of the University of Florida in Partial Fulfillment of the. Requirements for the Degree of Master of Science. CAROTENOID SYNTHESIS AND RETENTION IN ...
CAROTENOID SYNTHESIS AND RETENTION IN MANGO (Mangifera indica) FRUIT AND PUREE AS INFLUENCED BY POSTHARVEST AND PROCESSING TREATMENTS
By JENNIFER PITTET MOORE
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 2003
ACKNOWLEDGMENTS I would like to take this opportunity to thank my major advisor, Dr. Stephen Talcott, for his advice, encouragement, and direction during my graduate studies here at the University of Florida. I would also like to extend my thanks to my committee members, Dr. Maurice Marshall and Dr. Jeffrey Brecht, for their time and guidance. I extend a special thanks to my husband, Aaron Moore; and parents, Tom and Carol Pittet, for their undying support, understanding, and encouragement to complete this process. To my brother, Tom Pittet, you are my best friend, and I could not have done this without you. To my sister, Michele Hochstetler, for giving me a roll model in strength and courage. To my grandparents, Carl and Shirley Ault and Rene and Emily Pittet, thank you for always believing in me. To my In-laws, Charles and Duane Hollingsworth and Michael and Barbara Moore, I appreciate the support and words of encouragement more than you know. To my sister-in-law, Holly Davis, thank you for your friendship and advice along the way. Lastly, I extend a special thanks to Danielle, Joon, David, Melanie, and Angela for making the lab such an enjoyable and productive work environment.
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TABLE OF CONTENTS page ACKNOWLEDGMENTS………………………………………………………………...ii LIST OF TABLES………………………………………………………………………...v LIST OF FIGURES……………………………………………………………………...vii ABSTRACT…………………………………………………………………………...…iv CHAPTER 1 INTRODUCTION…...………………………………………………………………...1 2 LITERATURE REVIEW….....………………………………………………………..4 3 RIPENING-ASSOCIATED CHANGES IN CAROTENOID CONTENT AND ANTIOXIDANT CAPACITY OF MANGO FRUIT (Mangifera indica)…….…….22 Introduction………………………………………………..…………………………22 Methods and Materials………………………...……………...………………….......23 Fruit Preparation and Treatment…………………………………………………23 Carotenoid Extraction…..………………………………………………………..24 Quantification of Carotenoid Concentration……………………………………..24 Quantification of Antioxidant Capacity………………………………………….25 Analysis of Carotenoid Composition...…………………………………………..25 Evaluation of Pulp Color………………………………………………………...26 Moisture Determination………………………………………………………….26 Statistical Analysis Methods……………………………………………………..26 Results and Discussion………………………………………………………..……..27 Subjective Fruit Quality………………………………………………………….27 Effects on Carotenoid Concentration…………………………………………….29 Effects on Antioxidant Capacity………………………………………………....32 Effects on Color Characteristics………………………...……………………….38 Predictive Equations……………………………………………………………..39 Effects on Carotenoid Composition……………………………………………...40 Effects on Moisture Content……..………………………………………………45 Overall Effects of Cold Temperature Storage…………………………………...46 Conclusions………………………………………………………………………48
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RETENTION OF CAROTENOID CONTENT AND ANTIOXIDANT CAPACITY IN MANGO (Magnifera indica) PUREE AS INFLUENCED BY PROCESSING AND FORTIFICATION……..………………....…………..………50 Introduction…………………………………………………………………………..50 Methods and Materials……………………………………………………………….51 Mango Puree Fortification and Processing…………………………............……51 Extraction of Carotenoids………………………………………………........…..52 Quantification of Carotenoid Concentration……………………………………..52 Quantification of Antioxidant Capacity………………………………………….53 Statistical Analysis……………………………………………………………….53 Results and Discussion…………………………………………………………..…..53 Overall Quality and Intrinsic Mango Characteristics……………………………53 Effects of Processing and Accelerated Storage on Carotenoid Concentration………………………………………………………………...54 Effects of Processing and Accelerated Storage on Antioxidant Capacity……………………………………………………………………...58 Conclusions………………………………………………………………………69
5 SUMMARY AND CONCLUSION………………...………………………………..71 APPENDIX A CAROTENOID CONCENTRATION…..…………………………………………73 B UBIQUITOUS ANTIOXIDANT CAPACITY……………………………….……...74 C CAROTENOID ANTIOXIDANT CAPACITY………………………….………….75 D LIGHTNESS COLOR VALUES.……………………………………………....……76 E HUE ANGLE VALUES………………………………………….……….………….77 F CHROMA COLOR VALUES………………...…………..…………………………78 G MOISTURE VALUES……………………………………………………..………..79 REFERENCE LIST……………………………………………………………………...80 BIOGRAPHICAL SKETCH……………………………………………………………..85
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LIST OF TABLES Table
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A-1 Analysis of variance for carotenoid concentration…………..…………………….74 A-2 Effect tests of carotenoid concentration……………………………..…………….74 A-3 Carotenoid concentration of control and heat-treated mango stored at 5°C and 20°C (SM = 0.51) ………………………………………………………………….74 B-1 Analysis of variance for ubiquitous antioxidant capacity………………………….75 B-2 Effect tests for ubiquitous antioxidant capacity……………………………………75 B-3 Ubiquitous antioxidant capacity of control and heat-treated mango stored at 5°C and 20°C (SM = 0.18)…………..…………………………………………….75 C-1 Analysis of variance for carotenoid antioxidant capacity………………………….76 C-2 Effect tests for carotenoid antioxidant capacity……………………………………76 C-3 Carotenoid antioxidant capacity of control and heat-treated mango stored at 5°C and 20°C (SM = 0.17)……....………………………………………………...76 D-1 Analysis of variance for L color values……………………………………………77 D-2 Effect tests for L color values…………….………………………………………..77 D-3 Lightness color values of control and heat-treated mango stored at 5°C and 20°C (SM = 0.36)..……………………………………………….………………...77 E-1 Analysis of variance for hue angle values……………..…………………………...78 E-2 Effect tests for hue angle values……………...…………………………………….78 E-3 Hue angle values of control and heat-treated mango stored at 5°C and 20°C (SM = 0.54)..………………………………………………………….……...78
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F-1 Analysis of variance for chroma color values……………………………………...79 F-2 Effect tests for chroma color values………..………………………..……………..79 F-3 Chroma color values of control and heat-treated mango stored at 5°C and 20°C (SM = 0.17)……………………………………………………………...…...79 G-1 Analysis of variance for moisture values…………………………………………..80 G-2 Effect tests for moisture values………………….…………………………………80 G-3 Moisture values of control and heat-treated mango stored at 5°C and 20°C (SM = 0.36)...…………………………………………………………….………...80
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LIST OF FIGURES Figure
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2-1
Chemical structure of β-carotene……………………………….…………….…...6
2-2
Chemical structure of zeaxanthin…………………………………………….…...6
3-1
Total carotenoids (mg/L; є = 2500) present in control and heat-treated mango stored at 5°C and 20°C……..………………..…………………………….….….30
3-2
Antioxidant capacity (µM Trolox equivalents/mL) of phytochemical fractions obtained from mango stored at 20°C as affected by heat treatment ……...….….33
3-3
Antioxidant capacity (µM Trolox equivalents/mL) of phytochemical fractions obtained from mango stored at 5°C as affected by heat treatment…………....…35
3-4
Percent contribution of carotenoids to ubiquitous antioxidant capacity…………37
3-5
Percentage loss in lab color values in control and heat-treated mango stored at 5°C and 20°C…………………………………………....……………….……39
3-6
RP-HPLC chromatogram of carotenoids in Tommy Atkins mango allowed to ripen at ambient temperature for 16 days………..…………………...……….41
3-7
Total peak area of carotenoids in control and heat-treated mango stored at 5°C and 20°C as Determined By HPLC Analysis………………….…….……...42
3-8
Percentage of total peak area measured through HPLC Analysis Contributed by β-Carotene…………………………….………………………………………44
3-9
Percentage of moisture loss during ripening in control and heat-treated mango stored at 5°C and 20°C……..…………….……………………………..………..46
3-10
Antioxidant capacity (µM Trolox equivalents/mL) of phytochemical fractions obtained from mango as affected by cold temperature storage………....……….47
4-1
Total loss of carotenoid concentration (mg/L; є = 2500) of ripe mango puree………………….…………….……………………………………………56
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4-2
Total Loss of carotenoid concentration (mg/L; є = 2500) of unripe mango puree…………………………………………………………….……………56
4-3
