against an external standard of gallic acid. Unknown compounds were characterized based on retention time and UV spectral similarities to authentic standards (Sigma Chemical Co., St. Louis, MO) using Millennium 32R workstation. 3-2-3. Quantification of Total Soluble Polyphenolics Total soluble phenolic (TSP) concentration (a measure of total metal ion reducing capacity) including contributions from ascorbic acid was determined by Folin-Ciocalteau assay (Swain and Hillis, 1959). For each clarified ju ice isolate, 50 L mango juice was added to 1 ml of 0.25 N Folin-C iocalteu reagent and mixed by vortex for 30 sec. After 3 minutes reaction time, 1 ml of 1N sodium carbonate was added to form a water-soluble

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23 chromophore for a distinguishable blue colo r. After standing for 7 minutes, all the extractions were transferred to a Spectr amax 96-well and absorbance (Softmax PRO, Sunnyvale, CA) was read at 726 nm after 2 hour s. TSP was quantified in mg/L gallic acid equivalents (GAE). 3-2-4. Quantification of Antioxidant Capacity Total antioxidant capacity of ma ngo phytochemicals (from polyphenolics and ascorbic acid) was measured using the ORAC assay (oxygen radical absorbance capacity) run according to Talcott et al. (2002) and adapted to work with a 96-well Molecular Devices fmax fluorescent microplate reader (485 nm excitation and 538 nm emission). This method is based on the principle of the inhibition of oxidation of a fluorescent compound (fluorescence) in the presence of th e peroxyl radical gene rator 2,2-azobis (2amidinopropane) dihydrochloride (AAPH). A stock solution of fluorescein was produced by mixing 10 l of stock fluorescent solution and 50 ml of phosphate buffer (61.6:38.9 v/v, 0.75 M K2HPO4 and 0.75M NaH2PO4, pH 7.2) as described by Ou et al. (2001) and the peroxyl radical generator was made by mixi ng 350mg of AAPH in 5 ml of phosphate buffer (pH 7.0). The rate of fluorescence decay was recorded every 2 min for 70 min by calculating the area under the fluorescence decay curve for a standard curve (0-50 a blank (ORAC = 0 ), and mango juice following appr opriate dilution in phosphate buffer. Antioxidant capacity affected by tr eatments was quantified against a standard curve of Trolox (6hydroxy,5,7,8tetramethylchromancarboxylic acid), a watersoluble analog of vitamin E (ORAC = 1 Trolox) with potent antioxidant properties (Javanovic et al., 1994). Trolox was used at the concentrations of 1.65, 3.125, 6.25, 12.5, 25, and 50 M by serial dilution with the phosphate buffer. Each extract was diluted 50

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24 fold in pH 7.0 phosphate buffer before pipet ting into a 96-well microplate. Data was represented in Trolox equivalents per mL of clarified mango juice. 3-2-5. Statistical Analysis Method Changes in phytochemicals and antioxidant capacity affected by HWT time and fruit ripening were analyzed by analysis of variance (ANOVA) usi ng JMP 5 statistical software (SAS Institute, 2001). Mean separation was conducted using the LSD test, P<0.05. 3-3. Results and Discussion 3-3-1. Individual Polyphenolic Concentration Free gallic acid and hydrolyzable tannin s were the major pol yphenolics identified in mango and quantified against an authentic st andard of gallic acid (Talcott et al., 2005). Average gallic acid concentr ation during the 4 days storag e was changed as a result of HWT (Fig 3-1). Gallic acid concentration at HW70 did not show significant difference from the control while concentrations at HW90 and HW110 were significantly lower (41% and 42% less, respectively) than the cont rol. Synthesis of gallic acid was inhibited by HWT only when treatment time is longer th an that required for quarantine treatment depending on cultivar, weight, shape, and origin (APHIS, 2002; Lurie, 1998). When fruits were treated with hot wate r for extended time, heat-shock protein might have been induced. Heat shock protei ns (HST) are unique proteins produced in response to heat-shock treatment (high temp erature and/or long time) (Saltveit, 1998). Heat-shock inhibited synthesis of p henylalanine ammonia-lyase (PAL), which is the first enzyme of primary phenolic pathway (phenylpropanoid pathway) (Loaiza-Velarde et al., 2003; Rivero et al., 2001; Saltveit, 1998; Saltveit, 2000a). Since PAL activity induces accumulation of phenolic compounds, gallic acid reduction might be affected by

PAGE 37

25 decreased PAL activity when mangos were treated with hot water for extended time (Saltveit, 2000b). Average gallic acid concentration for all HWT increased by 24% during 4 days storage (Fig 3-1). During the 4-day storage, gallic acid concentrations of control and HW70 did not change while the concentr ations of HW90 and HW110 significantly increased. At both day 0 and day 4, they showed a similar result, in which gallic acid significantly decreased only when they were treated for longer times (HW90 and HW110). Reduced gallic acid concentrat ion of HW90 and HW110 by extended time treatment might continue to recover during 4 day storage. Hot Water Treatment ControlHW70HW90HW110 Gallic Acid ( g/g) 2 4 6 8 10 12 14 16 Day 0 Day 4 A A A AB AB B C C Figure 3-1. Changes in average gallic acid concentration ( g/g GAE) for two ripeness stages (day 0 and day 4) affected by HW T with varying length of treatment times (0, 70, 90, and 110 min). Average values and standard error bars of triplicate samples for all treatments are represented.

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26 S duced phenolic compounds, but also induc d stages (day 0 and day 4) as a result of different lengths of HWT (0, 70, 90, 110 Four hydrolyzable tannins were tenta tively identified based on UV spectral simila tt et al., ns at ince extended hot water treatment not only re ed heat injury, it might be an undesire d process for fruit and vegetables storage. Reported symptoms of heat injury are a bnormal color development, odd softening, the lack of starch breakdown, and the developmen t of internal hollows in mango (Jacobi an Wong, 1992; Lurie, 1998). Figure 3-2. Changes in total hydro lyzable tannin concentration ( g/g) for two ripeness 0 10 20 30 40 50 min). Average values and standard erro r bars of triplicate samples for all treatments are represented rities to gallic acid due to the diversity of hydrolyzable tannin present (Talco 2005). As a result of the four different durations of HWT, the concentration of hydrolyzable tannins increased (33% more) only at HW70, but the concentratio Hot Water Treatment Day 0 Day 4 Hydrolyzable Tannin ( g/g GAE) A B C D E EF F EF ControlHW70HW90HW110

PAGE 39

27 HW90 and HW110 were lower (37 and 34% less, re spectively) than cont rol (Fig 3-2) was a similar result as gallic acid except th e concentration of hydr olyzable tannins at HW70 increased and was highest among all th e HWT. Since heat stress causes PAL activity that is a core enzyme of phenylpropanoid pathway in ca talyzing synthesis of phenolic compounds, hydrolyzable tannin concentration could be increased by therma stress (Rivero et al., 2001). Reduction of hydr olyzable tannin concentration at HW90 an HW110 may be induced by heat-shock as explained in gallic acid reduction. Phenolic compounds of mango were found to decrease dur ing ripening It l d with a loss of ast sari, 1970). f on in zable tannin peaks were eluted ot ringency related to loss of phe nolic content (El-Ansari et al., 1971; Lakshminarayana et al., 1970; Mitra and Ba ldwin, 1997; Saleh and El-An However, in this study, total hydrolyzable tannin concentration did not change while gallic acid concentration significantly increa sed during the 4 day stor age. No change o hydrolyzable tannin concentra tion could be explained from the fact that since HWT inhibits ethylene synthesi s, ripening might be delayed (Lurie, 1998). Thus, delayed ripening may be the reason for the lack of ch ange in hydrolyzable tannin concentrati mango even though there was a 4-da y storage time difference. As shown in Fig 3-3, identified gallic acid and four hydroly by HPLC at Day 0 and Day 4. According to peak area, about 30% of gallic acid increased during the 4-day stor age while on average the four hydrolyzable tannins did n show any difference. Changes of gallic acid and four hydrolyzable tannins were shown in Table 3-2.

PAGE 40

28 AU -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40Minutes 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40Minutes 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Day1 Day4 Gallic Acid Gallic Acid HT 1 HT 1 HT 2 HT 2 HT 3 HT 3 HT 4 HT 4 Figure 3-3. HPLC chromatogram of polyphenolics, gallic acid and four hydrolyzable tannins (HT), present in mango flesh at Day 0 and Day 4. Peaks were tentatively identified based on retention time and spectral similarities against an authentic standard of gallic acid Table 3-2. Gallic acid (g/g) and average of four hydrolyzable tannins (g/g GAE) identified and quantified using HPLC as a result of different duration of HWT (0, 70, 90, 110 min) at two ripeness stages (Day 0 and Day 4) Ripeness Stages HotWater Treatment Gallic Acid HT 1 HT 2 HT 3 HT 4 Total HT HW0 11.59a 4.88b* 8.69b* 6.76a 1.27b 21.6 HW70 9.39a 7.27a* 21.7a 7.42a* 1.13b 37.5 HW90 5.15b 5.26b 6.04c 1.10b 2.27a 14.7 Day 0 HW110 4.70b 5.17b 5.92c 1.87b 1.04b 14.0 HW0 10.84a 3.49a 12.84b 7.03a 1.50c 24.9 HW70 11.42a 3.79a 22.42a 2.18c 2.66a* 31.1 HW90 8.18b* 3.77a 6.01d 2.81bc* 2.14b 14.7 Day 4 HW110 9.16ab* 3.89a 7.59c 3.86b* 1.64c* 17.0 1. *Indicates a significant difference between varying length of treatment times at each day of ripeness. 2. Different letters within column indicate significant difference at each day of ripeness.. 3. HT represents hydrolyzable tannin.

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29 3-3-2. Total Soluble Polyphenolics (TSP) TSP concentration including contribution from ascorbic acid, reducing sugar and likely small amount of soluble protein in mango decreased in re sponse to HWT. The average TSP concentrations in HWT mangos (70, 90 and 110 min) for both ripeness stages (225 g/g GAE) showed about 25% dec line compared to control (294 g/g GAE) (Fig 3-4). At day 4, non-HWT mangos show ed higher TSP concentration (36% more) than HWT mangos. Although no significant di fference was found between control and HWT mangos at day 0, the concentration of c ontrol was still higher than those of other mangos. Hot Water Treatment ControlHW70HW90HW110 Total Soluble Phenolics ( g/g) 0 50 100 150 200 250 300 350 400 450 500 Day 0 Day 4 A B BC BCD CDE DE DE E Figure 3-4. Changes in TSP (Total Soluble Phenolic) concentration ( g/g GAE) for two different ripeness stages (day 0 and day 4) effected by four different durations of HWT (0, 70, 90, and 110 min). Average va lues and standard error bars of triplicate samples for all treatments are represented

PAGE 42

30 Varying the length of HWT was not a si gnificant factor in fluencing the TSP concentration in mango. Comparing TSP c oncentrations between three different treatments excluding control, the average T SP concentration for tw o ripeness stages was not significantly different (F ig 3-4). Since polyphenolics we re shown to decrease during ripening, lower TSP concentration was expected at day 4 than day 0 (Haard and Chism, 1996). However, no significant difference was observed between day 0 and day 4. Technically, 6 9 days are required to transform an unripe mango to a physiologically ripe mango (developed color and flavor) at 25 C, which was the same temperature used for mango storage in this study (Jacobi et al ., 2001). Moreover, ascorbic acid which has reducing capacity is unstable to heat. Ev en required duration of HWT could decrease ascorbic acid content in mango. Therefore, the 4 day storage was a somewhat short period to expect ripening changes such as TSP reduction and reduced ascorbic acid content by thermal stress might affect TSP in HWT mangos. 3-3-3. Antioxidant Capacity Antioxidant capacity, evaluated based on peroxyl radical scav enging activity of water-soluble mango constituents, was not a ffected by varying lengths of HWT. Although average antioxidant capacity of cl arified mango juice for two ripeness stages tended to increase as durati on of heating increased, it was not significantly different compared to control (Fig 3-5). During the 4-day storage, average antioxidant capacity for different duration of HWT significantly decreased (7%) (P < 0.05). The radical scavenging ability of mango did not correlate with TSP in this study because TSP did not change during the 4-day storage. These observations were unexpected since gallic acid, which was the major

PAGE 43

31 polyphenolic compound and a strong antioxi dant in mango, increased during ripening although TSP and hydrolyzable tannins were not significantly cha nged. Since ascorbic acid, which is another contribu tor to antioxidant capacity wa s reduced by heat and during ripening, this might influence the retention of antioxidant capac ity (Bashir and AbuGoukh, 2003; SotoZamora et al., 2005; Yen and Hung, 2000) Hot Water Treatment ControlHW70HW90HW110 Antioxidant Capacity (Trolox Equivalent M TE/g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Day 0 Day 4 A B B BC BC BC BC C Figure 3-5. Changes in tota l antioxidant capacity ( M TE/g) for two different ripeness stages day 0 and day 4) affected by four different lengths of HWT (0, 70, 90, and 110 min). Average values and standard e rror bars of triplicate samples for all treatments are represented 3-4. Conclusion Gallic acid concentration significantly decreased as a result of extended HWT (HW90 and HW110) while the treatment dur ation required for quarantine treatment

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32 (HW70) did not change gallic acid concentr ation compared to control. Consequently, total hydrolyzable tannin concentration was elevated by HWT only with required treatment time by the water treatment manual while extended time of treatment decreased HT concentration. Average TSP decreased as a result of HWT regard less of duration of HWT while antioxidant capacity was not aff ected by HWT (Table 3-3). Since mangos imported from other countries must go th rough a HWT, finding optimum conditions to improve and/or keep the quality of the fruit is important. According to the results of this study, the required hot water immersion treatment time for mango quarantine showed beneficial effects such as retaining galli c acid and increasing h ydrolyzable tannins. Table 3-3. Total soluble phenolics ( g/g GAE) and total antioxidant capacity ( M TE/g) of mango treated with hot wa ter with varying length of treatment times (0, 70, 90, and 110 min) at two different ripe ning stages (Day 0 and Day 4) Ripening Stages Hot Water Treatment Total Soluble Polyphenolics Total Antioxidant Capacity HW0 252.6a 2.67b HW70 248.7a* 2.66b* HW90 237.2ab 2.69b Day 0 HW110 226.2b 2.97a* HW0 335.4a* 2.60a HW70 218.9b 2.59a HW90 212.1b* 2.59a Day 4 HW110 213.8b 2.41a 1. Indicates a significant difference between varying length of treatment times at each day of ripening. 2. Different letters within column indicate significant difference at each day of ripening.

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CHAPTER 4 TO EVALUATE POLYPHENOLICS, ANTIOXIDANT CAPACITY, AND PHYSIOLOGICAL CHANGES IN MANGOS ( MANGIFERA INDICA L .) FOLLOWING CONTROLLED AT MOSPHERE STORAGE COMBINED WITH HOT WATER IMMERSION TREATMENT 4-1. Introduction Even though several states such as Hawaii, California, and Texas grow mangos Florida is the only state to report mango produc tion in the US (Mossler and Mosheim, 2002). However, according to latest record s, domestic mango pr oduction in Florida (2,800 MT) accounted for less than 1% of tota l consumption in the US in 2004, so more than 99% of mangos are imported from other countries such as Mexico, Brazil, Peru, Haiti, and Guatemala to meet the consumer demands (FAO, 2004; Sauco, 2004). Since many mangos are imported from othe r countries (278,422 MT in 2003) and it takes a few days up to a few weeks for the im ported mangos to reach retail markets, CA storage is sometimes used to maintain the quality of fruit during transportation and storage (FAO, 2004). Especially, mangos importe d from other countries must be treated with hot water to eliminate invasive pest in sects. Studies relating CA storage and HWT of fruits have focused on physiological changes such as firmness, color, moisture content, acidity and sugar content, so limited info rmation is available relating phytochemical changes during CA storage with or without HWT to postharvest quality factors during fruit ripening. Therefore, this study focuse d on the changes in pol yphenolics and resultant antioxidant capacity during CA st orage associated with HWT. 33

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34 4-2. Materials and Methods 4-2-1. Fruit Preparation and Treatment For this study, 300 Tommy Atkins mangos were procured from Fresh King. Inc., Homestead, Florida in June, 2004 at the peak of the harvest season for Tommy Atkins. Mango fruits with uniform size, oval shape, a nd green color were pr e-selected to reduce variation among treatments. Fruits were held at 10C during the 6-hour transport to Gainesville. Mangos were stored at 10C at the Horticultu ral Sciences Department in University of Florida for 2 days until hot wa ter treatment and CA storage were applied. Of the 300 mangoes originally obtained, 234 mangos were selected based on their uniform size, shape (oval), and weight (average weight >500g). Prior to treatments, eighteen mangos were se lected as controls. This initial set of fruit, representative of the population was analyzed for phytochemical and antioxidant content as previously described in chapter 3. Additionally, soluble solids content (SSC) and titratable acidity (TA) values were quan tified as additional indices for fruit quality. USDA-APHIS guidelines were followed, whic h involve immersing fruit in water at 45.6 46.1C for 75 min. The remaining 216 mangos were divided into two groups, half for HWT and the remaining half immersed in water at 25 C as a non-heated control. Following these treatments, the fruits were held at 25oC until the skin was completely dry (about 60 min) and the center of the heated mangos returned to 25oC. The fruit were then transferred to their respective CA chambers with the following gas contents: 1. CA 1 (Control): Air (about 21% O2 + 79% N2) 2. CA 2: 3% O2 + 97% N2 3. CA 3: 3% O2 + 10% CO2 + 87% N2

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35 Fruits were placed in the chambers and gas composition regulated by four external gas tanks (air, O2, CO2, and N2) for 2 weeks at 10 The gas composition in each chamber was checked twice daily and regulat ed to desired composition. After 2 weeks, half of the mangos were removed and immediately analyzed for phytochemical attributes, antioxidant capacity, and quality index such as titratable acidity, soluble solids content, and fruit pulp color. The remaining fruits were moved into a 25 C environment to complete their ripening in an air (21% O2) environment. All the mangos stored in CA chamber were analyzed as described in Chapte r 3. Additionally, the inte rnal flesh color of the peeled fruits (CIE color values L*, a*, b*) was measured for all the mangos. Table 4-1. Mango CA storage with 1 control (a ir) and 2 different air compositions (CA2 and CA3) (CA1=Air, CA2=3% O2, CA3=3% O2 + 10% CO2) at 10oC for 2 weeks. One half of the mangos were treated with hot water and the other half was treated with 25 C water. CA storage chamber No HWT HWT Total 1. Air (Control) 36 2. 3% O2 + N2 36 3. 3% O2 + 10% CO2 + N2 36 4-2-2. Phytochemical Change s and Antioxidant Capacity Total soluble phenolic concentration and an tioxidant capacity were determined as previously described in Chapter 3. Identif ication and quantification of individual

