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Phytochemical and Antioxidant Stability of Thermally Processed Guava (Psidium guajava) and Guava Juice Blends


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PHYTOCHEMICAL, ANTIOXIDANT, AND STORAGE STABILITY OF THERMALLY PROCESSED GUAVA ( Psidium guajava ) AND GUAVA JUICE BLENDS By LANIER FENDER 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 LANIER FENDER

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iii ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Stephen Talcott, for giving me the opportunity to further my education, and es pecially for all of his help, suggestions, guidance and wisdom throughout my gradua te studies. I also thank my advising committee members, Dr. Charles Sims and Dr. Donald Huber, for their time and assistance with my research. I would also like to say thank you to my past and present lab and lunchmates: Chris, David, Flor, Jorge, Joon, Kim, Kris tine, Lisbeth, Maria, Sara, and Lorenzo for helping me in the lab and for helping to make graduate school an enjoyable experience.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION.........................................................................................................1 2 LITERATURE REVIEW..............................................................................................3 Guava Production and Market Potential.......................................................................3 Thermal Processing of Guava.......................................................................................4 Guava Antioxidants......................................................................................................5 Polyphenolics........................................................................................................6 Phenolic thermal stability...............................................................................9 Ascorbic Acid........................................................................................................9 Carotenoids/Lycopene.........................................................................................11 Anthocyanins..............................................................................................................13 3 EFFECTS OF THERMAL PROCESSING AND STORAGE ON THE PHYTOCHEMICALS AND ANTIOXIDANTS IN GUAVA NECTAR..................17 Materials And Methods..............................................................................................18 Materials And Processing....................................................................................18 Physicochemical Analysis...................................................................................19 Vitamin C.....................................................................................................19 Total soluble phenolics.................................................................................19 Antioxidant activity......................................................................................20 Lycopene......................................................................................................20 Statistical Analysis..............................................................................................21 Results And Discussion..............................................................................................21 Effects On L-Ascorbic Acid................................................................................21 Effects On Lycopene...........................................................................................24 Effects On Polyphenolics....................................................................................26

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v Effects On Antioxidant Activity..........................................................................27 Conclusions.........................................................................................................31 4 THE EFFECTS OF ANTHOCYANINCONTAINING JUICE BLENDS ON THE PHYSIOCHEMICAL AND ANTIOXIDANT CAPACITY OF GUAVA JUICE....32 Introduction.................................................................................................................32 Materials and Methods...............................................................................................34 Materials And Processing....................................................................................34 Chemical Analysis...............................................................................................35 L-Ascorbic Acid..................................................................................................35 Phenolics by HPLC......................................................................................35 Total anthocyanins.......................................................................................36 Percent monomeric and pol ymeric anthocyaninins.....................................36 Statistical Analysis..............................................................................................36 Results And Discussion..............................................................................................37 Effects On Anthocyanins.....................................................................................37 Guava juice blends.......................................................................................37 synthetic L-ascorbic acid models.................................................................42 anthocyanin controls....................................................................................47 Anthocyanin Degradation Kinetics.....................................................................48 Effect On Naturally Occurring A nd Fortified L-Ascorbic Acid.........................50 Effects On Antioxidant Activity..........................................................................51 Effects On Polyphenolics....................................................................................57 Conclusions.........................................................................................................59 5 SUMMARY AND CONCLUSIONS...........................................................................61 LIST OF REFERENCES...................................................................................................63 BIOGRAPHICAL SKETCH.............................................................................................69

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vi LIST OF TABLES Table page 3-1. Correlations (R2) of ORAC vs. L-ascorbic aci d, total soluble phenolics, and lycopene during storage for 28 days at 4 and 25oC..................................................31 4-1. Effects of storage temperature and Lascorbic acid on first order regression, rate, and half life of total an thocyanins. Different lett ers represent differences between sample types with or without L-asco rbic acid...........................................48 4-2. Effects of storage temperature and Lascorbic acid on first order regression, rate, and half life of total an thocyanins. Different lett ers represent differences between sample types with or without L-ascorb ic acid. N/A = not applicable due to high stability or fluctuation in the data................................................................51 4-3. Tentative identification of guava ju ice polyphenolics, meas ured by HPLC/PDA....58 4-4. Percent of initial polyphenolics afte r four weeks of storage at 6 and 25 oC..............59

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vii LIST OF FIGURES Figure page 3-1. Effects of processing and storage on Lascorbic acid. Bars represent the standard error of the mean, n=3..............................................................................................23 3-2. Effects of processing and storage on ly copene. Bars represent the standard error of the mean, n=3.......................................................................................................25 3-3. Effects of processing and storage on total soluble phenolics. Bars represent the standard error of the mean, n=3...............................................................................28 3-4. Percent initial of total soluble phenolics after water extraction and dilutions. J2: 100% guava juice, J3: puree from J2, dilu ted 1:1 with water, J4: puree from J3, diluted 1:1 with water, J5: puree from J 4, diluted 1:1 with water, J6: puree from J4, heated for 1 minute at 100oC. Bars represent the sta ndard error of the mean, n=3............................................................................................................................ 29 3-5. Effects of processing and storage on antioxidant activity. Bars represent the standard error of the mean, n=3...............................................................................30 4-1. Percent initial of tota l anthocyanins stored at 25 o C for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3......................38 4-2. Percent initial of tota l anthocyanins stored at 6oC for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3......................39 4-3. Percent initial of to tal anthocyanins stored at 25oC for 28 days. Control isthe unpasteurized juice. Bars represent standard error of the mean, n=3......................40 4-4. Percent initial of tota l anthocyanins stored at 6oC for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3......................41 4-5. Percent polymeric anthocyanins of acai based samples stored at 25oC. Bars represent standard error of the mean, n=3................................................................44 4-6. Percent polymeric anthocyanins of acai based samples stored at 6oC. Bars represent standard error of the mean, n=3................................................................44 4-7. Percent polymeric anthoc yanins of black currant ba sed samples stored at 25oC. Bars represent standard error of the mean, n=3.......................................................45

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viii 4-8. Percent polymeric ant hocyanins of black currant based samples stored at 6oC. Bars represent standard error of the mean, n=3.......................................................45 4-9. Percent initial of L-ascorbic ac id in acai based samples stored at 25oC. Control is the unpasteurized juice. Bars represen t standard error of the mean, n=3.................52 4-10. Percent initial of Lascorbic acid in acai based samples stored at 6oC. Control is the unpasteurized juice. Bars represen t standard error of the mean, n=3.................53 4-11. Percent initial of L-asco rbic acid in black currant based samples stored at 25oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 53 4-12. Percent initial of L-asco rbic acid in black currant based samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 54 4-13. Percent initial of anti oxidant activity in acai ba sed samples stored at 25oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 55 4-14. Percent initial of anti oxidant activity in acai ba sed samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 56 4-15. Percent initial of anti oxidant activity in black cu rrant samples stored at 25oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 56 4-16. Percent initial of anti oxidant activity in black cu rrant samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3............................................................................................................................ 57 4-17. HPLC chromatogram (at 280nm) of polyphenolic compounds found in guava juice. 1) gallic acid 2) (+)-catechin 3) e llagic acid 4) ellagic acid derivative 5) unknown characteristic guava polyphenolic............................................................58

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ix 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 PHYTOCHEMICAL AND ANTIOXIDANT STABILITY OF THERMALLY PROCESSED GUAVA ( Psidium guajava ) AND GUAVA JUICE BLENDS By Lanier Fender December 2005 Chair: Stephen T. Talcott Major Department: Food Science and Human Nutrition Due to limited availability of fresh guava most fruit destined for US markets is processed into juice, puree, jams, jellies, and syrup, and juice ble nds. Therefore, it is pertinent to study the effects of pasteurization and storage conditions on the health and quality aspects of guava juices and juice blends. The objectiv es of this study were to assess the thermal stability and shelf life properties of phytochemicals antioxidants in guava nectar and to determine the juice blen ding/color fortificati on potential of guava juice with an anthocyanin containing sour ce. Guava nectar was pasteurized at two different time/temperatures and stored in orde r to determine the stab ility of antioxidant phytochemicals and shelf life properties. Cl arified guava juice was blended with anthocyanin containing juices a nd isolates (aai and black cu rrant), pasteurized and then stored in order to determine the stabilit y of antioxidant phytochemicals and shelf life properties.

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x For the initial study, guava nectar was pasteurized at 82oC for 20 seconds (Process 1) and 87oC for 90 seconds (Process 2) and stored at 4 and 25oC. Pasteurization at 82oC for 20 seconds resulted in no significant cha nges in L-ascorbic aci d, lycopene, or total antioxidant activity, while total soluble phe nolics increased 77%. Pasteurization at 87oC for 90 seconds resulted in no significant di fferences in L-ascorb ic acid and total antioxidant activity, while lycopene decrease d 40% and total solubl e phenolics increased 72% as a result of pasteurization. After 42 days of storage at 4oC, L-ascorbic acid was completely degraded in both Process 1 and 2 nectars, lycopene decreased 40% in Process 1 and did not significantly decrease in Process 2, and total soluble phenolics and antioxidant activity were not significantly diffe rent for either process. After 42 days of storage at 25oC, L-ascorbic acid was completely degraded in both Process 1 and 2 nectars, lycopene decreased 40 % in Proce ss 1 and did not decrease in Process 2, while total soluble phenolics decrease d 28 and 17% for Process 1 a nd 2 respectively, and total antioxidant activity decreased 47 and 39% respectively. Clarified guava juice was blended with aai and black currant juice and C18 isolates pasteurized and stored for 28 days. Storage at 6oC significantly protected antioxidant phytochemicals in the guava juice blends, compared to those stored at 25oC. Guava juice blends stored at 6oC retained between 64-83% of their total anthocyanins, while those stored at 25oC retained between 39-74%. Overal l, C18 isolates + guava juice were more stable than the anthocyanin jui ce + guava juice treatments, with Aai C18 + guava juice being the most stable in terms of total anthocyanins. All L-ascorbic acid was lost after 28 days at 25oC, while juices held at 4oC retained between 20-65% of their initial L-ascorbic acid.

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1 CHAPTER 1 INTRODUCTION Diets high in fruits and vege tables have been shown to reduce the risks of certain chronic diseases such as cardiovascular diseas e, stroke, and atherosclerosis. Antioxidants found in fruits and vegetables, including vitamins A and C, lycopene, and other phytochemicals may be a contributing factor to the reduction of these diseases (Block and Langseth 1994). Tropical fruits are gaining popularity in the United States due to their potential health benefits and exotic flavors. Guava, one such tropical fruit, is unusual in that the fresh fruit are characterized by its so ft skin, short shelf life, and intense flavor, which makes it a prime candidate for tropical fruit blends. Therefore, many guavas are processed into juices, puree, jams, jellies, and syrup (Jimenez-Escrig and others 2001). Few studies have been conducted on th e phytochemistry and total antioxidant capacity of guava, especially processed gua va juice or puree. However, it is well documented that guava contains a very high amount of vitamin C. Vitamin C, as an antioxidant, has the capability to stabilize phytochemicals in processed fruits and is often an indicator compound for thermal stability. Th ermally processed fru its and fruit juices are often fortified with vitamin C to help stabilize more oxidizable or thermolabile compounds present. On the other hand, v itamin C has been shown to decrease the stability of anthocyanins in fr uits such as muscadine grapes and strawberries, but certain phenolics may result in an intermolecular copigmentation with the anthocyanins to increase overall color stability. Anthoc yanins may also polymerize with other polyphenolic compounds to increase color stabil ity. Color stability of anthocyanins is

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2 also increased by storage at refrigerated temperatures (4-7oC) as seen in blood orange juice (Choi et al. 2001, Bonave ntura and Russo 1993). Therefore, the polyphenolics in guava juice as well as low temperature storage may allow for the natural color fortification of guava juice, which is currently achieved by the addition of red dye #40. This may also allow for the mixing of guava puree with anthocyanin containing fruit, despite the high vitamin C content in guava. Fruit juices are pasteurized in order to achieve a reduction of microbes and to inactivate undesirable enzymes. The most popular way to achieve these goals is by thermal pasteurization. Thermal processing wa s initially thought to cause an overall decrease in the antioxidant act ivity of the fruit or vegeta ble. However, several recent studies have concluded that some antioxidants may be quite stable, depending on the food matrix (Dewanto et al. 2002). Since no st udies exist on the relationship of guava phytochemistry, and thermal processing, and guava/anthocyanin mixing, the results of this study will be of benefit to research ers as well as industry and consumers.

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3 CHAPTER 2 LITERATURE REVIEW Guava Production And Market Potential Guava ( Psidium guajava ) is a ditcotyledon from the family myrtaceae and is a tropical fruit known for its exotic flavor a nd potent aroma. The fruit is grown throughout the tropical regions of the world, including I ndia, Brazil, Mexico, Columbia, Venezuela, and the United States. In the U.S., it can be found in Florida, Califor nia, and Hawaii. In Hawaii, most of the guava is prepared as pasteurized puree and stored frozen or kept aseptic in plastic-lined cans or bags (Nagy et al.1993). As th e major producer of guava in the U.S., Hawaii harvested 550 acres in 2002 and processed 6.7 million pounds of guava in 2003. This is a 37% decrease from th e amount of guavas processed in 2001, and follows the declining trend in guava pr oduction over the last 12 years (National Agriculture Statistics Serv ice [NASS] 2004). Many Hawaii gua va growers have reported that this decrease is due to low guava pric es and sales, and ther efore less acreage of guavas being grown. Guavas soften quickly during ripening and therefore have a relatively short shelf life that limits the distribution of fresh guava fr uit in the United States (Nagy et al. 1993). Due to these fragile conditions, most guavas ar e processed into juice, puree, jam, jelly, syrup, nectar, fruit paste, or canned as halv es (Jimenez-Escrig and others 2001; Nagy et al.1993). However, unlike other tropical fruits such as mango and papaya, guava based products have not yet reached a high degree of popularity in the United States. This lack of interest may be due to consumer’s lack of knowledge about the fr uit’s exotic taste and

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4 nutritional benefits since corresponding fresh fruit are not available to help educate consumers. Further research and education on this subject may increase the production of guava containing products in the United States. Thermal Processing Of Guava Fruit juices, purees, and similar products mu st be pasteurized before being sold in order to inactivate oxidative enzymes and re duce the number of pathogens. This process is traditionally accomplished by rapid heating an d cooling of the puree to help preserve quality factors. Industrially, gua va puree may be heated to 90o C for 60 seconds or frozen immediately after mechanical processing (Gaetano 1997; Nagy et al.1993) for storage and/or distribution. These purees are then sold for the manufacture of juice, juice blends, jelly, jam, and many confectionary products For the juice and beverage industry, a second pasteurization is needed following blending to render the product commercially sterile. Although pasteurization is a necessity for juice safety, thermal treatments have their drawbacks including adverse effects on fl avor, color, and overa ll nutritional quality loss. Possible degradation of the numerous phytochemicals in guava may occur during thermal processing and storage. This include s loss of ascorbic acid, isomerization of lycopene and other carotenoids, and decrease s in overall polyphenolics and antioxidant activity. Guava puree is highly viscous due to its high insoluble solids content primarily from pectin, which makes up approximately 705-804 mg/100g of the frui t (Jagtiani et al. 1988). Pectin is found in the cell wall and middl e lamella layers of fruits and vegetables and is composed of linear chains of -D-galactopyranosyluronic acid units with neutral sugars and various side chains attached (BeMiller and Whistler 1996), and nutritionally

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5 functions as dietary fiber. Du e to its viscous nature, pectin must be broken down in guava puree in order to obtain a clarified juice. Th is is achieved by adding additional pectinase, an enzyme that hydrolyzes cell walls and larg e pectin molecules and allows for an easier juice extraction. Pectinas e, as well as other enzymes such as cellulase and amylase, are commonly added to fruit purees in order to obtain greater juic e yields. A 0.1% w/v addition of pectinase to guava puree was f ound to be effective in the juice making process (Nagy et al.1993) and resulted in greater juice yi elds. Brasil et al. (1995) found that a 600 ppm treatment of pectinase at 45oC for 120 minutes displayed the greatest yield (84.7%). One disadvantage of using clarified juice, rather than unclarified puree, is the loss of lycopene in the juice. Lycopene is found bound to the pectin in the fruit and the process of clarifyi ng removes the pectin, and therefor e the lycopene as well. This may result in artificial color fortification of guava juices, since lycopene gives guava its characteristic pink color. Although, guava puree is used for juices often, rather than clarified juice, the ad dition of pectinase will also allow for greater yields in compounds of interest during extractions. For example, one study found that pectinase treatments increased total soluble phenolics 11.5% and ascorbic acid 38.8% compared to the untreated guava puree (Brasil et al. 1995). The pectinase br eaks down more cell walls and therefore more phenolics are extracted and detected. Guava Antioxidants Biologically, antioxidants may be defined as compounds that prevent free radicals from destroying host cells. Free radicals resu lt from reactive oxygen sp ecies that contain an unpaired electron rather than the pair ed electrons found in stable functioning molecules. These free radicals can attach to cells in the body a nd cause destructive damage that could lead to an array of chr onic health problems, including cardiovascular