Antioxidant capacity of ubiquitous fractions containing all extractible phytochemicals in ripe mango puree ………………………………………..60
4-4
Antioxidant capacity of carotenoid fraction in ripe mango puree...…………62
4-5
Antioxidant capacity of ubiquitous fractions containing all extractible phytochemicals in unripe mango puree………………………………..…….66
4-6
Antioxidant capacity of carotenoid fraction in unripe mango puree……...…69
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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 CAROTENOID SYNTHESIS AND RETENTION IN MANGO (Mangifera indica) FRUIT AND PUREE AS INFLUENCED BY POSTHARVEST AND PROCESSING TREATMENTS By Jennifer Pittet Moore May 2003 Chair: Stephen T. Talcott Department: Food Science and Human Nutrition Mangos are a tropical, climacteric fruit produced domestically in Florida and imported into the US. Mango skin and flesh color is due to a class of naturally occurring plant pigments, carotenoids. Nearly all mangos available to US consumers have been treated with a thermal quarantine treatment and stored under cold temperatures to allow importation and maintain quality during marketing. In addition, mango fruit are often processed and incorporated into juices and purees. Two studies were conducted to provide a comprehensive overview of how mango carotenoids are affected by ripening conditions and processing. The first objective was to quantify carotenoid concentrations, carotenoid composition, carotenoid antioxidant capacity, and whole mango antioxidant capacity as
affected by postharvest handling regimes. Domestic mango fruit were treated with a simulated quarantine treatment (46°C for 60 minutes). Mango were then stored at 5°C
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and 20°C, sampled at Day 0, 4, 8, 12, 16, 20, and 24; and analyzed for total carotenoids, carotenoid antioxidant capacity, pulp color, and moisture. It was determined that heattreated mango stored at 20°C reached similar peak values as control mango for carotenoid concentration (32 mg/L), carotenoid antioxidant capacity (17 Trolox equivalents µM/mL), and color 4 to 6 days earlier in ripening. Cold temperature storage delayed ripening until transfer and all attributes reached levels similar to those of control fruit in 20 days. Heat treatment and storage at 5˚C resulted in inhibited carotenoid development and moisture loss, while antioxidant capacity was largely unaffected. Throughout this study, carotenoid concentration was positively correlated with carotenoid antioxidant capacity (R2 = 0.72) and inversely correlated with Hue angle (R2 = 0.86). The second objective was to quantify changes in mango antioxidant capacity, carotenoid concentration, and carotenoid antioxidant capacity as influenced by fortification and thermal processing. Two maturity stages of mango were blended into a puree and fortified with vitamin C (250 ppm and 1000 ppm), vitamin E (1 IU/g and 4 IU/G), fructose (2% and 8%). All control and fortified purees were pasteurized at 100°C for 15 minutes and then were stored in accelerated storage (37°C). It was determined that fortification with vitamin C acted to prevent carotenoid concentrations losses between 20 % and 30 % respectively when compared to control losses. Fortification with vitamin E acted to preserve carotenoid antioxidant capacity through 45days of storage, while control puree exhibited losses after Day 15.
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CHAPTER 1 INTRODUCTION Mango, often referred to as the king of tropical fruits, is an important fruit crop cultivated in tropical regions (Boghrma, 2000). Mango fruit is also becoming an important commodity in the United States. Florida currently has 1,675 acres and approximately 168,000 trees devoted to mango production, 88% of which occurs in Miami-Dade county (Mossler and Nesheim 2001). Increase in importation, availability, and consumer knowledge has caused the U.S. market for fresh mango to expand over 11% in recent years (HAP 2002). Color pigments, termed carotenoids, are responsible for the characteristic color of mango skin and pulp. Carotenoids have very diverse roles in biological functions of animals and plants including provitamin A activity, antioxidant activity, cell communication, immune function enhancement, UV skin protection, accessory pigments for light harvesting, and protection against photo-oxidative damage (Van de Berg et al. 2000). Evidence has been
presented that individuals with low carotenoid intake and/or low carotenoid blood levels have an increased risk of degenerative diseases. In a number of these diseases, free radical damage is thought to play a role in pathophysiology (Van de Berg et al. 2000). Carotenoids can help prevent free radical damage by acting as antioxidants because of their ability to quench radical species. Thus, carotenoids have been noted as being the most
abundant micronutrients found in cancer-preventative foods (Cano and Ancos 1994). Mango carotenoids are synthesized in mango fruit during ripening (Fennema 1996). The edible portion turns from pale yellow to yellow to deep yellow to orange-
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yellow during ripening due to carotenoid development. Carotenoid pigments are fairly stable within intact plant cells, but they become much more labile when fruits are subjected to postharvest handling practices and processing methods. Mangos are sensitive to oxygen, peroxides, temperature, light, type of packaging (modified atmosphere packaging), and length of storage (Van de Berg et al. 2000). Exposure to any of these elements may cause undesirable alterations in structure and bioactivity of carotenoids (in terms of isomerization, oxidation, or degradation). These alterations may in turn alter the UV-VIS properties of carotenoids, yet color appearance to the eye is mostly unaffected. In addition, antioxidant activity may be altered extensively (Fennema 1996). Many different ripening conditions, postharvest treatments, and processing methods are used to prepare mangos for marketing to consumers. Any of these processes can affect the synthesis of mango carotenoids during ripening (Fennema 1996). Previous studies have focused on antioxidant activity of carotenoids; or on how different ripening conditions, postharvest treatments, and processing methods affect mango carotenoids in terms of aesthetic quality, such as color development and quality maintenance. Yet, no research has been done to quantify changes in antioxidant activity as a result of ripening conditions, postharvest treatments, or processing methods. Thus, the effects of handling and processing on nutritional value considering antioxidant activity have been largely ignored by the mango-production and mango-processing industries. It is essential to preserve carotenoid structural and functional integrity of mangos throughout ripening, handling, postharvest treatment, processing, and storage. Although mangos rank among the top ten fruits in total world production (as cited in Van de Berg et al. 2000), studies evaluating ripening, postharvest treatments, processing, and storage
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conditions have largely neglected structural and functional changes to carotenoids. Identifying and defining conditions that optimize carotenoid quality and antioxidant activity will provide Florida mango producers with a highly valued commodity that is an appealing and wholesome source of disease-fighting antioxidants for consumers. Effects of ripening, postharvest treatments, and processing on mangos need to be considered from four perspectives: retention of carotenoids, chemical changes (isomerization or degradation) to carotenoids, effects on nutritional value quantified by antioxidant activity, and aesthetic quality of fruit flesh. The hypothesis was that ripening conditions, postharvest treatments, and processing methods of fresh mangos may affect carotenoid composition by destroying, isomerizing, or preventing synthesis of individual carotenoids; these changes will alter antioxidant properties of mango with little or no change to aesthetic quality. The specific objectives of this study were •
To identify major carotenoids present in mango fruit during ripening and to measure their antioxidant activity as affected by various postharvest handling regimes
•
To quantify physiochemical changes in mango carotenoids as affected by fortification and thermal pasteurization.