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36 polyphenolics were conducted using Waters 2690 HPLC as previously outlined in Chapter 3. 4-2-3. Evaluation of Flesh Color The internal flesh color of peeled fruit expressed as CIE color values (L*, a*, and b*) was measured by Minolta Chroma Me ter CR 200 Series with a 8mm aperture (Minolta Co., Ltd., Osaka, Japan). Hue angle an d chroma values were calculated from a* and b* using the method described by Lp ez and Gmez (2004). The colorimeter was calibrated by a standard flat white tile before measuring the flesh color of mangos. The following equations were used to calculate hue angle and chroma values of mango flesh color. Hue Angle = 14.3/180*)/*(1abTan +180 (180 is needed only if a* is negative) Chroma = 22** ba 4-2-4. Quantification of Titratable Acidity (TA) Titratable acidity was measured by titrat ing clarified juice against 0.1N NaOH to pH 8.2 using phenolphthalein indicator and a Corning model 140 digital pH meter as described by Jacobi et al. (2000). The acidity was expre ssed as anhydro citric acid equivalents, the predominant organic acid in mango. Mango juices (3g) were diluted 5fold in a small beaker on a stir plate. The sample was titrated using 0.1M NaOH until pH 8.2 was obtained. Finally, the amount of 0.1M Na OH used for the titration was recorded. Acid content (%) of each sample was calcula ted based on the following formula. In the formula, 0.064 was the miliequivalent factor for anhydrous citric acid 100 )( 064.0)(1.0 % gjuice NaOH NaOHml acid

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37 4-2-5. Quantification of So luble Solids Content (SSC) Soluble solids contents ( Brix) were quantified by clarifying fruit puree by centrifuging using a Beckman Optima TL 100 tabletop ultracen trifuge (Beckman Instrument Inc. Palo Alto, CA) at the speed of 15,000 rpm for 15 min. The supernatant was measured using a digital Leica Mark Abbe refractometer (L eica Inc. Buffalo, NY) at 20 C. Before using the instrument, it wa s calibrated using distilled water. Approximately 100 L of juice was placed on the prism of the refractometer, and the refractive index was read. 4-2-6. Statistical Analysis Method Statistical analysis was performed as previously described in Chapter 3 using JMP 5 statistical software (SAS In stitute, 2002). Analysis of variance (ANOVA) and LSD (P<0.05) test were used to determine the eff ect of CA storage combined with or without HWT on phytochemicals and other quality chan ges of fruit during storage and ripening. Pearson correlation test was used for relati on between TSP concentration and antioxidant capacity. 4-3. Result and Discussion 4-3-1. External Fruit Quality Hot water treatment was effective in preven ting physiological disorders on the fruit. Darkened lenticels (black spots) are the sy mptoms of anthracnose, the most common surface postharvest disease on mangos, and a ppeared only on the skin of the non-HWT mangos after 2 weeks of CA storage. C onsequently, the deterioration of non-HWT mangos was faster than that of HWT ma ngos during ripening. Non-HWT mangos started to deteriorate at 25 C 3 days after CA storage was finished. Their flesh started to sink and

PAGE 50

38 more darkened lenticels were detected on th e skin while development of anthracnose on HWT mangos was delayed until 1 week after CA storage was complete. On average, one of six repetitions was rotten at 25 C. Thus, one mango was re moved from each set of mangos before analyzing. CA storage could prevent anthracnose on mangos as well. Even though black spots are also symptom of CO2 injury, the spots on the mango sk in were probably not induced by CO2 in this study because less black spots were shown in CA3 (the only CA storage that contained CO2). Mangos stored in CA1 (air) deve loped more anthracnose compared to mangos in CA2 (3%O2) and CA3 (3%O2 + 10%CO2) after CA storage. Consequently, when comparing mangos between CA2 and CA3, more anthracnose was detected on the mangos stored in CA2 than CA3 because CA storage with low O2 and high CO2 was more effective in preventing fr uit decay than with only low O2 (Tian et al., 2004). After all the mangos were moved to 25 C, non-HWT mangos started to deteriorate 3 days after transfer. The mangos without HWT showed an thracnose on the skin without regard to CA storage conditions after 3 days at 25 C. With the exception of mangos stored in CA1, no fruit disorders were found on HWT mangos Through this result, it was hypothesized that a synergistic effect occurred in in hibiting anthracnose when CA storage was combined with HWT. 4-3-2. Individual Polyphenolic Concentration Two major polyphenolics (gallic acid and hydr olyzable tannin (HT )) and four minor polyphenolics (p-OH-benzoic acid, m -coumaric acid, p-coumairc acid, and ferulic acid) in mango were identified and quantified by HPLC. Gallic acid and six HTs were found about 98% more than four minor polyphe nolics found in mango. Since the minor

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39 polyphenolic concentrations were somewhat neg ligible, details of th eir changes were not analyzed. Two CA storages containing different ga s compositions and one air control were applied to mangos. CA1 was consisted of 21% O2 and 79% N2 (normal air composition) and was considered the control for the study. Technically, CA storage is based on lowered O2 and increased CO2 levels at low temperature and optimum O2 and CO2 levels for mango are 3-5 and 5-10 % depending on degr ee of ripeness (Kader, 2002; Mitra and Baldwin, 1997). Therefore, CA2 was 3% O2 and 97% N2 in an effort to monitor the effect of CA storage with reduced O2 (usually less effective than O2+CO2) (Tian et al., 2004). CA3 was 3% O2, 10% CO2, and 87% N2 to determine the effect of CA storage with lowered O2 and elevated CO2 in the optimum range for mango CA storage. The temperature for CA storage of fully mature mangos must be at least 10 C to prevent chilling injuries such as discoloration, pitting, poor colo r, and uneven ripe ning (Mitra and Baldwin, 1997). Thus, storage temperature for this study was fixed as 10 C regardless of storage gas compositions. Gallic acid concentrations for CA1, CA2 and CA3 were measured at three different stages of ripening (green, mid, and full) in effort to determine treatment effect during ripening. It was reported that phenolic compounds such as gallic acid decreased as fruit ripened (Haard and Chism, 1996; Mitra and Baldwin, 1997), and likewise in this study gallic acid concentrations of CA1, CA2 and CA3 significantly decreased by 16, 17 and 8% during ripening from green to full ripeness stage (Fig 4-1). Gallic acid concentration significantly decreased only be tween green and mid ripe at CA1 while the concentrations at CA3 decreased only between mid and fu ll ripe. Gallic acid concentration at CA2

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40 gradually declined throughout fruit ripeni ng. Even though it was a relatively short time between mid and full ripeness stages, there were several types of evid ence to support that mangos ripened during this period such as titratable acidity decrease and color development occurred between mid and full ri peness stage in this study. Thus, it could be described as a ripening rela ted change of gallic acid. Ripeness Green Mid Full Gallic Acid (mg/L) 100 110 120 130 140 150 160 170 180 190 200 210 220 230 CA1 CA2 CA3 Figure 4-1. Changes in average gallic acid concentration (mg/L) for CA1, CA2 and CA3 during ripening for fruit samples at gr een, mid, and full ripeness. The ripeness stages were green (on the day of harvest) mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe. In conclusion, gallic acid concentration significantly decreased during storage (P < 0.05), and then the concentration was held constant at CA1. This was supported by several studies, in which polyphenolic com pounds in mango were reported to gradually

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41 decrease then maintain fairly steady during ripening (Lakshininaray ana et al., 1970; Mitra and Baldwin, 1997). However, CA storage, especially CA3, inhibited reduction of gallic acid. After CA storage, the concentration con tinued to decreased, but it was still higher than that of CA1 and CA2. Gallic acid concentration was not affected by HWT. In Chapter 3, gallic acid concentration was unaffected only when HW T was applied with required temperature developed by USDA-APHIS for the sample ma ngos while the concentration decreased as a result of extended treatment times. Since HWT time for this study was 75 min and it was the required treatment time for the mangos used according to the USDA-APHIS treatment manual, no effect by HWT showed a similar result with the first study. In conclusion, HWT did not affect gallic acid concentration in this study because mangos were subjected to a HWT for the required time for insect quarantine found in the USDAAPHIS treatment manual. Each storage contained differe nt gallic acid and total HT concentrations. Average gallic acid concentration of mango in CA2 was not higher co mpared to concentration in CA1. The average concentration in CA3 (172 mg /L) was significantly higher than that of CA1 (159 mg/L) and CA2 (151 mg/L) (Fig 4-2) a nd it was 9% higher in CA3 compared to that of CA1 (P < 0.05). According to Ag rawal and Deepak (2002), Castells et al. (2002), Davey et al. (2004), Penuelas et al. ( 1996) and Saxon et al (2004), elevated CO2 increases synthesis of phenolic comp ounds in plants. Furthermore, high CO2 concentration could induce abiotic stress, wh ich increases phenolic compounds in plants. Even though low O2 could be a factor to induce abio tic stress as well, it was not enough to increase polyphenolics in this study. Ther efore, it was hypothesized that combination

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42 of O2 and CO2 significantly increased gallic acid concentration in mango by an effect of CO2 and abiotic stress while O2 did not increase the concentration In studies conducted by Sanchez-Mata et al. (2003) a nd Tian et al. (2004), CA storage was more effective in preventing or reducing fruit decay and prolonging shelf-life if reduced O2 and elevated CO2 levels were applied together when comp ared with normal atmospheric conditions. In conclusion, increasing phenolic compounds in mango could be an additional benefit of CA storage with reduced O2 and elevated CO2 as was found through this study. CA storage CA1 CA2 CA3 Gallic Acid (mg/L) 50 75 100 125 150 175 200 225 250 No HWT HWT A AB B D D C Figure 4-2. CA storage induced effects in aver age gallic acid concen tration (mg/L) with or without HWT. Control was expr essed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2). Hydrolyzable tannin is a polyphenolic co mpound mainly found in mango and total HT decreased by 46, 49 and 42% at CA1, CA 2 and CA3 (green through full) during

PAGE 55

43 ripening (Fig 4-3). Total HT concentrations at CA1 and CA2 did not change significantly while the concentration at CA3 increased during storage at 10 C. However, total HT concentrations at all the storages significantly decreased in air at 25 C following CA storage, and the average reduc tion was about 45%. According to this result, it was hypothesized that storage condition (low temper ature) prevented or increased total HT reduction because total HT con centration during storage at 10 C (green to mid) was fairly stable and then its concentration si gnificantly decreased in air at 25 C after CA storage (P < 0.05). Ripeness Green Mid Full Total Hydrolyzable Tannin (mg/L GAE) 60 80 100 120 140 160 180 200 220 240 CA1 CA2 CA3 Figure 4-3. Changes in average total hydroly zable tannin concentr ation (mg/L GAE) for CA1, CA2, and CA3 during mango ripening. Ea ch stage represents green (on the day of harvest), mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe. Average total HT concentration increased as a result of HWT (Fig 4-4). HWT induced an increase of 17% HT compar ed to NHW mangos. In this study, only

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44 hydrolyzable tannin concentrati on was affected by HWT. Other parameters like gallic acid, total soluble phenolics and antioxidant capacity were not affected by hot water treatment. In Chapter 2, HT concentration was elevat ed by required duration of HWT, and likewise HT significantly increased in response to heat treatment in this study. Thus, it could be hypothesized that HT concentra tion increased when mangos were treated with required treatment time. Furthermore, CA2 and CA3 also contained more HT in mango than CA1 (Fig 4-4). CA3 induced a significant increa se of 15% total HT in mango compared to control while the concentrations in CA2 a nd CA3 were not significantly different from each other (P < 0.05). It was a similar result wi th gallic acid change by CA storage. CA storage CA1 CA2 CA3 Total Hydrolyzable Tannin (mg/L GAE) 40 60 80 100 120 140 160 180 200 220 No HWT HWT A B B C D E Figure 4-4. CA storage induced change in average HT concentration (mg/L GAE) in mango with or without HWT. Control wa s expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2).

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45 Gallic acid attaches to glucose via me ta-depside bond by esterification, and it becomes B-Glucogallin with one gallic acid which is the simplest gallotannin (one of HTs). Depening on the number of gallic acid to glucose, gallot annin have different structure (Fig 4-5). Thus, more free gallic ac ids are synthesized, more gallotannins could be produced. In conclusion, it could be hypothesi zed that increase of total HT was caused by elevated CO2 that resulted in increased synthesis of phenolic compounds in the fruit. Elevated CO2 affects synthesis of phenolic co mpounds by increasing PAL (phenylalanine ammonia lyase) which is the first step of the phenylpropanoid pathway to be related to the synthesis of phenolic compounds such as lignin and tannins (Assis et al., 2001; Castells et al., 2002; Davey et al., 2004). Figure 4-5. Gallotannin biosynt hesis via meta-depside bond by esterification. Depending on the number of gallic acid to glucose, gallotannin have 5 different structure such as -glucogallin, 1-6-Digalloylglucos e, 1,2,6-Trigalloylglucose, 1,2,3,6Tetragalloylglucose, and 1,2,3,4,6-Pentag alloylglucose. (Grundhfer et al., 2001).

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46 4-3-3. Total Soluble Phenolics (TSP) Total soluble phenolics determined by th e Folin-Ciocalteau assay decreased by 58, 53 and 41% at CA1, CA2 and CA3 during fruit ri pening (Fig 4-6). In general, it has been reported that polyphenolic compounds decrease in many climacteric fruits such as mangos, bananas, tomatoes and guavas dur ing ripening (Haard and Chism, 1996; Lakshminarayana et al., 1970; Mitra and Baldwin, 1997; Selvaraj and Kumar, 1989), and likewise in this study TSP concentration significa ntly declined as fruit ripened (P < 0.05). Since major phenolic compounds (gallic acid and total HT) in mango also significantly decreased during ripening (12 and 46%, re spectively), reduction of TSP might be influenced by changes of those two phenolic compounds. Controlled atmosphere storage delayed the reduction of average TSP concentration for HWT during ripening (Fig 4-6). When CA st orage was finished (Mid point at Fig 4-6), the average concentrations in CA2 and CA 3 were both 11% higher than that in CA1. After 2 week CA storage, it was found that both CA storage c onditions effectively delayed TSP reduction in mango. Both CA2 and CA3 delayed 12% of average TSP reduction for HWT compared to control during 2 weeks CA storage. After storage, when the fruits were moved to air at 25C to complete ripening, TSP concentration was significantly higher (30%) in ma ngos previously stored in CA3 than in CA1. This was because the CA storage mangos were less ripe than the air control when the analysis was performed. In conclusion, CA2 as well as CA3 were effective in slowing the reduction of average TSP concentration as a result of delayed ripening. However, more appropriate gas composition for mango storage (CA3) to maintain the quality of the fruits and vegetables better and longer was more effectiv e at decreasing the reduction rate than CA2

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47 because CA3 combination delayed 30% of TSP reduction while CA2 prolonged only 12% of TSP concentration after CA storage. Ripeness Green Mid Full Total Soluble Phenolics ( g/g GAE) 150 175 200 225 250 275 300 325 350 375 400 425 450 CA1 CA2 CA3 Figure 4-6. Change in average to tal soluble phenolics (TSP) ( g/g GAE) in mango for CA1, CA2 and CA3 during ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA stor age), and full (the time fruit started deterioration) ripe. Average TSP for all CA treatments did not ch ange as a result of HWT (Fig 4-7). As previously shown in Chapter 3, the required durati on of HWT did not have an effect on average TSP in mango. Otherwise, average T SP concentration for HWT was affected by CA storage (Fig 4-7). In CA2 and CA3, av erage TSP was 11 and 18% higher than CA1, respectively. Even though average TSP in CA 2 and CA3 were significantly higher than control (P < 0.05), the concentr ation in CA3 was 9% higher than in CA2. Since gallic acid and total HT concentr ations for HWT increased due to an effect of CO2, increase of

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48 average TSP concentration for HWT might be di rectly affected by changes of gallic acid and total HT. CA storage CA1 CA2 CA3 Total Soluble Phenolics ( g/g GAE) 100 125 150 175 200 225 250 275 300 325 350 375 400 No HWT HWT A A A BC BC C Figure 4-7. Changes in averag e total soluble phenolics ( g/g GAE) as a result of CA storage (Air, O2, and O2+CO2) with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2). 4-3-4. Antioxidant Capacity Overall, antioxidant capacity changes of mango, which were thought to originate from polyphenolics and ascorbic acid showed a very good correlation with changes in TSP (r=0.98). Even though other antioxidant compounds such as carotenoids and tocopherol were found in mango, the ORAC assa y does not detect antioxidant capacity from carotenoid and tocopherol. During fr uit ripening, average antioxidant capacity

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49 decreased from 5.43 to 3.26 ( M TE/g) (Fig 4-8). The reduction in antioxidant capacity was 45, 43 and 32% during ripening (from the mature green stage to the final ripe stage). Ripeness Green Mid Full Antioxidant Capacity (Trolox Equivalent M/g) 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 CA1 CA2 CA3 Figure 4-8. Change in average antioxidant capacity (Trolox Equivalent M TE/g) for CA1, CA2 and CA3 determined by ORAC (Oxygen Radical Absorbance Capacity) assay during fruit ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA stor age), and full (the time fruit started deterioration) ripe. During ripening, antioxidant capacity of ma ngo decreased as a result of loss of phenolic compounds. Since antioxidant capacity is a desirable process in quenching harmful free radicals, the method to prevent or slow loss of compounds to be related to antioxidant capacity should be determined. In this study, it was found that CA storage slowed the reduction of antioxidant capacity as shown in TSP concentration during fruit ripening. After CA storage, both CA2 and CA 3 showed higher antioxidant capacity (4.96

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50 and 4.92 M TE/g, respectively) co mpared to CA1 (4.60 M TE/g). At 25 C, antioxidant capacity was 20% higher in CA3 than in CA 1 while antioxidant capacity in CA1 and CA2 was not different (Fig 4-8). CA storage CA1 CA2 CA3 Antioxidant Capacity (Trolox Equivalent M TE/g) 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 No HWT HWT A AB BCD CD D ABC Figure 4-9. Antioxidant capaci ty (Trolox equivalent M TE/g) effected by CA storage with or without HWT. It is measur ed by ORAC (Oxygen Radical Absorbance Capacity) assay. Hot water treatment did not affect antioxidant capacity of mangos while antioxidant capacity was affected by CA storage as s hown in TSP concentration. However, the mangos kept in CA3 showed 13% higher antioxidant capacity than that in CA1, and antioxidant capacity was not different in CA1 and CA2 (Fig 4-9). Antioxidant capacity was highest (4.31 M TE/g) in CA3 while the lowest value was CA1 (3.79 M TE/g).

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51 Since phenolic compounds and TSP concentrat ion was higher at CA3 than CA1 and CA2, antioxidant capacity might be infl uenced by those concentrations. 4-3-5. Fruit Flesh Color The color change of fruit is a reliable parameter to determine the extent of fruit ripening (Ninio et al., 2003). The internal flesh color of peeled mango fruit was determined based on CIE color values (L*, a* and b*). L* denotes relative darkness (0) and lightness (100), a* represents green or red, and b* means blue or yellow of the samples (Lpez and Gmez, 2004) (Fig 4-10). Hue angle is the numeric description of how yellow (90 ), or green (180 (in the case of mango fruit) th e object is and chroma is the value to describe the vividness to dulln ess of a color and (Jeong et al., 2003; Lpez and Gmez, 2004). In this study, color value wa s presented as L*, hue angle, and chroma by conversion from L*, a* and b*. Ethylene is a naturally occurring compound related to many biological changes such as growth, development, and ripening in fruits and vegetables (Saltveit, 1999). Since ethylene synthesis increases in climacteric fruits duri ng ripening, fruit ripening is accelerated. Synthesis of carotenoi ds increases as fruits ripen because ethylene stimulates synthesis of pigments such as anthocya nin and carotenoids and degradation of chlorophyll (Hatlen et al., 1998; Saltveit et al., 1999; Vila vicencio et al., 2001). As a result of increased carotenoid concentration during ripening, the L* value and hue angle decrease, but a* (redness), b* (yellowness), and chroma in crease (Hatlen et al., 1998).