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6 disease, stroke, atherosclerosis, and cancer (Block and Langseth 1994). Antioxidants, in turn, help to reduce the number of free radicals and in the process may reduce the risks of such diseases. Chemically speaking, antio xidants are reducing agents which donate electrons and cause a substance to be redu ced. An oxidizer is a compound which accepts electrons and therefore oxidizes another. Antio xidants can also be de fined as a substance, that when present in small amounts compared to the substrate, prevents or greatly decreases a pro-oxidant initiation oxidat ion of the substrate (Prior & Cao 1999). Therefore, antioxidants work to redu ce pro-oxidants through a redox reaction. Antioxidants not only work in vivo, but in vitr o as well. Antioxidants play a crucial role in both enzymatic and non-enzymatic browning reactions and help to prevent lipid oxidation in foods as well. Diet ary antioxidants include vitami ns A, C, and E as well as numerous non-nutritive compounds such as polyphenolics, fl avonoids, carotenoids, and thiol-containing compounds. Polyphenolics Polyphenolics are synthesized by numer ous plants as secondary metabolites. Polyphenolic compounds serve as both functiona l components to the plant as well as the consumer. For example, some polyphenols serv e as pigments (anthocyanins), compounds to ward off insects and other herbivores such as astringent tannins, and UV light protectants (Herrmann 1995). These compounds are also important in foods for their sensory attributes such as color, astringe ncy, and bitterness; as well as their possible nutritional properties. Polyphe nolics are products of 3 majo r plant metabolic pathways. Phenolic acids are secondary metabolites of the shikimate pathway; the phenylpropenoid pathway produces the cinnamic acid derivativ es which are precursors of flavonoids and ligans, and the ‘flavonoid route’ produ ces the numerous and diverse flavonoid

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7 compounds (De Bruyne et al., 1999). Pheno lic compounds consist of a phenol, an aromatic ring with at least one hydroxyl group attached. This structure relates to phenolics antioxidant ability, which depends on many factors. Some of these factors include: 1) location of hydroxyl groups 2) number and arrang ement of phenolic subunits and 3) and the number of hydroxyl groups (R ice-Evans et al. 1995). Phenolic compounds can be classified into several categories based on their structures. Polyphenols, which include flavonoids, have at least two phenol groups. The largest polyphen ols are the tannins, which have a molecular weight of 500-3,000 and also have the ability to bind proteins (Bate-Smith & Swain 1962). Tannins ca n be classified into two subgroups, the hydrolysable and the condensed tannins. Hydrolysable tannins are those readily hydrolyzed by acids or enzymes into gallic or ellagic acid (Hagerman et al. 1992). Hydrolysable tannins are commonly found in foods such as guava, grapes, and wine. Both condensed and hydrolysable tannins have been shown to have antioxidant, enzyme inhibiting, and antimicrobial properties (De Br uyne et al. 1999). Condensed tannins, also called “vegetable tannins” or proanthocyanidins, are fla vonoid polymers, which can be degraded in the presence of aci d and heat to form either cyan idin or delphinidin and are relatively stable, compared to the hydrolys able tannins (Hagerman et al.1992). Common condensed tannins include polymers of catechin and epicatechin (Fi gure 2-1) which can be found in guava, teas, and numerous fruits and vegetables. Several studies have demonstrated the antioxidant activity of conde nsed tannins. For example procyanidins B1 and B3 have been shown to have a stronger an tioxidant activity for li noleic acid then both ascorbic acid and -tocopherol (De Bruyne et al., 1999) Condensed tannins have also

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8 been shown to have anti-inflammatory, an ti-diarrhoeal, and anti-ulcer activity (De Bruyne et al. 1999). The third phenolic group is the phenolic acids that cons ist of a benzene ring and least one carboxylic acid group. Examples of phenolic acids are ca ffeic, chlorogenic, p coumaric, gallic, and ellagic acids. Many pheno lic acids are linked through ester, ether, and acetal bonds to either structural component s of the plant such as cellulose, proteins, or lignin; to larger polyphenols (tannins); or to smaller orga nic molecules such as glucose (Robbins 2003). This leads to considerable diversity among the various classes of polyphenolics and therefore i nherent difficulty in anal ysis and identification. Limited information exists on the topic of guava phenolics, however, previous studies have shown guava to contain a variety of polyphenolics including flavonols, phenolics acids, flavan-3-ols, and condensed tannins. (Miean and Mohamed 2001; Misra and Seshadri 1967 ). Miean and Mohamed (200 1) reported guava to contain 550 and 579 mg/kg of dry weight of the fl avonols myricetin and apigenin, respectively, while absent in other major flavonoids such as quercetin, luteolin, and kaempfer ol. Other phenolics detected in guava include ellagic and gallic acid and numerous is omers of catechin and epicatechin (Misra and Seshadri 1967). K ondo and others (2005) reported the amount of total soluble phenolics in guava skin and fles h, as measured by the Folin-Ciocalteu assay, to be 915 and 637 mol/kg FW, respectively, de monstrating that the majority of the polyphenolics are found the skin. Through HPLC analysis, they also found guava skin to contain gallic acid (120020mo l/kg), catechin (45 mol/kg), epicatechin (16 mol/kg), and chlorogenic acid (10 mol/kg) (Figure 2-1).

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9 Phenolic thermal stability Phenolic compounds are abundant in fres h and processed fruits and vegetables. However, current studies report that the thermal stability of these compounds in processed foods depends on the matrix in which they are found. A study on Figure 2-1. Structures guava polyphenolics: ca techin (left) and ellagic acid (right). tomatoes concluded that thermal processing de creased ascorbic acid levels, but increased lycopene, total phenolics, flavonoids, and an tioxidant content (Dewanto et al. 2002). Studies on sweet corn and table beets also showed an increase in antioxidants after thermal processing (Dewanto et al 2002). The sweet corn had an increase in total free phenolic content and free ferulic acid conten t after thermal processing. Therefore, since no published data on guava phenol ics related to processing exists, there is a need to research the thermal processing effects on guava polyphenolics. Like other phytochemicals, polyphenolic compounds will oxidize and degrade with exposure to oxygen, heat, and light. The extent of this degradation depends on the type of polyphenolic as well as the presence of other pol yphenolics and antioxidants such as vitamin C, which may help to stabili ze the polyphenolic compounds (Fennema 1996). Ascorbic Acid Ascorbic acid, a required nutrient for humans, is one of the most abundant antioxidants consumed, with fruits being the ma in source of the nutrient. L-ascorbic acid is an excellent reducing agent and large quantities may help stabilize pheno lics and other

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10 antioxidants during processing by the donation of hydrogen atoms. This reducing ability is due to its 2,3-enediol moie ty (Fennema 1996). Ascorbic aci d is often considered an index of nutrient quality during processing and storage of foods because of this stabilizing nature (Fennema 1977). Guavas contain approximately 230mg of total ascorbic acid / 100 g of edible portion of fruit (U nited States Department of Agriculture [USDA] nutrient database 2005), fi ve times more than a serving of orange. It is second in vitamin C content among all fruit, with the ace rola cherry containi ng the most (Uddin et al. 2002). Numerous studies exist on the relationship between vitamin C and thermal processing. Although few studies have been don e on the effects of thermal processing on guava puree, Uddin et al. (2001) studied the e ffects of degradation of ascorbic acid in dried guava during storage. The results show ed that as storage time and temperature increased, ascorbic acid content decreased in the dried guava samples (Uddin et al. 2001). A study conducted on yellow passion fruit (Talcott et al. 2003) demonstrated that vitamin C fortification preserved more phytochemical s during processing compared to the nonfortified control. Since guava already contai ns extremely high levels of vitamin C, the phytochemicals and antioxidants in the matrix may be heat stable. However, vitamin C itself is extremely heat and light sensitive and therefore may be greatly decreased during thermal processing. This has been widely de monstrated in previous studies on various fruits and vegetables, including tomatoes (Dew anto et al. 2002) and orange juice (Lian et al. 2000). The degradation of L-ascorbic acid de pends on many factors including heat, light, metals, and water activity. The degradation of L-ascorbic acid involves its oxidation into

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11 dehydroascorbic acid (DHAA), which is active as vitamin C, but not as an antioxidant, into degradation products such as 2,3-diket ogulonic acid, unsaturat ed carboxylic acids, and furfural, among others (Fennema 1996, Bren es 2005). These end products are neither active as vitamin C nor antioxidants and have be en linked to juice quality issues such as non-enzymatic browning and anthocyani n destruction (Dallas 1996, Brenes 2005). Carotenoids/Lycopene Carotenoids are abundant in the red, yellow, orange, and green colored vegetables and fruits. They are, after chlorophyll, the second most widely occurring plant pigment found in nature (MacDougall 2002). Carotenoids are tetraterpenes that can be classified into two major groups including carotenes (hydrocarbons) and xant hophylls (oxygenated hydrocarbons). Carotenoids may be straight chai ned, such as lycopene, or contain a 5 or 6 carbon ring on one or both ends, such as -carotene (MacDougall 2002 ). The high degree of hydration and long carbon chain length of these molecules makes them hydrophobic and therefore fat soluble molecules. The majo r purpose of carotenoid s in the human diet is to serve as precursors to provitamin A, a required nutrient for humans. In order to serve this purpose, the carotenoid must contain a B-ionone ring (Fennema 2000). Carotenoids containing this structure include and -carotene, and -crypt oxanthin. Carotenoids without this structure, such as lycopene, do not possess pro-vitamin A activity yet serve as dietary antioxidants. As an antioxidant, carotenoids are known to quench singlet oxygen and protect against cellular oxidative damage (Deshpande et al. 1995). This ability has linked carotenoid consumption w ith decreased risks of cancer, cataracts, atherosclerosis, and the pro cess of aging (von Elbe and Sc hwartz 1996). A wide variety

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12 of carotenoids have been identifi ed in guava, including phytofluene, -carotene, lycopene, cryptoflavin, cryptoxanthin, a nd lutein (Mercadante et al. 1999). Lycopene is a fat soluble carotenoid res ponsible for the red or pink pigment in several fruits and vegetables such as to matoes, watermelon, pink grapefruit, and guava. Structurally, lycopene is a linear, 40 carbon hydrocarbon containing 11 conjugated and 2 non-conjugated double bonds (Rao & Agarwal 1999). Of all the dietary carotenoids, lycopene has the highest singlet oxygen que nching ability in the body. However, since lycopene lacks a B-ionone ring it does not pr ovide vitamin A activity (Rao & Agarwal 1999). Lycopene has been associated with decr eased risks of cardiovascular disease, as well as prostate, pancreas, and stomach cancers (Gerster 1997). Several studies (Gonzalez-Abreu et al. 1985; Me rcadante et al. 1999) have identified lycope ne in fresh guava fruit. Although no studies have been published observing processed guava and lycopene, numerous studies abound on the relationship between thermally processed tomatoes and lycopene. Many of these report an increase in lycopene concentration in thermally processed tomatoes compared to fresh tomatoes. Although this may be due to the breakage of cell walls and loss of water during the thermal treatment, some processed tomato products have shown an increase in bioavailable lycopene compared to fresh tomatoes. This increase in lycopene bioavailability may be due to the isomerization of the carotenoid from its transto cis form, occurring as a result of the presence of oxygen and heat during processing. Oxidation of car otenoids occurs as a result of either autooxidation, which is a spontaneous free ra dical chain reaction in the presence of oxygen, or by photooxidation in the presence of light (MacDougall 2002 ). Overall, the

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13 rate of carotenoid oxidation depends on carote noid structure, oxygen, te mperature, light, water activity, pH, and the presence of proand antioxidants (Chou & Breene 1972). Oxidation of carotenoids resu lts in the formation of colo rless end products such as compounds with epoxy, hydroxyl, and carbonyl groups (MacDougall 2002), as well as a variety of cis -isomers, which still retain some color. Lycopene, as well as other carotenoids occur mostly in their trans form in natural, unprocessed foods. However, during proces sing and storage, conversion into the cis form of one or more double bonds is likely. This isomerization may results in color changes because the spectral properties of the cis vs. the trans form are different. The addition of a cis -double bond to an all trans carotenoid results in a hypsoc hromic shift of 2 to 5 nm (MacDougall 2002). The cis -double bond also adds an additio nal peak in the near-ultra violet absorbance spectrum, which results in a lighter colored hue (MacDougall 2002). Although the trans-carotenoids and color may decrease during proces sing and storage, many studies have shown the cis -carotenoid to have a highe r antioxidant capacity (Bohm 2001, Levin et al. 1994). For example, Bo hm and others (2001) found that the cis -isomers of lycopene -carotene had higher TEAC values than their trans-isomers. Therefore, identification and quantification of the antioxi dant properties of the lycopene isomers in processed guava puree would provide novel data for this field. Anthocyanins Anthocyanins are flavonoids responsible for the blue, purple, and red colors in many fruits and vegetables. Six main anthoc yanidin base structur es are found in foods including pelargonidin, cyan idin, delphinidin, peonidin, pe tunidin, and malvidin with hundreds of derivatives of these six from various sugar and/or acylated moieties. Anthocyanin color is extremely unstable and overall stability may be affected by pH,

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14 light, heat, oxygen, iron, copper, tin, ascorb ic acid, and copigmentation (MacDougall 2002, Fennema 2000) In most cases, color stability of an ant hocyanin containing food is greatly reduced by the presence of ascorbic acid. This may o ccur as the result of hydroperoxide formation that forms during the oxidation of ascorbic acid (von Elbe and Schwartz 1996).The hydroperoxides formed then act to degrade the anthocyanin molecule and pigment (von Elbe and Schwartz 1996). This degrada tion has shown to me mutual with both anthocyanins and ascorbic acid decreasing in each others presen ce. Although the exact mechanism of this degradation is not known, several have been proposed. Several researchers have proposed a condensation reaction between the two compounds (PoeiLangston et al.1981, Garzon et al 2002). Degradation products of L-ascorbic acid and dehydroascorbic acid such as 2,3-diketogulonic acid, furfurals such as HMF, and other aldehydes have been shown to increase an thocyanin destruction (Es-Safi et al. 1999, 2002). Previous studies have shown a decrease of anthocyanins with increasing amounts of ascorbic acid in many fruits including cranberry, strawberry, grapes, and blood orange juices (Choi et al. 2001). Therefore, this limits the extent to which anthocyanin containing products, such as fruit juices, can be fortified with vitamin C. It also may limit the amount of fruit juice blends that can be made with anthocyanin containing fruits. The kinetics of anthocyani n stability has also been pr eviously studied with total anthocyanin concentration over time concl uded to follow first order kinetics by many authors (Brenes et al. 2005, Turker et al. 2004, Garzon et al. 2002). Fi rst order kinetics, including rate of degradation and half lif e (the time necessary for a 50% reduction in concentration) are determined by changing conc entration into a natural log (ln) scale,

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15 over storage time. A high correlation of this data represents first order kinetics. First order kinetics has also explai ned anthocyanin + L-ascorbic ac id, as well as anthocyanin + polyphenolic (copigmentation) kine tics (Brenes et al. 2005). Copigmentation is a weak association be tween an anthocyanin molecule and other molecules such as other anthocyanins, meta ls, or polyphenolic compounds, which results in color enhancement or greater color stab ility of the matrix during processing and storage. Copigmentation usually results in an increase in red color intensity (hyperchromic shift) and a bathochromic shif t from reddish to blui sh hues (Gutierrez et al. 2004). Copigmentation can be demonstrated by comparing different varieties of wines. For example, Gutierrez et al. 2004 conducted a study on the phenolic and anthocyanin compositions of the wine varieties Cabern et Sauvignon, Cencibel, and Syrah and found that Cencibel demonstrated the lowest exte nt of copigmentation with aging that was attributed to its low flavanol concentration, compared to the other two varieties. This shows the effect of intermol ecular copigmentation, in which an anthocyanin molecule forms a copigment with a colorless mol ecule, usually a poly phenolic compound through weak hydrophobic forces (Mazza et al. 1990) Although copigmentation would most likely not occur in an anthocyanin juice/ tropical juice blend, the added polyphenolics such as catechin and epi catechin found in guava juice may help to protect the anthocyanins during storage. Several studies have shown that anthocyanins will condense with proanthocyanidins such as (+)-catech in and (-)-epicatechin to form polymeric compounds which in turn are more stab le during storage (D allas 1996, Bishop 1984). Anthocyanin stability in fruits such as grapes, strawberries, and aai has been previously studied in model systems (Del Po zo et al. 2004; Brenes et al. 2005). These

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16 studies have looked at the effects of vitamin C fortification, as well as added substances in hope to form copigments with the anthoc yanins. For example Del Pozo et al. (2004) studied the effects of vitamin C fortifica tion of aai juice on co lor stability. They concluded that the added ascorbic acid decr eased the anthocyanin stability in the aai juice. However, no studies exist on the effect s on color stability of adding an anthocyanin extract to a fruit juice with high vitamin C and polyphenolics content, such as guava. As demonstrated by the wine example (Gutierrezz 2004), polyphenolics may help to stabilize anthocyanins in a real juice system.