CHAPTER 2 LITERATURE REVIEW Mango Market Mango is a tropical fruit that originated in Southeast Asia and has been cultivated for at least 4,000 years. Mango has become one of the most important commercial fruit crops in the world, being the second largest tropical crop next to bananas in terms of production, acreage, and popularity (Desai and Salunkhe 1984). Worldwide mango production was estimated at 22.4 million tons in 2000 (FAO 2001). Because of worldwide distribution of mango production and technologies that control flowering, it is possible to supply fresh mango to worldwide markets year round (Sauco 1996). The increase in production, availability, and consumer knowledge of mangos to US consumers has caused markets for fresh mangos to expand. In 1995, US mango consumption exceeded that of many other tropical fruits, including pineapples, papayas, avocados, guavas, and passion fruit. The US is the single largest importer of mangos, importing 237,953 MT of mangos estimated at $165 million in 2001 alone. This is an increase of over 11% from 1998. In fact, imported mango accounts for approximately 99% of all US domestic sales (HAP 2002). The mango fruit varies in size, the smallest being no larger than a hen's egg. The largest can weigh up to 1.1 kg (2 pounds; Litz 1997). Mangos may be oval, round, heart-shaped, kidney-shaped, or long and slender. The skin colors of the ripe fruit
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vary and can be red, yellow-red, yellow, yellow green, or green. Each mango has a single flat seed surrounded by yellow-orange flesh. Mango Carotenoids The characteristic color of the mango skin and edible flesh is mostly due to the presence of carotenoids, with the exception of some red anthocyanins present in the skin. The term carotenoid summarizes a class of structurally related 40-carbon compounds made of eight repeating isoprene units. Mangos contain both provitamin A carotenoids (carotenes) such as α-carotene, β-carotene, and γ-carotene; and oxygenated carotenoids (xanthophylls) such as β-cryptoxanthin, lutein, zeaxanthin, violaxanthin, antheraxanthin, auroxanthin, and neoxanthin (Ben-Amotz and Fishler 1998, Cano and Ancos 1994, John et al. 1970). Carotenoids have a diverse role in the biological functioning of living organisms, including provitamin A activity, antioxidant activity, modulation of detoxifying enzymes, regulating gene expression, cell communication, immune function enhancement, UV skin protection, and visible color (Clevidence et al. 2000). The diverse functions of carotenoids contribute to their distribution in plants. Carotenoids are among the widest distributed class of pigments. It is estimated that 108 tons of carotenoids are produced each year in nature and are present in photosynthetic and nonphotosynthetic organisms. In all photosynthetic organisms, carotenoids serve two major functions: accessory pigments for light harvesting and prevention of photooxidative damage (Van de Berg et al. 2000). Nonphotosynthetic animals also need carotenoids, yet they are unable to synthesize them on their own. Therefore, it is necessary for animals to acquire carotenoids through their diet. As of 1992, 600 different
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carotenoids had been identified, 40 to 50 of which the human body can readily absorb, metabolize, and use (Van de Berg et al. 2000). Carotenoids are tetraterpenoids with a basic structure consisting of eight isopenoid residues arranged in two, 20-carbon units formed by head-to-tail condensation (de Mann 1999). This symmetrical molecule provides the basic C40 skeleton structure of all carotenoids. On this basic structure, carotenoids may contain various functional groups that distinguish them from one another. Carotenoids are subdivided into two major subgroups based on chemical structure. Carotenoids composed solely of carbon and hydrogen are termed carotenes (Figure 2-1); Oxygenated carotenoids, having at least one oxygen molecule in addition to carbon and hydrogen, are termed xanthophylls (Figure 2-2). The deep yellow, orange, and red characteristic colors of carotenoids are due to the large amounts of conjugated carbon-carbon double bonds, exhibiting absorption maxima in the region of 400 to 500 nm.
Figure 2-1. Chemical structure of β-carotene (carotenoid subgroup carotenes)
OH
HO
Figure 2-2. Chemical structure of zeaxanthin (carotenoid subgroup xanthophylls)
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Carotenoids As Antioxidants The extensive conjugated double bonds of carotenoids contribute functional properties in addition to the physical color characteristics. Conjugated double bonds provide a reactive electron rich system that is susceptible to attack from electrophilic compounds (Van de Berg et al. 2000). This structural characteristic contributes largely to carotenoid antioxidant functions. An antioxidant can be broadly defined as "any substance that when present at low concentrations compared to those of an oxidisable substrate indefinitely delays or prevents oxidation" (Halliwell and Gutteridge 1999). Carotenoid molecules have nine or more conjugated double bonds that can transfer energy from excited species, such as triplet oxygen, peroxyl radicals, and singlet molecular oxygen (1O2), to the carotenoid very efficiently and thus prevent harmful radical damage to other compounds (Beutner et al. 200; Lowe and Young 2000; Mascio et al. 2000; Stahl and Seis 1996). Carotenoids are among the most effective naturally occurring quenchers of singlet oxygen (as cited in Van de Berg et al. 2000), which is known to be capable of damaging DNA and having mutagenic effects on biological systems. Carotenoids can also inhibit lipid peroxidation, which can lead to the formation of damaging radicals (Fennema 1996). Therefore, retention of carotenoids during postharvest treatments and processing operations is critical for quality and nutritional purposes. Carotenoid conjugated chains and chain end groups are important for their functioning as antioxidants. In general, greater conjugation results in greater antioxidant activity (Yamoto et.al 2001). Also, the polarity of functional groups in carotenoids with terminal rings (β-ionone rings) can also influence antioxidant activity. Increasing polarity
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of these functional end groups, such as the presence of carbonyl and hydroxyl groups, will result in a change in solubility and a potential change in chemical and biological associations compared with those of unoxygenated carotenes (Miller et. al 1996). Therefore, antioxidant activity of carotenoids depends on many factors including degree of conjugation and functional end groups. This also suggests that carotenoids will have different antioxidant activities between the subgroups of carotenes and xanthophylls, as well as, within the two subgroups. Complete mechanisms by which carotenoids quench free radical species are not completely understood. However, their potential antioxidant activity can be assessed by measuring their stability against a radical, their ability to participate in electron-transfer reactions, and their interactions with other antioxidants and oxygen. The antioxidant mechanisms observed generally involve electron donation, H● donation, or radicaladduct formation (Bohm et al. 2002), but mechanisms employed tend to vary depending upon the carotenoid and radical species present. Carotenoid Stability Carotenoid pigments are fairly stable in their natural environment in intact cells, but they become much more labile when fruits are subjected to postharvest treatments or processing (Forget et al. 2000). The high degree of unsaturation in carotenoid structures, which is responsible for color and antioxidant properties in biological systems, also renders carotenoids susceptible to isomerization, oxidation, and degradation. Thus, the conjugated double bond system of the carotenoids is largely responsible for carotenoid instability. Carotenoids are sensitive to oxygen, peroxides, temperature, light, modified
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atmosphere packaging, and length of storage (Maini and Sudhakar 1994). Exposure to any of these elements may cause destruction or isomerization. The carbon-carbon double bonds of carotenoids all lie in the same plane. Hydrogen atoms at each double bond of the carotenoid molecule can exist in one of two geometrical isomer forms: cis- or trans- (Britton et.al 1995). The cis- or transdesignation reflects the location of the substituent groups with respect to the main chain. The cis- designation is given to those substituents that are on the same side of the carboncarbon double bond axis. The trans- designation is given to those substituents that are on the opposite side of the main chain. The number of theoretical geometrical isomers possible for each carotenoid is large because every carbon-carbon double bond of the carotenoid chain could exist in two different forms, either cis- or trans-. However, very few carotenoid isomers are found in nature. Most natural carotenoids occur in the all trans- form, since the bent structure of cis- carotenoid isomers are generally sterically hindered due to the close association of end rings and functional end groups with the main chain. The cis- orientation at a double bond would render the cis- isomer less stable than the trans-configuration. Therefore, the most highly hindered carotenoid molecules are those with cyclohenenyl end groups, with large interactions occurring between the rings and the carotenoid main chain (Britton 1995). The occurrence of the cis-isomers increases as carotenoid molecules are exposed to light, oxygen, and heat. However, specific cis- isomers occur more often than others as attributed to steric hindrance within each compound. Cis- configuration about the 9, 13, 15, 15’, 13’, and 9’ are encountered more often than any others. Carotenoid cis- isomers at 7, 11, 7’, and 11’ are rarely encountered even after iodine-catalyzed stereomutation due