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52 Fig 4-10. The CIELAB color space system. L* represents lightness and a*, and b* show the strength of own colors (a: red to green and b: yellow to blue) (Lpez and Gmez, 2004) Ripeness Mid Full L* 68 69 70 71 72 73 74 75 76 CA1 CA2 CA3 A AB ABC BC BC C Figure 4-11. Average rate of reducing lightness (L*) for HWT during fruit ripening. Each stage represents mid (after 2 week CA st orage) and full (the time fruit started deterioration) ripe.

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53 Since color of yellow pulp fr uit such as mango develops from greenish-white to yellow during ripening, pulp color changes from lighter (higher L* value) to deeper color (lower L* value). As shown in Fig 4-11, L* was 4% higher in CA3 than in CA1 after CA storage, but L* values in a ll CA storage did not show diffe rences at 25C. According to this result, ripening was inhibi ted when the fruit was under CO2 environment (during CA storage at CA3) and then no difference was found at 25 C after CA storage. Ripeness Mid Full Hue Angle (o) 86 88 90 92 94 96 98 100 102 104 CA1 CA2 CA3 A A B B B B Figure 4-12. Reduction rate of average hue angle (o) for HWT depends on CA storage air composition during ripening. Each stage repres ents mid (after 2 week CA storage) and full (the time fruit started deteriorat ion) ripe. Hue angle was defined by the coordination a* and b*. Average hue angle and chroma for HWT tende d to decrease without regard to CA storage during mango ripening (Fig 4-12, 13). Th e decline in hue angle represented the change from green (180 ) to yellow (90 ) as fruits lost their chlorophyll (green pigment

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54 in the skin) and synthesis of carotenoid (yellow pigment in the flesh) was stimulated by ethylene during ripening (Gueva ra and Pardo-Gonzlez, 1996; Saltveit, 1999). After CA storage, hue angles were hi gher in CA2 and CA3 (8 and 10%, respectively) than in CA3. However, all hue angles were not significantly different at 25 C. This result showed that ripening was delayed and/or caro tenoid synthesis was inhibited as a result of CA storage, but there was no residual effect after transfer to air at 25 C. Since CA storage condition (lowered O2 and elevated CO2) inhibits respiration of fruits (consuming O2 and producing CO2) and decreases ethylene ac tivity, higher hue angle in CA 2 and CA3 was an evidence of delayed ripening. Ripening Mid Full Chroma 50 52 54 56 58 60 62 64 CA1 CA2 CA3 A A B B BC C Figure 4-13. Increase in average chro ma for HWT depends on CA storage air composition during ripening. Each stage repres ents mid (after 2 week CA storage) and full (the time fruit started deterior ation) ripe. Chroma was defined by the coordination a* and b*.

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55 As yellow pulp fruits such as mango, st ar fruit, and pepino ripen, pulp color changes from green to yellow and this appear s as an increase in chroma and mixture of color is a reason of low chroma (Prono-Wida yat et al., 2003). Consequently, chroma was highest at CA1 through mango ripening because mangos in CA1 were riper than in CA2 and CA3 that inhibited mango ripening. Chroma in CA2 rapidly decreased after storage while chroma in CA3 did not change even after storage. CA storage CA1 CA2 CA3 Hue Angle (o) 70 75 80 85 90 95 100 105 110 No HWT HWT A A A A B B Figure 4-14. Changes of average hue angle (o) affected by CA storage with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2). Neither CA storage nor HWT affected th e change of L* value of mango flesh (data not shown). However, hue angle significantly increase d by (7 and 5%, respectively) as a result both CA storages (CA2 and CA3) compared to control (CA1), and chroma was

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56 higher only in CA2 and CA3 (8 and 4%, respec tively) compared to control (CA1) (Fig 415, 16). CA storage CA1 CA2 CA3 Chroma 4 6 8 10 12 14 16 18 No HWT HWT A AB AB AB B B Figure 4-15. Changes of average chroma aff ected by CA storage with or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA storages were shown as CA2 (3% O2 and 97% N2), and CA3 (3% O2, 10% CO2 and 87% N2). 4-3-6. Titratable Acidity (TA) Titratable acidity (TA) represents the to tal concentration of titratable acid in a sample and is considered an important attribut e in determining the taste quality of fruits and vegetables (Berezin et al ., 1994). According to Jacobi et al. (2000) and Tovar et al. (2001), TA in mango decreases as mango ripens because two major or ganic acids (citric and malic acid) used in respiration decrease during mango ripening. Titratable acidity for CA1, CA2 and CA3 decreased during ripening (F ig 4-16). Since CA storage delays fruit

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57 ripening, reduction rate of TA at CA2 a nd CA3 was inhibited dur ing the CA storage period (green to mid ripe) compared to th e period from mid to full ripe after CA treatment. However, reduction rate of TA at CA1 was not inhibited during CA storage period. Ripeness Green Mid Full Titratable Acidity (%) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 CA1 CA2 CA3 Figure 4-16. Change of average titratabl e acidity (%) for CA1, CA2 and CA3 during mango ripening. Each stage represents green (on the day of harvest), mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe. Average TA was higher at CA2 and CA3 as a result of CA storage (CA2-35% and CA3-32%) compared to control (CA1) and TA of mango decreased by 18% HWT (Fig 417). This change has been reported previous ly that TA of fruits including mango are reduced by heat treatment (Jacobi et al., 2000; Neven and Mitcham, 1994; Ninio et al., 2003).

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58 CA storage CA1 CA2 CA3 Titratable Acidity (%) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 NHW at 10oC HW at 10oC NHW at 25oC HW at 25oC Figure 4-17. Change of titratable acidity ( %) in mango affected by CA storage with or without HWT. Titratable acidi ty was measured twice at 10 C (mid ripe) and 25 C (full ripe). 4-3-7. Soluble Solids Content (SSC) Soluble solids content is one of the most reliable parameters in judging fruit quality as consumers consider quality factors such as SSC and TA as much as visible quality (e.g. color, size and firmness) (Hoehn et al., 2002; Lu, 2004). In this study, average SSC decreased by 50% during CA storage at 10 C and then increased by 35% at 25 C. Especially, a degree of reduction was higher at CA2 than at CA1 and CA3 (Fig 4-18).

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59 Ripeness Green Mid Full Soluble Solids Content (oBrix) 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 CA1 CA2 CA3 Figure 4-18. Effect of the fruit ripe ning on average soluble solids content ( Brix) for all CA storage and HWT in mangos. Each st age represents green (on the day of harvest), mid (after 2 week CA stor age) and full (the time fruit started deterioration) ripe. Since SSC is known to increase during fr uit ripening as insoluble starch is transformed into soluble solids (Martise n and Schaare, 1998; McGlone and Kawano, 1998; Vela et al., 2003), reduction of SSC dur ing storage was not anticipated result. However, several studies have shown se veral examples in decreasing SSC during ripening. Numerous studies ha ve reported that low O2 storage suppresses SSC increase (Hoehn et al., 2003; Lopez et al., 2000; Taylor et al., 1995). According to Taylor et al. (1995), SSC in plum, which is a climacter ic fruit like mango also decreased during ripening, and when apples were stored with high relative humidity to prevent drying out, SSC decreased during ripening (Saad et al., 200 4). Therefore, it could be assumed that

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60 decrease of SSC might be influenced by low O2 composition or naturally decreased or affected by high relative humidity during stor age. After 2 weeks CA storage, SSC started to increase as shown in Fig 4-18. Comparing SSC of CA1, CA2 and CA3, SSC in CA2 at 10 C was significantly lower compared to CA1 and CA3 (Fig 4-19). This might be because lowered O2 suppressed SSC synthesis as expl ained above. After storage at 10 C, SSC in CA2 became higher than CA1 at 25 C. CA storage CA1 CA2 CA3 Soluble Solids Content (oBrix) 0 2 4 6 8 10 12 14 16 18 20 NHW at 10oC HW at 10oC NHW at 25oC HW at 25oC Figure 4-19. Average soluble solids content aff ected by CA storage with or without HWT. Soluble solids content was measured twice at 10 C (mid ripe) and 25 C (full ripe). 4-4. Conclusion The external quality of mango fruit during ripening was affected by CA storage and HWT as both postharvest treatments inhibited external disorders such as anthracnose on

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61 the mango skin. CA storage was effective to increase phenolic co mpounds and resultant antioxidant capacity in this study. The CA with reduced O2 and elevated CO2 concentration (CA3) was more e ffective than CA with low O2 (CA2) and the former increased TSP by 18%, antioxidant capacity by 12%, gallic acid by 9%, and total hydrolyzable tannin by 15% compared to cont rol. However, HWT increased only total HT (17%) and did not affect to the other parameters. The TSP, antioxidant capacity, gallic acid, and total HT significantly decreased by 50%, 40%, 12% and 46%, respectively during ripening (from green to full ripe). In addition, CA3 significantly inhibited the reduction rate of TSP and antioxi dant capacity compared to CA1. At the end of fruit ripening, TSP and antioxidant capac ity in CA3 fruits were higher by 30% and 20% than those in CA1. St rictly speaking, since CO2 in CA3 increased synthesis of phenolic compounds, reduction of TSP and antio xidant capacity in CA3 was delayed during ripening. Unlike imported mangos, domestic mangos grown in Florida such as Tommy Atkins are not usually subjected to HWT b ecause of the possibility that they were infested by dangerous tropical fruit flies and/ or larvae is negligible Through this study, it was confirmed that domestic mangos do not have any disadvantages compared to imported mangos, although they ar e not treated with hot water. Furthermore, since it is proved that CA storag e with elevated CO2 was effective to increase polyphenolics and resultant antioxidant capacity, using CA stor age will be good choice even for domestic transportation.

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CHAPTER 5 SUMMARY AND CONCLUSION As the popularity and consumption of mango in the US have increased, the necessity of postharvest treatments for maintain ing the quality and exte nding shelf-life of fruits during transportation and storage has al so increased. Numerous studies related to mango postharvest treatment have stressed physiological changes during this time. Therefore, these studies were designed to investigate phytochemical changes and resultant antioxidant capacity in man go by hot water immersion and controlled atmosphere storage, which are most im portant postharvest treatments on mangos. Hot water immersion treatment (HWT), legally required thermal quarantine treatment for imported mangos was applied to mangos with varying le ngths of treatment times (0, 70, 90 and 110 min) followed by 4 days of storage at 10 C. When mangos were subjected to the required le ngth of treatment time (70 min) for quarantine, polyphenolic compounds were stable or el evated. However, when mangos were treated for extended times (90 and 110 min), polyphenolics significan tly decreased compared to control (0 min). Antioxidant capacity of mangos was not affected by HWT. Controlled atmosphere (CA) storage based on reduced O2 and/or elevated CO2 at low temperature was a factor to change phytochemicals in mango. CA2 (lowered O2) did not change phytochemicals and antioxidant capacity compared to CA1 (normal air) while CA2 (lowered O2 and elevated CO2) increased polyphenolics and resultant antioxidant capacity as a result of CO2 effect. HWT used in this study had no affect on changes of polyphenolics and antioxidant capacity. 62

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63 In conclusion, selected postharvest treat ments in this study were effective to prevent anthracnose and increase shelf-lif e. Moreover, CA storage with lowered O2 and elevated CO2 induced increases in polyphenolics a nd resultant antioxidant capacity in mangos. Domestically produced mangos are usua lly not subject to HWT and CA storage is not used due to relatively short tran sportation and storage compared to imported mangos. Therefore, applications of HWT and CA storage should be considered even for domestic mango varieties such as Tommy Atki ns to get advantages in preserving fruit quality and extending shelf-lif e with beneficial effect s on phytochemical contents.

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BIOGRAPHICAL SKETCH Youngmok Kim was born on September 4, 1976, in Seoul, South Korea. He graduated from Dae-il High School in February 1995 and majored in food engineering at Kyungwon University in March 1995. In 2001, he came to the US to study hotel and restaurant management at Lewi s-Clark State College in Idah o for 9 months and returned to Korea with three hotel and restaurant field certificates. Af ter graduating from Kyungwon University in 2003, he came to Universi ty of Florida to join the Food Science and Human Nutrition Department graduate program. He recei ved a Master of Science in August 2005 and he will continue his study fo r a doctorate degree in the Food Science and Human Nutrition Department at the University of Florida. 73


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CHANGES IN POLYPHENOLICS AND RESULTANT ANTIOXIDANT CAPACITY
IN 'TOMMY ATKINS' MANGOS (Mangifera indica L.) BY SELECTED
POSTHARVEST TREATMENTS















By

YOUNGMOK KIM


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

Youngmok Kim

































This thesis is dedicated to the people I love: my parents, my teachers, and my friends.
















ACKNOWLEDGMENTS

First of all, I would like to express my heartfelt thanks to my supervisory

committee chair, Dr. Stephen Talcott who is an amazing mentor and teacher. His warm

personality, professional guidance, and profound knowledge encouraged me to complete

this work. Continuing as a doctorate student with him is a glorious opportunity and I feel

extremely fortunate to have this opportunity.

I would also like to express my gratitude to my supervisory committee members,

Dr. Jeffrey K. Brecht and Dr. Susan Percival for their thoughtful advice, consideration,

and support. I am grateful for all their help and encouragement in completing this work.

I thank you my beloved parents from the bottom of my heart for supporting me

intellectually, psychologically and physically throughout my life. I will keep trying to be

a son they can be proud of. I also want to thank my special fellow laboratory members Dr.

Joonhee Lee, Chris, David, Flor, Jorge, Kristine, Lanier, and Lisbeth. I really appreciate

their advice, help, and time. I want to give my special thanks to Dr. Lee for her valuable

advice and instruction. I am grateful to Sunghee for her encouragement, patience,

understanding, and love. I love you so much.

Finally, I would like to thank my country, South Korea, for allowing me to

complete my master's degree here at University of Florida by financially supporting me

for two years.
















TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS ........................................ iv

LIST OF TABLES.. .. ................ .............. .............. vii

LIST OF FIGURES .............. .....................viii

ABSTRACT ................ .................................................. .......... xi

CHAPTER

1 INTRODUCTION ........................ ..... ................... 1

2 LITERATURE REVIEW .............. ...................... ............................ ........ 4

2-1 M ango M market ............. .................................................. 4
2-2 Mango Cultivars ........... ................................................. 7
2-3 M ango Ripening ........ ................................ 8
2-4 Mango Postharvest Handling ......................................... .... ......... 10
2-4-1 Hot Water Immersion Treatment (HWT) .................................... 10
2-4-2 Controlled Atmosphere (CA) Storage ................. ........... ...... 12
2-5 M ango Polyphenolics ............................. ............... .............. 14
2-6 Polyphenolics as Antioxidants ...................................... 17

3 PHYTOCHEMICAL CHANGES AND RESULTANT ANTIOXIDANT
CAPACITY IN MANGOS DURING 4 DAY STORAGE AFFECTED BY
VARYING LENGTHS OF HOT WATER IMMERSION TREATMENT .............. 19

3-1 Introduction ...... ........................................ 19
3-2 M materials and M methods .................................. ....................... .. ......... 20
3-2-1 Fruit Preparation and Treatm ent........................................... ................ 20
3-2-2 Identification and Quantification of Individual Polyphenolics............. 22
3-2-3 Quantification of Total Soluble Polyphenolics..................... 22
3-2-4 Quantification of Antioxidant Capacity............ .................................. 23
3-2-5 Statistical Analysis Method ................................... 24
3-3 Results and Discussion.................................. ......... 24
3-3-1 Individual Polyphenolic Concentration ........................................ 24
3-3-2 Total Soluble Polyphenolics (TSP).............................. 29
3-3-3 Antioxidant Capacity .............................................. 30









3-4 C conclusion ........................................ 31

4 TO EVALUATE POLYPHENOLICS, ANTIOXIDANT CAPACITY, AND
PHYSIOLOGICAL CHANGES IN MANGOS (Mangifera Indica L.)
FOLLOWING CONTROLLED ATMOSPHERE STORAGE COMBINED
WITH HOT WATER IMMERSION TREATMENT .......................................... 33

4-1 Introduction ...... ........................................ 33
4-2 M materials and M ethods ................................. ......................... .. ...... 34
4-2-1 Fruit Preparation and Treatm ent ......................................................... ....... 34
4-2-2 Phytochemical Changes and Antioxidant Capacity............................. 35
4-2-3 Evaluation of Flesh Color................ ........................ 36
4-2-4 Quantification of Titratable Acidity (TA).................................... .... 36
4-2-5 Quantification of Soluble Solids Content (SSC) ..................................... 37
4-2-6 Statistical Analysis M ethod ........................................ 37
4-3 R esult and D discussion ........................................ 37
4-3-1 External Fruit Quality ......................... ............................. 37
4-3-2 Individual Polyphenolic Concentration ........................................ 38
4-3-3 Total Soluble Phenolics (TSP)............................................................... 46
4-3-4 Antioxidant Capacity .............................................. 48
4-3-5 Fruit Flesh Color. ........................ ... ................ 51
4-3-6 Titratable Acidity (TA) ....................................... .............. 56
4-3-7 Soluble Solids Content (SSC)............... .............. .............. 58
4-4 Conclusion .. .............................................. 60

5 SUMMARY AND CONCLUSION ............................................. 62

LIST OF REFERENCES ..........................................................................64

B IO G R A PH ICA L SK E TCH ....................................................................... 73
















LIST OF TABLES


Table page

2-1. Main mango producing countries in 2004 (www.fao.org) ....................................... 5

2-2. Global imports of mangos (MT) (Sauco 2004) .............. .............. 6

2-3. Producing countries, selected cultivars, and main marketing season (Nakasone
and Paull, 1998)..................... ............... ......... 8

2-4. Determined dip time based on origin, shape, and weight of fruit. (USDA-APHIS,
2002)............................................ 11

3-1. HWT with four different lengths of time (0, 70, 90, and 110 min) at 460C with
different ripeness stages (day 0 and day 4) at 230C............................................. 21

3-2. Gallic acid (jig/g) and average of four hydrolyzable tannins (ig/g GAE) identified
and quantified using HPLC as a result of different duration of HWT (0, 70, 90,
110 min) at two ripeness stages (Day 0 and Day 4)........................... 28

3-3. Total soluble phenolics (ig/g GAE) and total antioxidant capacity (yM TE/g) of
mango treated with hot water with varying length of treatment times (0, 70, 90,
and 110 min) at two different ripening stages (Day 0 and Day 4).................... 32