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17 CHAPTER 3 THE EFFECTS OF THERMAL PROCESSING AND STORAGE ON THE PHYTOCHEMICALS AND ANTIOXIDANTS IN GUAVA NECTAR Antioxidant phytochemicals are steadily gaining the intere sts of American consumers, expanding markets and increasing sales of fruits, vegetables, botanicals, dietary supplements, and numerous f ood and related products containing these compounds. Likewise, tropical fruits are gain ing popularity in the United States due to their exotic flavors and potentially unique phytonutrient composition. Of these tropicals, guava has yet to reach the popularity of other tropicals such as banana, mango, and papaya yet products that cont ain processed forms of the fr uit are gaining in popularity. Fresh guavas suffer from a relatively short shelf life, which effectually prevent US imports and limited domestic fruit production. Ho wever, due to its diverse phytochemical composition and unique flavor, guava has the po tential for market expansion in the area of juices, purees, and other processed products. Investigations into the thermal stabi lity and shelf life properties of antioxidant phytochemicals present in gua va puree are limited despite the fact that thermally processed purees, juices, and jellies are the most predomin ant guava-based products in US markets (Jimenez-Escrig et al. 2001). Many of these products are shelf stable while others are lightly pasteurized and held refr igerated creating cond itions for significant phytonutrient degradation during storage. Guav a is known to contain the second highest concentration of ascorbic acid among all fruits with only acerola ch erry containing more on average. Ascorbic acid is well know n for its oxidative protection of other

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18 phytochemicals including carotenoids, folic acid, tocopherols, and polyphenolic compounds and serves to retard oxidation and stabilize phytochemical compounds during thermal processing and storage (Dewanto et al. 2003, Gregory 2000) The objectives of this study were to determine the effects of two thermal pasteurization time and temperature combinations and two storage te mperatures on the stab ility of vitamin C, lycopene, polyphenolics, and antioxi dant capacity of guava puree. Materials And Methods Materials And Processing Mature guava fruit were obtained from Sardinia Farms in Homestead, Florida and transported to the Food Science and Human Nutr ition Department in Gainesville, Florida. The whole guava fruit were mechanically pres sed into puree and imme diately frozen at 20C. On the day of processing, the puree wa s thawed under cold running water and diluted 50% with a 0.5M citric acid buffer at pH 3.8. This dilution served to thin the puree for ease of pasteurization and to mimic a commercial puree. The nectar was then divided into two equal portions for processing at 82C for 20 seconds (Process 1) or 87C for 90 seconds (Process 2) in accordance with commercial processors in Hawaii (Dr. Alvin Huang, University of Hawaii). The nectar was pumped by a peristaltic pump (Cole Parmer, Chicago, IL) through a stainless steel tube into a temperature controlled water bath (Hart Scientific, American Fork, UT) were it was held at the time a nd temperatures mentioned above. The juice was then passed through a cooli ng tube and chilled to 10 C where it was collected into sterile glass containers. Following pasteurization, sodium azide (50 mg/L) was added to the nectars to insure inhibition of microbial gr owth during storage. The pasteurized samples were then individually filled in triplicate in to 30 ml test tubes a nd stored at 4 and 25o C in

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19 the dark for 0, 14, 28, and 42 days. A non-pasteuri zed nectar that also contained sodium azide was retained and stored until identical cond itions as a control for the experiment. Aliquots of the nectar were removed for extr action of lycopene and the remaining nectar was treated with 0.1% v/w of pectinas e (100,000 AJDU/ml) and incubated at 32o C for 30 minutes. The nectar was then clarified by cen trifugation to obtain a clear serum that was used for the remaining chemical analyses. Physicochemical Analysis The clarified nectars were assayed fo r vitamin C, antioxidant capacity, total soluble phenolics, individual phe nolic acids, and the original n ectar for lycopene with all assays conducted within 48 hrs of their respective pro cessing and storage times. Vitamin C Vitamin C was determined in all samples as previously described by Talcott et al. (2003) by extracting in a 3% aqueous citric acid solution, filtering through a 0.45 m Whatman syringe filter, and injecting into a Waters Alliance 2695 HPLC separation unit (Waters Corp. Miflord, MA) with an isocrati c flow at a rate of 1 mL/min with 0.2M K2H2PO4 as the mobile phase. Separations were made on a Supelcosil LC-18 column (Supelco, Bellefonte, PA) with detection at 280 nm with a Waters 996 PDA detector. Vitamin C characterized based on retenti on time and spectroscopic determinations against an authentic standard (Sigma Chemical Co., St. Louis, MO). Total soluble phenolics Total soluble phenolics were analyzed using the Folin-Ciocalteu metal reduction assay, previously described by Talcott et al 2000, using gallic acid as a standard. For analysis, 100 L of clarified nectar wa s reacted with a commerci al preparation of 0.25N Folin-Ciocalteu reagent and after a 3 min reaction was neutralized with 1N sodium

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20 carbonate. After 7 minutes, water was added to bring total volume to 10 mL and the sample mixed using a vortex. Approximately 30 minutes later, the samples and standard curve was pipetted in to a mi crotiter plate and the absorban ce at 726 nm read using a Spectra Max 190 spectrophotometer (Molecular devices, Sunnyville, CA). Total soluble phenolics were reported in mg/L eq uivalents of gallic acid. Antioxidant activity Antioxidant capacity of hydrophilic co mpounds was determined by the oxygen radical absorbance capacity (ORAC) assay as previously described by Prior and others (2001). This assay measures the peroxyl radical scavenging properties of guava phytochemicals generated by 2,2’-azobis (2 -amidinopropane) dihydrochloride in the presence of fluorescein, th e fluorescent probe used as an indicator of oxidation. Antioxidant capacity was calculated by integr ating the area under the fluorescence decay curve in the presence of guava phytochemical s and calibrated using a standard curve of Trolox, a water soluble vitamin E analogue with data reported in M Trolox equivalents/mL of guava nectar. Lycopene Lycopene was extracted from the initial guava nectar using an equal mixture of hexane and acetone. Upon extraction, water was added to the solvent mixture to partition lycopene into the hexane layer, which was co llected and filtered for for HPLC analysis. Lycopene was separated by HPLC using a YM C C30 Carotenoid TM column (250 X 4.6 mm) with detection at 470 nm. Sample con centration was determined from a standard curve of lycopene calculated based its mo lar extinction coefficient in hexane.

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21 Statistical Analysis This study consisted of a 2 x 2 x 4 x 3 full factorial design where the factors included 2 processing time/temperatures, 2 st orage temperatures, and 4 storage times following pasteurization with analysis fo r each treatment conducted in triplicate. Regression analyses, Pearson correlation, and analysis of variance were conducted using JMP5 software (SAS Institute 2002), with mean separation performed by the least significant difference (LSD) test ( P < 0.05). Results And Discussion Effects On L-Ascorbic Acid Guava is well documented as having one of the highest concentrations of vitamin C among all fruits, typically containi ng between 200-300 mg of vitamin C/100 g. Previous studies on the effects of storag e time on freeze dried guava demonstrated an inverse relationship between vitamin C concen tration and storage time and temperature (Uddin et al. 2002). No other st udies exist on thermal pasteuri zation or storage of guava nectar. However, L-ascorbic acids low ther mal and storage stability has been widely studied in other fruits. Talcott et al. (2003) found that pasteurization (85oC for 30 min) of passion fruit juice resulted in a 25% loss of Lascorbic acid and all L-ascorbic acid was lost after 14 days of storage at 37oC. Likewise, this study demons trated similar results for changes in L-ascorbic acid over time wher eby it decreased as a function of storage time/temperature and processing conditions (82oC for 20 seconds and 89oC for 90 seconds). Both L-ascorbic acid and dehydroascor bic acid are functional as vitamin C in vivo, yet a goal of this study was to focu s on those factors infl uencing antioxidant properties and phytochemical retention. It is likely that L-ascorbic acid was initially

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22 converted to dehydroascorbic ac id, which has no antioxidant capacity, and then into other degradation products that have negative implicat ions for nectar quality such as off-colors and flavors. Immediately after thermal pr ocessing, no significant differences in Lascorbic acid were observed between Proce ss 1 and Process 2 and the non-pasteurized nectars (Figure 3-1). This indi cated that the conditions of processing did not create an environment suitable for ascorbic acid oxida tion or degradation. Ho wever, during storage ascorbic acid concentration rapidly decreased in response to the past eurization conditions temperature of storage. Guava nectar pasteu rized with Process 1, lower temperature and shorter time, retained 26% more as corbic acid during storage at 4oC than Process 2 and Process 1 nectar stored at 25oC retained 35% more L-ascorbic acid than the Process 2 nectar, which lost all L-ascorb ic acid after just 14 days. The results of this study suggest that thermolabile, oxidative or other de gradation products formed during thermal processing that did not originate from ascorbic acid itself affected the stability of the Lascorbic acid during storage. The greatest effect on the retention of ascorbic acid during storage was storage temperature, irregardless of pasteurization conditions, with nectars at 4C retaining Lascorbic acid for a longer period of time than nectars stored at 25C. This effect of storage temperature can be seen previous stud ies on citrus fruit. Burdurlu et al. (2005) studied ascorbic acid degrad ation in oranges, tangerines lemons, and grapefruits and found that ascorbic acid retenti on after storage at 28, 37, and 45oC was about 54.5-83.7%, 23.6-27%, 15.1-20%, respectively. L-Ascorbic acid was completely destroyed in Process 2 after 14 days of storage at 25o C whereas nectars stored at 4o C still contained 43% of

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23 the initial concentrati on. Likewise complete loss was obs erved for Process 1 after 28 days at 25o C but still retained 26% at 4C. Factors that influence the oxidation of ascorbic acid include pH, oxygen, water activity, and the presence of cer tain metal ions such as ir on and copper and is generally day 0day 14day 28day 42 mg/L L-ascorbic acid 0 50 100 150 200 250 300 350 Processed: 82o C/20 sec, stored: 4o C Processed: 82o C/20 sec, stored: 25o C Processed: 87o C/90 sec, stored: 4o C Processed: 87o C/90 sec, stored: 25o C unprocessed control Figure 3-1. Effects of processing and storag e on L-ascorbic acid. Bars represent the standard error of the mean, n=3. exacerbated with light and elevated temper atures (Fennema 2000). Although the guava nectars were stored in the dark, minimizing their exposure to light, the effect of the storage temperature differential was signifi cant indicating the majo r cause for oxidation of L-ascorbic acid. Another pot ential factor contributing to L-ascorbic acid degradation was the presence of oxygen. No steps to cont rol atmospheric oxygen in the headspace or dissolved in the nectar were made, and lik ely was causative towa rds oxidation. Despite no observable change in L-ascorbic acid follo wing pasteurization, th e higher temperature

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24 and longer processing time of Process 2 along with storage at 25C for both processes created an environment for rapid oxidation of L-ascorbic acid. This temperature dependence is import ant since many guava products, such as nectars, purees, and juice blends are currently shelf stable products a nd therefore stored at room temperature. In conclusion, a recomme ndation to guava processors would be to optimize pasteurization conditions to avoi d excessive heat exposure followed by refrigerated storage in effort to retain the greatest amount of L-ascorbic acid. Effects On Lycopene The main distinguishing characteristic fo r pink guavas is the presence of the red carotenoid lycopene. Lycopene concentrati ons have been well documented in other commodities such as tomatoes, watermelon, grap efruit, and papaya but guava is an often over-looked source for this important phytochemical. All trans -lycopene was measured by HPLC before and after pro cessing with a 40% decrease observed for Process 2, yet no difference was observed between Process 1 a nd the unpasteurized control (Figure 3-2). After the initial loss du e to pasteurization, Process 2 did not result in subsequent loss in lycopene with storage at either 4 or 25C. However, Process 1 exhibited a large decrease during storage and by Day 14 a 34 and 28% d ecrease occurred when stored at 4 and 25o C, respectively. Through 28 days of storag e, lycopene concentration was mainly a function of the processing parameters rath er than storage temperature and by Day 42 there were no differences among the nectars (Figure 3-2). Carotenoids, including lycopene are se nsitive to oxygen, temperature, light, peroxides, and storage (Maini and Sudhakar 1994). The appreciab le loss of lycopene after Process 2 may be explained by the conversion of trans -lycopene into cis -lycopene through heat and oxidation. Cis -lycopene is more easily oxidized than trans -lycopene

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25 into degradation products which in turn do not act as antioxidants (Bohmet al. 2004). Cis lycopene isomers may also re-isomerize to trans -lycopene day 0day 14day 28day 42 mg/L Lycopene 12 14 16 18 20 22 24 26 28 30 Processed: 82o C/20 sec, stored: 4o C Processed: 82o C/20 sec, stored: 25o C Processed: 87o C/90 sec, stored: 4o C Processed: 87o C/90 sec, stored: 25o C unprocessed control Figure 3-2. Effects of processing and storage on lycopene. Bars represent the standard error of the mean, n=3. throughout storage (Lovric et al. 1970, MacDougall 2002), which may explain the increase in trans -lycopene observed at Day 28 for Process 2 stored at 4o C. Previous studies (Giovanelli and Paradi so 2002, Lovric et al. 1970) with tomatoes have also demonstrated re-isomerization from cis to trans during processing and storage. Lovric et al. concluded that alltrans -lycopene was more stable at 20o C compared to -10, 2 and 37 o C and attributed this to the fact that re-i somerization is favored at this temperature. Lycopene stability in products such as guava nectar is also a function of light, water activity, oxygen, pH, temperature, and the presence of pro-ox idants or antioxidants (Chou & Breene 1972; Minguez-Mosavera 1995; Yanislieva et al.1998). Among the

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26 antioxidants available in guava, ascorbic aci d, despite the disparity in polarity, is known to stabilize lycopene (Mortensen et al. 2001). Although this protecting effect was not observed during pasteurization, it may help e xplain the stability of lycopene during storage. Effects On Polyphenolics Total soluble phenolics were measur ed by the Folin-Ciocalteu assay, which measures the ability of phytochemical compounds to reduce an oxidized metal ion. Although the target compounds for this assa y were polyphenolics, compounds such as ascorbic acid, certain soluble proteins, melonoidins, and reducing sugars give a measurable interference in the assay. Results of the assay reveal ed that following pasteurization with both conditions the tota l soluble phenolics significantly increased from 806 mg/L gallic acid equivalents ( GAE) to 1046 mg/L (30% increase) and 1108 mg/L (37% increase) for Process 1 and 2, re spectively (Figure 3-3). These increases following thermal processing may be explai ned by several factor s, including the conditions of the assay itself. Due to the br oad range of compounds detected by the assay, changes in these compounds following pasteuri zation can easily influence the ability to reduce metal ions in solution. Additionally th e use of guava puree to create the nectar likely contained intact plant cells whereby the conditions of heating helped to release cell-wall or vacuole-bound com pounds with metal reducing ca pabilities. Any of these compounds present or their effect on the assa y following processing could account for the increase. Dewanto et al. (2002) observed similar results in sw eet corn. In this study, total soluble phenolics in corn, as measured by the Folin-Ci ocalteu assay increased 24, 32, and 36 % when heated at 115oC for 10, 25, and 50 minutes, respectively.

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27 Additional evidence for the influence of heat on the breakdown or release of cellular constituents was obtained by a dding pectinase to 100% guava puree and incubating at 35o C for 45 minutes and centrifuged to obtain a clear serum (Figure 3-4). The residual solids were then extracted a second time with water, centrifuged, and a second serum obtained. This pro cess was repeated 5 times with the final sample placed in boiling water for 5 min to determine the rel ease of cellular-bound co mpounds with metal reducing capabilities. Results indicated that significant am ounts of compounds with metal reducing capabilities persisted with a 35% increase obs erved after heating. Results may indicate that increased total soluble pheno lics after processing ma y be related to the rupturing of cells and resulted in better extrac tability. Findings also indicate that that the use of clarified guava is like ly underestimating the total c oncentration of phytochemicals present. It may be necessary in the future to explore other extraction techniques such as alcoholic solvents to insure comp lete phytochemical solubilization. During storage, the residual concentrati on of total soluble phenolics was mainly a function of storage temperature, whereas nectars stored at 4o C retained higher concentrations than samples stored at 25o C for both pasteurization parameters and each decreased continuously at a similar rate ove r time. Throughout the 42 days of storage at 4o C, there were no differences between th e samples processed under the two different conditions, whereas only small differences were observed for n ectars stored at 25o C. For Process 1 and Process 2 samples stored at 25o C there was a 28% and 17% decrease in total soluble phenolics afte r 42 days, respectively. Effects On Antioxidant Activity Antioxidants are important in foods in term s of their potential health benefits as well as their radical scavenging abilities which can prevent oxidation in foods.