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to the highly unfavored stereo arrangement (Britton 1995). Iodine is often used to induce the isomerization of carotenoids as it acts as a catalyst in carotenoid isomerization. Mango Ripening Since mango is a climacteric fruit, ripening takes place on or off the tree. Mango fruit are often harvested by hand at the mature green stage and then allowed to ripen on their own or after postharvest treatment with ethylene to initiate and coordinate ripening. Maturity is determined by the texture, shape, size, and external color of the fruit (Agbo and Inyang 1995). The physiology of ripening involves numerous physical activities that result in loss of fruit weight and volume, changes in specific gravity, peel and pulp color development, and a decline in fruit firmness. These physical changes are followed by a series of chemical changes, such as a decline in acidity, starch, and insoluble solids as well as an increase in total sugars and aroma forming compounds (Gowda and Huddar 2001). Studies have found that mango's characteristic peel and pulp color development involves a progressive loss of chlorophyll in addition to an increase in carotenoid composition and content (Lizada 1991). This indicates that transition of mango peel and edible pulp from green to pale yellow to yellow to deep yellow to orange-yellow does not involve a simple unmasking of carotenoids, but synthesis as well (Fennema 1996). The increase in carotenoids involves an increase in maximum concentration as well as number of identifiable carotenoids (Cama et al. 1970). A major change in carotenoids during ripening involves development of β-carotene, the most prevalent carotenoid present at 50% of total carotenoids, the concentration of which is highest in fully ripe fruit (John et al. 1970). However, carotenoid synthesis generally produces many xanthophylls during
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early ripening, with carotenes, such as β-carotene, forming later in maturation. Predominant xanthophylls synthesized early in ripening include violaxanthin, antheraxanthin, and auroxanthin, while carotenes synthesized later in ripening include βcarotene, α-carotene, ζ-carotene, and γ-carotene (Gomez-Lim 1997; John et al. 1994). All of these carotenoids serve as chemoprotectants. Other antioxidant compounds, such as ascorbic acid and polyphenolics are also synthesized during ripening. As a climacteric fruit, mango exhibits a respiratory climacteric during ripening. The first signs of ripening are peel and pulp yellowing and a softening of mango flesh. These changes are accompanied by an onset of the climacteric period and an increase in ethylene production. Ethylene levels in mango before the onset of the climacteric period are low, 0.01 ppm (Lizada 1991). At the height of the climacteric, ethylene reaches levels of 0.100 ppm for 24 – 48 hours (Kader 2002). Ethylene is responsible for the onset of ripening as well as senescence and eventual death. Therefore, time from harvest to onset of the climacteric period has been used as a measure of potential storage life. Postharvest Treatments Once harvested, fully mature mangos are typically stored under a variety of postharvest conditions depending on the producing country and the ultimate intent for the fruit, such as fresh or processed markets. If ripe fruit is the final goal, then proper storage conditions that allow for optimal fruit ripening are necessary. Postharvest treatments and extended storage time may affect carotenoid stability both directly and indirectly. For example, exposure to light, oxygen, and heat can directly destroy and/or isomerize carotenoids (Britton et al. 1995). Carotenoids may also be indirectly affected by postharvest treatments and extended storage due to increases in metabolic pathways that
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produce lipoxygenase (Biacs et al. 1989). This enzyme catalyzes the oxidation of polyunsaturated fatty acids. Lipoxygenase, when oxidizing unsaturated fatty acids, cooxidizes carotenoids (Biacs et al. 1989). Once oxidized, carotenoids lose many or all of their biological functions. Therefore, assessment of postharvest treatments to preserve and protect carotenoids is important. Postharvest treatments and storage conditions of mangos are aimed at altering ripening processes in the fruit in an effort to maximize quality at the consumer level. It is during fruit ripening that carotenoids are synthesized. Alterations to the ripening process through postharvest treatments and storage conditions can have considerable effects on carotenoids. Some of the most common postharvest treatments include hot water baths, cold temperature storage, and controlled atmosphere storage. Cold Temperature Storage Mangos ripen with good quality characteristics at 25˚C within 6 to 7 days and become overripe and spoiled within 15 days (Vazquez-Salinas et. al 1985). However, refrigeration is often used during storage and transportation of mature green mangos in an effort to extend shelf life and improve quality, and it has met some success. Cold temperature storage affects several quality characteristics of mango during storage and during subsequent ripening at normal temperatures. Low temperature storage effects carotenoid composition by inhibiting development of total carotenoids. In a study conducted by Thomas (1975), mangos stored at 7°C for 16 days and then allowed to ripen at room temperature produced 22-53% less carotenoids than those mangos allowed to ripen normally. Low temperature storage has also been found to decrease aroma,
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flavor, and overall quality upon ripening (Medlicott et al. 1990). Low temperature storage is also associated with an increase in physiological disorders. Mango fruit stored at temperatures between 5°C and 10°C for extended periods of time exhibit chilling injury. Chilling injury is characterized by surface and internal browning, pitting, water soaking, uneven ripening, failure to ripen, development of offflavors and off-aroma, and increased incidence of surface mold and decay (Kader 2002). Although chilling injury is the result of cold temperature storage, symptoms of this physiological disorder often do not appear until after the commodity has been returned to room temperature and normal ripening begins. The extent of chilling injury symptom development is influenced by temperature, length of storage, and maturity of the fruit (Lizada 1991). Immature mango fruit have been found to exhibit a higher tolerance to cold temperature storage and chilling injury than mature fruit, however, chilled immature fruit fail to develop full ripeness characteristics such as color development and loss of firmness once transferred to normal ripening temperatures (Medlicott et al. 1990). Cold temperature storage may influence ripening characteristics through the ethylene controlled climacteric period. Lederman et al. (1997) found no direct connection between mango chilling injury and changes in ethylene production, but concluded that cold temperature stored mangos displayed an increased ability to converted added ACC, the biosynthesis precursor of ethylene, to ethylene. Quarantine Treatments Mango fruit produced outside of the United States must undergo a thermal or chemical quarantine treatment to ensure that any pests, such as fruit fly adults, larvae, or eggs, are killed before they are allowed for import. Mango fruit undergoing heat
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treatment must reach and maintain a pulp temperature between 45.6°C and 46.1°C for 10 min (Kader 2002). Typically, this involves a 46.4 °C (115.5°F) hot water bath for 60 to 90 min depending on country of origin, cultivar, and fruit size. Both beneficial and adverse side effects associated with thermal quarantine treatments for mangos have been reported. Increasing fruit temperature increases fruit respiration rates and decreases shelf life; this effect can be controlled through heating temperature, length of heat treatment, and time taken to subsequently cool fruit to normal temperatures. External injury symptoms due to heat treatments include reactions leading to peel discoloration, lenticel spotting, and skin scalding occurring in patches or over the whole fruit (Nyanjade et al. 1998; Lizada 1991). In a study by Agbo and Inyang (1995), assessing the hot water treatment of the mango cultivar Julie from Nigeria, carotenoids were shown to increase 6.78%, indicating enhanced synthesis during ripening. This study also found an increase in total sugars (1.89%), reducing sugars (7.69%), pH (4.34 to 4.50), ascorbic acid (9.74% mg/100 g). A decrease in titratiable acidity was also found (20%). In addition, there was no significant difference in flesh color, taste, or mouth-feel between heat-treated and control samples. Heat-treated mangos are also associated with increases in L and chroma ratings and a decrease in hue angle compared to non-heattreated fruit. In addition, mango fruit exposed to the hot water treatments have shown greater resistance to postharvest diseases (Hetherington et al. 2000). Postharvest techniques are often used in combination. Combining a quarantine heat treatment with subsequent cold temperature storage has been found to adversely affect mango fruit by increasing the incidence of lenticel spotting, skin scald, internal flesh breakdown, formation of a spongy pulp near the distal end, formation of off-flavors,
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inhibition of characteristic color development, and failure to ripen normally (Nyanjage et al. 1998). Additionally, L and chroma values are lower and hue angles are higher compared to heat-treated fruit that remained at room temperature. Processing Treatments In many countries where mangos are grown, mango fruit is typically consumed fresh. The types of mangos that are not consumed fresh and reserved for processing are considered nonpulpy mangos. Mangos are harvested in both ripe and unripe stages before processing depending on the desired characteristics of a finished product. Mangos are used in a wide variety of food products, and are typically processed by one of the following four methods: freezing, canning, juicing, and dehydration (Nanjundaswamy 1997). Many mango products receive a thermal treatment (canning or pasteurization) that is calculated based on thermal inactivation time for microorganisms and/or enzymes responsible for deleterious sensory reactions such as peroxidase (POD). Mild treatments involving short times at high temperatures can reduce adverse effects on carotenoids such as degradation or isomerization. However, severe heat treatments or prolonged exposure can compromise fruit color (Gomez-Lim 1997). Exposure to light, oxygen, and/or thermal processing will cause a significant loss and isomerization of xanthophylls. Carotenes, however, tend to be more resistant to isomerization via light and oxygen (Nguyen and Schwartz 1999), but both xanthophylls and carotenes are readily isomerized and/or destroyed through processing because of extreme time-temperature processing involved in a typical pasteurization (Godoy and Rodriguez-Amaya 1987). Carotenoid isomerization as a result of processing has been observed in a variety of commodities. A study by Lessin et al. (1997), reported that