4-1. Mango CA storage with 1 control (air) and 2 different air compositions (CA2 and
CA3) (CAl=Air, CA2=3% 02, CA3=3% 02 + 10% C02) at 10-C for 2 weeks.
One half of the mangos were treated with hot water and the other half was
treated with 250C water...... .................................. 35
















LIST OF FIGURES


Figure page

2-1. Structure of gallotannin (p-1,2,3,4,6-pentagalloyl-O-D-glucose) (Zalewska et al.
1995) ............................................................ 16

2-2. A depside bond which is formed between the phenolic group of the upper and the
acid group of the lower gallic acid units (Mueller-Harvey 2001)........................... 16

3-1. Changes in average gallic acid concentration (ig/g GAE) for two ripeness stages
(day 0 and day 4) affected by HWT with varying length of treatment times (0,
70, 90, and 110 min). Average values and standard error bars of triplicate
samples for all treatments are represented. ................................................. ...... 25

3-2. Changes in total hydrolyzable tannin concentration (jg/g) for two ripeness stages
(day 0 and day 4) as a result of different lengths of HWT (0, 70, 90, 110 min).
Average values and standard error bars of triplicate samples for all treatments
are represented .............................................. 26

3-3. HPLC chromatogram of polyphenolics, gallic acid and four hydrolyzable tannins
(HT), present in mango flesh at Day 0 and Day 4. Peaks were tentatively
identified based on retention time and spectral similarities against an authentic
standard of gallic acid.. ..... .. ...... .. .... ......... ......... .. .......... ..... 28

3-4. Changes in TSP (Total Soluble Phenolic) concentration (ig/g GAE) for two
different ripeness stages (day 0 and day 4) effected by four different durations of
HWT (0, 70, 90, and 110 min). Average values and standard error bars of
triplicate samples for all treatments are represented ........................................ 29

3-5. Changes in total antioxidant capacity (jM TE/g) for two different ripeness stages
day 0 and day 4) affected by four different lengths of HWT (0, 70, 90, and 110
min). Average values and standard error bars of triplicate samples for all
treatm ents are represented .............................................................................. 3 1

4-1. Changes in average gallic acid concentration (mg/L) for CA1, CA2 and CA3
during ripening for fruit samples at green, mid, and full ripeness. The ripeness
stages were green (on the day of harvest), mid (after 2 week CA storage), and
full (the time fruit started deterioration) ripe.............................. 40









4-2. CA storage induced effects in average gallic acid concentration (mg/L) with or
without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA
storage were shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2
and 87% N2). ......................................... 42

4-3. Changes in average total hydrolyzable tannin concentration (mg/L GAE) for CA1,
CA2, and CA3 during mango ripening. Each stage represents green (on the day
of harvest), mid (after 2 week CA storage), and full (the time fruit started
deterioration) ripe. .......................................... 43

4-4. CA storage induced change in average HT concentration (mg/L GAE) in mango
with or without HWT. Control was expressed as CAl (21% air + 97% N2) and
two CA storage were shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02,
10% C O 2 and 87% N 2). ...................................................... 44

4-5. Gallotannin biosynthesis via meta-depside bond by esterification. Depending on
the number of gallic acid to glucose, gallotannin have 5 different structure such
as P-glucogallin, 1-6-Digalloylglucose, 1,2,6-Trigalloylglucose, 1,2,3,6-
Tetragalloylglucose, and 1,2,3,4,6-Pentagalloylglucose. (Grundhofer et al.,
2001)....................................... ................... .... ....................... 45

4-6. Change in average total soluble phenolics (TSP) (ig/g GAE) in mango for CA1,
CA2 and CA3 during ripening. Each stage represents green (on the day of
harvest), mid (after 2 week CA storage), and full (the time fruit started
deterioration) ripe. .......................................... 47

4-7. Changes in average total soluble phenolics (ig/g GAE) as a result of CA storage
(Air, 02, and 02+C02) with or without HWT. Control was expressed as CAl
(21% air + 97% N2) and two CA storage were shown as CA2 (3% 02 and 97%
N2), and CA3 (3% 02, 10% CO2 and 87% N2)................ ................ .... 48

4-8. Change in average antioxidant capacity (Trolox Equivalent yM TE/g) for CA1,
CA2 and CA3 determined by ORAC (Oxygen Radical Absorbance Capacity)
assay during fruit ripening. Each stage represents green (on the day of harvest),
mid (after 2 week CA storage), and full (the time fruit started deterioration) ripe. 49

4-9. Antioxidant capacity (Trolox equivalent yM TE/g) effected by CA storage with or
without HWT. It is measured by ORAC (Oxygen Radical Absorbance Capacity)
assay. .................................................... 50

4-10. The CIELAB color space system. L* represents lightness and a*, and b* show
the strength of own colors (a: red to green and b: yellow to blue) (L6pez and
G6mez, 2004) ...................... ............. .................. 52

4-11. Average rate of reducing lightness (L*) for HWT during fruit ripening. Each
stage represents mid (after 2 week CA storage) and full (the time fruit started
deterioration) ripe. .......................................... 52









4-12. Reduction rate of average hue angle (0) for HWT depends on CA storage air
composition during ripening. Each stage represents mid (after 2 week CA
storage) and full (the time fruit started deterioration) ripe. Hue angle was defined
by the coordination a* and b*. ........................................ 53

4-13. Reduction in average chroma for HWT depends on CA storage air composition
during ripening. Each stage represents mid (after 2 week CA storage) and full
(the time fruit started deterioration) ripe. Chroma was defined by the
coordination a* and b*. ....................................... .................... 54

4-14. Changes of average hue angle (0) affected by CA storage with or without HWT.
Control was expressed as CAl (21% air + 97% N2) and two CA storage were
shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2 and 87% N2).... 55

4-15. Changes of average chroma affected by CA storage with or without HWT.
Control was expressed as CAl (21% air + 97% N2) and two CA storage were
shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2 and 87% N2).... 56

4-16. Change of average titratable acidity (%) for CA1, CA2 and CA3 during mango
ripening. Each stage represents green (on the day of harvest), mid (after 2 week
CA storage), and full (the time fruit started deterioration) ripe. ............................. 57

4-17. Change of titratable acidity (%) in mango affected by CA storage with or
without HWT. Titratable acidity was measured twice at 100C (mid ripe) and
250C (full ripe).................................... ................... 58

4-18. Effect of the fruit ripening on average soluble solids content ('Brix) for all CA
storage and HWT in mangos. Each stage represents green (on the day of harvest),
mid (after 2 week CA storage) and full (the time fruit started deterioration) ripe.. 59

4-19. Average soluble solids content affected by CA storage with or without HWT.
Soluble solids content was measured twice at 100C (mid ripe) and 250C (full
ripe). ........................................ ........ 60
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

CHANGES IN POLYPHENOLICS AND RESULTANT ANTIOXIDANT CAPACITY
IN 'TOMMY ATKINS' MANGOS (Mangifera indica L.) BY SELECTED
POSTHARVEST TREATMENTS

By

Youngmok Kim

August 2005

Chair: Stephen T. Talcott
Major Department: Food Science and Human Nutrition

Mango (Mangifera indica L.) is one of the most important tropical fruits

worldwide and is gaining popularity in the US. The demand of mangos far exceeds

domestic supply thus extending markets for fruit import, whereby a hot water treatment

(HWT) is required against invasive pests. Additionally, controlled atmosphere (CA)

storage is sometimes used to preserve quality and extend shelf-life of mango fruit during

transportation and storage. Postharvest studies with fresh mangos have primarily focused

on quality characteristics and limited data exist for fruit phytochemicals and antioxidant

content and their resultant changes during postharvest handling and ripening. Therefore,

these studies were conducted to investigate phytochemical and antioxidant changes in

fresh mangos as influenced by the stage of fruit ripeness for the application of CA in

combination with a HWT.

The first objective was to quantify phytochemical changes and resultant antioxidant

capacity in mangos during 4 days storage by varying lengths of hot water immersion









treatment before storage. In this study, unripe mangos (Tommy Atkins) were first

subjected to three different HWT (0, 70, 90, and 100 min all at 46C) and then stored at

25C for 4 days. Phytochemical and antioxidant content was monitored after each HWT

and throughout storage. Hot water treatment at 460C for 70 min increased total

hydrolyzable tannin (30%) but did not induce significant changes in gallic acid, total

soluble phenolics and antioxidant content. Hot water treatments of >70 min did not affect

fruit antioxidant content but resulted in decreases of gallic acid (40%), total hydrolyzable

tannins (35%) and total soluble phenolics (25%). No remarkable phytochemical changes

associated with ripening were detected during the 4 days storage.

The second objective was to identify and quantify polyphenolics and resultant

antioxidant capacity in mango following controlled atmosphere (CA) storage combined

with HWT. In this study, unripe mangos were held under three CA conditions with or

without a HWT (460C for 75 min). Controlled atmosphere treatments included CAl

(21% 02 + 79% N2), CA2 (3% 02 + 97% N2,) and CA3 (3% 02 + 10% CO2 + 87% N2)

for 2 weeks at 10C. Half of the fruit was immediately collected for analysis after CA

storage while remaining fruits were held at 250C until fully ripe. Analyses for this study

included individual and total phenolics by HPLC, total soluble phenolics (Folins assay),

antioxidant capacity (ORAC assay), soluble solid content (SSC), titratable acidity (TA),

and flesh color. Hot water treatment at 460C for 75 min before CA storage did not change

the phytochemical and antioxidant content of mangos. Fruit in CAl and CA2 contained

equivalent phytochemical and antioxidant levels while CA3 fruit contained significantly

higher amounts of gallic acid (12%), hydrolyzable tannins (15%), total soluble phenolics

18%) and antioxidant content (13%) compared to CAl (P < 0.05).














CHAPTER 1
INTRODUCTION

Mango (Magnifera Indica.L) is a popular tropical fruit all over the world due to its

luscious color, characteristic taste, and excellent nutritional value. Since mango imports

to the US have grown due to greatly increased demand and mangos have come down in

price, the mango market has shown a rapid growth. For example, between 1996 and 2004,

there was 40% increase in mango imports to the US (Sauco, 2004). Even though several

cultivars such as Tommy Atkins, Keitt and Kent are commercially cultivated in Florida

and Hawaii, the production covers less than 1% of domestic consumption (2,800 MT) in

2004 (FAO, 2004). Now, more than 99% of mangos (278,422 MT) are imported mainly

from Mexico (about 70% in 2000) followed by Brazil, Ecuador, Peru, Haiti and

Guatemala (Sauco, 2004).

Mango is a valuable source of phenolic compounds that have bitter and astringent

taste that improve the characteristic taste of foods and develop food quality such as color

and flavor (Grundhofer et al., 2001; Haard and Chism, 1996). Phenolic compounds are

the most widely distributed secondary metabolites in all higher plant, which have a

phenol with one or more hydroxy substitutions (Hagerman et al., 1998; Robbins, 2003).

Several studies reported phenolic compounds found in mango such as quercetin, iso

querectin, kaempferol, mangiferin, gallic acid, m-digallic acid, m-trigallic acid, ellagic

acid, and hydrolyzable tannin such as gallotannin (El-Ansari et al., 1969; El-Sissi and

Saleh, 1965). Especially, gallic acid and hydrolyzable tannins are known as major

phenolic compounds found in mango.









Oxidation is a fundamental process for the cells in the body. However, there is a

critical side effect of oxidation, generation of free radicals and other reactive oxygen

species (ROS) such as peroxyl radical (ROO-), hydroxyl radical (HO-), superoxide ion

(02 -) and singlet oxygen (102) (L6pez et al., 2003). Free radicals involving oxygen are

considered ROS and most ROS are classified as free radicals (Karakaya et al., 2001).

Since free radicals have unpaired electron(s) and continuously find another electron to be

stable, important cellular components in the body such as DNA and cell membrane could

be damaged because cellular respiration is inhibited (Antolovich et al., 2001). However,

antioxidant compounds mainly found in fruits and vegetables protect the body from free

radicals and other ROS. Polyphenolic compounds abundantly found in mango not only

provide various functionalities in foods but also act as a strong antioxidant in the body.

Phenolic compounds have a strong antioxidant capacity in quenching and/or neutralizing

free radicals by donating hydrogen to reactive free radicals (Karakaya et al., 2001;

Femandez-Pach6n et al., 2004).

Since mango is a climacteric fruit, which implies a highly perishable nature, several

postharvest treatments have been used to keep the quality and extend shelf-life. Mangos

from other countries can be infested by fruit flies such as Tephripid fruit flies, so all the

imported mangos must go through a thermal quarantine treatment such as a hot water

treatment (HWT) to eliminate invasive pests before importing to the US (Jacobi et al.,

2001). Controlled Atmosphere (CA) storage is used for fruits and vegetables to prolong

their shelf-life and preserve them from quality deterioration (Sanchez-Mata et al., 2003).

Since most of mangos consumed in the US (99%) are imported and transportation usually

takes a few days to a few weeks, application of CA storage must be considered.









Limited study has shown the relationship between postharvest treatments and

polyphenolics in mango, but mostly physiological changes such as color, firmness, shelf-

life and ripening rate. Thus, two studies were designed to investigate how postharvest

treatments affect polyphenolics and antioxidant capacity in mango. The first study was

conducted to figure out the effect of HWT on mango polyphenolics and resultant

antioxidant capacity when mangos were subjected to HWT with three different treatment

times. The second study was conducted to find out phytochemical changes and resultant

antioxidant capacity by CA storage of mango combined with HWT. Controlled

atmosphere storage having two different gas compositions was applied to the mangos

with a control for 2 weeks. The hypothesis was hot water immersion treatment with

varying lengths of time and CA storage with different gas composition combined with

HWT will induce phytochemical and antioxidant capacity changes in mango.

The specific objectives in this study were as follows:

1. To quantify phytochemical changes and resultant antioxidant capacity in mangos

during 4 days storage by varying lengths of hot water immersion treatment (0, 70, 90 and

110 min) applied before storage.

2. To identify and quantify polyphenolics and resultant antioxidant capacity in mangos

following different CA treatments combined with or without hot water immersion

treatment.














CHAPTER 2
LITERATURE REVIEW

2-1. Mango Market

Mango (Magnifera indica. L) is widely considered as the "king" of tropical fruits

due to its world-wide popularity, production, and acreage. Additionally, it is a valuable

and economically important tropical fruit throughout the world due to its vibrant colors,

characteristic taste, and nutritional composition. Fresh fruit imports to the US and Europe

have increased due to increased consumer demands for fresh and processed mango

products. In turn, mangos have become an affordable tropical fruit that has found favor

with consumers in a variety of foods and beverages.

Mango has been cultivated for about 4,000 years and its production and

consumption has gradually increased as its popularity grows. Originating over 4,000

years ago in India and Burma, its cultivation has spread to Malaysia, Eastern Asia, and

Eastern Africa (Mitra and Baldwin, 1997). According to history records in the US,

mangos began to be cultivated in Cape Sable, Florida in 1833 (Crane and Campbell,

1994; CRFG, 2001). Now, at least 87 countries are known to grow over 26,286,255 MT

in 2004 throughout the world (Food and Agricultural Organization [FAO], 2004; Sauco,

2004). Mango production is highest in India, the leading mango producing country at

41% of the world's production (10,800,000 MT), followed by China, Thailand, Mexico,

Pakistan, Indonesia, the Philippines, Nigeria, and Brazil (FAO, 2004) (Table 2-1). Other

mango producing countries such as Australia, Peru, Venezuela, Haiti, and Dominican

Republic also produce and export mangos (DA-AMAS., 2003; Jacobi et al., 2001;









Mossler et al., 2002; Sauco, 2004) (Table 2-1). Asia produces 76.9% of the total world

production followed by the Americas (13.8%) and Africa (9%) (Sauco, 2004). Mexico is

the leading mango exporter country at 41% of the world market (102,500 MT), followed

by Philippines (7.8%) and Pakistan (7.6%) (Sauco 2004). The world's largest mango

importing country is the US (Table2-2) and the mango market in the US has steadily

grown in response to increasing demand. Mexico is also the leading mango exporter to

the US market followed by Brazil, Ecuador, Peru, Haiti and Guatemala (Sauco, 2004).

Even though the history of mango production and consumption in the US is relatively

short compared to other countries such as Europe and China, now the US is the largest

mango importing country due to great demand in the domestic mango market.

Table 2-1. Main mango producing countries in 2004 (www.fao.org)
Production
Rank Country
1 India 10,800,000
2 China 3,400,000
3 Thailand 1,750,000
4 Mexico 1,503,010
5 Pakistan 1,072,000
6 Philippines 890,000
7 Brazil 845,000
8 Indonesia 800,000
9 Nigeria 730,000
10 Vietnam 337,000
11 Egypt 327,000
12 Haiti 261,000
13 Bangladesh 243,000
14 Cuba 235,000
15 Madagascar 210,000
16 Democratic Republic of the Congo 200,000
17 Sudan 195,000
18 United Republic of Tanzania 195,000
19 Guatemala 187,000
20 Dominican Republic 180,000









Table 2-2. Global imports of mangos (MT) (Sauco 2004)
Country 1998 1999 2000
USA 197,000 219,000 235,000

Europe 115,000 170,000 172,000

Hong Kong 47,000 33,000 33,000

UAE 39,000 38,000 38,000

Malaysia 21,000 25,000 25,000

Saudi Arabia 14,000 14,000 14,000

Singapore 11,000 14,000 15,000

Japan 9,000 9,000 10,000


Since mangos are grown in tropical climate, they are also cultivated in the southern

states in the US. However, Florida is the only state in the United States where agricultural

statistics are reported for mangos even though mangos are also cultivated to various

extents in Hawaii, California, Texas, and Puerto Rico (Mossler and Nesheim, 2002).

More than 80% of Florida mangos are cultivated in Miami Dade County with the

remaining acreage in Lee, Palm Beach, and scattered other counties where adequate

conditions for growth exist (Mossler et al., 2002). Recently, new mango trees are being

planted in Merritt Island in Florida (Mossler and Nesheim, 2002).

Before Hurricane Andrew in 1992, mango production in Florida was over 10,000

MT from 1172 ha (Crane and Balerdi, 1997). However, the production was reduced to

1,250 MT from 648 ha. Recovery from damage was poor and slow because many

remaining trees continued dying, damaged bark was uncovered from excessive sunlight

and roots of trees got damaged (Crane and Balerdi, 1997). As a result of poor recovery,

Florida production is still less than 1% of total consumption in the US (HAP, 2002;

Sauco, 2004).









2-2. Mango Cultivars

Among over 1,000 different mango cultivars throughout the world, about 800

mango cultivars have been named (Pandey, 1986). Since each mango growing country

possesses different climate, geological feature, harvest time, and marketing season, each

country generally has its own major cultivars for commercial use (Nakasone and Paul,

1998; Sauco, 2004) (Table 2-3). For example, Australian production is dominated >95%

by a single variety "Kensington" (also known as 'Kensington pride') and 'Tommy

Atkins' and 'Keitt' are the major varieties grown in Florida (Crane and Campbell, 1994;

Jacobi et al., 1998). Since many cultivars have their own characteristics, each mango is

cultivated according to their regional and climatic conditions in their respective countries.