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28 Therefore, it is important to have an overall measurement of the antioxidant ability of fruits and vegetables, which are the leading s ource of antioxidants in the American diet. Many antioxidant capacity assays are currently used in the food industry, day 0day 14day 28day 42 mg/L Total Soluble Phenolics (gallic acid eq) 600 800 1000 1200 1400 1600 1800 2000 Processed: 82o C/20 sec, stored: 4o C Processed: 82o C/20 sec, stored: 25o C Processed: 87o C/90 sec, stored: 4o C Processed: 87o C/90 sec, stored: 25o C unprocessed control Figure 3-3. Effects of processing and storage on total soluble phenolics. Bars represent the standard error of the mean, n=3. including the total radical trapping antioxi dant parameter (TRAP), Trolox equivalence antioxidant capacity (TEAC), ferric ion re ducing antioxidant power (FRAP), and the oxygen radical absorbance capacity (ORAC) method (Huang et al. 2005). Antioxidant activity was measured in this study, using the ORAC assay for hydrophilic antioxidants, which excludes compo unds such as lycopene, tocopherol, or other carotenoids that may be present in the nectars. However, a study on the antioxidant capacity of lycopene found low radical scavengi ng activities for lycopene (Bangalore et al. 2005), therefore th e hydrophilic compounds likely represented the majority of antioxidant compounds in guava such as ascorbic acid and polyphenolics.

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29 J2J3J4J5J6 % of initial 0 20 40 60 80 100 Total Soluble Phenolics Figure 3-4. Percent initial of total soluble phenolics after water extraction and dilutions. J2: 100% guava juice, J3: puree from J 2, diluted 1:1 with water, J4: puree from J3, diluted 1:1 with water, J5: puree from J4, diluted 1:1 with water, J6: puree from J4, heated for 1 minute at 100oC. Bars represent the standard error of the mean, n=3. Antioxidant capacity of the guava ne ctars was unchanged immediately after pasteurization in all samples when compared to the unpasteurized control (Figure 3-5). Overall, loss of antioxidant capacity was a function of stor age temperature rather than processing conditions, with nectars stored at 4 o C retaining a higher antioxidant capacity than nectars stored at 25 o C as previously observed for ascorbic acid and total soluble phenolics. For those nectars stored at 25 o C, there was a significant decrease (22%) in antioxidant capacity after 14 days, while the samples stored at 4 o C remained unchanged throughout the 42 day study. Nectars pasteurized with Process 1 and 2 and stored at 25 o C lost 47 and 39% of the antioxidant capacity after 42 days, respectively and followed a similar decrease over time.

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30 Table 3-1 shows correlations between ORAC and L-ascorbic acid, total soluble phenolics, and lycopene. Antioxidant capacity in guava puree was attributed mainly to the hydrophilic antioxidants Lascorbic acid and the va rious polyphenolic compounds. Higher correlations were seen between OR AC vs. L-ascorbic acid, followed by total soluble phenolics, while ORAC vs. lycopene was poorly correlate d. Overall, ORAC was better correlated to all phytochemicals during 25oC storage, due to increased stability of and fluctuations observed in the samples stored at 4oC. Leong and Shui (2002) contributed 50% of guava’s anti oxidant activity to ascorbic acid, which is similar to the results of this study. 010203040 Antioxidant capacity (M TE/mL) 4 6 8 10 Processed: 82o C/20 sec, stored: 4o C Processed: 82o C/20 sec, stored: 25o C Processed: 87o C/90 sec, stored: 4o C Processed: 87o C/90 sec, stored: 25o C unprocessed control Figure 3-5. Effects of processing and storage on antioxidant activity. Bars represent the standard error of the mean, n=3.

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31 Table 3-1. Correlations (R2) of ORAC vs. L-ascorbic aci d, total soluble phenolics, and lycopene during storage for 28 days at 4 and 25oC Sample Set L-as corbic acid Total soluble phenolics Lycopene Process 1, 4o C 0.26 0.22 0.20 Process 1, 25o C 0.73 0.40 0.45 Process 2, 4o C 0.18 0.16 0.01 Process 2, 25o C 0.45 0.49 0.00 Conclusions Overall, the stability of antioxidants in guava nectar was a function of storage temperature and time with the conditions of processing a less important factor following pasteurization. Storage temperature was the majo r factor in stability of L-ascorbic acid, total soluble phenolics, and total antioxidant capacity, while processing time/temperature was the major factor in stability for lycope ne. Guava nectar can be thermally processed and stored without affecting the majority of its antioxidants as long as strict temperature control is maintained during storage. All L-as corbic acid was lost and total antioxidant capacity was significantly decreased while pol yphenolic compounds were the most stable compounds during processing and storag e for 42 days at both 4 and 25 o C. A nitrogen purge may be a sufficient way to increase the stability of L-ascorbic acid and therefore total antioxidant capacity in guava puree.

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32 CHAPTER 4 THE EFFECTS OF ANTHOCYANIN-CONTAINING JUICE BLENDS ON THE PHYSIOCHEMICAL AND ANTIOXIDANT CAPACITY OF GUAVA JUICE Introduction Due to limited availability of fresh guava, most fruit destined for US markets are processed into juice, puree, jams, jellies, and syrup (Jim enez-Escrig and others 2001), with a majority utilized in juice blends. Nume rous fruit blends that incorporate guava and other tropical fruits exist in retail markets, but few of these blends contain anthocyanincontaining juices and may even be colored with artificial colorants to give an impression of red or pink colors. Anthocyanins are polyphenolic compounds that give fruit their blue, purple, and red colors and contribute to the health promo ting properties and the appearance of the fruit and ju ice. Anthocyanins are commonly extracted from natural sources such as grapes, red radish, black car rots, purple sweet potatoes, and red cabbage and may be added to juices, yogurts, a nd ice cream among other products to add nutritional benefits and as a natural color substitute for artificial pigments. However, issues surrounding natural color fortification or blending ju ices containing anthocyanins is their inherent instability during processing and storage. Anthocyanin stability is dependent on pH, temperature, light, a nd oxygen which tend to adversely impact retention as well as the pres ence of other polyphenolic compounds that act as cofactors and serve to enhance overall retention. A diversity of polyphenolic compounds such as catechin and rosmarinic acid were shown to serve as cofactors to anthocyanins which help to increase their stability. Anthocyanins are also affected by the presence of ascorbic

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33 acid which has shown to degrade anthocyanins in a mutually destructive process in fruit juices such as grapes, strawberries, and acai (Choi et al 2001, De l Pozo et al. 2004; Brenes et al. 2005). Despite th e high concentration of na turally occurring polyphenolics present in guava that may serve as cof actors to anthocyanins, the unusually high concentration of ascorbic acid present in guava may be a primary reason why anthocyanin concentrates and juices natura lly containing anthocyanins are not commonly blended with guava juice. Two cyanidin based anthocyanin sources, a ai and black current, were chosen for this study. Cyanidin-based anthocyanin pigmen ts were chosen due to their abundance in natural products including blac kberry, elderberry, bl ack carrot, sweet po tato, red cabbage, black currant, and aai (Malien-Aubert et al 2000, Stintzing et al 2002, Del Pozo et al. 2004). Cyanidin 3-glucoside, which is the pr edominant anthocyanin in black currant and aai (Del Pozo et al. 2004, Nielsen et al. 2003) is the most commonly occurring anthocyanin in nature (Mazza et al. 1993). Aai and black currant juices vary mostly in their non-anthocyanin polyphenolic composition. Aai pulp has been previously reported to contain predominantly ferulic acid, epicatechin, p -hydroxy benzoicv c acid, gallic acid, protocatechuic acid, and (+)-catechin, (Del Pozo et al. 2004) while black currant has been reported to contain predominantly m -coumaric acid, p -courmaric acid, salicylic acid, ferulic acid, and caffeic acid (Zadernowski 2005). Preliminary studies were performe d on aai juice and C18 isolates under accelerated conditions (37oC). Total anthocyanins were measured in samples every 30 minutes for a total of 4 hours and results indi cated aai juice anthocyanins to be more stable than aai C18 bound anthocyanins in the presence of L-ascorbic acid. This

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34 suggested that an unknown co mpound or compounds in the aai juice matrix has a protecting effect on the anthocyanins and th erefore was the justification for further investigations of the difference between the st ability of anthocyanin containing juices and their C18 bound isolates. This study will also answer the question to whether clarified guava juice, which is naturally yellow in color, can be color fortified with natural anthocyanin pigments for better marketability, and if clarifie d guava juice, which is often used in juice blends, can be blended with anthocyanin containing juice, despite the high ascorbic acid content. This study was designed to explore the e ffects of anthocyanin-containing juices (black current and aai) and anthocyanin concen trates blended with clarified guava juice, which is high in both ascorbic acid and polyphenolic compounds. The juices and anthocyanin isolates were mixed into a pH buffered solution and evaluated with and without the addition of ascorb ic acid. Juices were pasteu rized and stored at 6 and 25o C for a total of 4 weeks to determine degr adation kinetics of the antioxidant and phytochemical constituents. Materials And Methods Materials And Processing Clarified guava juice was prepared as previously described in Chapter 3 and adjusted to pH 3.5 with citric acid. Looza brand black currant juice (30% juice, 15o Brix) was purchased from a local market and adjusted to pH 3.5. Fr ozen aai puree was obtained from Bolthouse Farms (Bakersfiel d, CA) and clarified by centrifugation and filtered though a bed of diatomaceous earth. The resultant juice was adjusted to 15 Brix with the addition of sucrose and diluted to 30% juice using pH 3.5 buffer. Anthocyanin isolates were prepared from the black current and aai juices by partitioning from

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35 Waters C-18 5g Sep Pak Vac cartridges, colle cting anthocyanins and non-anthocyanin polyphenolics with acidified methanol. The acidi fied methanol was then evaporated and re-dissolved in pH 3.5 citric acid buffer. Isol ation in this manner removed sugars, organic acids, and other small, polar molecule s leaving behind a C18 bound isolate. Juices and reconstituted C18 isolates were blended 1:1 with guava juice with respective controls processe d by blending each treatment 1:1 with pH 3.5 buffer, with and without L-ascorbic acid fortified at th e concentration present in the diluted guava juice (400 mg/L). The juices were placed into 50 mL glass containers and pasteurized in a hot water bath to 80 o C then immersed in an ice wate r bath until the samples reached 25C. The juices were then placed into labeled test tubes, purged with nitrogen, capped and stored at either 6 or 25C in the dark. Pre and post pasteurized juices and controls were immediately analyzed for ascorbic aci d, antioxidant capacity, a nd total anthocyanins as previously described in Chapter 3 while analysis samples for individual polyphenolics by HPLC were stored at -20 o C until time for analysis (<30 days). Total anthocyanins, Lascorbic acid, antioxidant activity, and polypheno lics will be reported as percent of initial concentration (Day 0, post-pasteurization). Chemical Analysis L-ascorbic acid L-ascorbic acid was assayed as described in Chapter 3. Phenolics by HPLC Predominant flavonoids and phenolic acids were analyzed by HPLC according to conditions of Talcott et al (2002), using a Waters 2695 Alliance HPLC system with a Waters Spherisorb 5 m ODS2 column (250 X 4.6 mm) with detection at 280 nm using a Waters 996 PDA detector. Compounds were characterized or identified by

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36 spectroscopic interpretation, re tention time, and comparison to authentic standards (Sigma Chemical Co., St. Louis, MO). Total anthocyanins Total anthocyanins were measured us ing a pH shift method as previously described by Talcott et al. 2002. Samples we re diluted 10x with a hydrochloric acid buffer at pH 1.0. An aliquot of the buffer was pipetted into a 96 well microplate and absorbance read using a Spectra Max 190 sp ectrophotometer at 520 nm with absorbance values adjusted to a 1 cm light path. An extinction coefficient of 29,600 was used to quantify total anthocyanins in mg/L equi valents of cyanidin 3-glucoside. Percent monomeric and polymeric anthocyaninins The proportion of monomeric/polymeric anthocyanins was determined as previously described by Brenes et al. 2005, based on color re tention at 520nm at pH 3.5 in the presence of sodium bisulfite, which bleaches mono meric anthocyanins with remaining color attributable to polymeric fo rms. Samples were read against a blank that consisted of sample diluted with buffe r at pH 3.5 and read on a Spectra Max 190 spectrophotometer. Statistical Analysis This study consisted of a 4 x 2 x 5 x 3 full factorial design. The factors studied were 4 guava juice blends, 2 storage temperatures, and 4 storage times, with each sample run in triplicate. Regression analyses, Pears on correlation coefficients, and analysis of variance were conducted using JMP software Ve rsion 5 (SAS Institute 2002), with mean separation performed by the least signif icant difference (LSD) test (P < 0.05).

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37 Results And Discussion Effects On Anthocyanins Guava juice blends Pasteurization had a small, but statistically significant effect on total anthocyanin concentration in all guava juice blends. The acai juice + guava juice anthocyanins were the most stable during pasteurization with onl y a 4% decrease in their total anthocyanins (Figure 4-1). The other guava juice blends, bl ack currant juice + guava, black currant C18 + guava, and acai C18 + guava showed no differe nces from each other due to processing with an average decrease of 6% of thei r total anthocyanins Figure 4-1 and 4-2). Pasteurization also increased the concentrati on of polymeric anthocyanins in the juice blends. When comparing the unpasteurized juic es, all juice blends containing guava juice had slightly higher polymeric anthocyanins when compared to the juices without guava juice, with acai C18 + guava juice c ontaining the highest amount of polymeric anthocyanins before pasteurization (7 1.2%). The high proportion of polymeric anthocyanins in all of the C18 bound juices most likely resulted from the extraction process which involved slight heating (< 40 oC) during evaporation. The guava juice blend that started with the least amount of polymeric anthocyanins was black currant juice + guava at 39.9%. Pasteuri zation significantly increased the polymeric anthocyanins 6% in aai juice while having no significan t effect on aai C18 + guava juice blends. Pasteurization also significantly increased the polymeric anthocya nins by 8% in both black currant juice and C18 blends. This again demonstrates the stabili ty of the aai C18 + guava juice blend. Storage temperature, anthocyanin source, and the anthocyanin-based food matrix all attributed differences in to tal anthocyanin concentration over a total storag e period of

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38 control17142128 Total Anthocyanins (% of intial) 40 60 80 100 Acai Juice + Buffer, 25 o C Storage Acai Juice + Buffer + AA, 25 o C Storage Acai C18 + Buffer, 25 o C Storage Acai C18 + Buffer + AA, 25 o C Storage Acai Juice + Guava Juice, 25 o C Storage Acai C18 + Guava Juice, 25 o C Storage Days Figure 4-1. Percent initial of to tal anthocyanins stored at 25 o C for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3. 28 days. When comparing only the juice ble nds with added guava juice, all were significantly different in their total anthocyanin concentratio n at 28 days of storage at both 6 and 25 o C except for acai C18 + guava juice and acai juice + guava juice which showed no differences between each other at 6 oC storage after 28 days. The juices in order of decreasing anth ocyanin retention at 6 oC storage were acai C18 + guava juice (83%), acai juice + guava juic e (80%), black currant juice + guava juice (72%), and the least stable being black curra nt C18 + guava juice (64%). The juices stored at 25 o C in order of decreasing percent of initial of tota l anthocyanin concentra tion were acai C18 + guava juice (74%), acai juice + guava juic e (47%), black currant C18 + guava juice

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39 Days control17142128 Total Anthocyanins mg/L (% of initial) 60 70 80 90 100 110 120 Acai Juice + Buffer, 6o C Storage Acai Juice + Buffer + AA, 6o C Storage Acai C18 + Buffer, 6o C Storage Acai C18 + Buffer + AA, 6o C Storage Acai Juice + Guava, 6o C Storage Acai C18 + Guava, 6o C Storage Figure 4-2. Percent initial of to tal anthocyanins stored at 6oC for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3. (42%), and black currant jui ce + guava juice (39%). This demonstrates a large storage temperature dependence, with those stored at 4oC retaining significantly more total anthocyanins than those stored at 25oC. These results also indicate that aai anthocyanins were are more stable in the presence of guava than black currant anthocyanins. The proportion of monomeric anthocyanins decreased over time in each juice treatment and as a result of st orage temperature. Turker et al. (2004) observed a similar trend in percent monomeric/polymeric anthoc yanins during storage time in a fermented black carrot beverage, showing that the % monomeric anthocyanins decreased17.8, 49.2,

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40 control17142128 Total Anthocyanins (% of initial) 20 40 60 80 100 120 Black Currant Juice + Buffer, 25 oC Storage Black Currant Juice + Buffer + AA, 25 oC Storage Black Currant C18 + Buffer, 25 oC Storage Black Currant C18 + Buffer + AA, 25 oC Storage Black Currant Juice + Guava Juice, 25 oC Storage Black Currant C18 + Guava Juice, 25 oC Storage Days Figure 4-3. Percent initial of to tal anthocyanins stored at 25o C for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3. and 93.3% in the carrot beverages st ored for 90 days at 4, 25, and 40 o C, respectively, with a respective incr ease in % polymeric fo r the carrot beverages over the 90 day period. An increase in polymeric anthocyanins has b een extensively inves tigated for red wine aging, where polymeric anthocyanins are a desired quality aspect (Vidal et al. 2002, Arnous et al. 2001). Likewise, storage studi es with blood orange juice found that anthocyanins could polyermize with hydroxycinnamic acids to form larger pyranoanthocyanins (Hillebrand et al. 2004) si milar to a model system where malvidin 3,5-diglucoside condensed with catechin to form a colo rless condensation product