16
canning fresh tomatoes increased cis-β-carotene isomer from 12.9% to 31.2%. In a similar study by Stahl and Sies (1992), preparation of spaghetti sauce increased the concentration of lycopene cis isomers. In addition to aesthetically altering processed mango products, isomerization or degradation of carotenoids due to thermal processing; light or oxygen may affect the nutritional and functional properties of mango in two ways: by reducing provitamin A activity and by reducing antioxidant activity. Processing and Provitamin A Activity Vitamin A, or retinol, is a fat-soluble vitamin used to maintain ocular health in humans. Vitamin A can be consumed directly from foods such as cod and tuna fish liver oils, mammalian liver, egg yolk, milk, and milk products (de Mann 1999). Another source of dietary vitamin A is provitamin A carotenoids. Provitamin A carotenoids serve as precursors to vitamin A as they are used to synthesize vitamin A during their absorption in the intestinal mucosa. Mango vitamin A content is estimated at 235 RE/100g (Godoy and Rodriquez-Amaya 1994). The structure of a provitamin A carotenoid must have at least one unsubstituted βionone ring and a polyene side chain as β-carotene, α-carotene, γ-carotene, and βcryptoxanthin. Substitution of hydroxyl or ketone groups, dehydration, removal of the βionone ring, or hydrogenation eliminates provitamin A activity expressed by that given carotenoid. Carotenoids containing oxygen in one end group may also exhibit provitamin A activity, however, oxygenation in both end groups eliminates activity. The all-trans configuration of provitamin A carotenoids yields the greatest provitamin A activity. Isomerization into cis configurations compromises activity through molecular
17
rearrangements that influence enzymatic interactions responsible for conversion to vitamin A. Therefore, a cis configuration at one or more double bond reduces provitamin A activity of carotenoids. For example, in rats, all-trans-β-carotene has a potency of 100%, while that of 15,15’-mono-cis- β-carotene is only 30-50% (De Ritter and Purcell 1981). Processing and Antioxidant Activity The antioxidant activity of thermally processed foods has recently been reported to be higher than that of the fresh or unprocessed foods. In several studies, the antioxidant activity was greater than expected as a result of processing treatments (Takeoka et al. 2001; Lavelli et al. 2000; Nguyen and Schwartz 1999; Stahl and Seis 1992). Several factors may contribute to this observed increase in antioxidant activity: increased availability of carotenoids after processing and isomerization of the carotenoids from the all-trans to cis-isomer configuration. Carotenoids may also be more bioavailable from processed products since some studies have indicated greater carotenoid uptake after processing as compared with fresh foods. For example, lycopene from a processed tomato paste was found to be higher in serum blood levels than that of lycopene from fresh tomatoes (Gartner et al. 1997). Increased bioavailability of carotenoids may be due largely to the fact that carotenoids are naturally associated with lipids, proteins (carotenoproteins), and the plant matrix in whole fresh fruits (Klein and Kurilich 2000). These carotenoids may be liberated during processing by disruption of cell walls and cleavage of the carotenoids from proteins and lipids. Unassociated carotenoids are more available to be digested and absorbed and more available to interact with radical species (Clevidence et al. 2000).
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Carotenoid isomerization is observed during and after thermal processing treatments and can be attributed to exposure to heat and light. Due to the development of C30 reversed phase HPLC, carotenoids and carotenoid isomers are more successfully isolated and identified. Isolation and identification of cis-isomers have allowed study of individual carotenoid isomers and quantification of their potential antioxidant activity. A recent study by Bohm et al. (2002), found that cis isomers of lycopene, α-carotene, and zeaxanthin exhibited higher antioxidant activity than all trans- forms as measured by the Trolox equivalent antioxidant capacity (TEAC) assay against a peroxyl radical. Therefore, increases in antioxidant activity observed in processed foods may be attributable to cis-isomer formation. The increase in antioxidant activity of cis-isomers may be due to the bent shape of cis-isomer configurations compared to that of the straight-chained, rigid all-trans configurations. A bent shape would allow for greater solubility and allow for more interaction of cis-isomers with radical species. Evidence of higher antioxidant activity of certain carotenoid isomers has been documented in vivo as well. Several studies have explored the potential of cis isomers as more efficient antioxidants in both animal and human studies (Werman et al. 1999; Levin and Mokady 1994; Jinenez and Pick 1993). Carotenoid Shelf Life Mango purees and nectars are added to a wide variety of products including jams, preserves, and beverages followed by thermal pasteurization before (aseptic) or after packaging to reduce microbial contamination. These products are often fortified with vitamin A and/or vitamin E to help improve oxidative stability and quality of the product. An antioxidant, such as vitamin A or vitamin E, added to any food system will help
19
reduce rates of oxidative damage to unsaturated molecules, such as unsaturated fatty acids. Sweeteners are also added to help improve consumer acceptance and may act as reducing agents to prevent changes to phytochemical components. These fortifications will interact with the carotenoid content of the mango juice or puree differently. Vitamin E (α-tocopherol) is an antioxidant that is an effective chemoprotectant agent against lipid oxidation. Recent studies have shown α-tocopherol to have a positive influence on carotenoid retention in foods that are processed or stored for long periods of time due to its protective action against lipid oxidation (De Ritter and Purcell 1981). Although a synergistic relationship between α-tocopherol and carotenoids has been observed, this relationship has not been completely quantified in many food products such as mango puree/juice and its addition may serve to better retain all classifications of carotenoids. α-tocopherol is a fat soluble, lipophilic antioxidant. It does not generally interact with radical species in polar environments. Carotenoids, which are fairly nonpolar and lipophilic, associate with α-tocopherol in nonpolar environments. In vitro model studies observing interactions between carotenoids and α-tocopherol have shown that oxidized radicals of α-tocopherol interact with carotenoids to generate a radical cation of the carotenoid and reduced α-tocopherol. However, this process is not likely to be important in vivo because the α-tocopherol radical would be deprotonated to the natural radical α-TO● under biological conditions (Mortensen et al. 2001). Therefore, interactions between carotenoid radical cations and α-tocopherol can regenerate the parent carotenoid and the stable natural radical α-TO●, preserving color and antioxidant activity of carotenoids.
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Vitamin C (ascorbic acid) is a water-soluble antioxidant that occurs in all living tissues and is widely distributed in many fruits and vegetables. It acts as a strong antioxidant and immune enhancer. Vitamin C is often added to processed fruit products to aid color and flavor retention. These benefits are attributed to the reducing and oxygen scavenging abilities of added ascorbic acid (Nanjundasuamy et al. 1997). Carotenoid radical cations, produced during carotenoid oxidation, are effectively reduced to the parent carotenoid by vitamin C (Mortensen et al. 2001). Vitamin C has been shown to regenerate highly hydrophobic hydrocarbon carotenes in addition to the oxygenated xanthophylls. β-carotene radical cations, for example, are effectively reduced by Vitamin C. Vitamin C is able to regenerate carotenoid radical cations due to structural changes involved in carotenoid oxidation. The structural properties of radical cations are different than those of parent carotenoid structures. These structural changes cause the cation radicals to move and orient differently than parent carotenoids. Thus, carotenoid radical cations are able to interact with water-soluble species even though the parent hydrocarbon carotenoid is entirely hydrophobic, allowing vitamin C to regenerate parent carotenoids (Mortensen et al. 2001). Regeneration of carotenoids helps preserve the color quality and antioxidant activity of carotenoids. Fructose, an effective sweetener, is present naturally in high concentrations in mangos. Fructose, a reducing sugar, has a free hydroxyl group that may interact with surrounding molecules. This structural feature allows fructose to exhibit limited antioxidant behavior against weak radicals. Fructose’s reducing power may serve to regenerate and/or protect carotenoids from oxidation, thus protecting the color quality and antioxidant activity of carotenoids.
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Many processed mango products are fortified with antioxidants to help preserve the carotenoid structure and functions. However, Mortensen et al. (2001) reported that a delicate balance exists between co-antioxidants and radical species. They reported that the synergistic antioxidant activity of carotenoids and other co-antioxidants, such as vitamin C and vitamin E, is sensitive to the concentrations of these components. A large increase in one component disrupts this balance and reduces the antioxidant activity of the system and possibly promotes pro-oxidative behavior.