Mango may be divided into two types, which are Indian and Indo-Chinese. Since

two types have quite different features, this is a good indicator to classify mangos. For

example, Indo-Chinese type mangos have polyembryonic seeds that have multiple

embryos while Indian type mangos have monoembryonic seeds (Crane and Campbell,

1997). Indo-Chinese type mango has better resistance to anthracnose caused by

Colletotrichum gloeosporioides Penz, which is the most important fruit disease in Florida

(Crane et al., 1997; Gamagaea et al., 2004; Vivekananthan et al., 2004). Since 'Tommy

Atkins' mango is an Indo-Chinese type, it is relatively resistant to anthracnose.

Even though it has been reported that about eighty mango cultivars are found in

Florida, only a few varieties are commercially cultivated. 'Tommy Atkins', 'Keitt', 'Van

Dyke', 'Palmer', 'Kent', 'Haden', 'Sensation', and 'Parvin' are commercially grown varieties

in Florida (Crane and Campbell, 1994; Sauco, 2004). Especially, 'Tommy Atkins' and

'Keitt' accounts for >50% of the total production in Florida (Campbell, 1992; Morton,

1987). 'Tommy Atkins' is oval in shape (8 to 15 cm in diameter) and its size ranges from









medium to large at 450 to 750g with a thick, tough adherent skin that is yellow to orange

in color (Gilman and Watson, 1993). Even though 'Tommy Atkins' has relatively poor

eating quality (rated fair to good) and has fibrous pulp, it is the most widely distributed

and commercially variety produced in Florida due to great resistance to anthracnose,

tolerance to handling damage and endurance to hot water immersion treatment (Campbell

and Campbell, 1993).

Table 2-3. Producing countries, selected cultivars, and main marketing season (Nakasone
and Paull, 1998)
Country Selected Cultivar Marketing Season
USA-Florida Keitt, Irwin, Tommy Atkins, Kent, Van Dyke, Palmer Jul-Aug

Australia Kensington Pride, Keitt, Kent, Palmer, Irwin Oct-Mar

India Alphonso, Banganpalli, Dashehari, Bangalora, Langra, Apr-Jul
Mulgoa, Neelum, Pairi
Brazil Haden, Tommy Atkins, Kent, Keitt, Palmer, Bourbon, Oct-Feb
Espada, Itamarco, Caco, Rosa, Carlota
Indonesia Arumanis, Dodol, Gedong, Golek, Cengkir Sep-Jan
Israel Keitt, Tommy Atkins, Kent, Maya, Haden Jul-Aug

Malaysia Harumanis, Golek, Maha 65, MA 200 (Malgoa) Jun-Aug

Mexico Haden, Manila, Esmeralda, Kent, Keitt, Tommy Atkins, Apr-Oct
Jan Dyke, Palmer
The Philippines Carabao, Pico, Julie Jun-Sep

South Africa Peach, Zill, Fascell, Sensation, Tommy Atkins, Keitt Nov-Jan
Spain Tommy Atkins, Keitt, Lippens, Osteen Jul-Aug
Taiwan Irwin, Yellow No.1, Haden Jul-Oct
Thailand Nan Dok Mai, Rad, Tongdum, Okrong Mar-May


2-3. Mango Ripening

Ripening is a complex process that involves not only many physiological changes

but many chemical changes due to the fact that postharvest fruits continue to respire.

Mango is a climacteric fruit, which means that its respiration rate rises as fruit ripening

proceeds in response to ethylene (Alexander and Grierson, 2002; Silva et al., 2003).









Physiological changes occur as a result of chemical alterations in climacteric fruits during

ripening such as a change in pulp and skin color (loss of chlorophylls and synthesis of

carotenoids), loss of weight and volume (loss of moisture), decline of firmness (softening

caused by pectin breakdown), altered taste (loss of acidity and increased soluble solids

due to conversion of starch to sugar) and synthesis of more ethylene (a ripening hormone)

(Jacobi et al., 1998; Kader, 1997; Lizada, 1991; Modi and Reddy, 1967).

Since mangos are harvested when they are still green as a climacteric fruit, they are

expected to withstand postharvest handling, and then complete their ripening (riper stages

generally preferred by consumers) at the fresh market, determining exact ripening indices

are important for market consideration. Chemical parameters may include soluble solids

contents, acidity, starch content, and phenolic constituents while physical parameters may

include shape, size, surface color, and shoulder growth (Mitra and Baldwin, 1997). The

most definitive indicator of a mango's maturity is to determine shoulder growth (from

green to mid-ripe) and color development (almost all yellow color pulp) (Holmes et al.,

1990; Jacobi et al., 1998). Even if mangos are of the same variety and harvested on the

same day, they could be different from each other, for example, their shape, color

development, and weight. This is because the amount of sunlight, rain, and other

environmental factors are different, depending on location on a tree even though fruit was

grown on the same tree. In conclusion, accurate standards to distinguish the degree of

maturity as well as the degree of ripeness are important for mangos to adjust time for the

fruit market.









2-4. Mango Postharvest Handling

2-4-1. Hot Water Immersion Treatment (HWT)

Thermal quarantine treatment is legally required for fresh mangos to be accepted by

several importing countries such as the United States and Japan because imported

mangos are highly susceptible to infection by fruit flies in the form of adults, larvae, or

eggs. In the US, mangos from other countries must go through a thermal quarantine

treatment to eliminate invasive pests (e.g. Ceratitis capitata (Mediterranean fruit fly) and

Anastrepha spp., Anastrepha ludens (Mexican fruit fly)) before importing to the US

(APHIS, 2002). Several methods for quarantine treatments are available for fruits

including irradiation, vapor heat, forced hot-air, and hot water immersion treatments

(APHIS, 2002). However, only hot water immersion treatment (HWT) is approved in the

US to disinfect mango; not only is HWT the most effective treatment, but it has several

direct advantages (Jacobi et al., 2001). Advantages include ease of handling and strict

control of treatment environments, which allows for exact temperature control during

treatment, and less cost as an additional benefit of HWT (about 90% less than vapor heat

treatment) (Sharp and Hallman, 1994; Fallik, 2004).

Required HWT conditions such as treatment time, temperature, and water purity

are rigidly and specifically developed and controlled by USDA-APHIS (Animal and

Plant Health Inspection Service) because fruit fly and/or its larvae and eggs may remain

under a variety of storage conditions. The target disinfestation probability in eliminating

pests in fruit is over 99.9968% ("probit 9") in many countries (Jacobi et al., 2000). Since

the profit value is so high, every condition for thermal quarantine must be controlled

strictly. Legally required time and temperature for HWT in the US is 70, 90, or 110 min

immersion at 460C, so the guidelines published by USDA-APHIS sets these times based









on cultivar, weight, size, shape, and country of origin (USDA-APHIS 2002) (Table 2-4).

However, hot water treatment may damage the quality of mangos and cause more

internal and external injury than vapor treatment if the treatment temperature is out of

optimum range (Jacobi and Wong, 1992).

Table 2-4. Determined dip time based on origin, shape, and weight of fruit. (USDA-
APHIS, 2002)
Fruit weight
Origin of fruit Fruit shape Fruit weight Dip time (minutes)
(grams)
Flat, elongated Up to 400 65
varieties1 400 to 570 75
Puerto Rico, U.S. Virgin
Up to 500 75
Islands, or West Indies Rounded
2 500-700 90
varieties
701-900 110
Flat, elongated Up to 375 65
Mexico or Central America varieties1 400 to 570 75
(North of and including Costa Up to 500 75
Rounded
Rica) 2 500 to 700 90
varieties
701 to 900 110
Flat, elongated Up to 375 65
Panama, South America or varieties 375 to 570 75
West Indies islands of Aruba, varieties 375 to 570 75
Bonaire, Curacao, Margarita, Rounded Up to 425 75
Tortuga, or Trinidad and Tobago varieties2 425 to 650 90

*'Cultivars: 'Frances', Carrot', 'Zill', 'Ataulfo', 'Carabao', 'Irwin', and 'Manila'.
*2Cultivars: 'Tommny Atkins', 'Kent', 'Heyden', and 'Keitt'.

An organized process for HWT was also set as a standard by USDA-APHIS.

According to the Hot Water Treatment Schedule (APHIS, 2002), mangos must be sorted

by origin, weight and shape before HWT. Water quality for washing, dipping, or

showering the fruit should be considered before treatment and the water must be

chlorinated (50-200 ppm). During the treatment, the fruit must be submerged at least 4

inches (10.16 cm) below the water level at the target temperature. In addition, the water

must be constantly circulated and the temperature of the water should be kept at 460C

0.30C throughout the treatment. After HWT, mangos should stand for at least 30 minutes









prior to refrigeration to ensure that all fruit fly larvae would be completely killed during

this time. Recommended temperatures for refrigeration are 55-570F (12.8-13.90C) at 85

to 90% RH. This storage condition will delay softening and extend storage life to 2 to 3

weeks.

2-4-2. Controlled Atmosphere (CA) Storage

During transport from an exporting country to an importing country by trucks

and/or ships, fruits can easily perish unless they are held under proper storage conditions

because transport usually takes from a few days to a few weeks (usually 2-3 weeks).

Since the main mango exporter for the US market is Mexico followed by Brazil, Ecuador,

Peru, Haiti and Guatemala, shipping mangos may vary according to the targeted countries.

In order to protect quality, delay ripening, reduce ethylene production and respiration rate,

prevent fruit disease, and extend shelf-life during shipping, CA storage is sometimes

employed (Abd. Shukor et al., 2000; Rattanapanone and Watada, 2000). Domestic mango

production satisfies a small portion (about 1%) of total mango consumption while the

mango market is getting larger in the US. Since about 99% of mangos are imported, CA

storage should easily be applied to the US mango market as a means to extend shelf-life

and keep the quality during transportation and storage.

CA storage is a common method used to preserve fruits and vegetables from quality

deterioration and to extend shelf-life. CA storage, an artificially increased CO2 and

decreased 02 level environment at reduced temperatures, decreases ethylene (ripening

hormone) production, reduces sensitivity to ethylene and lessens respiration rate. Thus, it

can prolong shelf-life of fruits, postpone chlorophyll degradation, and keep the texture of

fruit (Mitra and Baldwin, 1997; Kader et al., 1989; Kader, 2002). Since fruits are still









'alive' even after harvest by continuing to respire (consuming 02 and producing C02),

CA storage conditions are effective to inhibit respiration. CA storage also has additional

benefits such as maintaining titratable acidity, preserving fruit firmness, and reducing

pitting (Wang and Vestrheim, 2002; Chen et al., 1981; Sanchez et al., 2003). Therefore,

CA storage can help maintain fruit quality deterioration and it also gives additional

advantages to fruits such as extent postharvest shelf-life and delayed ripening.

The optimum 02 and CO2 composition and temperature in a CA chamber varies

with the cultivar and growing region of fruits. In general, optimum atmosphere

composition for mature-green mangos is 3 to 5% of 02 and 5 to 8% of CO2 at 12 to 130C

for up to 3 weeks and for tree-ripe mangos is 3 to 5% of 02 and 5 to 8% of CO2 at 80C or

5% 02 and 10% CO2 at 50C for 3 weeks (Bender et al., 2000). These recommended

conditions vary due to natural variability of fruit and dynamic response to storage

conditions (Saltveit, 2003). Too low 02 and too high CO2 levels in CA storage can induce

anaerobic respiration and fruit injury (Kader et al., 1989). Anaerobic respiration can

cause shortening of shelf-life, increasing susceptibility to decay, producing off-flavors,

and leading to physiological disorders (Brecht, 1980; Kader et al., 1989). Furthermore,

the symptoms of CO2 injury (too high C02) are development of abnormal and grayish

color, prevention of normal aroma development, production of off-flavors, and generation

of grey spots. The symptom of 02 injury (too low 02) is irreversible inhibition of ripening

(chlorophyll decline) (Bender et al., 2000.; Mitra and Baldwin, 1997). Keeping the

atmosphere concentration in the optimum range in the CA chamber is important to

prevent anaerobic respiration and fruit injury. Thus, atmosphere composition has to be

monitored and adjusted to maintain optimum gas concentration.









2-5. Mango Polyphenolics

Mango is a source of phenolic compounds that improve food quality, and prevent

food deterioration. Those are gallic acid, gallotannin, p-OH-benzoic acid, and ferulic acid,

which are found in mango pulp and skin (Schieber et al., 2000). Phenolic compounds are

the most widely distributed plant secondary metabolites and are found in all higher plants

(Hagerman et al., 1998). Basically, they have one common structural feature, which is a

phenol (an aromatic ring possessing at least one hydroxyl substituent) (Robbins, 2003).

Phenolics may be divided into two categories that include polyphenols and simple

phenols based on the number of phenol subunits present, for example, polyphenolics have

at least two phenol subunits and tannins have at least three phenol subunits. Phenolic acid

is a phenol that has one carboxylic acid functional group and is related to color, sensory

quality, and antioxidant capacity of foods (Robbins, 2003). Polyphenolics are abundantly

found in fruits and vegetables and are important compounds in reducing risks of human

diseases and oxidative damage to biological membrane (Kelly et al., 2002; Robbins,

2003). Thus, it is important for human to increase consumption of polyphenolics with

antioxidant properties by eating fruits and vegetables (Kang and Saltveit, 2002).

Moreover, polyphenolics provide various functionalities, such as color, flavor, and

astringency in foods, and polyphenolics are likely to be involved in the plant defense

mechanism as a chemical barrier, helping the plant to recover from injured surface

(Grundhofer et al., 2001; Haard and Chism, 1996).

Technically, phenolics that have three or more phenol subunits and have an ability

to precipitate proteins are categorized as "tannin" and are categorized as either

"condensed" or "hydrolyzable" tannins (Hagerman, 2002; Robbins, 2003). Tannins are a

specific class of polyphenolics that have been identified, but poorly characterized in









mangos. Generally, tannins have high molecular weights (>1000 amu) and can be found

in abundance in oak, tea, sumac, nuts, and some fruits. Several valuable functions of

tannin include protein precipitation, cell wall stabilization, insecticides, herbicides, and

anti-carcinogenic agents due to their antioxidant capacity (Hartzfeld et al., 2002; Osslpov

et al., 1997; Werner et al., 1999). Tannin has about 15-30 times better peroxy radical

quenching capacity than simple phenol and Trolox because it has relatively many

hydroxyl groups and it is highly polymerized (Hagerman et al., 1998). Condensed tannins

are polymeric forms of (+)-catechin and (-)-epicatechin and/or vary from dimers to

considerably larger polymeric procyanidins, due to their ability to form cyanidin or

delphinidin (prodelphinidins) in the presence of acid and heat. There are two kinds of

hydrolyzable tannins: gallotannin and ellagic acid which are derivatives of gallic acid

(Hagerman, 2002).

Gallic acid (3,4,5-trihydroxybenzoic acid) is an abundant polyphenolic compound

found in mango with gallotannins and it is derived from quinic acid via the shikimic acid

pathway (also known as the phenylpropanoid pathway). Gallotannin is the simplest form

of hydrolyzable tannin. As a polygalloyl ester of glucose, gallotannin has five hydroxyl

groups attached to the core sugar (glucose) (Hagerman, 2002) (Fig 2-1). Gallotannin is

divided into two parts, glucose and gallic acid, upon hydrolysis with strong acid and is

extended by adding galloyl esters via meta-depside bonding to glucose (Hagerman, 2002;

Niemetz et al., 1999) (Fig 2-3).


























HO
Figure 2-1. Structure of gallotannin (p-1,2,3,4,6-pentagalloyl-O-D-glucose) (Zalewska et
al. 1995)

sugar

upper



OH

Depside bond --- O
lower
Ko j gallic acid


HO OH
H
Figure 2-2. A depside bond which is formed between the phenolic group of the upper and
the acid group of the lower gallic acid units (Mueller-Harvey 2001)

Many polyphenolics in mango were previously identified. Quercetin and

kaempferol were the first polyphenolics to be tentatively identified in mango using 2-

dimensional paper chromatography (El- Ansari et al., 1969). Subsequently, El-Ansari et

al. (1969) reported the presence of ellagic acid, gallic acid (GA), m-digallic acid, m-

trigallic acid, isoquercetin, mangiferin, quercetin and gallotannin in mango. Gallic acid

and gallotannin are known as the major polyphenolic compounds found in mango, which

are important antioxidants to quench harmful free radicals (Saleh and El-Ansari, 1970).









2-6. Polyphenolics as Antioxidants

Oxygen is necessary for human beings and other living organisms to function and

the oxidative mechanism is necessary for the cells to survive in the body. However, when

oxygen is transformed to free radicals and ROS (reactive oxygen species), which then act

as oxidants that have a tendency to donate their oxygen to other materials, oxygen plays a

harmful role in our body (Karakaya et al., 2001). Many ROS are free radicals. Free

radicals and other ROS such as peroxy radical, hydroxyl radical, singlet oxygen derived

from normal essential metabolic process or other external sources can harm all kinds of

biological materials such as proteins, carbohydrates, lipids, and nucleic acid (Karakaya et

al., 2001; Femrnndez-Pach6n et al., 2004). In the human body, ROS and free radicals can

affect the formation of biological membranes, change enzyme activity, induce abnormal

organ metabolism, and influence the generation of cataracts, atherosclerosis, and

degenerative diseases (Karakaya and Nehir, 1999; Leitao and Mensor, 1999). ROS and

free radicals cause oxidation that induces deterioration of food, resulting in rancidity,

changes in color, and declines in nutritional quality, flavor, texture and safety

(Antolovich et al., 2002).

Fortunately, antioxidants such as ascorbic acid, tocopherol, carotenoid, and

polyphenolics can quench and neutralize free radicals (Shadidi and Naczk, 1995).

Consequently, antioxidant capacity also reduces the possibility of several diseases like

cancer, stroke, atherosclerosis, and cardiovascular diseases (Karakaya et al., 2001;

Shadidi and Naczk, 1995). Antioxidant capacity of phenolic compounds comes from their

redox properties in acting as a reducing agents, hydrogen donator, metal chelator and

singlet oxygen quencher (Pyo et al., 2004). Even though there are other antioxidant

compounds in fruits and vegetables such as ascorbic acid, tocopherol, carotenoids, and






18


lycopenes, phenolic compounds have stronger radical quenching capacity (Pyo et al.,

2004; Toit et al., 2001). For example, flavonoid including multiple functional phenolic

groups has 2 to 5 fold stronger radical quenching capacity than ascorbic acid and

tocopherol (Toit et al., 2001).














CHAPTER 3
PHYTOCHEMICAL CHANGES AND RESULTANT ANTIOXIDANT CAPACITY IN
MANGOS DURING 4 DAY STORAGE AFFECTED BY VARYING LENGTHS OF
HOT WATER IMMERSION TREATMENT

3-1. Introduction

Mango (Magnifera indica L.) has been a popular and economically important

tropical fruit throughout the world due to its excellent eating quality (bright color, sweet

taste and luscious flavor) and nutritional composition (diverse amount of vitamins,

minerals, fiber and various antioxidant compounds). Fresh fruit imports to the US,

Europe, and Japan have increased due to increased consumer demand for fresh and

processed mango products. In turn, mangos have become an affordable tropical fruit that

has found favor with consumers in a variety of foods and beverages. In the US, although

mangos are commercially produced in Florida and Hawaii, the amount of production is

not enough to meet domestic demands. Thus, mango importation in the US has gradually

increased in the market. Since imported mangos can easily be a host for Ceratitis capitata

(Mediterranean fruit fly) and Anastrepha spp., Anastrepha ludens (Mexican fruit fly)

causing quarantine risks in the form of adults, larvae, or eggs (Jacobi et al., 2001; USDA-

APHIS, 2002), all mangos imported to the US must be subjected to a thermal quarantine

treatment to eradicate invasive pests. Several methods for quarantine are available such as

irradiation, fumigation, vapor heat, forced hot-air, and hot water immersion treatments.