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41 Days Days control17142128 Total Anthocyanins mg/L (% of initial) 50 60 70 80 90 100 110 120 Black Currant Juice + Buffer, 6 oC Storage Black Currant Juice + Buffer + AA, 6 oC Storage Black Currant C18 + Buffer, 6 oC Storage Black Currant C18 + Buffer + AA, 6 oC Storage Black Currant Juice + Guava Juice, 6 oC Storage Black Currant C18 + Guava Juice, 6 oC Storage Figure 4-4. Percent initial of to tal anthocyanins stored at 6oC for 28 days. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3. (Bishop and Nagel 1984). After 28 days of storage at 6 o C Aai C18 + Guava and Aai C18 + Buffer + L-Ascorbic Acid had the highe st polymeric anthocyani ns within the juice blends stored at this temperature, at 77.8 and 76.2 % polymeric anthocyanins, respectively. This is a slight increase from the pre-processed control, which started with 71% polymeric. The Black Currant C18 + Guava had the second highest amount of polymeric anthocyanins out of all guava c ontaining samples (53.6% compared to its Lascorbic acid control at 45.5%). Black Curra nt Juice + Guava had the lowest amount of polymeric anthocyanins compared to the othe r guava juice blends, and consisted of 41.5 % after 28 days at 4 o C. At 25 o C storage, the Aai C18 + Guava and the Black Currant

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42 C18 + Guava had the most polymeric anthocyanins (83.8 and 79.7 % respectively. Similarly, the Aai Juice + Guava and Black Currant Juice + Guava were not different from each other (68.0 and 69.2 % respectively). These results show a strong indication that the concentration of polym eric anthocyanins were instru mental in preventing the loss of total anthocyanins, which is based on a co lorimetric assay. Since consumers tend to make purchasing decisions based on visual pe rception of anthocyani n-based color, this stability is important in cons umers’ perception of fruit juices. Anthocyanins, as found in fresh fruit and juices are in their monome ric form. Over storage time, the monomeric anthocyanins in fruit juices may undergo a condensation reaction to form polymeric pigments as previously discussed (Bis hop and Nagel 1984, Hillebrand et al. 2004, Wang 2003). Several mechanisms have been theori zed for this condensation, reaction which include: a reaction between proanthocyanidin and anthocyanin, which results in colorless or orange xanthylium salts, or an acetalde hyde, glyoxylic acid, furfural, or HMF related condensation between anthocyanin and proant hocyanidin resulting in the formation of purple pigments (Bishop et al 1984, Dallas et al. 1996, Es-Safi et al 2000). Despite the mechanism of reaction, polymeric anthocyanins are more stable than their monomeric counterparts. This may explain the high stabil ity of the aai C18 samples, which started with the highest degree of anthocyanin polymerization. Synthetic L-ascorbic acid models Pasteurization also had a small, but st atistically significant effect on total anthocyanin concentration in the model juices containing L-ascorbic acid, resulting in a decrease in total anthocyanins in all juices Aai juice and C18 anthocyanins decreased 8 and 4%, respectively while black currant ju ice and C18 anthocyani ns decreased 5 and 9%, respectively. Synthetic ascorbic acid jui ce models had a lower increase of polymeric

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43 anthocyanins overall than the guava juice blends, suggesting that guava polyphenolics may be polymerizing with the anthocyanins while in the juice matrix. Also, the concentration of polymeric ant hocyanins in the juice matrix was higher than those in the C18 isolate, which may be attributed to th e higher concentration of HMF in the juice system. As previously explained, one s uggested mechanism for the formation of polymeric anthocyanins is a condensation of non-anthocyanin pol yphenolics, such as condensed tannins (catechin and epicatechin ) with the anthocyanin pigments in the presence of HMF. The polymeric anthocyani ns in the Aai Juice + L-Ascorbic Acid blend increased 3% post pasteurization; compar ed to the Aai Juice + Guava blend which increased 6%. The polymeric anthocyanins in Aai C18 + L-Ascorbic Acid did not significantly change, as in the Aai C18 + Guava blend. The polymeric anthocyanins in the Black Currant Juice + L-Ascorbic Acid in creased 3% compared to the Black Currant Juice + Guava which increased 8%, while th e Black Currant C18 + L-Ascorbic Acid increased 10%, which was not significantly di fferent than the 8% increase in the guava blend. In order to determine if the guava ma trix had an effect on the anthocyanin containing juices during storage, juices consisting of ant hocyanin source, buffer, and Lascorbic acid were compared to those jui ces containing the anthocyanin source and guava juice. Guava juice showed a protecting effect on several of the juice blends, which is attributed to the guava polyphenolics formi ng more stable polymeric compounds with the anthocyanins. Aai C18 + Guava and Black Currant C18 + Guava, compared to their counter parts with equal amounts of L-ascorbic acid however la cking the guava juice, had

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44 Acai Juice + Buffer % Polymeric Anthocyanins 0 20 40 60 80 100 Unprocessed Control Post-Processing, Day 0 Day 7 at 25o C Storage Day 28 at 25o C Storage Acai Juice + Buffer + AA Acai Juice + Guava Acai C18 + Buffer Acai C18 + Buffer + AA Acai C18 + Guava Figure 4-5. Percent polymeric anthocyanins of acai based samples stored at 25oC. Bars represent standard er ror of the mean, n=3. Acai Juice + Buffer % Polymeric Anthocyanins 0 20 40 60 80 100 Unprocessed Control Post-Processing, Day 0 Day 7 at 6o C Storage Day 28 at 6o C Storage Acai Juice + Guava Acai C18 + Buffer Acai C18 + Guava Acai C18 + Buffer + AA Acai Juice + Buffer + AA Figure 4-6. Percent polymeric anthocyanins of acai based samples stored at 6oC. Bars represent standard er ror of the mean, n=3.

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45 Black Currant Juice + Buffer % Polymeric Anthocyanins 0 20 40 60 80 100 120 Unprocessed Control Post-Processing, Day 0 Day 7 at 25o C Day 28 at 25o C Black Currant Juice + Buffer + AA Black Currant Juice + Guava Black Currant C18 + Buffer Black Currant C18 + Buffer + AA Black Currant C18 + Guava Figure 4-7. Percent polymeric anthocyanins of black currant based samples stored at 25oC. Bars represent standard error of the mean, n=3. Black Currant Juice + Buffer % Polymeric Anthocyanins 0 20 40 60 80 Unprocessed Control Post-Processing, Day 1 Day 7 at 6 oC Storage Day 28 at 6 oC Storage Black Currant Juice + Buffer + AA Black Currant Juice + Guava Black Currant C18 + Buffer Black Currant C18 + Buffer + AA Black Currant C18 + Guava Figure 4-8. Percent polymeric anthocyanins of black currant based samples stored at 6oC. Bars represent standard error of the mean, n=3.

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46 a greater percent of initial total anthocyanins after 28 days at 4 o C of storage. The Black Currant Juice + Guava however, had a lower final concentration compared to the black currant juice fortified synthetically, while the Aai Juice + Guava showed no difference compared to the aai juice fortified synthetical ly. This demonstrated that the C18 fraction was more stable then their respective juices and that these C18 isolates, together with guava juice, may be more stable as well. Th is aspect will be further discussed in the effects on polyphenolics section. Polymeric anthocyanins significantly increased in all synthetically fortified ascorbic acid blends. However, afte r 28 days of storage at both 6 and 25oC, guava juice blends had a greater concentration of polym eric anthocyanins than the synthetically fortified L-ascorbic acid blends. After 28 days of storage at 25oC storage, Aai Juice + LAscorbic Acid had 72% polymer ic anthocyanins (9% less than Aai Juice + Guava), Aai C18 + L-Ascorbic Acid had 91% polymeric an thocyanins (no significant difference than Aai C18 + Guava), Black Currant Juice + L-Ascorbic Acid had 75% polymeric anthocyanins (8% less than Black Currant Juice + Guava) and Black Currant C18 + LAscorbic Acid had 86% polymeric anthocya nins (4% less than Black Currant C18 + Guava). After 28 days at 6oC storage, Aai Juice + L-Ascorbic Acid had 49% polymeric anthocyanins (6% less than Aai Juice + Gu ava), Aai C18 + L-Ascorbic Acid had 78% polymeric anthocyanins (no significant difference than Aai C18 + Guava), Black Currant Juice + L-Ascorbic Acid had 46% pol ymeric anthocyanins (9% less than Black Currant Juice + Guava), and Black Currant C18 + L-Ascorbic Acid had 67% polymeric anthocyanins (no significant differen ce than Black Currant C18 + Guava).

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47 Anthocyanin controls For those samples lacking L-ascorbic aci d, only the black curr ant juice and C18 bound black currant juice decreased (5 and 7 %, respectively) as a result of thermal processing. The aai juice and C18 bound aai ju ice without added L-ascorbic acid were the most stable during thermal processing. Unde r similar processing conditions, Brenes et al. (2005) reported a 12% d ecrease in total anthocyani ns in a grape juice ( Vitis vinifera ) model system with and without added ascorbic acid, which is similar to the results of this study. Overall, the effect of pasteurizati on on total anthocyanin concentration was minimal (<5 or 7%) in all samples. Pasteu rization had no significant effect polymeric anthocyanins on all controls expect black currant juice + buffer, which increased 5%. This demonstrates the stability of the anthoc yanins in a system lacking ascorbic acid. As expected, anthocyanin/buffer control m odel juice anthocyanins were the most stable throughout the 28 days of storage. At 25oC storage, Aai Juice + Buffer and Aai C18 + Buffer retained 76 and 90% of thei r total anthocyanins, respectively. Black Currant Juice + Buffer and Black Currant C18 + Buffer retained 60 and 64% of their total anthocyanins, respectively. At 6oC storage, Aai Juice + Bu ffer and Aai C18 + Buffer retained 93 and 94%, respectiv ely and Black Currant Juice + Buffer and Black Currant C18 + Buffer retained 88 and 85%, respect ively. This demonstrates an anthocyanin source (aai versus black currant) dependence and for the aai model juices, and anthocyanin matrix dependen ce (juice versus C18 isolat e). The anthocyanin/buffer controls also retain ed the most monomeric anthocyani ns, and therefore had the least amount of polymeric anthocyanins, after 28 days of storage, contai ning in the range of 54-70% polymeric anthocyani ns when stored at 25oC and 42-65% when stored at 6oC.

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48 Anthocyanin Degradation Kinetics Regression analysis was performed on th e total anthocyanin concentration in a natural log (ln) scale versus time in order to determine the appropriate kinetics model. The total anthocyanin stability during storage followed first order kinetics as previously concluded by several other researchers (Bre nes et al. 2005, Turker et al. 2004, Garzon et al. 2002). From first order kine tic calculations, the half lif e was determined for total anthocyanins in the guava juice containing sa mples. Storage temperature had the greatest effect on anthocyanin half life, with the samples stored at 6 oC having significantly greater half lives than those stored at 25 oC for 28 days (Table 4-1.) Table 4-1. Effects of storage temperature a nd L-ascorbic acid on fi rst order regression, rate, and half life of total anthocyanins Different letters re present differences between sample types with or without L-as corbic acid. 6o C Storage 25o C Storage Sample R2 k t1/2 R2 k t1/2 Acai Juice + Buffer + AA 0.91 -0.0063 112.6 a,b 0.96 -0.0291 23.85 c Acai Juice + Guava 0.89 -0.0074 97.73 a,b 0.98 -0.0271 25.58 b,c Acai C18 + Buffer + AA 0.82 -0.0097 71.99 b 0.92 -0.0226 30.65 b Acai C18 + Guava 0.76 -0.0055 131.9 a 0.76 -0.0111 62.78 a Black Currant Juice + Buffer + AA 0.68 -0.0068 110.2 a 0.98 -0.0352 19.69 b.c Black Currant Juice + Guava 0.85 -0.0107 65.48 b 0.87 -0.0342 21.36 a,b Black Currant C18 + Buffer + AA 0.89 -0.0178 39.43 b 0.92 -0.0374 18.55 c Black Currant C18 + Guava 0.93 -0.0148 46.98 b 0.82 -0.0298 23.38 a The influence of storage temperature on anthocyanin stability was generally in agreement with previously published data on the subject (Garzon et al. 2002). The juices lacking L-ascorbic acid however showed high stability of to tal anthocyanins during the 28 day storage period, especially those held at 6 oC, and therefore kinetics were not run due to the lack of fit of kinetic models (zero, first, or second order). ANOVA analyses on total anthocyanin half lives were performed on sample sets consisting of anthocyanin juice/C18 + gua va, anthocyanin juice/C18 + buffer, and

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49 anthocyanin juice/C18 + buffer + L-ascorbic acid. Within the aai set stored at 6 oC, Aai C18 + Guava had the highest half life, 131.9 days (P<0.05), which was significantly greater than its L-ascorbic acid control, wh ich had a half life of 72 days. However, the aai C18 samples showed no differences at 6 oC when compared to the aai juice samples. The Aai Juice + Guava and Aai Juice + L-Ascorbic Acid also showed no statistical differences be tween each other. The aai samples stored at 25 oC showed similar results, with the A ai C18 + Guava again having a greater half life (62.8 days) compared to the others, with its L-ascorbic acid control ha ving a half life of 30.7 days. The half lives of the Aai Juice + L-Ascorb ic Acid and the Aai Juice + Guava samples were 23.9 and 25.6 days, respectively, which were not statistically different from one another. Therefore, when comparing acai samples, the aai C18 samples had the highest half life and the guava jui ce had a protecting effect on the aai C18 samples. Black currant samples did not show this protecti ng effect. When comparing the black currant samples stored at 6 oC, Black Currant Juice + L-Ascorbic Acid at the greatest half life (110 days compared to the ju ice + guava sample at 65 days ) and was significantly greater than all black currant juice blends stored at 6 oC The black currant samples with guava juice demonstrated slightly greater stability than the black currant juice synthetically fortified with L-ascorbic acid when stored at 25 oC with Black Currant C18 + Guava having the greatest half life of 23 days. The Black Currant Juice + Gu ava half life was not different than the black currant C18 + guava juices nor the Black Currant Juice + LAscorbic Acid juices. These results again indicate that storage temperature plays the greatest role in the protection of the guava juice blend stability.

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50 Effect On Naturally Occurring And Fortified L-Ascorbic Acid As previously discussed in chapter 3, guava contains very high concentrations of L-ascorbic acid and when combined with anthocyanins in solution has shown to be mutually destructive in a va riety of food products (Brenes et al. 2005, Choi et al. 2001). This destruction has been linked to the formation of dehydroascorbic acid breakdown products, but the exact mechanis m for their adverse interacti on has not been completely elucidated. This study demonstrated similar re sults with juices cont aining anthocyanins losing L-ascorbic acid at a much greater rate than the guava juice control which contained no anthocyanins. All L-ascorbic acid containing samples in th is study started with similar amounts of L-ascorbic acid, containing betw een 350-400 mg/L. The clarified guava juice used in the study was assayed to contain 700 mg/L L-ascorbic acid which is in the range of previously published data on guava ascorbic acid (USDA nutrient handbook 2005, Uddin 2002). Unlike the results of Chapter 3 where guava nectar was found to be impervious to losses during pasteurization, seve ral juice treatments lost L-ascorbic acid as a result of pasteurization. The L-ascorbic acid in the Aai C18 + Guava, Black Currant Juice + Guava, and Black Currant C18 + Guava all significantly decreased as a result of pasteurization while none of th e fortified treatments on anthocyanin control treatments significantly decreased. Therefore, it is conc luded that guava juice, which naturally contains L-ascorbic acid, had a negative e ffect on the L-ascorbic acid stability of anthocyanin containing juices compared to those fortified with L-ascorbic acid. In agreement with previous studies (Brenes 2005, Garzon 2002), the L-ascorbic acid in this study followed firstorder kinetics and half life a nd rate were calculated as previously described. Storage temperature pl ayed the major role in L-ascorbic acid stability during storage time, with samples stored at 6o C having much greater half lives

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51 than those stored at 25o C. The L-ascorbic acid source (natural versus fortified) was not significant in terms of L-ascorbic acid stabil ity over storage time. This is an important result, and demonstrates that guava juice ha d no protecting effect on the L-ascorbic acid. The average L-ascorbic acid half life for ant hocyanin containing samples stored at 6 and 25 oC were 29 and 2 days, respectively. By 28 days, all juices stored at 25 oC contained no detectable L-ascorbic acid. The guava juice c ontrol was the most stable at both storage temperatures compared to all other samples w ith half lives of 67 and 5 days at 6 and 25o C, respectively. This demonstrates the mutual de struction of ascorbic acid in the presence of anthocyanins. This is an important finding for the food industry and suggests that juices must be kept at refrigerated temperatures (4-7oC) in order to protect the L-ascorbic acid. Lascorbic acid is extremely important in fruit due to its antioxidant pr operties, as well as its protecting effect on other phytochemicals such as phenolic acids and carotenoids. Its stability is especially importa nt in anthocyanin containing ju ice blends since it is evident that the degradation products of L-ascorbic acid degrade ant hocyanins as well (Es-Safi et al. 1999, 2002; Brenes et al. 2005). L-ascorbic acid is therefor e often used as an indicator of fruit juice quality and actions taken to improve its stability may also improve the overall juice shelf life in te rms of nutrition and quality. Effects On Antioxidant Activity Hydrophilic antioxidant capacity was measured using the oxygen radical absorbance capacity assay as described in Chapter 3, primarily measuring contributions from anthocyanins, non-anthocyanin polyphenolic s, and ascorbic acid. This section will discuss the antioxidant activity of guava jui ce blends. Prior to past eurization, the samples