CHAPTER 3 RIPENING-ASSOCIATED CHANGES IN CAROTENOID CONTENT AND ANTIOXIDANT CAPACITY OF MANGO FRUIT (Mangifera indica) Introduction Mangos are a tropical, climacteric fruit produced domestically in Florida and imported into the US. In 2001, it was estimated that 99% of all mango fruit consumed in the US were imported from Mexico and other South American countries (HAP 2002). This being the case, nearly all mangos available to US consumers have been treated with a thermal quarantine treatment and stored under cold temperatures to allow importation and maintain quality during marketing. Aesthetic consequences of both heat treatment and cold storage have been widely studied and are associated with both beneficial and adverse effects. Cold temperature storage has been found to successfully delay ripening, however, temperatures too low result in physiological disorders and premature senescence. Heat treatments are an effective means of insuring sanitation and have been reported to increase carotenoid synthesis and ascorbic acid, but heat treatment can also result in peel discoloration, lenticel spotting, and skin scalding (Hetherington et al. 2000; Nyanjade et al. 1998; Lizada 1991). Any effects heat treatment or cold temperature storage exhibit in subsequent ripening of mango may do more than simply alter aesthetic quality. To date, effects on carotenoid composition, carotenoid concentrations, and carotenoid antioxidant capacity of ripening mango, and compounds responsible for overall mango antioxidant capacity have not been evaluated. The objectives of this study were to quantify carotenoid
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composition, carotenoid concentration, and carotenoid antioxidant capacity of heattreated and cold temperature stored mangos compared to non-heat-treated mangos and mangos allowed to ripen normally at room temperature. Methods and Materials Fruit Preparation and Treatment Mature green mangos (cv. Tommy Atkins) were provided through contact with Dr. Jonathan Crane in Homestead, Florida, on June 11, 2002. Fruit were harvested from a single grove of uniform maturity to reduce variation. Fruit were transported on the day of harvest to the Food Science and Human Nutrition Department at the University of Florida and divided randomly into two groups for postharvest treatment application. The first group was immersed in a hot water bath of 50°C for 60 minutes to simulate required quarantine treatments for imported mango fruit (Litz 1997), while the second group remained untreated as a control. After treatment, both control and heat-treated mangos were divided further for storage at either 5°C or 20°C. After 8 days of storage in 5°C, both heat-treated and control mangos were transferred to 20°C and allowed to ripen. From each of the four treatment groups, five fruit were analyzed after 0, 4, 8, 12, 16, 20, and 24 days of ripening. Treatment groups stored at 20°C were analyzed through day 20 while treatment groups stored at 5°C initially were analyzed through day 24. Fruit were manually peeled and pulp was cut away from the seed and blended in a kitchen scale food processor to the smallest attainable particle size. These samples were analyzed for total carotenoids, individual carotenoid content, carotenoid antioxidant capacity, antioxidant capacity of whole fruit, pulp color, and moisture.
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Carotenoid Extraction A 5 g sample of fruit from each treatment was homogenized with a tissue homogenizer in an extraction solution of 50:50 acetone:ethanol for 2 minutes or until smooth and uniform in consistency, filtered through No. 4 filter paper, and washed with 50:50 acetone:ethanol until the residue was colorless. This ubiquitous extraction solution, containing all extractable phytochemicals, was used to measure the total carotenoid concentration and antioxidant capacity of whole mangos. Carotenoids were further isolated from the ubiquitous extract solution by adding 5 mL of 50:50 petroleum ether:ethyl ether, mixing, and waiting for phase separation. An aliquot of the carotenoid extract (upper phase) was evaporated under nitrogen and redissolved in 50:50 acetone:ethanol to maintain consistency of solvent for subsequent analysis. Carotenoids were also analyzed by HPLC after saponification with 10% KOH at room temperature for 24 h to remove carotenoid esters and injected into a Waters 2695 HPLC. Quantification of Carotenoid Concentration Carotenoid concentration was determined by scanning 2 mL of ubiquitous extract on a Beckman DU 640 Spectrophotometer between 350 nm and 550 nm and recording absorbance values at 425 nm, 447 nm, and 470 nm. These wavelengths correspond to predominant carotenoids present in mango, for example β-carotene (429, 452, 478), antheraxanthin (422, 445, 472), and violaxanthin (420, 443, 470) (Britton 1995; De Ritter and Purcell 1981). Concentration was calculated using methods outlined by Gross (1991) using 2500 as an average extinction coefficient for all carotenoids and reported in parts per million using Eq. 3-1.
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Carotenoid Concentration (mg/L) =
Absorbance x extraction volume_____ Sample weight x 100 x extinction coefficient
Eq. 3-1
Quantification of Antioxidant Capacity The procedure for the oxygen radical absorbance capacity (ORAC) assay was conducted as described initially by Cao et al. (1995) and later modified by Ou et al. (2001) with the use of fluorescein as the fluorescent probe and as later described by Talcott et al. (2003). An 11x dilution of both ubiquitous and carotenoid fractions was used throughout the study and compared to standard curve representing 6.25, 12.5, 25, and 50 µM of α-Trolox, a water soluble form of vitamin E. Two different blanks were prepared, one blank containing only buffer and one blank containing 50:50 acetone:ethanol to account for solvent effects when determining antioxidant capacity. The experiment was carried out at 37°C and fluorescence readings were taken every 2 min for 70 min. Fluorescence readings were measured on a Molecular Devices fmax® 95well fluorescent microplate reader (535 nm excitation and 560 nm emission) and fluorescent decay curves were calculated using MS Excel. Analysis of Carotenoid Composition Saponified carotenoid extracts were analyzed on a Waters Alliance 2690 system and a C30 column and detected using a Waters 996 photodiode array detector. The mobile phase consisted of a gradient between mobile phase A (methonal:acetonitrile 75:25 (v/v) and mobile phase B (100% methyl-tert-butyl-either) (MTBE). The flow rate was 1mL min-1, column temperature was set at 25oC, injection volume was 100 µL, and detection was set at 450 nm. The following gradient program was used: •
Initial conditions: 97% A / 3% B
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• • • • •
From 0 to 10 min: 95% A, 5% B From 11 to 19 min: 86% A, 14% B From 20 to 29 min: 75% A, 25% B From 30 to 54 min: 50% A, 50% B From 55 to 66 min: 26% A. 74% B
The column re-equilibrated to initial conditions prior to next sample injection. Carotenoids were tentatively identified by comparing previously identified compounds separated on a similar C30 column (Clinton et al. 1996; Craft, 1992) and comparison to previous studies of mango carotenoids through HPLC analyses (Lee 2001, Cano and Ancos 1994). Evaluation of Pulp Color A 5 g aliquot of each mango pulp puree sample was placed in a plastic sample cup, covered with a black nonreflective cover, and evaluated on a Gardner Colorgard System 2000 colorimeter in triplicate for lightness, hue angle, and chroma. Colorimeter was standardized between treatments with blank and white tiles. Moisture Determination Mango pulp puree was evaluated for moisture content by placing a 5 g sample of each in a Precision Economy Oven at 135°C for 2 hours. Weight was recorded before and after to determine percent moisture of each sample. Equation 3-2 was used to calculate moisture content for all samples. Moisture Content = Initial sample weight – final sample weight x 100 Eq. 3-2 Initial sample weight – pan weight Statistical Analysis Methods All analyses were evaluated in triplicate and data points represent mean and standard deviation. LSD test (P < 0.05) was employed to determine the effect of heat
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treatment and storage temperature on assay values by using JPM software (SAS Institute, 2001). Results and Discussion Subjective Fruit Quality Storage at 20°C Mango fruit stored at 20°C had a shelf life of approximately 20 days, which was 4 days shorter than that of mango stored at 5°C for 8 days before transfer to 20°C. This suggests that cold temperature storage extended shelf life of mangos by approximately 4 days. By Day 8, both heat-treated and control mango stored at 20°C began to display visible color development as the green surface had become more yellow and orange with little difference between the two groups. By Day 12 and Day 16, surface color of heattreated mango stored at 20°C had turned completely yellow, orange, and red with no green color. These mango showed bruising accentuated during heat treatment. Control mango stored at 20°C had also developed characteristic red and orange surface color. By day 20, heat-treated mango stored at 20°C had turned more yellow in surface color, while control mangos still maintained a red and orange peel. The pulp of both groups was similar in visual color development and quality. From these subjective quality observations, it can be concluded that heat treatment and subsequent ripening at 20°C results in characteristic mango qualities similar to control mango stored at 20°C. Therefore, heat treatment and ambient storage does not adversely affect subjective aesthetic quality.