However, only the hot water immersion treatment is legally allowed in the US because it

is the most effective quarantine method for mangos (Jacobi et al., 2001).









Numerous studies have addressed physiological changes (e.g. heat injury, heat

tolerance, ripening velocity, and shelf-life) caused by hot water immersion treatment

(HWT). Polyphenolic compounds such as gallic acid and hydrolyzable tannin are found

in mango and are important because these phytochemicals have a strong antioxidant

capacity and therefore serve to improve food quality. However, limited information

concerning phytochemical changes after HWT is available. This study concentrated on

phenolic compound changes caused by HWT with varying length of treatment time and

resultant antioxidant capacity. The objective of this study was to identify and quantify

polyphenolic compounds and antioxidant capacity in fresh mangos after three HWT times

for two ripeness stages and its affect on fruit quality.

3-2. Materials and Methods

3-2-1. Fruit Preparation and Treatment

Mangos for this study were obtained from Lyons Farms in Homestead, FL in June

2002. Forty mature-green mangos were chosen based on the uniformity of color, size, and

weight. Fruits were transported on the day of harvest to the University of Florida and

subjected to HWT with a laboratory scale fruit heating system (Model HWH-2, Gaffney

Engineering, Gainesville, FL). All mangos were stored for 2 days at 140C prior to the

treatment. The mangos were randomly divided into four groups and the first three groups

were immersed into a hot water bath for 70 min (HW70), 90 min (HW90), or 110 min

(HW110) at 460C. The fourth group remained untreated to use as a control (HWO). Since

median weight of sample mangos was 497.5g, required treatment time was 75 min in

accordance with the direction of APHIS treatment manual for mangos (USDA-APHIS,

2002). In this study, 70 min, the nearest time to 75 min, was determined as required

treatment time for a regular 20 min interval between treatments (70, 90 and 110 min).









Because treatment time usually varies within a range of 10 min according to

hydrocooling rate and the median was less than 500g, 5 min difference might not

influence on the result caused by HWT. Samples were acquired for immediate analysis

after HWT while remaining fruit was held at 230C for an additional 4 days prior to

obtaining analysis samples. A total of eighteen mangos were evaluated for each of the

four treatments and included a control, divided into five groups with the edible flesh of

three mangos pooled for analysis. The sixth group was utilized to measure fruit core

temperature and they were not included in the dataset. Core temperature was obtained by

placing a thermocouple inside the fruit during treatment.

Table 3-1. HWT with four different lengths of time (0, 70, 90, and 110 min) at 460C with
different ripeness stages (day 0 and day 4) at 230C
Treatment
Period HW 0 (control) HW 70 HW 90 HW 110
Period

C-1 H70-1 H90-1 H110-1

C-2 H70-2 H90-2 H110-2

Day 0 C-3 H70-3 H90-3 H110-3

& Day 4 C-4 H70-4 H90-4 H110-4

C-5 H70-5 H90-5 HI 10-5

C-6 H70-6 H90-6 H1 10-6

Total DO-18, D4-18 DO-18, D4-18 DO-18, D4-18 DO-18, D4-18


Mango skins were manually peeled and flesh blended using a laboratory blender

(Waring commercial, Model 31BL91) for 5 min to obtain a consistent puree and held at -

200C until analysis whereby the samples were thawed for chemical analysis. A 5g puree

was treated with 20[tl of pectinase (Pectinex Ultra SP-L from Aspergillus aculeatus.

SIGMA chemical Co.), then incubated at 350C for 3 hours, and centrifuged until a clear









supernatant (clarified mango juice) was obtained from which phytochemical analyses

were conducted.

3-2-2. Identification and Quantification of Individual Polyphenolics

Individual polyphenolics were identified and quantified using HPLC as described

by Talcott et al. (2000). Clarified juice isolates were filtered through a 0.45 [im PTFE

filter (Whatman, Clifton, NJ) and then injected into a Waters 2695 Alliance

chromatography system. Compounds were separated on a Waters Spherisorb ODS 2

column using a gradient elution program. Mobiles phases consisted of Phase A (98%

H20: 2% Acetic acid) and Phase B (68% H20: 30% Acetonitrile: 2% Acetic acid) run at

0.8 mL/min. Polyphenolics were separated using a gradient elution system that kept

mobile phase B 0% for 5 min and then changed phase B 0% to 10% in 20 min; 10% to

25% in 30 min; 25% to 50% in 40 min; 50% to 75% in 50 min; 75% to 100% in 70 min

and returned to original condition in 2 min for the next injection. Polyphenolics were

detected and quantified at 280 nm using a Waters 996 photodiode array (PDA) detector

against an external standard of gallic acid. Unknown compounds were characterized

based on retention time and UV spectral similarities to authentic standards (Sigma

Chemical Co., St. Louis, MO) using Millennium 32i workstation.

3-2-3. Quantification of Total Soluble Polyphenolics

Total soluble phenolic (TSP) concentration (a measure of total metal ion reducing

capacity) including contributions from ascorbic acid was determined by Folin-Ciocalteau

assay (Swain and Hillis, 1959). For each clarified juice isolate, 50 yL mango juice was

added to 1 ml of 0.25 N Folin-Ciocalteu reagent and mixed by vortex for 30 sec. After 3

minutes reaction time, 1 ml of IN sodium carbonate was added to form a water-soluble









chromophore for a distinguishable blue color. After standing for 7 minutes, all the

extractions were transferred to a Spectramax 96-well and absorbance (Softmax PRO,

Sunnyvale, CA) was read at 726 nm after 2 hours. TSP was quantified in mg/L gallic acid

equivalents (GAE).

3-2-4. Quantification of Antioxidant Capacity

Total antioxidant capacity of mango phytochemicals (from polyphenolics and

ascorbic acid) was measured using the ORAC assay (oxygen radical absorbance capacity)

run according to Talcott et al. (2002) and adapted to work with a 96-well Molecular

Devices fmax fluorescent microplate reader (485 nm excitation and 538 nm emission).

This method is based on the principle of the inhibition of oxidation of a fluorescent

compound (fluorescence) in the presence of the peroxyl radical generator 2,2'-azobis (2-

amidinopropane) dihydrochloride (AAPH). A stock solution of fluorescein was produced

by mixing 10yl of stock fluorescent solution and 50 ml of phosphate buffer (61.6:38.9 v/v,

0.75 M K2HPO4 and 0.75M NaH2PO4, pH 7.2) as described by Ou et al. (2001) and the

peroxyl radical generator was made by mixing 350mg of AAPH in 5 ml of phosphate

buffer (pH 7.0). The rate of fluorescence decay was recorded every 2 min for 70 min by

calculating the area under the fluorescence decay curve for a standard curve (0-50 yM), a

blank (ORAC = 0 yM), and mango juice following appropriate dilution in phosphate

buffer. Antioxidant capacity affected by treatments was quantified against a standard

curve of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-

soluble analog of vitamin E (ORAC = 1 yM Trolox) with potent antioxidant properties

(Javanovic et al., 1994). Trolox was used at the concentrations of 1.65, 3.125, 6.25, 12.5,

25, and 50 IM by serial dilution with the phosphate buffer. Each extract was diluted 50









fold in pH 7.0 phosphate buffer before pipetting into a 96-well microplate. Data was

represented in pM Trolox equivalents per mL of clarified mango juice.

3-2-5. Statistical Analysis Method

Changes in phytochemicals and antioxidant capacity affected by HWT time and

fruit ripening were analyzed by analysis of variance (ANOVA) using JMP 5 statistical

software (SAS Institute, 2001). Mean separation was conducted using the LSD test,

P<0.05.

3-3. Results and Discussion

3-3-1. Individual Polyphenolic Concentration

Free gallic acid and hydrolyzable tannins were the major polyphenolics identified

in mango and quantified against an authentic standard of gallic acid (Talcott et al., 2005).

Average gallic acid concentration during the 4 days storage was changed as a result of

HWT (Fig 3-1). Gallic acid concentration at HW70 did not show significant difference

from the control while concentrations at HW90 and HW110 were significantly lower

(41% and 42% less, respectively) than the control. Synthesis of gallic acid was inhibited

by HWT only when treatment time is longer than that required for quarantine treatment

depending on cultivar, weight, shape, and origin (APHIS, 2002; Lurie, 1998).

When fruits were treated with hot water for extended time, heat-shock protein

might have been induced. Heat shock proteins (HST) are unique proteins produced in

response to heat-shock treatment (high temperature and/or long time) (Saltveit, 1998).

Heat-shock inhibited synthesis of phenylalanine ammonia-lyase (PAL), which is the first

enzyme of primary phenolic pathway (phenylpropanoid pathway) (Loaiza-Velarde et al.,

2003; Rivero et al., 2001; Saltveit, 1998; Saltveit, 2000a). Since PAL activity induces

accumulation of phenolic compounds, gallic acid reduction might be affected by










decreased PAL activity when mangos were treated with hot water for extended time

(Saltveit, 2000b).

Average gallic acid concentration for all HWT increased by 24% during 4 days

storage (Fig 3-1). During the 4-day storage, gallic acid concentrations of control and

HW70 did not change while the concentrations of HW90 and HW1 10 significantly

increased. At both day 0 and day 4, they showed a similar result, in which gallic acid

significantly decreased only when they were treated for longer times (HW90 and

HW1 10). Reduced gallic acid concentration of HW90 and HW1 10 by extended time

treatment might continue to recover during 4 day storage.




16
Day 0
14 4 Day 4

A
12 A A

=LAB
10 AB





6 F A

4-F




Control HW70 HW90 HW110

Hot Water Treatment

Figure 3-1. Changes in average gallic acid concentration (ig/g GAE) for two ripeness
stages (day 0 and day 4) affected by HWT with varying length of treatment times
(0, 70, 90, and 110 min). Average values and standard error bars of triplicate
samples for all treatments are represented.









Since extended hot water treatment not only reduced phenolic compounds, but also

induced heat injury, it might be an undesired process for fruit and vegetables storage.

Reported symptoms of heat injury are abnormal color development, odd softening, the

lack of starch breakdown, and the development of internal hollows in mango (Jacobi and

Wong, 1992; Lurie, 1998).


Control HW70 HW90 HW110

Hot Water Treatment


Figure 3-2. Changes in total hydrolyzable tannin concentration (ig/g) for two ripeness
stages (day 0 and day 4) as a result of different lengths of HWT (0, 70, 90, 110
min). Average values and standard error bars of triplicate samples for all
treatments are represented

Four hydrolyzable tannins were tentatively identified based on UV spectral

similarities to gallic acid due to the diversity of hydrolyzable tannin present (Talcott et al.,

2005). As a result of the four different durations of HWT, the concentration of

hydrolyzable tannins increased (33% more) only at HW70, but the concentrations at









HW90 and HW110 were lower (37 and 34% less, respectively) than control (Fig 3-2). It

was a similar result as gallic acid except the concentration of hydrolyzable tannins at

HW70 increased and was highest among all the HWT. Since heat stress causes PAL

activity that is a core enzyme of phenylpropanoid pathway in catalyzing synthesis of

phenolic compounds, hydrolyzable tannin concentration could be increased by thermal

stress (Rivero et al., 2001). Reduction of hydrolyzable tannin concentration at HW90 and

HW110 may be induced by heat-shock as explained in gallic acid reduction.

Phenolic compounds of mango were found to decrease during ripening with a loss

of astringency related to loss of phenolic content (El-Ansari et al., 1971;

Lakshminarayana et al., 1970; Mitra and Baldwin, 1997; Saleh and El-Ansari, 1970).

However, in this study, total hydrolyzable tannin concentration did not change while

gallic acid concentration significantly increased during the 4 day storage. No change of

hydrolyzable tannin concentration could be explained from the fact that since HWT

inhibits ethylene synthesis, ripening might be delayed (Lurie, 1998). Thus, delayed

ripening may be the reason for the lack of change in hydrolyzable tannin concentration in

mango even though there was a 4-day storage time difference.

As shown in Fig 3-3, identified gallic acid and four hydrolyzable tannin peaks were

eluted by HPLC at Day 0 and Day 4. According to peak area, about 30% of gallic acid

increased during the 4-day storage while on average the four hydrolyzable tannins did not

show any difference. Changes of gallic acid and four hydrolyzable tannins were shown in

Table 3-2.











0 40
035
SDayl
0300
0 25- 3





0 05
0 00

500 1000 1500 2000 2500 305 3500 4000 4500 5000 5500 6000


040
035 Day4

0 305

020
015
010
IV ;
0 0
0 00

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000
Minutes



r *:::; 3-3. HPLC chromatogram of .. i :enolics, gallic acid and four hydrolyzable
tannins (HT), present in mango flesh at Day 0 and Day 4. Peaks were tentatively
identified based on retention time and spectral similarities against an authentic
standard of gallic acid



Table 3-2. Gallic acid (pg/g) and average of four hydrolyzable tannins (lg/g GAE)
identified and quantified using HPLC as a result of different duration of HWT (0,
70, 90, 110 min) at two ;:1 j ... stages (Day 0 and Day 4)
Ripeness HotWater Gallic UT I T 2 UT 3 UT 4 Total
Stages Treatment Acid HT
Day 0 HWO 11.59a 4.88b* f > 1.' 6.76a 1.27b 21.6
HW70 9.39a 7.27a* 21.7a 7.42a* 1.13b 37.5
HW 90 5.15b 5 :.:. 6. :. 1.10b 14.7
HW110 4.70b 5.17b 5.92c 1.87b 1.04b 14.0


.,4 HWO 10. : 3. 1'- 1 7.03a 1.:::- 24.9
HW70 11.42a 3.79a 22.42a 2.18c 31.1
HW90 8.18b* 3.77a 6.01d 2.81bc* 2.14b 14.7
HWI10 9.16ab* 3.89a 7.59c 3.86b* 1.64c* 17.0
a ...* ..... =.: ... between .. "* i.. :1. of treatment times at each day of ripeness.
. .:::. ..: letters ::1. column inn..: .: significant *:'" --* at each day of ripeness..
3. HT represents hydrolyzable tannin.










3-3-2. Total Soluble Polyphenolics (TSP)

TSP concentration including contribution from ascorbic acid, reducing sugar and

likely small amount of soluble protein in mango decreased in response to HWT. The

average TSP concentrations in HWT mangos (70, 90 and 110 min) for both ripeness

stages (225 [tg/g GAE) showed about 25% decline compared to control (294 yg/g GAE)

(Fig 3-4). At day 4, non-HWT mangos showed higher TSP concentration (36% more)

than HWT mangos. Although no significant difference was found between control and

HWT mangos at day 0, the concentration of control was still higher than those of other

mangos.




500
Day 0
450 WIm Day 4

400-

A
S350- A

300 -
B BC
S250 BCD CDE
DE E DE
200 -

150 -

100 -

50 M

0
Control HW70 HW90 HW110

Hot Water Treatment

Figure 3-4. Changes in TSP (Total Soluble Phenolic) concentration (ig/g GAE) for two
different ripeness stages (day 0 and day 4) effected by four different durations of
HWT (0, 70, 90, and 110 min). Average values and standard error bars of
triplicate samples for all treatments are represented









Varying the length of HWT was not a significant factor influencing the TSP

concentration in mango. Comparing TSP concentrations between three different

treatments excluding control, the average TSP concentration for two ripeness stages was

not significantly different (Fig 3-4). Since polyphenolics were shown to decrease during

ripening, lower TSP concentration was expected at day 4 than day 0 (Haard and Chism,

1996). However, no significant difference was observed between day 0 and day 4.

Technically, 6-9 days are required to transform an unripe mango to a physiologically

ripe mango (developed color and flavor) at 250C, which was the same temperature used

for mango storage in this study (Jacobi et al., 2001). Moreover, ascorbic acid which has

reducing capacity is unstable to heat. Even required duration of HWT could decrease

ascorbic acid content in mango. Therefore, the 4 day storage was a somewhat short

period to expect ripening changes such as TSP reduction and reduced ascorbic acid

content by thermal stress might affect TSP in HWT mangos.

3-3-3. Antioxidant Capacity

Antioxidant capacity, evaluated based on peroxyl radical scavenging activity of

water-soluble mango constituents, was not affected by varying lengths of HWT.

Although average antioxidant capacity of clarified mango juice for two ripeness stages

tended to increase as duration of heating increased, it was not significantly different

compared to control (Fig 3-5).

During the 4-day storage, average antioxidant capacity for different duration of

HWT significantly decreased (7%) (P < 0.05). The radical scavenging ability of mango

did not correlate with TSP in this study because TSP did not change during the 4-day

storage. These observations were unexpected since gallic acid, which was the major









polyphenolic compound and a strong antioxidant in mango, increased during ripening

although TSP and hydrolyzable tannins were not significantly changed. Since ascorbic

acid, which is another contributor to antioxidant capacity was reduced by heat and during

ripening, this might influence the retention of antioxidant capacity (Bashir and Abu-

Goukh, 2003; Soto- Zamora et al., 2005; Yen and Hung, 2000)


Control HW70 HW90 HW110

Hot Water Treatment

Figure 3-5. Changes in total antioxidant capacity (yM TE/g) for two different ripeness
stages day 0 and day 4) affected by four different lengths of HWT (0, 70, 90, and
110 min). Average values and standard error bars of triplicate samples for all
treatments are represented

3-4. Conclusion

Gallic acid concentration significantly decreased as a result of extended HWT

(HW90 and HW110) while the treatment duration required for quarantine treatment









(HW70) did not change gallic acid concentration compared to control. Consequently,

total hydrolyzable tannin concentration was elevated by HWT only with required

treatment time by the water treatment manual while extended time of treatment decreased

HT concentration. Average TSP decreased as a result of HWT regardless of duration of

HWT while antioxidant capacity was not affected by HWT (Table 3-3). Since mangos

imported from other countries must go through a HWT, finding optimum conditions to

improve and/or keep the quality of the fruit is important. According to the results of this

study, the required hot water immersion treatment time for mango quarantine showed

beneficial effects such as retaining gallic acid and increasing hydrolyzable tannins.

Table 3-3. Total soluble phenolics (ig/g GAE) and total antioxidant capacity (yM TE/g)
of mango treated with hot water with varying length of treatment times (0, 70, 90,
and 110 min) at two different ripening stages (Day 0 and Day 4)
Ripening Hot Water Total Soluble Total Antioxidant
Stages Treatment Polyphenolics Capacity
HWO 252.6a 2.67b
Day 0 HW70 248.7a* 2.66b*
Day 0
HW90 237.2ab 2.69b
HW110 226.2b 2.97a*

HWO 335.4a* 2.60a
HW70 218.9b 2.59a
HW90 212.1b* 2.59a
HW110 213.8b 2.41a
S* Indicates a significant difference between varying length of treatment times at each day of ripening.
2 Different letters within column indicate significant difference at each day of ripening.