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52 Table 4-2. Effects of storage temperature and anthocyanins on first order regression, rate, and half life of L-ascorbic acid. Different letters represent differences between sample types with or without L-ascorb ic acid. N/A = not applicable due to high stability or fluc tuation in the data. 6o C Storage 25o C Storage Sample R2 k t1/2 R2 k t1/2 Acai Juice + Buffer + AA 0.86 -0.0211 32.94 b 0.89 -0.2951 2.36 b,c Acai Juice + Guava N/A N/A N/A 0.82 -0.2652 2.69 b Acai C18 + Buffer + AA 0.84 -0.0388 24.06 b 0.88 -0.3000 2.36 b,c Acai C18 + Guava N/A N/A N/A 0.96 -0.3725 1.86 c Guava Juice 0.65 -0.0118 66.79 a 0.67 -0.1453 4.77 a Black Currant Juice + Buffer + AA 0.65 -0.0411 31.31 b 0.83 -0.2963 2.44 b Black Currant Juice + Guava N/A N/A N/A 0.96 -0.3725 1.86 b Black Currant C18 + Buffer + AA 0.76 -0.0258 28.67 b 0.97 -0.2863 2.45 b Black Currant C18 + Guava 0.78 -0.0545 14.87 b 0.99 -0.3533 1.96 b Guava Juice 0.65 -0.0118 66.79 a 0.67 -0.1453 4.77 a ControlDay 1Day 7Day 17Day 21Day 28 L-Ascorbic Acid (% of initial) 0 20 40 60 80 100 120 140 160 Acai Juice + Buffer + AA, 25 oC Storage Acai C18 + Buffer +AA, 25 oC Storage Acai Juice + Guava Juice, 25 oC Storage Acai C18 + Guava Juice, 25 oC Storage Guava Juice, 25 oC Storage Figure 4-9. Percent initial of L-ascorbic ac id in acai based samples stored at 25oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3.

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53 ControlDay 1Day 7Day 14Day 21Day 28 L-Ascorbic Acid (% of initial) 0 20 40 60 80 100 120 140 160 Acai Juice + Buffer + AA, 6 oC Storage Acai C18 + Buffer + AA, 6 oC Storage Acai Juice + Guava Juice, 6 oC Storage Acai C18 + Guava Juice, 6 oC Storage Guava Juice, 6 oC Storage Figure 4-10. Percent initial of L-ascorbic acid in acai based samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3. ControlDay 1Day 7Day 14Day 21Day 28 L-Ascorbic Acid (% of initial) 0 20 40 60 80 100 120 140 160 Black Currant Juice + Buffer + AA, 25 oC Storage Black Currant C18 + Buffer + AA, 25 oC Storage Black Currant Juice + Guava Juice, 25 oC Storage Black Currant C18 + Guava Juice, 25 oC Storage Guava Juice, 25 oC Storage Figure 4-11. Percent initial of L-ascorbic acid in black currant based samples stored at 25oC. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3.

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54 ControlDay 1Day 7Day 14Day 21Day 28 L-Ascorbic Acid (% of initial) 20 40 60 80 100 120 140 160 180 Black Currant Juice + Buffer + AA, 6 oC Storage Black Currant C18 + Buffer + AA, 6 oC Storage Black Currant Juice + Guava Juice, 6 oC Storage Black Currant C18 + Guava Juice, 6 oC Storage Guava Juice, 6 oC Storage Figure 4-12. Percent initial of L-ascorbic acid in black currant based samples stored at 6oC. Control is the unpasteur ized juice. Bars represen t standard error of the mean, n=3. had an antioxidant capacity between the rang es of 9-14 uM Trolox eq/mL, with the black currant and aai C18 + guava samples having the lowest antioxidant activity (9.17 and 10.11 uM Trolox eq/mL respectively). Aai pulp ha s previously been reported to have an antioxidant activity of 48.6 uM TE/mL (Del-Pozo Insfran et al. 2004) and black currant berries between 54.4 and 101.4, depending on cultivar (Wu et al. 2004), while no published data exists on guava antioxidant ac tivity as measured by the ORAC assay. The lower antioxidant activity of the C18 samples wa s attributed to the lower concentration of total anthocyanins and the hi gher degree of polymeric anthoc yanins which occurred as a result of the isolation proce ss. Tsai and Huang (2003) reporte d the antioxidant capacity in the roselle flower, as measured by the FR AP, TEAC, and DPPH scavenging effect, to decrease with increasing polym erization, similar to the re sults of this study. Each

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55 treatment acted independently of each other as a result of pasteurization. Guava juice, Aai C18 + Guava Juice, and Black Curra nt C18 + Guava Juice all had increasing antioxidant values after processing, while the Aai Juice + Guav a Juice ORAC value decreased and the Black Currant Juice + Guava Juice ORAC value did not change. Similar to the previous phytochemicals discussed, the antioxidant activity in the juices was temperature dependent, with the samples stored at 6 oC retaining more antioxidant activity than the samples stored at 25 oC. When comparing antioxidant activities after 28 days of storage to the ini tial post-processing values, differences were only seen in C18 samples at both storage te mperatures. After 28 days of storage at 6 oC, the aai C18 + guava and black currant C18 + guava retained 88.55 and 90.96 % of their initial antioxidant activity and when stored at 25 oC, retained 87.16 and 83.98 % of their antioxidant activity, respectively. controlday 0day 7day 14day 21day 28 Antioxidant Activity (uM Trolox eq/ml) 60 70 80 90 100 110 120 130 Acai Juice + Guava Juice, 25 oC Storage Acai C18 + Guava Juice, 25 oC Storage Guava Juice, 25 oC Storage Figure 4-13. Percent initial of antioxidant activity in acai based samples stored at 25oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3.

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56 controlday 0day 7day 14day 21day 28 Antioxidant Activity (uM Trolox eq/mL) 70 80 90 100 110 120 130 140 Acai Juice + Guava Juice, 6 oC Storage Acai C18 + Guava Juice, 6 oC Storage Guava Juice, 6 oC Storage Figure 4-14. Percent initial of antioxidant activity in acai based samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3. controlday 0day 7day 14day 21day 28 Antioxidant Activity (uM Trolox eq/mL) 60 80 100 120 140 Black Currant Juice + Guava Juice, 25 oC Storage Black Currant C18 + Guava Juice, 25 oC Storage Guava Juice, 25 oC Storage Figure 4-15. Percent initial of antioxidant ac tivity in black currant samples stored at 25oC. Control is the unpasteurized juice. Bars represent standard error of the mean, n=3.

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57 controlday 0day 7day 14day 21day 28 Antioxidant Activity (uM Trolox eq/mL) 70 80 90 100 110 120 130 Black Currant Juice + Guava Juice, 6 oC Storage Black Currant Juice + Guava C18, 6 oC Storage Guava Juice, 6 oC Storage Figure 4-16. Percent initial of antioxidant ac tivity in black currant samples stored at 6oC. Control is the unpasteu rized juice. Bars represent standard error of the mean, n=3. Effects On Polyphenolics This discussion will identify the non-anthocyanin polyphenolics in guava containing samples, determine their storag e stability, and determ ine their effects on anthocyanin stability. Major polyphenolic compounds were evaluated in pure guava juice. Figure (4-17) shows a typical polyphe nolic chromatogram of guava juice at 280 nm, separated by HPLC conditions as previously outlined. Due to the numerous compounds separated, only the compounds found in the greatest concen tration and/or the resolved compounds were evaluated. Few st udies exist that iden tify and quantify the polyphenolics present in guava, but have incl uded ellagic acid and condensed tannins (Misra and Seshadri, 1967), and gallic acid, catechin, epicatechin, and chlorogenic acid (Kondo et al., 2005). Based on retention times, spectral properties, and comparison to authentic standards, gallic acid, catechin, and ellagic acid were positively identified in

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58 guava juice in this study (Table 4-3), while several compounds were tentatively identified and classified into general groups, such as benzoic acid esters, procyanidins, and ellagic acid derivatives. These compounds, listed in Table 4-3, were evaluated in all guava containing samples in terms of thei r storage stabilit ies at 6 and 25 oC (Table 4-4). Figure 4-17. HPLC chromatogram (at 280nm) of polyphenolic compounds found in guava juice. 1) gallic acid 2) (+)-cate chin 3) ellagic acid 4) ellagic acid derivative 5) unknown characte ristic guava polyphenolic. Table 4-3. Tentative identification of guava juice polyphenolics, measured by HPLC/PDA. Peak No. Retention Time (min) Spectral Properties (nm) Tentative Identification 1 17.06 215, 271 gallic acid 2 31.28 281 catechin 3 49.89 252, 369 ellagic acid 4 51.38 252, 365 ellagic acid derivative 5 54.36 219, 276 unknown Individual peaks (1-5) were summed for each sample after four weeks of storage at both 6 and 25 o C and reported as percent initial of day 0 concentrations (Table 4-4). ANOVA analysis was performed, comparing the different samp les at their respective

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59 Table 4-4. Percent of initial polyphenolics after four weeks of storage at 6 and 25 oC. Letters represent statistical differences (P<0.05, n=3). Sample 6 oC 25 oC Guava 80.8 b 73.0 a Acai Juice + Guava 98.6 a 74.6 a Acai C18 + Guava 72.4 c 51.1 c Black Currant Juice + Guava 93.3 a 61.2 b Black Currant C18 + Guava 94.4 a 65.2 b storage temperatures. At 6 o C of storage, Aai Juice + Guava, Black Currant Juice + Guava and Black Currant C18 + Guava re tained the most polyphenolics (98.6, 93.3, and 94.4 %, respectively). Guava retained the s econd greatest amount (80.8%), while Acai C18 + Guava retained the leas t amount of polyphenolics (72.4 %). Polyphenolic retention at 25 o C was lower than those stored at 6 o C. Aai C18 + Guava and the pure Guava sample retained 74.6 and 73 % of initial, respectively, at 25 o C storage. Both black currant samples, Black Currant Juice + Guav a and Black Currant C18 + Guava retained 61.2 and 65.2 percent while the Aai C18 + Guav a again retained the least amount of the polyphenolic compounds measured (51.1%). Thes e results are unexpect ed since the Aai C18 + Guava had the highest anthocyanin st ability compared to the other samples. However, this may be a result of the so me of the polyphenolic compounds, such as procyanidins (catechin) polymeriz ing with the anthocyanins in the presence of furfurals such as HMF, as seen in blood orange juic e (Hillebrand 2004) and black carrot juice (Wu 2004). HMF was detected in the juice blends with a chromatographic peak at retention time 8.6 minutes at 285 nm. Conclusions Only slight decreases in total anthoc yanins, L-ascorbic acid, and antioxidant activity were seen as a result of pasteuriza tion in the juice ble nds. Phytochemical and

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60 antioxidant stability in guava juice and guava juice/anthocyanin ju ice and C18 mixtures were storage temperature dependent. It is recommended that juice manufactures keep strict temperature control unde r refrigerated conditions for anthocyanin an d L-ascorbic acid containing juices. Further investigations on aai C18 + guava juice mixtures are needed in order to determine exact reasons for its stability. Possible studies include HPLC/MS determinations of exact anthocyani ns and other polyphenolics that may form as a result of polymerization. However the result s of this study indica te that the aai C18 isolate may be polymerizing with the guava ju ice therefore increasing the stability of the anthocyanins. This is evident from the hi gh degree of polymeric anthocyanins, the increased stability of total anthocyanins compar ed to the synthetic ascorbic acid fortified blend, and the overall decrease of non-anthocyanin polyphenolics.

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61 CHAPTER 5 SUMMARY AND CONCLUSIONS Guava products such as juice, nectar, pur ees, and jams are steadily increasing in the United States market. Few studies are ava ilable pertaining to guava juices in relation to pasteurization and storage, especially thos e relating to the stability of its health and quality aspects such as the color and an tioxidant phytochemicals. To provide an overview, these studies consis ted of two pasteurization cond itions on guava nectar with two subsequent storage temperatures. This study concluded that st orage temperature is the biggest factor relating to increasing the stability of L-ascorbic acid, lycopene, polyphenolics, and antioxidant activit y, with the nectar stored at 4oC better retaining Lascorbic acid, polyphenolics and antioxidant activity, and the nectar stored at 25oC better retaining lycopene. The second study was conducte d in order to determine the effects of blending clarified guava juice with anthocyanin containing ju ices and concentrates, for the purpose of juice blending and color for tification, respectively. The L-ascorbic acid found in guava juice was found to be mutually destructive in the presence of the added anthocyanins. This study demonstrated temper ature dependence as we ll, with the juices stored at 6oC retaining more color overall (anthocy anins) and L-ascorbic acid, than those stored at 25oC. Guava polyphenolics were also found to have a protecti ng effect on aai and black currant C18 concentr ates. This suggests possible interactions between guava phenolics (catechin) and anthocyanins, due to antioxidant protection and formation of more stable polymers. Overall conclusions a nd suggestions to industry are to keep juice

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62 blends at refrigerated temperatures (4-7oC) in order to protect overall quality and antioxidant phytochemicals

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66 Jimnez-Escrig, A.; Rincn, M.; Pulido, R.; Sa ura-Calixto, F. 2001. Guava fruit (Psidium guajava L.) as a new source of antioxidant dietary fiber. J. Agric Food Chem 49:5489-5493. Kondo, S.; Kittikon, M.; and Kanlayanarat, S. 2005. Preharvest antioxi dant activities of tropical fruit and the effects of low temperature storage on antioxidants and jasmonates. Postharv. Bio. Tech. Kondo, S.; Tsuda, K.; Muto, N.; Ueda, J. 2002. Antioxidative activity of apple skin or flesh extracts associated with fruit de velopment on selected apple cultivars. Scientia Horticulterae 96:177-185. Leong, L; Shui, G. 2002. An investigation of anti oxidant capacity of fruits in Singapore markets. Food Chem. 76: 69-75. Levin G., Mokady S. Antioxidant activity of 9cis compared to alltrans -carotene in vitro. Free Radic. Biol. Med 1994; 17:77-82. Lian, Y. S.; Liang, G. H.; Deng, X. Q.; Dong, G.; Chen, X. H.; Xie, L. 2000. Stability of vitamin C in beverage of orange juice. Food Ind 2:14-15. Lovric, T., Sablek, Z., Boskovic, M. 1970. Cistrans isomerization of lycopene and color stability foam-mat dried tomato powder during storage. J. Sci. Food Ag. 21: 641647. Macdougall, D. 2002. Colour in Food. Woodhead publishing. Abington, Cambridge. P. 1 90-211; 278-286. Malien-Aubert, C.; Dangles, O.; Amiot, M. J. 2000. Color stability of commericial anthocyanin-based extracts in relatio n to the phenolic composition. Protective effects by intraand in termolecular copigmentation. J. Agric. Food Chem 49: 170-176. Mazza, G.; Brouillard, R. 1990. The mechanism of co-pigmentation of anthocyanins in aqueous solutions. Phytochemistry 29: 1097-1102. Mazza, G.; Miniati, E. 1993. Anthocyanins in fruits, vegetables, and grains. CRC Press. Boca Raton, FL. Mercadante, A.Z.; Steck, A.; Pfander, H. 1999. Carotenoids from guava: isolation and structure elucidation. J. Agric. Food Chem 47:145-151. Miean, Koo Hui and Mohamed, Suhaila. 2001. Flavonoid (Myric etin, Quercetin, Kaempferol, Luteolin, and Apigenin) content of edible tropical plants. J Agric. Food Chem 49: 3106-3112.