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Storage at 5°C Subjective evaluations of overall quality were made throughout the study and used to generally assess the degree of ripeness or postharvest defects. Characteristics evaluated included surface color, texture, aroma, firmness, bruising, incidence of anthracnose, skin scalding, and chilling injury symptoms. Heat-treated and control mango fruit stored initially at 5°C showed no visible differences in surface defects, pulp color, or firmness at transfer to 20°C (Day 8). At Day 12, heat-treated mangos initially stored at 5°C began to display minor surface discoloration with brown spotting and bruising incurred during handling became evident. Both heat-treated and control mangos initially stored at 5°C displayed very little characteristic color (yellows, orange, red) development and remained mostly green. By Day 16, the surface of heat-treated mango stored at 5°C had begun to develop chilling injury symptoms in terms of surface browning and pitting. However, mango fruit exposed to thermal quarantine treatments may also develop heat injury, and symptoms also include skin scald, blotchy discoloration, and uneven ripening (Kader 2002). Control mango stored at 5°C began to develop characteristic mango color as visible surface color changed from green to yellow-orange to red. The surface quality of control mango stored at 5°C was visibly higher than that of the heat-treated mango stored at 5°C with little incidence of surface blemishing. However, pulp color and quality of both heat-treated and control mango was similar. By day 20, heat-treated mango stored at 5°C was displaying further heat and chilling injury symptoms such as extreme surface browning (60% - 70% of fruit surface), pitting, shriveling, wilting, and limited characteristic color development (yellows, oranges, or reds). Pulp of this heat-treated mango began to display spots of browning in fruit tissue as well. According to Kader
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(2002), mango fruit that have developed severe chilling injury will develop grayish scalding on skin surface and flesh browning. By Day 20, control mango stored at 5°C had developed considerable more surface color (yellows, oranges, and reds) and displayed limited surface browning (~15%). On Day 24, heat-treated mango stored at 5°C was of poor surface quality, with the whole fruit surface turning brown (70% - 80%) and very little visible color development (yellows, oranges, or reds). These fruit had also increased amounts of wilting and shriveling as well as developed spots of anthracnose. The pulp still displayed brown spotting throughout its tissue (25% - 30%). Surface of control mango stored at 5°C had begun to develop small amounts of surface browning (~10%), but displayed no signs of wilting, shriveling, anthracnose, or flesh browning. It can be concluded from these subjective evaluations that heat-treated mango stored at 5°C had developed severe physiological disorders of either heat or chilling injury, and as a result, suffered poor color development, surface pitting, skin browning due to scalding, and flesh browning. Control mango stored at 5°C showed only slight signs of any disorder at termination of the study on Day 20. The increased severity of physiological disorder symptoms suffered by heat-treated and cold temperature stored mangos versus none suffered by heat-treated mangos stored at 20°C suggests that heat treatments applied in this study resulted in heat injury and symptoms were enhanced by subsequent cold temperature storage, resulting in physical symptoms similar to those observed in chilling injured fruit. Nyanjage et al. (1998) also suggested that rapid heat gain experienced during heat treatment followed by rapid heat loss (cold storage) stresses and weakens mango fruit cell structure, causing increased incidence and severity of postharvest physiological disorders, including chilling injury.
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Affects on Carotenoid Concentration Storage at 20°C Carotenoid concentration was measured at three different wavelengths: 420nm, 443nm, and 470nm. These wavelengths correspond to overlapping absorption wavelengths of three major carotenoids (β-carotene, violaxanthin, and antheraxanthin) found in mango (Cano and Ancos 1994). Concentrations at 420nm and 443nm (є = 2500) during ripening for all treatment and storage temperatures were positively correlated to concentrations at 470nm (є = 2500), R2 = 0.88 and R2 = 0.97 respectively. Thus, carotenoid concentrations were reported as determined at 470nm. Carotenoid concentration, measured as total carotenoids at 470 nm (mg/L; є = 2500), increased during ripening in both heat-treated and control mango (Figure 3-1). 35
Carotenoid Concentration (mg/L)
30
Average Standard Error = 0.79 Average Standard Error Bar = l
25
20
15
10
Control 5C Control 20C Hot Water 5C Hot Water 20C
5
0 Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
Day 24
Figure 3-1. Total carotenoids (mg/L; є = 2500) present in control and heat-treated mango stored at 5°C and 20°C
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Day 0 carotenoid concentrations were found to be significantly different (7 ppm versus 10 ppm), but this difference was attributed to fruit variation as no treatments had been applied. Heat-treated and control mango carotenoid concentration remained the same through Day 8, after which carotenoids in heat-treated mango exceeded those of control mango, 15 ppm and 6 ppm respectively. Heat-treated mango carotenoid concentration exceeded that of control mango until termination of the study at Day 20 when these two treatments were no longer different. Heat-treated mango attained a maximum carotenoid concentration on Day 16 at 33.4 ppm, while control mango did not reach maximum carotenoid concentration until Day 20 at 31.8 ppm. Previous studies conducted by Jacobi and Giles (1997) and Vazquez-Salinas et al (1985) reported that heat treatments acted to accelerate ripening, resulting in increased color and carotenoid development. These data presented here, however, suggest that application of heat treatments do not necessarily increase carotenoid concentrations, but that heat treatment increases the rate of carotenoid synthesis so that carotenoid concentration maximums are reached 4 to 6 days earlier in ripening. Storage at 5°C Heat-treated and control mango stored at 5°C showed an increase in carotenoid concentration during ripening, however, this increase was delayed while fruit were subjected to 5°C storage (Figure 3-1). Carotenoids maintained levels similar to that of Day 0 through Day 8. Upon transfer to room temperature, concentrations began to increase. By Day 12, heat-treated mango contained significantly more carotenoids than control mango. However, at day 16 and through the remainder of the study, heat-treated mango carotenoid concentration fell significantly below control mango though
32
concentrations in both treatments continued to increase through Day 20. Concentrations of control mango were increased throughout ripening to a maximum of 29.5 (mg/L), while heat-treated mango concentrations peaked earlier at Day 20 with only 23.8 (mg/L). These data suggest that heat-treated mango stored at 5°C exhibits an initial increase in carotenoid concentration earlier in ripening than control mango, similar to the trend observed for heat-treated mango stored at 20°C. However, as ripening progressed at 20°C after Day 16, heat-treated mango failed to develop peak carotenoid concentrations similar to that of control mango. In addition, carotenoid concentrations of heat-treated mango were found to decrease between Day 20 and Day 24, suggesting accelerated carotenoid breakdown. These data can also be attributed to development of heat and chilling injury in heat-treated mango stored at 5°C. As was observed for color data, combining heat treatment with cold storage resulted in a hindered development of characteristic carotenoid concentrations despite higher initial concentrations. Similar trends were reported by Jacobi and Giles (1997) and Medlicott et al (1990). These studies found that heat treatment combined with cold temperature storage can result in an inhibition of ripening and low carotene formation determined by visual assessment of peel and pulp color and subjective color rating respectively. Effects on Antioxidant Capacity Storage at 20°C This study evaluated antioxidant capacity of both carotenoids and ubiquitous fractions. Carotenoid antioxidant capacity was measured from the carotenoid fraction and reflects antioxidant capacity of carotenoids only. The ubiquitous fraction included both a hydrophobic fraction composed of carotenoids and a hydrophilic fraction composed
Ubiquitous ORAC (Trolox uM/mL)
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25 20
A
15 10 5
Control 20C Hot Water 20C
0
Carotenoid ORAC (Trolox uM/mL)
Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
25 20
B
15 10 5
Control 20C Hot Water 20C
0 Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
Figure 3-2. Antioxidant capacity (µM Trolox equivalents/mL) of phytochemical fractions obtained from mango stored at 20°C as affected by heat treatment Measurements were taken at Day 0, 4, 8, 12, 16, and 20. A) Ubiquitous fraction containing all extractable phytochemicals B) Carotenoid fraction of 20°C containing only carotenoids mainly of polyphenolics and ascorbic acid. During ripening at 20°C for 20 days, ubiquitous fractions (all extractable phytochemicals) of both heat-treated and control mango increased in ORAC values indicating a rise in antioxidant capacity (Figure 3-2A). Both heat-treated and control mango reached maximum ORAC values on Day 16 at 22.7 and 21.5 Trolox equivalents µM/mL respectively. Throughout ripening, no significant difference was detected between the heat-treated and control mango, suggesting that