CHAPTER 4
TO EVALUATE POLYPHENOLICS, ANTIOXIDANT CAPACITY, AND
PHYSIOLOGICAL CHANGES IN MANGOS (MANGIFERA INDICA L.)
FOLLOWING CONTROLLED ATMOSPHERE STORAGE COMBINED WITH HOT
WATER IMMERSION TREATMENT

4-1. Introduction

Even though several states such as Hawaii, California, and Texas grow mangos

Florida is the only state to report mango production in the US (Mossler and Mosheim,

2002). However, according to latest records, domestic mango production in Florida

(2,800 MT) accounted for less than 1% of total consumption in the US in 2004, so more

than 99% of mangos are imported from other countries such as Mexico, Brazil, Peru,

Haiti, and Guatemala to meet the consumer demands (FAO, 2004; Sauco, 2004).

Since many mangos are imported from other countries (278,422 MT in 2003) and it

takes a few days up to a few weeks for the imported mangos to reach retail markets, CA

storage is sometimes used to maintain the quality of fruit during transportation and

storage (FAO, 2004). Especially, mangos imported from other countries must be treated

with hot water to eliminate invasive pest insects. Studies relating CA storage and HWT of

fruits have focused on physiological changes such as firmness, color, moisture content,

acidity and sugar content, so limited information is available relating phytochemical

changes during CA storage with or without HWT to postharvest quality factors during

fruit ripening. Therefore, this study focused on the changes in polyphenolics and resultant

antioxidant capacity during CA storage associated with HWT.









4-2. Materials and Methods

4-2-1. Fruit Preparation and Treatment

For this study, 300 'Tommy Atkins' mangos were procured from Fresh King. Inc.,

Homestead, Florida in June, 2004 at the peak of the harvest season for 'Tommy Atkins'.

Mango fruits with uniform size, oval shape, and green color were pre-selected to reduce

variation among treatments. Fruits were held at 100C during the 6-hour transport to

Gainesville. Mangos were stored at 100C at the Horticultural Sciences Department in

University of Florida for 2 days until hot water treatment and CA storage were applied.

Of the 300 mangoes originally obtained, 234 mangos were selected based on their

uniform size, shape (oval), and weight (average weight >500g).

Prior to treatments, eighteen mangos were selected as controls. This initial set of

fruit, representative of the population was analyzed for phytochemical and antioxidant

content as previously described in chapter 3. Additionally, soluble solids content (SSC)

and titratable acidity (TA) values were quantified as additional indices for fruit quality.

USDA-APHIS guidelines were followed, which involve immersing fruit in water at

45.6- 46.10C for 75 min. The remaining 216 mangos were divided into two groups, half

for HWT and the remaining half immersed in water at 250C as a non-heated control.

Following these treatments, the fruits were held at 250C until the skin was completely dry

(about 60 min) and the center of the heated mangos returned to 250C. The fruit were then

transferred to their respective CA chambers with the following gas contents:

1. CA 1 (Control): Air (about 21% 02 + 79% N2)

2. CA 2: 3% 02 + 97% N2

3. CA 3: 3% 02 + 10% C02 + 87% N2









Fruits were placed in the chambers and gas composition regulated by four external

gas tanks (air, 02, C02, and N2) for 2 weeks at 100C. The gas composition in each

chamber was checked twice daily and regulated to desired composition. After 2 weeks,

half of the mangos were removed and immediately analyzed for phytochemical attributes,

antioxidant capacity, and quality index such as titratable acidity, soluble solids content,

and fruit pulp color. The remaining fruits were moved into a 250C environment to

complete their ripening in an air (21% 02) environment. All the mangos stored in CA

chamber were analyzed as described in Chapter 3. Additionally, the internal flesh color of

the peeled fruits (CIE color values L*, a*, b*) was measured for all the mangos.

Table 4-1. Mango CA storage with 1 control (air) and 2 different air compositions (CA2
and CA3) (CAl=Air, CA2=3% 02, CA3=3% 02 + 10% C02) at 10oC for 2 weeks.
One half of the mangos were treated with hot water and the other half was treated
with 250C water.

CA storage
No HWT HWT Total
chamber


1. Air (Control) 36




2.3% 02+ N2 36



3.3% 02+ 36

10% CO2 + N2


4-2-2. Phytochemical Changes and Antioxidant Capacity

Total soluble phenolic concentration and antioxidant capacity were determined as

previously described in Chapter 3. Identification and quantification of individual









polyphenolics were conducted using Waters 2690 HPLC as previously outlined in

Chapter 3.

4-2-3. Evaluation of Flesh Color

The internal flesh color of peeled fruit expressed as CIE color values (L*, a*, and

b*) was measured by Minolta Chroma Meter CR 200 Series with a 8mm aperture

(Minolta Co., Ltd., Osaka, Japan). Hue angle and chroma values were calculated from a*

and b* using the method described by L6pez and G6mez (2004). The colorimeter was

calibrated by a standard flat white tile before measuring the flesh color of mangos. The

following equations were used to calculate hue angle and chroma values of mango flesh

color.

Hue Angle = [(Tan'(b*/a*))x(180 /3.14)]+180 (180 is needed only if a* is
negative)
Chroma = a*2 +b*2

4-2-4. Quantification of Titratable Acidity (TA)

Titratable acidity was measured by titrating clarified juice against 0. IN NaOH to

pH 8.2 using phenolphthalein indicator and a Corning model 140 digital pH meter as

described by Jacobi et al. (2000). The acidity was expressed as anhydro citric acid

equivalents, the predominant organic acid in mango. Mango juices (3g) were diluted 5-

fold in a small beaker on a stir plate. The sample was titrated using 0. 1M NaOH until pH

8.2 was obtained. Finally, the amount of 0. 1M NaOH used for the titration was recorded.

Acid content (%) of each sample was calculated based on the following formula. In the

formula, 0.064 was the miliequivalent factor for anhydrous citric acid

.acid ml NaOH x 0.1. (NaOH) x 0.064
%acid = x 100
juice(g)









4-2-5. Quantification of Soluble Solids Content (SSC)

Soluble solids contents ('Brix) were quantified by clarifying fruit puree by

centrifuging using a Beckman Optima TL 100 tabletop ultracentrifuge (Beckman

Instrument Inc. Palo Alto, CA) at the speed of 15,000 rpm for 15 min. The supernatant

was measured using a digital Leica Mark I Abbe refractometer (Leica Inc. Buffalo, NY)

at 200C. Before using the instrument, it was calibrated using distilled water.

Approximately 100L of juice was placed on the prism of the refractometer, and the

refractive index was read.

4-2-6. Statistical Analysis Method

Statistical analysis was performed as previously described in Chapter 3 using JMP 5

statistical software (SAS Institute, 2002). Analysis of variance (ANOVA) and LSD

(P<0.05) test were used to determine the effect of CA storage combined with or without

HWT on phytochemicals and other quality changes of fruit during storage and ripening.

Pearson correlation test was used for relation between TSP concentration and antioxidant

capacity.

4-3. Result and Discussion

4-3-1. External Fruit Quality

Hot water treatment was effective in preventing physiological disorders on the fruit.

Darkened lenticels (black spots) are the symptoms of anthracnose, the most common

surface postharvest disease on mangos, and appeared only on the skin of the non-HWT

mangos after 2 weeks of CA storage. Consequently, the deterioration of non-HWT

mangos was faster than that of HWT mangos during ripening. Non-HWT mangos started

to deteriorate at 250C 3 days after CA storage was finished. Their flesh started to sink and









more darkened lenticels were detected on the skin while development of anthracnose on

HWT mangos was delayed until 1 week after CA storage was complete. On average, one

of six repetitions was rotten at 250C. Thus, one mango was removed from each set of

mangos before analyzing.

CA storage could prevent anthracnose on mangos as well. Even though black spots

are also symptom of CO2 injury, the spots on the mango skin were probably not induced

by CO2 in this study because less black spots were shown in CA3 (the only CA storage

that contained C02). Mangos stored in CAl (air) developed more anthracnose compared

to mangos in CA2 (3%02) and CA3 (3%02 + 10%C02) after CA storage. Consequently,

when comparing mangos between CA2 and CA3, more anthracnose was detected on the

mangos stored in CA2 than CA3 because CA storage with low 02 and high CO2 was

more effective in preventing fruit decay than with only low 02 (Tian et al., 2004). After

all the mangos were moved to 250C, non-HWT mangos started to deteriorate 3 days after

transfer. The mangos without HWT showed anthracnose on the skin without regard to

CA storage conditions after 3 days at 250C. With the exception of mangos stored in CA1,

no fruit disorders were found on HWT mangos. Through this result, it was hypothesized

that a synergistic effect occurred in inhibiting anthracnose when CA storage was

combined with HWT.

4-3-2. Individual Polyphenolic Concentration

Two major polyphenolics (gallic acid and hydrolyzable tannin (HT)) and four minor

polyphenolics (p-OH-benzoic acid, m-coumaric acid, p-coumairc acid, and ferulic acid)

in mango were identified and quantified by HPLC. Gallic acid and six HTs were found

about 98% more than four minor polyphenolics found in mango. Since the minor









polyphenolic concentrations were somewhat negligible, details of their changes were not

analyzed.

Two CA storage containing different gas compositions and one air control were

applied to mangos. CAl was consisted of 21% 02 and 79% N2 (normal air composition)

and was considered the control for the study. Technically, CA storage is based on

lowered 02 and increased CO2 levels at low temperature and optimum 02 and CO2 levels

for mango are 3-5 and 5-10 % depending on degree of ripeness (Kader, 2002; Mitra and

Baldwin, 1997). Therefore, CA2 was 3% 02 and 97% N2 in an effort to monitor the effect

of CA storage with reduced 02 (usually less effective than 02+C02) (Tian et al., 2004).

CA3 was 3% 02, 10% C02, and 87% N2 to determine the effect of CA storage with

lowered 02 and elevated CO2 in the optimum range for mango CA storage. The

temperature for CA storage of fully mature mangos must be at least 100C to prevent

chilling injuries such as discoloration, pitting, poor color, and uneven ripening (Mitra and

Baldwin, 1997). Thus, storage temperature for this study was fixed as 100C regardless of

storage gas compositions.

Gallic acid concentrations for CA1, CA2 and CA3 were measured at three different

stages of ripening (green, mid, and full) in effort to determine treatment effect during

ripening. It was reported that phenolic compounds such as gallic acid decreased as fruit

ripened (Haard and Chism, 1996; Mitra and Baldwin, 1997), and likewise in this study

gallic acid concentrations of CA1, CA2 and CA3 significantly decreased by 16, 17 and

8% during ripening from green to full ripeness stage (Fig 4-1). Gallic acid concentration

significantly decreased only between green and mid ripe at CAl while the concentrations

at CA3 decreased only between mid and full ripe. Gallic acid concentration at CA2










gradually declined throughout fruit ripening. Even though it was a relatively short time

between mid and full ripeness stages, there were several types of evidence to support that

mangos ripened during this period such as titratable acidity decrease and color

development occurred between mid and full ripeness stage in this study. Thus, it could be

described as a "ripening related change" of gallic acid.




230
220 e CA1
-- CA2
210 CA3
200 -
190 -
S 180 -
170 -
160 -
S150
140 -
130 -
120 -
110 -
100 -
Green Mid Full

Ripeness

Figure 4-1. Changes in average gallic acid concentration (mg/L) for CA1, CA2 and CA3
during ripening for fruit samples at green, mid, and full ripeness. The ripeness
stages were green (on the day of harvest), mid (after 2 week CA storage), and full
(the time fruit started deterioration) ripe.

In conclusion, gallic acid concentration significantly decreased during storage (P <

0.05), and then the concentration was held constant at CAl. This was supported by

several studies, in which polyphenolic compounds in mango were reported to gradually









decrease then maintain fairly steady during ripening (Lakshininarayana et al., 1970; Mitra

and Baldwin, 1997). However, CA storage, especially CA3, inhibited reduction of gallic

acid. After CA storage, the concentration continued to decreased, but it was still higher

than that of CAl and CA2.

Gallic acid concentration was not affected by HWT. In Chapter 3, gallic acid

concentration was unaffected only when HWT was applied with required temperature

developed by USDA-APHIS for the sample mangos while the concentration decreased as

a result of extended treatment times. Since HWT time for this study was 75 min and it

was the required treatment time for the mangos used according to the USDA-APHIS

treatment manual, no effect by HWT showed a similar result with the first study. In

conclusion, HWT did not affect gallic acid concentration in this study because mangos

were subjected to a HWT for the required time for insect quarantine found in the USDA-

APHIS treatment manual.

Each storage contained different gallic acid and total HT concentrations. Average

gallic acid concentration of mango in CA2 was not higher compared to concentration in

CAl. The average concentration in CA3 (172 mg/L) was significantly higher than that of

CAl (159 mg/L) and CA2 (151 mg/L) (Fig 4-2) and it was 9% higher in CA3 compared

to that of CAl (P < 0.05). According to Agrawal and Deepak (2002), Castells et al.

(2002), Davey et al. (2004), Penuelas et al. (1996) and Saxon et al. (2004), elevated CO2

increases synthesis of phenolic compounds in plants. Furthermore, high CO2

concentration could induce abiotic stress, which increases phenolic compounds in plants.

Even though low 02 could be a factor to induce abiotic stress as well, it was not enough

to increase polyphenolics in this study. Therefore, it was hypothesized that combination










of 02 and CO2 significantly increased gallic acid concentration in mango by an effect of

CO2 and abiotic stress while 02 did not increase the concentration. In studies conducted

by Sanchez-Mata et al. (2003) and Tian et al. (2004), CA storage was more effective in

preventing or reducing fruit decay and prolonging shelf-life if reduced 02 and elevated

CO2 levels were applied together when compared with normal atmospheric conditions. In

conclusion, increasing phenolic compounds in mango could be an additional benefit of

CA storage with reduced 02 and elevated CO2 as was found through this study.



250
No HWT
225 W5W4 HWT

200 -
AB
175 B

Z 150 D D I











CA storage

Figure 4-2. CA storage induced effects in average gallic acid concentration (mg/L) with
or without HWT. Control was expressed as CA1 (21% air + 97% N2) and two CA
storage were shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2
and 87% N2).

Hydrolyzable tannin is a polyphenolic compound mainly found in mango and total

HT decreased by 46, 49 and 42% at CA1, CA2 and CA3 (green through full) during










ripening (Fig 4-3). Total HT concentrations at CAl and CA2 did not change significantly

while the concentration at CA3 increased during storage at 100C. However, total HT


concentrations at all the storage significantly decreased in air at 250C following CA

storage, and the average reduction was about 45%. According to this result, it was

hypothesized that storage condition (low temperature) prevented or increased total HT

reduction because total HT concentration during storage at 100C (green to mid) was fairly


stable and then its concentration significantly decreased in air at 25 C after CA storage (P

< 0.05).




240
-*- CA1
220 --- CA2
CA3
S200 -

180 -

I 160 -

I 140 -

120 -

100 -

80 -

60 -
Green Mid Full

Ripeness
Figure 4-3. Changes in average total hydrolyzable tannin concentration (mg/L GAE) for
CA1, CA2, and CA3 during mango ripening. Each stage represents green (on the
day of harvest), mid (after 2 week CA storage), and full (the time fruit started
deterioration) ripe.

Average total HT concentration increased as a result of HWT (Fig 4-4). HWT

induced an increase of 17% HT compared to NHW mangos. In this study, only










hydrolyzable tannin concentration was affected by HWT. Other parameters like gallic acid,

total soluble phenolics and antioxidant capacity were not affected by hot water treatment.

In Chapter 2, HT concentration was elevated by required duration of HWT, and likewise

HT significantly increased in response to heat treatment in this study. Thus, it could be

hypothesized that HT concentration increased when mangos were treated with required

treatment time. Furthermore, CA2 and CA3 also contained more HT in mango than CAl

(Fig 4-4). CA3 induced a significant increase of 15% total HT in mango compared to

control while the concentrations in CA2 and CA3 were not significantly different from

each other (P < 0.05). It was a similar result with gallic acid change by CA storage.



220 -
SNo HWT
W 200 W*W4 HWT

180 -

160- A
.B B B
140 C

120 -

100- E

S80 -

S60 -

40
CAl CA2 CA3

CA storage

Figure 4-4. CA storage induced change in average HT concentration (mg/L GAE) in
mango with or without HWT. Control was expressed as CAl (21% air + 97% N2)
and two CA storage were shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02,
10% CO2 and 87% N2).









Gallic acid attaches to glucose via meta-depside bond by esterification, and it

becomes B-Glucogallin with one gallic acid which is the simplest gallotannin (one of

HTs). Depening on the number of gallic acid to glucose, gallotannin have different

structure (Fig 4-5). Thus, more free gallic acids are synthesized, more gallotannins could

be produced. In conclusion, it could be hypothesized that increase of total HT was caused

by elevated CO2 that resulted in increased synthesis of phenolic compounds in the fruit.

Elevated CO2 affects synthesis of phenolic compounds by increasing PAL phenylalaninee

ammonia lyase) which is the first step of the phenylpropanoid pathway to be related to

the synthesis of phenolic compounds such as lignin and tannins (Assis et al., 2001;

Castells et al., 2002; Davey et al., 2004).


COOH
43P-0te HtaS tSQg
UOP M@ -











on the number of gallic acid to glucose, gallotannin have 5 different structure








Tetragalloylglucose, and 1,2,3,4,6-Pentagalloylglucose. (Grundhdfer et al.,
such as 001).-glucogallin 1-6-Digalloylglucose, 1,2,6-Trigalloylglucose, 1,2,3,6-
Tetragalloylglucose, and 1,2,3,4,6-Pentagalloylglucose. (Grundhdfer et al.,
2001).









4-3-3. Total Soluble Phenolics (TSP)

Total soluble phenolics determined by the Folin-Ciocalteau assay decreased by 58,

53 and 41% at CA1, CA2 and CA3 during fruit ripening (Fig 4-6). In general, it has been

reported that polyphenolic compounds decrease in many climacteric fruits such as

mangos, bananas, tomatoes and guavas during ripening (Haard and Chism, 1996;

Lakshminarayana et al., 1970; Mitra and Baldwin, 1997; Selvaraj and Kumar, 1989), and

likewise in this study TSP concentration significantly declined as fruit ripened (P < 0.05).

Since major phenolic compounds (gallic acid and total HT) in mango also significantly

decreased during ripening (12 and 46%, respectively), reduction of TSP might be

influenced by changes of those two phenolic compounds.

Controlled atmosphere storage delayed the reduction of average TSP concentration

for HWT during ripening (Fig 4-6). When CA storage was finished (Mid point at Fig 4-6),

the average concentrations in CA2 and CA3 were both 11% higher than that in CA1.