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68 Talcott, S., Percival, S., Pettet-Morre, J., Celoria, C. 2003 Phytochemical composition and antioxidant activity of fo rtified yellow passion fruit. J. Agric. Food Chem. 51(4): 935-941. Talcott, S. T.; Howard, L. R.; Brenes, C. H. 2000. Antioxidant changes and sensory properties of carrot puree processed with and without periderm tissue. J. Agric. Food Chem. 48, 1315-1321. Talcott, S.; Brenes, C.; Pires, D.; Del Pozo -Instran, D. 2003. Phytoche mical stability and color retention of copigmented and processed muscadine grape juice. J. Agric. Food Chem. 51(4): 957-963. Turker, N.; Aksay, S.; Ekiz, H.I. 2004. Effect of storage temperature on the stability of anthocyanins of a fermented bl ack carrot beverage: shalgam. J Agric. Food Chem 52: 3807-3813. Uddin, M.S., Hawlader, M.N.A., Ding, L uo, Mujumdar, A.S. 2002. Degradation of ascorbic acid in dried guava during storage. J. Food Engineering 51:21-26. Ou, B, Hampsch-Woodill, M, Prior, RL. 2001. Development and validation of an improved oxygen radical absorbance cap acity assay using fluorescein as a fluorescent probe. J. Agric. Food Chem. 49:4619-4626. Vidal, S.; Cartalade, D.; Souquet, J.; Fulcrand, H.; Cheynier, Y. 2002. Changes in proanthocyanidin chain length in winelike model systems. J. Agric. Food Chem. 50: 2261-2266. Von Elbe, J.H. and Schwartz, J.S. 1996. Colorants. In: Food chemistry Third Edition. Fennema, O.R. (Ed.), pp. 651-722. Marcel Dekker, Inc. NY. Wang, H.; Race, E.J.; Shrikhande, A.J. 2003. Anthocyanin transformation in cabernet sauvignon wine during aging. J. Agric. Food Chem 51: 7989-7994. Wilson, C.; Shaw, P.; Cambell, C. 1982. Determ ination of organic acids and sugars in guava cultivars by HPLC. J Sci. and Food Agric. 33:777-780. Wu, X.; Gu, L.; Prior, R.; Mckay, S. 2004. Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucas and their antioxidant capacity. J. Agric. Food Chem 52: 7846-7856. Yen, Gow-Chin & Lin, Hsin-Tang.1999. Changes in Volatile Flavor Components of Guava Juice with High-Pressure Treatm ent and Heat Processing during Storage. J. Agric. Food Chem 47: 2082-2087.

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69 BIOGRAPHICAL SKETCH Lanier Elaine Fender was born May 15, 1982 in Dothan, Alabama. She then moved to Ocala, Florida, where she was raised. After completion of high school in 2000, Lanier attended Central Florida Community College in Ocala, Florida, where she completed her Associate of Arts degree in May 2002. Lanier re ceived her Bachelor of Science degree in food science and human nutrition in April 2004 from the University of Florida. She continued at the University of Florida, where she completed her Master of Science degree, specializing in food science, in December 2005.


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Title: Phytochemical and Antioxidant Stability of Thermally Processed Guava (Psidium guajava) and Guava Juice Blends
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Copyright Date: 2008

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Title: Phytochemical and Antioxidant Stability of Thermally Processed Guava (Psidium guajava) and Guava Juice Blends
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PHYTOCHEMICAL, ANTIOXIDANT, AND STORAGE STABILITY OF
THERMALLY PROCESSED GUAVA (Psidium guajava) AND GUAVA JUICE
BLENDS















By

LANIER FENDER


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

LANIER FENDER















ACKNOWLEDGMENTS

I would like to thank my major advisor, Dr. Stephen Talcott, for giving me the

opportunity to further my education, and especially for all of his help, suggestions,

guidance and wisdom throughout my graduate studies. I also thank my advising

committee members, Dr. Charles Sims and Dr. Donald Huber, for their time and

assistance with my research.

I would also like to say thank you to my past and present lab and lunchmates:

Chris, David, Flor, Jorge, Joon, Kim, Kristine, Lisbeth, Maria, Sara, and Lorenzo for

helping me in the lab and for helping to make graduate school an enjoyable experience.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES .................................. .............................. ............ vi

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. vii

ABSTRACT .............. .......................................... ix

CHAPTER

1 IN TRODU CTION ...................... .... ............................................ 1

2 LITER A TU RE REV IEW ........................................... ................................. 3

Guava Production and Market Potential.......................... ......... ................3
T herm al Processing of G uava......................................................................... ........ 4
G uava A antioxidants ....................................................... 5
P olyphenolics ............... ........... ................................ ..................
Phenolic therm al stability ...................................................... .................. 9
A scorbic Acid ..................................... ........ ........................ 9
Carotenoids/Lycopene ........... .................. ..... ......................
A nthocyanins .............. ................................................................... .......... 13

3 EFFECTS OF THERMAL PROCESSING AND STORAGE ON THE
PHYTOCHEMICALS AND ANTIOXIDANTS IN GUAVA NECTAR ..................17

M materials A nd M ethods ...................................................................... ..................18
Materials And Processing......... .......... ............................................ 18
Physicochem ical A nalysis........................................................ ............... 19
Vitamin C .................................... ........ ................... 19
T otal soluble phenolics........................................... .......................... 19
Antioxidant activity ....... .................. ............ ......................... 20
L y co p en e ...............................................................2 0
Statistical A naly sis .......................... .......... ............... .... .. .....2 1
R results A nd D discussion .......... ... ... ........ ... ................ ..... .. .. ............ 21
Effects On L-A scorbic A cid ................................................... ............... ... 21
E effects O n L y cop en e ........................................ ............................................24
Effects On Polyphenolics ............................................................................ 26









Effects On Antioxidant Activity ................. ....... ..... ........................ ............... 27
C o n c lu sio n s ................................................................................................... 3 1

4 THE EFFECTS OF ANTHOCYANIN-CONTAINING JUICE BLENDS ON THE
PHYSIOCHEMICAL AND ANTIOXIDANT CAPACITY OF GUAVA JUICE ....32

In tro d u ctio n .......................................................................................3 2
M materials and M methods ....................................................................... ..................34
M materials A nd Processing........................................................ ................ 34
C hem ical A naly sis........... .......................................................... ...... .... ... ..35
L -A scorbic A cid ...................... ........ ............ ................. .... ... ....35
P henolics by H P L C ..................................... ........ ................ ..............35
Total anthocyanins ...................................... ............ ........36
Percent monomeric and polymeric anthocyaninins ...................................36
Statistical A analysis .......................... ............ ...........................36
R results A nd D discussion ......... ................................. ...................... ............... 37
Effects O n A nthocyanins...................................................... ............... 37
G uava juice blends ................ .......................... .. ............ ......... .... ............ .. 37
synthetic L-ascorbic acid m odels ...................................... ............... 42
anthocyanin controls ............................................................................. 47
A nthocyanin D egradation K inetics .......................... ... ......... ............... .... 48
Effect On Naturally Occurring And Fortified L-Ascorbic Acid .......................50
Effects On Antioxidant Activity............................... .....................51
Effects On Polyphenolics ............................................................................. 57
C o n c lu sio n s ..................................................................... 5 9

5 SUMMARY AND CONCLUSIONS ........................................ ....................... 61

L IST O F R E F E R E N C E S ....................................................................... ... ................... 63

B IO G R A PH IC A L SK E TCH ..................................................................... ..................69





















v















LIST OF TABLES


Table p

3-1. Correlations (R2) of ORAC vs. L-ascorbic acid, total soluble phenolics, and
lycopene during storage for 28 days at 4 and 25C ............................. ...............31

4-1. Effects of storage temperature and L-ascorbic acid on first order regression, rate,
and half life of total anthocyanins. Different letters represent differences
between sample types with or without L-ascorbic acid. ........................................48

4-2. Effects of storage temperature and L-ascorbic acid on first order regression, rate,
and half life of total anthocyanins. Different letters represent differences
between sample types with or without L-ascorbic acid. N/A = not applicable due
to high stability or fluctuation in the data. ........................................ ...................51

4-3. Tentative identification of guava juice polyphenolics, measured by HPLC/PDA....58

4-4. Percent of initial polyphenolics after four weeks of storage at 6 and 25 C..............59
















LIST OF FIGURES


Figure page

3-1. Effects of processing and storage on L-ascorbic acid. Bars represent the standard
error of the m ean, n=3 .............................................. .. ......... ............ 23

3-2. Effects of processing and storage on lycopene. Bars represent the standard error
of the mean, n=3 .................................... ............................. .. ........25

3-3. Effects of processing and storage on total soluble phenolics. Bars represent the
standard error of the m ean, n=3 ........... ....................................... .....................28

3-4. Percent initial of total soluble phenolics after water extraction and dilutions. J2:
100% guava juice, J3: puree from J2, diluted 1:1 with water, J4: puree from J3,
diluted 1:1 with water, J5: puree from J4, diluted 1:1 with water, J6: puree from
J4, heated for 1 minute at 1000C. Bars represent the standard error of the mean,
n = 3 ................... ........................................................ ................ 2 9

3-5. Effects of processing and storage on antioxidant activity. Bars represent the
standard error of the m ean, n=3 ........... ....................................... .....................30

4-1. Percent initial of total anthocyanins stored at 25 o C for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3 ....................38

4-2. Percent initial of total anthocyanins stored at 60C for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3 ..................39

4-3. Percent initial of total anthocyanins stored at 25oC for 28 days. Control isthe
unpasteurized juice. Bars represent standard error of the mean, n=3 ....................40

4-4. Percent initial of total anthocyanins stored at 6oC for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3 ..................41

4-5. Percent polymeric anthocyanins of acai based samples stored at 250C. Bars
represent standard error of the mean, n=3 ................ ......................................44

4-6. Percent polymeric anthocyanins of acai based samples stored at 60C. Bars
represent standard error of the mean, n=3............................. .......................44

4-7. Percent polymeric anthocyanins of black currant based samples stored at 250C.
Bars represent standard error of the mean, n=3 ....................... ........................45









4-8. Percent polymeric anthocyanins of black currant based samples stored at 60C.
Bars represent standard error of the mean, n=3 ................................................. 45

4-9. Percent initial of L-ascorbic acid in acai based samples stored at 250C. Control is
the unpasteurized juice. Bars represent standard error of the mean, n=3 .................52

4-10. Percent initial of L-ascorbic acid in acai based samples stored at 60C. Control is
the unpasteurized juice. Bars represent standard error of the mean, n=3 ...............53

4-11. Percent initial of L-ascorbic acid in black currant based samples stored at 250C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................................................................................................ . .5 3

4-12. Percent initial of L-ascorbic acid in black currant based samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................................................................................................ . .5 4

4-13. Percent initial of antioxidant activity in acai based samples stored at 250C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................ ......... ............... ............................................5 5

4-14. Percent initial of antioxidant activity in acai based samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................ ......... ............... ............................................56

4-15. Percent initial of antioxidant activity in black currant samples stored at 250C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................ ......... ............... ............................................56

4-16. Percent initial of antioxidant activity in black currant samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n = 3 ................ ......... ............... ............................................57

4-17. HPLC chromatogram (at 280nm) of polyphenolic compounds found in guava
juice. 1) gallic acid 2) (+)-catechin 3) ellagic acid 4) ellagic acid derivative 5)
unknown characteristic guava polyphenolic. ................................... ..................... 58














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


PHYTOCHEMICAL AND ANTIOXIDANT STABILITY OF THERMALLY
PROCESSED GUAVA (Psidium guajava) AND GUAVA JUICE BLENDS

By

Lanier Fender

December 2005

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

Due to limited availability of fresh guava, most fruit destined for US markets is

processed into juice, puree, jams, jellies, and syrup, and juice blends. Therefore, it is

pertinent to study the effects of pasteurization and storage conditions on the health and

quality aspects of guava juices and juice blends. The objectives of this study were to

assess the thermal stability and shelf life properties of phytochemicals antioxidants in

guava nectar and to determine the juice blending/color fortification potential of guava

juice with an anthocyanin containing source. Guava nectar was pasteurized at two

different time/temperatures and stored in order to determine the stability of antioxidant

phytochemicals and shelf life properties. Clarified guava juice was blended with

anthocyanin containing juices and isolates (acai and black currant), pasteurized and then

stored in order to determine the stability of antioxidant phytochemicals and shelf life

properties.









For the initial study, guava nectar was pasteurized at 820C for 20 seconds (Process

1) and 87C for 90 seconds (Process 2) and stored at 4 and 25C. Pasteurization at 820C

for 20 seconds resulted in no significant changes in L-ascorbic acid, lycopene, or total

antioxidant activity, while total soluble phenolics increased 77%. Pasteurization at 87C

for 90 seconds resulted in no significant differences in L-ascorbic acid and total

antioxidant activity, while lycopene decreased 40% and total soluble phenolics increased

72% as a result of pasteurization. After 42 days of storage at 40C, L-ascorbic acid was

completely degraded in both Process 1 and 2 nectars, lycopene decreased 40% in Process

1 and did not significantly decrease in Process 2, and total soluble phenolics and

antioxidant activity were not significantly different for either process. After 42 days of

storage at 250C, L-ascorbic acid was completely degraded in both Process 1 and 2

nectars, lycopene decreased 40 % in Process 1 and did not decrease in Process 2, while

total soluble phenolics decreased 28 and 17% for Process 1 and 2 respectively, and total

antioxidant activity decreased 47 and 39% respectively.

Clarified guava juice was blended with acai and black currant juice and C18

isolates pasteurized and stored for 28 days. Storage at 60C significantly protected

antioxidant phytochemicals in the guava juice blends, compared to those stored at 250C.

Guava juice blends stored at 60C retained between 64-83% of their total anthocyanins,

while those stored at 250C retained between 39-74%. Overall, C18 isolates + guava juice

were more stable than the anthocyanin juice + guava juice treatments, with Acai C18 +

guava juice being the most stable in terms of total anthocyanins. All L-ascorbic acid was

lost after 28 days at 250C, while juices held at 40C retained between 20-65% of their

initial L-ascorbic acid.














CHAPTER 1
INTRODUCTION

Diets high in fruits and vegetables have been shown to reduce the risks of certain

chronic diseases such as cardiovascular disease, stroke, and atherosclerosis. Antioxidants

found in fruits and vegetables, including vitamins A and C, lycopene, and other

phytochemicals may be a contributing factor to the reduction of these diseases (Block and

Langseth 1994). Tropical fruits are gaining popularity in the United States due to their

potential health benefits and exotic flavors. Guava, one such tropical fruit, is unusual in

that the fresh fruit are characterized by its soft skin, short shelf life, and intense flavor,

which makes it a prime candidate for tropical fruit blends. Therefore, many guavas are

processed into juices, puree, jams, jellies, and syrup (Jimenez-Escrig and others 2001).

Few studies have been conducted on the phytochemistry and total antioxidant

capacity of guava, especially processed guava juice or puree. However, it is well

documented that guava contains a very high amount of vitamin C. Vitamin C, as an

antioxidant, has the capability to stabilize phytochemicals in processed fruits and is often

an indicator compound for thermal stability. Thermally processed fruits and fruit juices

are often fortified with vitamin C to help stabilize more oxidizable or thermolabile

compounds present. On the other hand, vitamin C has been shown to decrease the

stability of anthocyanins in fruits such as muscadine grapes and strawberries, but certain

phenolics may result in an intermolecular copigmentation with the anthocyanins to

increase overall color stability. Anthocyanins may also polymerize with other

polyphenolic compounds to increase color stability. Color stability of anthocyanins is









also increased by storage at refrigerated temperatures (4-7C) as seen in blood orange

juice (Choi et al. 2001, Bonaventura and Russo 1993). Therefore, the polyphenolics in

guava juice as well as low temperature storage may allow for the natural color

fortification of guava juice, which is currently achieved by the addition of red dye #40.

This may also allow for the mixing of guava puree with anthocyanin containing fruit,

despite the high vitamin C content in guava.

Fruit juices are pasteurized in order to achieve a reduction of microbes and to

inactivate undesirable enzymes. The most popular way to achieve these goals is by

thermal pasteurization. Thermal processing was initially thought to cause an overall

decrease in the antioxidant activity of the fruit or vegetable. However, several recent

studies have concluded that some antioxidants may be quite stable, depending on the food

matrix (Dewanto et al. 2002). Since no studies exist on the relationship of guava

phytochemistry, and thermal processing, and guava/anthocyanin mixing, the results of

this study will be of benefit to researchers as well as industry and consumers.














CHAPTER 2
LITERATURE REVIEW

Guava Production And Market Potential

Guava (Psidium guajava) is a ditcotyledon from the family myrtaceae and is a

tropical fruit known for its exotic flavor and potent aroma. The fruit is grown throughout

the tropical regions of the world, including India, Brazil, Mexico, Columbia, Venezuela,

and the United States. In the U.S., it can be found in Florida, California, and Hawaii. In

Hawaii, most of the guava is prepared as pasteurized puree and stored frozen or kept

aseptic in plastic-lined cans or bags (Nagy et al. 1993). As the major producer of guava in

the U.S., Hawaii harvested 550 acres in 2002 and processed 6.7 million pounds of guava

in 2003. This is a 37% decrease from the amount of guavas processed in 2001, and

follows the declining trend in guava production over the last 12 years (National

Agriculture Statistics Service [NASS] 2004). Many Hawaii guava growers have reported

that this decrease is due to low guava prices and sales, and therefore less acreage of

guavas being grown.

Guavas soften quickly during ripening and therefore have a relatively short shelf

life that limits the distribution of fresh guava fruit in the United States (Nagy et al. 1993).