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antioxidant capacity of the ubiquitous fractions were not adversely affected by thermal quarantine treatment when fruit were stored at 20°C during ripening. Antioxidant capacity of carotenoid fractions of heat-treated and control mangos increased during ripening at 20°C similar to ubiquitous fractions (Figure 3-2B). After 4 days of ripening, values began to increase; yet no difference was detected between the two treatments through Day 8. By Day 12, however, carotenoid antioxidant capacity of heat-treated mango was significantly higher than that of the control. Heat-treated mango remained higher than control through Day 16, but at Day 20, ORAC values for control mango had reached that of heat-treated mango. This suggests that heat treatment triggers increases in antioxidant capacity earlier in ripening, however, similar antioxidant levels are eventually achieved in control fruit. These trends in antioxidant capacity of heattreated mango and control mango were positively correlated on average with the same trend in total carotenoids (R2 = 0.74), indicating that carotenoid concentration directly influences carotenoid antioxidant capacity. This suggests that it may be possible to predict carotenoid antioxidant capacity based on carotenoid concentration. The fact that no significant difference was observed in ubiquitous antioxidant capacity suggests that although heat treatment and subsequent 20°C storage increases total carotenoids and carotenoid fraction antioxidant capacity, no effect on overall antioxidant capacity of whole mango is imparted. Storage at 5°C Ubiquitous antioxidant capacity began to increase in both control and heat-treated mango after Day 4 (Figure 3-3A). No difference was measured between treatments until day 16, when ubiquitous antioxidant capacity of heat-treated mango exceeded that of control mango and peaked at 20.6 Trolox equivalents µM/mL, its highest value. By Day
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20, heat-treated mango fell below control mango, which peaked at 17.6 Trolox equivalents µM/mL and remained elevated through the study, while heat-treated mango began to fall through Day 24. This suggests that heat treatment initially results in an increase and peak in ubiquitous antioxidant capacity 4 days earlier in ripening than control mango, further suggesting that heat-treated mango subsequently stored in cold temperature exhibits a shorter shelf life. In addition, storage at 5°C resulted in a decrease or loss in ubiquitous antioxidant capacity after Day 16 that was unobserved in the control,
Ubiquitous ORAC (Trolox uM/mL)
which peaked and maintained its capacity throughout the study. 25
A 20
15
10
5
Control 5C Hot Water 5C
0
Carotenoid ORAC (Trolox uM/mL)
Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
Day 24
25
B 20
15
10
5
Control 5C Hot Water 5C
0 Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
Day 24
Figure 3-3. Antioxidant capacity (µM Trolox equivalents/mL) of phytochemical fractions obtained from mango stored at 5°C as affected by heat treatment. Measurements were taken at Day 0, 4, 8, 12, 16, 20, and 24. A) Ubiquitous fraction containing all extractable phytochemicals B) Carotenoid fraction containing only carotenoids.
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Heat-treated mango and control mango exhibited an increase in carotenoid antioxidant capacity during ripening at 5°C (Figure 3-3B). Increases were observed to begin at Day 8 for both treatments. No significant difference was detected between treatments until Day 16 when heat-treated mango had antioxidant capacity higher than that of control mango and peaked at 17.3 (µM Trolox/L). After Day 16, heat-treated mango steadily decreased and control mango became significantly higher and peaked at 16.8 (µM Trolox/L) on Day 20. A difference was no longer detected between treatments at day 24. These data also suggest that heat treatment resulted in an initial increase in antioxidant capacity earlier in ripening than in control mango. As observed in 20°C stored fruit, carotenoid concentration was positively correlated with carotenoid antioxidant capacity (R2 = 0.71), further suggesting that carotenoid concentration directly influences carotenoid antioxidant capacity and that either may be used to predict the other. Additionally, the trends in carotenoid antioxidant capacity were similar to those observed in the ubiquitous fraction suggesting that trends in carotenoid antioxidant capacity were significant enough to influence antioxidant capacity of the ubiquitous fraction, which was not observed in mango stored at 20°C. Antioxidant capacity of control and heat-treated mango stored at 5°C and 20°C exhibited a slight decline after maximum values were reached (Figures 3-2 and 3-3), while no decrease was observed in carotenoid concentrations (Figure 3-1) except in heattreated mango stored at 5°C. This decrease in antioxidant capacity in ubiquitous and carotenoid fractions while no changes were observed in carotenoid concentration can be attributed in part to the spectrophotometric method used to calculate carotenoid concentration. This method measured all carotenoids extractible from mango samples
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without distinguishing structural integrity. Thus, although carotenoid concentrations are high, structural alterations, such as degradation or isomerization, would compromise ability to reduce peroxyl radicals generated during the ORAC assay, decreasing antioxidant capacity while measured concentrations remained high. Ubiquitous fraction of all storage and treatment groups contained carotenoids as well as other phytochemicals (polyphenolics and ascorbic acid). Therefore, it was possible to calculate the influence carotenoids impart on antioxidant capacity of ubiquitous fractions by subtracting carotenoid ORAC from ubiquitous ORAC and then dividing by ubiquitous ORAC. This relationship is illustrated in Figure 3-4. ORAC ratio values and errors were calculated by averaging all treatments and storage temperatures together thereby accounting for differences between storage temperatures as well as heat treatment. Initially this ORAC ratio is low, approximately 50% through day 8 of the study, then it began to increase and peaked on day 20 at approximately 90%. 100
ORAC Ratio
80 60 40 20 0 Day 0
Day 4
Day 8
Day 12
Day 16
Day 20
Day 24
Figure 3-4. Percent contribution of carotenoids to ubiquitous antioxidant capacity calculated as ubiquitous ORAC – Carotenoid ORAC/Ubiquitous ORAC * 100 averaged for both control and heat-treated mango stored at 5°C and 20°C.
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This indicates that when carotenoid concentrations are low in mature green fruit, carotenoid antioxidant capacity has little influence on ubiquitous antioxidant capacity. However, as ripening continues, carotenoid concentration increases and so does the ORAC ratio to nearly 90%, illustrating that antioxidant capacity of mature mango is largely due to carotenoids. The decrease in ORAC contribution from carotenoids at day 8, suggests that ubiquitous antioxidant capacity increases earlier in ripening mango than that of carotenoids, potentially due to synthesis of polyphenolics and/or ascorbic acid. These data also suggest that trends influencing ORAC ratio values were largely unaffected by either heat treatment or storage temperature. Effects on Color Characteristics Pulp color of all mango fruit was evaluated with respect to lightness, hue angle, and chroma at each evaluation time. According to de Mann (1999), lightness is a measure of a color's lightness or brightness. Hue angle is a measure of the color angle or actual color. A decrease of hue angle in edible mango flesh represents a change from green to yellow to red. And finally, chroma is a measure of a color's intensity. Figure 3-5 illustrates the percentage changes in color attributes of all treatments from day 0 to day 20 in 20°C stored mango and day 24 in 5°C stored mango. It can be concluded that heattreated mango stored at 5°C failed to ripen normally and achieve characteristic quality of mature mango fruit. Heat-treated mango stored at 5°C exhibited the largest percentage loss in lightness. The large loss in lightness suggests browning of fruit pulp most likely due to heat or chilling injury or polyphenoloxidase enzymes. Heat-treated mango stored at 5°C also exhibited a smaller reduction in hue angle than all other treatments, indicating a failure to develop characteristic mango color due to inhibited carotenoid formation.
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40
% Loss in Lightness
A 30
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C o n tro l 2 0 C
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% Loss in Hue Angle
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% Loss in Chroma
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Figure 3-5. Percentage loss in lab color values in control and heat-treated mango stored at 5°C and 20°C from Day 0 to Day 45. A) Percentage loss in L color values. B) Percentage loss in hue angle. C) Percentage loss in chroma color values.
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Heat-treated mango stored at 5°C had, by far, the most significant loss in chroma, nearly 45% more than the next closest treatment (Control mango at 5°C ). This significant loss in chroma indicates a loss of pulp color intensity, also an indication of browning. Jacobi and Giles (1997) also found that heat treatment caused higher chroma and lightness and lower hue angle during ripening. Predictive Equations Highly correlated analyses can often be used to predict one another through predictive equations. Throughout this study, carotenoid antioxidant capacity and carotenoid concentration were highly correlated, R2 = 0.72. In addition, both carotenoid antioxidant capacity and carotenoid concentration were highly negatively correlated with hue angle, R2 = 0.75 and R2 = 0.86 respectively. Thus, it was possible to generate an equation to predict mango carotenoid antioxidant capacity from either hue angle, carotenoid concentration, or both. The predictive equation for carotenoid antioxidant capacity based upon both carotenoid concentration and hue (P