After 2 week CA storage, it was found that both CA storage conditions effectively

delayed TSP reduction in mango. Both CA2 and CA3 delayed 12% of average TSP

reduction for HWT compared to control during 2 weeks CA storage. After storage, when

the fruits were moved to air at 250C to complete ripening, TSP concentration was

significantly higher (30%) in mangos previously stored in CA3 than in CAl. This was

because the CA storage mangos were less ripe than the air control when the analysis was

performed. In conclusion, CA2 as well as CA3 were effective in slowing the reduction of

average TSP concentration as a result of delayed ripening. However, more appropriate

gas composition for mango storage (CA3) to maintain the quality of the fruits and

vegetables better and longer was more effective at decreasing the reduction rate than CA2










because CA3 combination delayed 30% of TSP reduction while CA2 prolonged only

12% of TSP concentration after CA storage.




450
425 0 CA1
v-- CA2
400 CA3
375 -
~J 350 -

325 -
300 -
275 -
250 -

7 225 -
200 -
175 -
150 -
Green Mid Full

Ripeness

Figure 4-6. Change in average total soluble phenolics (TSP) (ig/g GAE) in mango for
CA1, CA2 and CA3 during ripening. Each stage represents green (on the day of
harvest), mid (after 2 week CA storage), and full (the time fruit started
deterioration) ripe.

Average TSP for all CA treatments did not change as a result of HWT (Fig 4-7). As

previously shown in Chapter 3, the required duration of HWT did not have an effect on

average TSP in mango. Otherwise, average TSP concentration for HWT was affected by

CA storage (Fig 4-7). In CA2 and CA3, average TSP was 11 and 18% higher than CA1,

respectively. Even though average TSP in CA2 and CA3 were significantly higher than

control (P < 0.05), the concentration in CA3 was 9% higher than in CA2. Since gallic

acid and total HT concentrations for HWT increased due to an effect of C02, increase of









average TSP concentration for HWT might be directly affected by changes of gallic acid

and total HT.



400
375- No HWT
iaea HWT
W 350 -
325- A
L 300 A
BC
.X 275 BC
% 250 C, W 1|
225 -


175 -
A 150 -

125
100 1
CA1 CA2 CA3

CA storage

Figure 4-7. Changes in average total soluble phenolics (1g/g GAE) as a result of CA
storage (Air, 02, and 02+C02) with or without HWT. Control was expressed as
CA1 (21% air + 97% N2) and two CA storage were shown as CA2 (3% 02 and
97% N2), and CA3 (3% 02, 10% CO2 and 87% N2).

4-3-4. Antioxidant Capacity

Overall, antioxidant capacity changes of mango, which were thought to originate

from polyphenolics and ascorbic acid showed a very good correlation with changes in

TSP (r=0.98). Even though other antioxidant compounds such as carotenoids and

tocopherol were found in mango, the ORAC assay does not detect antioxidant capacity

from carotenoid and tocopherol. During fruit ripening, average antioxidant capacity










decreased from 5.43 to 3.26 (yM TE/g) (Fig 4-8). The reduction in antioxidant capacity

was 45, 43 and 32% during ripening (from the mature green stage to the final ripe stage).




6.0
-6- CA1
--- CA2
5.5- CA2
CA3

5.0 -


2 4.5 -


4.0 -


3.5 -


.2 3.0


2.5
Green Mid Full

Ripeness

Figure 4-8. Change in average antioxidant capacity (Trolox Equivalent IM TE/g) for
CA1, CA2 and CA3 determined by ORAC (Oxygen Radical Absorbance
Capacity) assay during fruit ripening. Each stage represents green (on the day of
harvest), mid (after 2 week CA storage), and full (the time fruit started
deterioration) ripe.

During ripening, antioxidant capacity of mango decreased as a result of loss of

phenolic compounds. Since antioxidant capacity is a desirable process in quenching

harmful free radicals, the method to prevent or slow loss of compounds to be related to

antioxidant capacity should be determined. In this study, it was found that CA storage

slowed the reduction of antioxidant capacity as shown in TSP concentration during fruit

ripening. After CA storage, both CA2 and CA3 showed higher antioxidant capacity (4.96









and 4.92 yM TE/g, respectively) compared to CAl (4.60 yM TE/g). At 250C, antioxidant

capacity was 20% higher in CA3 than in CAl while antioxidant capacity in CAl and

CA2 was not different (Fig 4-8).


CA1 CA2 CA3

CA storage

Figure 4-9. Antioxidant capacity (Trolox equivalent yM TE/g) effected by CA storage
with or without HWT. It is measured by ORAC (Oxygen Radical Absorbance
Capacity) assay.

Hot water treatment did not affect antioxidant capacity of mangos while antioxidant

capacity was affected by CA storage as shown in TSP concentration. However, the

mangos kept in CA3 showed 13% higher antioxidant capacity than that in CA1, and

antioxidant capacity was not different in CAl and CA2 (Fig 4-9). Antioxidant capacity

was highest (4.31 yM TE/g) in CA3 while the lowest value was CAl (3.79 yM TE/g).









Since phenolic compounds and TSP concentration was higher at CA3 than CAl and CA2,

antioxidant capacity might be influenced by those concentrations.

4-3-5. Fruit Flesh Color

The color change of fruit is a reliable parameter to determine the extent of fruit

ripening (Ninio et al., 2003). The internal flesh color of peeled mango fruit was

determined based on CIE color values (L*, a* and b*). L* denotes relative darkness (0)

and lightness (100), a* represents green or red, and b* means blue or yellow of the

samples (L6pez and G6mez, 2004) (Fig 4-10). Hue angle is the numeric description of

how yellow (900), or green (1800) (in the case of mango fruit) the object is and chroma is

the value to describe the vividness to dullness of a color and (Jeong et al., 2003; L6pez

and G6mez, 2004). In this study, color value was presented as L*, hue angle, and chroma

by conversion from L*, a* and b*.

Ethylene is a naturally occurring compound related to many biological changes

such as growth, development, and ripening in fruits and vegetables (Saltveit, 1999). Since

ethylene synthesis increases in climacteric fruits during ripening, fruit ripening is

accelerated. Synthesis of carotenoids increases as fruits ripen because ethylene stimulates

synthesis of pigments such as anthocyanin and carotenoids and degradation of

chlorophyll (Hatlen et al., 1998; Saltveit et al., 1999; Vilavicencio et al., 2001). As a

result of increased carotenoid concentration during ripening, the L* value and hue angle

decrease, but a* (redness), b* yellownesss), and chroma increase (Hatlen et al., 1998).











L* White


-b*Blue





-a* Green




Black


+a* Red





+b* Yellow


Fig 4-10. The CIELAB color space system. L* represents lightness and a*, and b* show
the strength of own colors (a: red to green and b: yellow to blue) (L6pez and
G6mez, 2004)

76 -
-*- CA1
75 -- CA2
CA3
74 -
A

73 AB

72 ABC

BC 'BC
71 -

70 -

69 -

68 -
Mid Full

Ripeness

Figure 4-11. Average rate of reducing lightness (L*) for HWT during fruit ripening. Each
stage represents mid (after 2 week CA storage) and full (the time fruit started
deterioration) ripe.










Since color of yellow pulp fruit such as mango develops from greenish-white to

yellow during ripening, pulp color changes from lighter (higher L* value) to deeper color

(lower L* value). As shown in Fig 4-11, L* was 4% higher in CA3 than in CAl after CA

storage, but L* values in all CA storage did not show differences at 250C. According to

this result, ripening was inhibited when the fruit was under CO2 environment (during CA

storage at CA3) and then no difference was found at 250C after CA storage.





104 CA1
v- CA2
102 -CA3

100 A

98- A

g 96 -

94 -

92- B

90 -

88 -

86 -


Mid Full


Ripeness

Figure 4-12. Reduction rate of average hue angle (0) for HWT depends on CA storage air
composition during ripening. Each stage represents mid (after 2 week CA storage)
and full (the time fruit started deterioration) ripe. Hue angle was defined by the
coordination a* and b*.

Average hue angle and chroma for HWT tended to decrease without regard to CA

storage during mango ripening (Fig 4-12, 13). The decline in hue angle represented the

change from green (1800) to yellow (900) as fruits lost their chlorophyll (green pigment










in the skin) and synthesis of carotenoid (yellow pigment in the flesh) was stimulated by

ethylene during ripening (Guevara and Pardo-Gonzalez, 1996; Saltveit, 1999). After CA

storage, hue angles were higher in CA2 and CA3 (8 and 10%, respectively) than in CA3.

However, all hue angles were not significantly different at 250C. This result showed that

ripening was delayed and/or carotenoid synthesis was inhibited as a result of CA storage,

but there was no residual effect after transfer to air at 250C. Since CA storage condition

(lowered 02 and elevated C02) inhibits respiration of fruits (consuming 02 and producing

C02) and decreases ethylene activity, higher hue angle in CA2 and CA3 was an evidence

of delayed ripening.





64 -4- CA1
-*- CA2
A CA3
62 -

60 -

2 58 -










50
Mid Full

Ripening

Figure 4-13. Increase in average chroma for HWT depends on CA storage air
composition during ripening. Each stage represents mid (after 2 week CA storage)
and full (the time fruit started deterioration) ripe. Chroma was defined by the
coordination a* and b*.
50 ---------------------------








coordination a* and b*.










As yellow pulp fruits such as mango, star fruit, and pepino ripen, pulp color

changes from green to yellow and this appears as an increase in chroma and mixture of

color is a reason of low chroma (Prono-Widayat et al., 2003). Consequently, chroma was

highest at CAl through mango ripening because mangos in CAl were riper than in CA2

and CA3 that inhibited mango ripening. Chroma in CA2 rapidly decreased after storage

while chroma in CA3 did not change even after storage.




110
SNo HWT
105 HWT

100 -
AA AA
95 -
B B
90 -

85 -

80 -

75 -

70
CAl CA2 CA3

CA storage

Figure 4-14. Changes of average hue angle (0) affected by CA storage with or without
HWT. Control was expressed as CAl (21% air + 97% N2) and two CA storage
were shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2 and 87%
N2).

Neither CA storage nor HWT affected the change of L* value of mango flesh

(data not shown). However, hue angle significantly increased by (7 and 5%, respectively)

as a result both CA storage (CA2 and CA3) compared to control (CAl), and chroma was










higher only in CA2 and CA3 (8 and 4%, respectively) compared to control (CA1) (Fig 4-

15, 16).




18
No HWT

16-


14 A
AB AB
B AB
< 12 B


S10 -


8-
'A





CA1 CA2 CA3

CA storage

Figure 4-15. Changes of average chroma affected by CA storage with or without HWT.
Control was expressed as CA1 (21% air + 97% N2) and two CA storage were
shown as CA2 (3% 02 and 97% N2), and CA3 (3% 02, 10% CO2 and 87% N2).

4-3-6. Titratable Acidity (TA)

Titratable acidity (TA) represents the total concentration of titratable acid in a

sample and is considered an important attribute in determining the taste quality of fruits

and vegetables (Berezin et al., 1994). According to Jacobi et al. (2000) and Tovar et al.

(2001), TA in mango decreases as mango ripens because two major organic acids (citric

and malic acid) used in respiration decrease during mango ripening. Titratable acidity for

CA1, CA2 and CA3 decreased during ripening (Fig 4-16). Since CA storage delays fruit










ripening, reduction rate of TA at CA2 and CA3 was inhibited during the CA storage

period (green to mid ripe) compared to the period from mid to full ripe after CA

treatment. However, reduction rate of TA at CAl was not inhibited during CA storage

period.




2.0
-*- CA1
1.8 -- CA2

1.6 CA3

1.4 -

S1.2-

S1.0-

0.8 -

0.6 -

0.4 -

0.2 -

0.0 -
Green Mid Full

Ripeness

Figure 4-16. Change of average titratable acidity (%) for CA1, CA2 and CA3 during
mango ripening. Each stage represents green (on the day of harvest), mid (after 2
week CA storage), and full (the time fruit started deterioration) ripe.

Average TA was higher at CA2 and CA3 as a result of CA storage (CA2-35% and

CA3-32%) compared to control (CAl) and TA of mango decreased by 18% HWT (Fig 4-

17). This change has been reported previously that TA of fruits including mango are

reduced by heat treatment (Jacobi et al., 2000; Neven and Mitcham, 1994; Ninio et al.,

2003).













2.2

2.0 NHW at 100C
HW at 10C
1.8 S NHW at 250C
1.6 \ HW at 2 5 C

1.4 -

1.2 -

I 10

0.8 -

0.6 -

0.4 -

0.2 -

0.0
CAl CA2 CA3

CA storage
Figure 4-17. Change of titratable acidity (%) in mango affected by CA storage with or
without HWT. Titratable acidity was measured twice at 100C (mid ripe) and 250C
(full ripe).

4-3-7. Soluble Solids Content (SSC)

Soluble solids content is one of the most reliable parameters in judging fruit quality

as consumers consider quality factors such as SSC and TA as much as visible quality (e.g.

color, size and firmness) (Hoehn et al., 2002; Lu, 2004). In this study, average SSC

decreased by 50% during CA storage at 100C and then increased by 35% at 250C.

Especially, a degree of reduction was higher at CA2 than at CAl and CA3 (Fig 4-18).












20.0
-- CA1
17.5 CA2
CA3
15.0-

S12.5-

10.0

r/) 7.5

5.0

2.5

0.0
Green Mid Full

Ripeness

Figure 4-18. Effect of the fruit ripening on average soluble solids content ('Brix) for all
CA storage and HWT in mangos. Each stage represents green (on the day of
harvest), mid (after 2 week CA storage) and full (the time fruit started
deterioration) ripe.

Since SSC is known to increase during fruit ripening as insoluble starch is

transformed into soluble solids (Martisen and Schaare, 1998; McGlone and Kawano,

1998; Vela et al., 2003), reduction of SSC during storage was not anticipated result.

However, several studies have shown several examples in decreasing SSC during

ripening. Numerous studies have reported that low 02 storage suppresses SSC increase

(Hoehn et al., 2003; Lopez et al., 2000; Taylor et al., 1995). According to Taylor et al.

(1995), SSC in plum, which is a climacteric fruit like mango also decreased during

ripening, and when apples were stored with high relative humidity to prevent drying out,

SSC decreased during ripening (Saad et al., 2004). Therefore, it could be assumed that









decrease of SSC might be influenced by low 02 composition or naturally decreased or

affected by high relative humidity during storage. After 2 weeks CA storage, SSC started

to increase as shown in Fig 4-18. Comparing SSC of CAl, CA2 and CA3, SSC in CA2 at

100C was significantly lower compared to CAl and CA3 (Fig 4-19). This might be

because lowered 02 suppressed SSC synthesis as explained above. After storage at 100C,

SSC in CA2 became higher than CAl at 250C.


NHW at 10OC
18 HW at 10OC
18886 8 NHW at 250C
S16 -6
*H HW at 2 5 C
o 14 -

S12-

U 10 -

a -





2-

0
CAl CA2 CA3

CA storage

Figure 4-19. Average soluble solids content affected by CA storage with or without HWT.
Soluble solids content was measured twice at 100C (mid ripe) and 250C (full ripe).

4-4. Conclusion

The external quality of mango fruit during ripening was affected by CA storage and

HWT as both postharvest treatments inhibited external disorders such as anthracnose on









the mango skin. CA storage was effective to increase phenolic compounds and resultant

antioxidant capacity in this study. The CA with reduced 02 and elevated CO2

concentration (CA3) was more effective than CA with low 02 (CA2) and the former

increased TSP by 18%, antioxidant capacity by 12%, gallic acid by 9%, and total

hydrolyzable tannin by 15% compared to control. However, HWT increased only total

HT (17%) and did not affect to the other parameters. The TSP, antioxidant capacity,

gallic acid, and total HT significantly decreased by 50%, 40%, 12% and 46%,

respectively during ripening (from green to full ripe). In addition, CA3 significantly

inhibited the reduction rate of TSP and antioxidant capacity compared to CAl. At the end

of fruit ripening, TSP and antioxidant capacity in CA3 fruits were higher by 30% and

20% than those in CAl. Strictly speaking, since CO2 in CA3 increased synthesis of

phenolic compounds, reduction of TSP and antioxidant capacity in CA3 was delayed

during ripening.

Unlike imported mangos, domestic mangos grown in Florida such as 'Tommy

Atkins' are not usually subjected to HWT because of the possibility that they were

infested by dangerous tropical fruit flies and/or larvae is negligible. Through this study, it

was confirmed that domestic mangos do not have any disadvantages compared to

imported mangos, although they are not treated with hot water. Furthermore, since it is

proved that CA storage with elevated CO2 was effective to increase polyphenolics and

resultant antioxidant capacity, using CA storage will be good choice even for domestic

transportation.














CHAPTER 5
SUMMARY AND CONCLUSION

As the popularity and consumption of mango in the US have increased, the

necessity of postharvest treatments for maintaining the quality and extending shelf-life of

fruits during transportation and storage has also increased. Numerous studies related to

mango postharvest treatment have stressed physiological changes during this time.

Therefore, these studies were designed to investigate phytochemical changes and

resultant antioxidant capacity in mango by hot water immersion and controlled

atmosphere storage, which are most important postharvest treatments on mangos.

Hot water immersion treatment (HWT), legally required thermal quarantine

treatment for imported mangos was applied to mangos with varying lengths of treatment

times (0, 70, 90 and 110 min) followed by 4 days of storage at 100C. When mangos were

subjected to the required length of treatment time (70 min) for quarantine, polyphenolic

compounds were stable or elevated. However, when mangos were treated for extended

times (90 and 110 min), polyphenolics significantly decreased compared to control (0

min). Antioxidant capacity of mangos was not affected by HWT. Controlled atmosphere

(CA) storage based on reduced 02 and/or elevated CO2 at low temperature was a factor to

change phytochemicals in mango. CA2 (lowered 02) did not change phytochemicals and

antioxidant capacity compared to CAl (normal air) while CA2 (lowered 02 and elevated

CO2) increased polyphenolics and resultant antioxidant capacity as a result of CO2 effect.

HWT used in this study had no affect on changes of polyphenolics and antioxidant

capacity.









In conclusion, selected postharvest treatments in this study were effective to

prevent anthracnose and increase shelf-life. Moreover, CA storage with lowered 02 and

elevated CO2 induced increases in polyphenolics and resultant antioxidant capacity in

mangos. Domestically produced mangos are usually not subject to HWT and CA storage

is not used due to relatively short transportation and storage compared to imported

mangos. Therefore, applications of HWT and CA storage should be considered even for

domestic mango varieties such as Tommy Atkins to get advantages in preserving fruit

quality and extending shelf-life with beneficial effects on phytochemical contents.
















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BIOGRAPHICAL SKETCH

Youngmok Kim was born on September 4, 1976, in Seoul, South Korea. He

graduated from Dae-il High School in February 1995 and majored in food engineering at

Kyungwon University in March 1995. In 2001, he came to the US to study hotel and

restaurant management at Lewis-Clark State College in Idaho for 9 months and returned

to Korea with three hotel and restaurant field certificates. After graduating from

Kyungwon University in 2003, he came to University of Florida to join the Food Science

and Human Nutrition Department graduate program. He received a Master of Science in

August 2005 and he will continue his study for a doctorate degree in the Food Science

and Human Nutrition Department at the University of Florida.