Due to these fragile conditions, most guavas are processed into juice, puree, jam, jelly,

syrup, nectar, fruit paste, or canned as halves (Jimenez-Escrig and others 2001; Nagy et

al. 1993). However, unlike other tropical fruits such as mango and papaya, guava based

products have not yet reached a high degree of popularity in the United States. This lack

of interest may be due to consumer's lack of knowledge about the fruit's exotic taste and









nutritional benefits since corresponding fresh fruit are not available to help educate

consumers. Further research and education on this subject may increase the production of

guava containing products in the United States.

Thermal Processing Of Guava

Fruit juices, purees, and similar products must be pasteurized before being sold in

order to inactivate oxidative enzymes and reduce the number of pathogens. This process

is traditionally accomplished by rapid heating and cooling of the puree to help preserve

quality factors. Industrially, guava puree may be heated to 900 C for 60 seconds or frozen

immediately after mechanical processing (Gaetano 1997; Nagy et al. 1993) for storage

and/or distribution. These purees are then sold for the manufacture of juice, juice blends,

jelly, jam, and many confectionary products. For the juice and beverage industry, a

second pasteurization is needed following blending to render the product commercially

sterile. Although pasteurization is a necessity for juice safety, thermal treatments have

their drawbacks including adverse effects on flavor, color, and overall nutritional quality

loss. Possible degradation of the numerous phytochemicals in guava may occur during

thermal processing and storage. This includes loss of ascorbic acid, isomerization of

lycopene and other carotenoids, and decreases in overall polyphenolics and antioxidant

activity.

Guava puree is highly viscous due to its high insoluble solids content primarily

from pectin, which makes up approximately 705-804 mg/100g of the fruit (Jagtiani et al.

1988). Pectin is found in the cell wall and middle lamella layers of fruits and vegetables

and is composed of linear chains of c-D-galactopyranosyluronic acid units with neutral

sugars and various side chains attached (BeMiller and Whistler 1996), and nutritionally









functions as dietary fiber. Due to its viscous nature, pectin must be broken down in guava

puree in order to obtain a clarified juice. This is achieved by adding additional pectinase,

an enzyme that hydrolyzes cell walls and large pectin molecules and allows for an easier

juice extraction. Pectinase, as well as other enzymes such as cellulase and amylase, are

commonly added to fruit purees in order to obtain greater juice yields. A 0.1% w/v

addition of pectinase to guava puree was found to be effective in the juice making

process (Nagy et al.1993) and resulted in greater juice yields. Brasil et al. (1995) found

that a 600 ppm treatment of pectinase at 450C for 120 minutes displayed the greatest

yield (84.7%). One disadvantage of using clarified juice, rather than unclarified puree, is

the loss of lycopene in the juice. Lycopene is found bound to the pectin in the fruit and

the process of clarifying removes the pectin, and therefore the lycopene as well. This may

result in artificial color fortification of guava juices, since lycopene gives guava its

characteristic pink color. Although, guava puree is used for juices often, rather than

clarified juice, the addition of pectinase will also allow for greater yields in compounds

of interest during extractions. For example, one study found that pectinase treatments

increased total soluble phenolics 11.5% and ascorbic acid 38.8% compared to the

untreated guava puree (Brasil et al. 1995). The pectinase breaks down more cell walls and

therefore more phenolics are extracted and detected.

Guava Antioxidants

Biologically, antioxidants may be defined as compounds that prevent free radicals

from destroying host cells. Free radicals result from reactive oxygen species that contain

an unpaired electron rather than the paired electrons found in stable functioning

molecules. These free radicals can attach to cells in the body and cause destructive

damage that could lead to an array of chronic health problems, including cardiovascular









disease, stroke, atherosclerosis, and cancer (Block and Langseth 1994). Antioxidants, in

turn, help to reduce the number of free radicals and in the process may reduce the risks of

such diseases. Chemically speaking, antioxidants are reducing agents which donate

electrons and cause a substance to be reduced. An oxidizer is a compound which accepts

electrons and therefore oxidizes another. Antioxidants can also be defined as a substance,

that when present in small amounts compared to the substrate, prevents or greatly

decreases a pro-oxidant initiation oxidation of the substrate (Prior & Cao 1999).

Therefore, antioxidants work to reduce pro-oxidants through a redox reaction.

Antioxidants not only work in vivo, but in vitro as well. Antioxidants play a crucial role

in both enzymatic and non-enzymatic browning reactions and help to prevent lipid

oxidation in foods as well. Dietary antioxidants include vitamins A, C, and E as well as

numerous non-nutritive compounds such as polyphenolics, flavonoids, carotenoids, and

thiol-containing compounds.

Polyphenolics

Polyphenolics are synthesized by numerous plants as secondary metabolites.

Polyphenolic compounds serve as both functional components to the plant as well as the

consumer. For example, some polyphenols serve as pigments (anthocyanins), compounds

to ward off insects and other herbivores such as astringent tannins, and UV light

protectants (Herrmann 1995). These compounds are also important in foods for their

sensory attributes such as color, astringency, and bitterness; as well as their possible

nutritional properties. Polyphenolics are products of 3 major plant metabolic pathways.

Phenolic acids are secondary metabolites of the shikimate pathway; the phenylpropenoid

pathway produces the cinnamic acid derivatives which are precursors of flavonoids and

ligans, and the flavonoidd route' produces the numerous and diverse flavonoid









compounds (De Bruyne et al., 1999). Phenolic compounds consist of a phenol, an

aromatic ring with at least one hydroxyl group attached. This structure relates to

phenolics antioxidant ability, which depends on many factors. Some of these factors

include: 1) location of hydroxyl groups 2) number and arrangement of phenolic subunits

and 3) and the number of hydroxyl groups (Rice-Evans et al. 1995). Phenolic compounds

can be classified into several categories based on their structures. Polyphenols, which

include flavonoids, have at least two phenol groups. The largest polyphenols are the

tannins, which have a molecular weight of 500-3,000 and also have the ability to bind

proteins (Bate-Smith & Swain 1962). Tannins can be classified into two sub- groups, the

hydrolysable and the condensed tannins. Hydrolysable tannins are those readily

hydrolyzed by acids or enzymes into gallic or ellagic acid (Hagerman et al. 1992).

Hydrolysable tannins are commonly found in foods such as guava, grapes, and wine.

Both condensed and hydrolysable tannins have been shown to have antioxidant, enzyme

inhibiting, and antimicrobial properties (De Bruyne et al. 1999). Condensed tannins, also

called "vegetable tannins" or proanthocyanidins, are flavonoid polymers, which can be

degraded in the presence of acid and heat to form either cyanidin or delphinidin and are

relatively stable, compared to the hydrolysable tannins (Hagerman et al. 1992). Common

condensed tannins include polymers of catechin and epicatechin (Figure 2-1) which can

be found in guava, teas, and numerous fruits and vegetables. Several studies have

demonstrated the antioxidant activity of condensed tannins. For example procyanidins B

and B3 have been shown to have a stronger antioxidant activity for linoleic acid then both

ascorbic acid and a-tocopherol (De Bruyne et al., 1999). Condensed tannins have also









been shown to have anti-inflammatory, anti-diarrhoeal, and anti-ulcer activity (De

Bruyne et al. 1999).

The third phenolic group is the phenolic acids that consist of a benzene ring and

least one carboxylic acid group. Examples of phenolic acids are caffeic, chlorogenic, p-

coumaric, gallic, and ellagic acids. Many phenolic acids are linked through ester, ether,

and acetal bonds to either structural components of the plant such as cellulose, proteins,

or lignin; to larger polyphenols (tannins); or to smaller organic molecules such as glucose

(Robbins 2003). This leads to considerable diversity among the various classes of

polyphenolics and therefore inherent difficulty in analysis and identification.

Limited information exists on the topic of guava phenolics, however, previous

studies have shown guava to contain a variety of polyphenolics including flavonols,

phenolics acids, flavan-3-ols, and condensed tannins. (Miean and Mohamed 2001; Misra

and Seshadri 1967 ). Miean and Mohamed (2001) reported guava to contain 550 and 579

mg/kg of dry weight of the flavonols myricetin and apigenin, respectively, while absent

in other major flavonoids such as quercetin, luteolin, and kaempferol. Other phenolics

detected in guava include ellagic and gallic acid and numerous isomers of catechin and

epicatechin (Misra and Seshadri 1967). Kondo and others (2005) reported the amount of

total soluble phenolics in guava skin and flesh, as measured by the Folin-Ciocalteu assay,

to be 915 and 637 [mol/kg FW, respectively, demonstrating that the majority of the

polyphenolics are found the skin. Through HPLC analysis, they also found guava skin to

contain gallic acid (120020[tmol/kg), catechin (45 [mol/kg), epicatechin (16 [mol/kg),

and chlorogenic acid (10 [mol/kg) (Figure 2-1).









Phenolic thermal stability

Phenolic compounds are abundant in fresh and processed fruits and vegetables.

However, current studies report that the thermal stability of these compounds in

processed foods depends on the matrix in which they are found. A study on

OH

HO 0 H 0

OH Cawchin OH
0 ElMgiC add


Figure 2-1. Structures guava polyphenolics: catechin (left) and ellagic acid (right).

tomatoes concluded that thermal processing decreased ascorbic acid levels, but increased

lycopene, total phenolics, flavonoids, and antioxidant content (Dewanto et al. 2002).

Studies on sweet corn and table beets also showed an increase in antioxidants after

thermal processing (Dewanto et al 2002). The sweet corn had an increase in total free

phenolic content and free ferulic acid content after thermal processing. Therefore, since

no published data on guava phenolics related to processing exists, there is a need to

research the thermal processing effects on guava polyphenolics.

Like other phytochemicals, polyphenolic compounds will oxidize and degrade

with exposure to oxygen, heat, and light. The extent of this degradation depends on the

type of polyphenolic as well as the presence of other polyphenolics and antioxidants such

as vitamin C, which may help to stabilize the polyphenolic compounds (Fennema 1996).

Ascorbic Acid

Ascorbic acid, a required nutrient for humans, is one of the most abundant

antioxidants consumed, with fruits being the main source of the nutrient. L-ascorbic acid

is an excellent reducing agent and large quantities may help stabilize phenolics and other









antioxidants during processing by the donation of hydrogen atoms. This reducing ability

is due to its 2,3-enediol moiety (Fennema 1996). Ascorbic acid is often considered an

index of nutrient quality during processing and storage of foods because of this

stabilizing nature (Fennema 1977). Guavas contain approximately 230mg of total

ascorbic acid / 100 g of edible portion of fruit (United States Department of Agriculture

[USDA] nutrient database 2005), five times more than a serving of orange. It is second in

vitamin C content among all fruit, with the acerola cherry containing the most (Uddin et

al. 2002).

Numerous studies exist on the relationship between vitamin C and thermal

processing. Although few studies have been done on the effects of thermal processing on

guava puree, Uddin et al. (2001) studied the effects of degradation of ascorbic acid in

dried guava during storage. The results showed that as storage time and temperature

increased, ascorbic acid content decreased in the dried guava samples (Uddin et al. 2001).

A study conducted on yellow passion fruit (Talcott et al. 2003) demonstrated that vitamin

C fortification preserved more phytochemicals during processing compared to the non-

fortified control. Since guava already contains extremely high levels of vitamin C, the

phytochemicals and antioxidants in the matrix may be heat stable. However, vitamin C

itself is extremely heat and light sensitive and therefore may be greatly decreased during

thermal processing. This has been widely demonstrated in previous studies on various

fruits and vegetables, including tomatoes (Dewanto et al. 2002) and orange juice (Lian et

al. 2000).

The degradation of L-ascorbic acid depends on many factors including heat, light,

metals, and water activity. The degradation of L-ascorbic acid involves its oxidation into









dehydroascorbic acid (DHAA), which is active as vitamin C, but not as an antioxidant,

into degradation products such as 2,3-diketogulonic acid, unsaturated carboxylic acids,

and furfural, among others (Fennema 1996, Brenes 2005). These end products are neither

active as vitamin C nor antioxidants and have been linked to juice quality issues such as

non-enzymatic browning and anthocyanin destruction (Dallas 1996, Brenes 2005).

Carotenoids/Lycopene

Carotenoids are abundant in the red, yellow, orange, and green colored vegetables

and fruits. They are, after chlorophyll, the second most widely occurring plant pigment

found in nature (MacDougall 2002). Carotenoids are tetraterpenes that can be classified

into two major groups including carotenes (hydrocarbons) and xanthophylls (oxygenated

hydrocarbons). Carotenoids may be straight chained, such as lycopene, or contain a 5 or 6

carbon ring on one or both ends, such as P-carotene (MacDougall 2002). The high degree

of hydration and long carbon chain length of these molecules makes them hydrophobic

and therefore fat soluble molecules. The major purpose of carotenoids in the human diet

is to serve as precursors to provitamin A, a required nutrient for humans. In order to serve

this purpose, the carotenoid must contain a B-ionone ring (Fennema 2000). Carotenoids

containing this structure include 3- and a-carotene, and f-cryptoxanthin. Carotenoids

without this structure, such as lycopene, do not possess pro-vitamin A activity yet serve

as dietary antioxidants. As an antioxidant, carotenoids are known to quench singlet

oxygen and protect against cellular oxidative damage (Deshpande et al. 1995). This

ability has linked carotenoid consumption with decreased risks of cancer, cataracts,

atherosclerosis, and the process of aging (von Elbe and Schwartz 1996). A wide variety









of carotenoids have been identified in guava, including phytofluene, P-carotene,

lycopene, cryptoflavin, cryptoxanthin, and lutein (Mercadante et al. 1999).

Lycopene is a fat soluble carotenoid responsible for the red or pink pigment in

several fruits and vegetables such as tomatoes, watermelon, pink grapefruit, and guava.

Structurally, lycopene is a linear, 40 carbon hydrocarbon containing 11 conjugated and 2

non-conjugated double bonds (Rao & Agarwal 1999). Of all the dietary carotenoids,

lycopene has the highest singlet oxygen quenching ability in the body. However, since

lycopene lacks a B-ionone ring it does not provide vitamin A activity (Rao & Agarwal

1999). Lycopene has been associated with decreased risks of cardiovascular disease, as

well as prostate, pancreas, and stomach cancers (Gerster 1997). Several studies

(Gonzalez-Abreu et al. 1985; Mercadante et al. 1999) have identified lycopene in fresh

guava fruit. Although no studies have been published observing processed guava and

lycopene, numerous studies abound on the relationship between thermally processed

tomatoes and lycopene. Many of these report an increase in lycopene concentration in

thermally processed tomatoes compared to fresh tomatoes. Although this may be due to

the breakage of cell walls and loss of water during the thermal treatment, some processed

tomato products have shown an increase in bioavailable lycopene compared to fresh

tomatoes.

This increase in lycopene bioavailability may be due to the isomerization of the

carotenoid from its trans- to cis- form, occurring as a result of the presence of oxygen

and heat during processing. Oxidation of carotenoids occurs as a result of either

autooxidation, which is a spontaneous free radical chain reaction in the presence of

oxygen, or by photooxidation in the presence of light (MacDougall 2002). Overall, the









rate of carotenoid oxidation depends on carotenoid structure, oxygen, temperature, light,

water activity, pH, and the presence of pro- and antioxidants (Chou & Breene 1972).

Oxidation of carotenoids results in the formation of colorless end products such as

compounds with epoxy, hydroxyl, and carbonyl groups (MacDougall 2002), as well as a

variety of cis-isomers, which still retain some color.

Lycopene, as well as other carotenoids occur mostly in their trans form in natural,

unprocessed foods. However, during processing and storage, conversion into the cis form

of one or more double bonds is likely. This isomerization may results in color changes

because the spectral properties of the cis vs. the trans form are different. The addition of

a cis-double bond to an all trans carotenoid results in a hypsochromic shift of 2 to 5 nm

(MacDougall 2002). The cis-double bond also adds an additional peak in the near-ultra

violet absorbance spectrum, which results in a lighter colored hue (MacDougall 2002).

Although the trans-carotenoids and color may decrease during processing and storage,

many studies have shown the cis-carotenoid to have a higher antioxidant capacity (Bohm

2001, Levin et al. 1994). For example, Bohm and others (2001) found that the cis-isomers

of lycopene P-carotene had higher TEAC values than their trans-isomers. Therefore,

identification and quantification of the antioxidant properties of the lycopene isomers in

processed guava puree would provide novel data for this field.

Anthocyanins

Anthocyanins are flavonoids responsible for the blue, purple, and red colors in

many fruits and vegetables. Six main anthocyanidin base structures are found in foods

including pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin with

hundreds of derivatives of these six from various sugar and/or acylated moieties.

Anthocyanin color is extremely unstable and overall stability may be affected by pH,









light, heat, oxygen, iron, copper, tin, ascorbic acid, and copigmentation (MacDougall

2002, Fennema 2000)

In most cases, color stability of an anthocyanin containing food is greatly reduced

by the presence of ascorbic acid. This may occur as the result of hydroperoxide formation

that forms during the oxidation of ascorbic acid (von Elbe and Schwartz 1996).The

hydroperoxides formed then act to degrade the anthocyanin molecule and pigment (von

Elbe and Schwartz 1996). This degradation has shown to me mutual with both

anthocyanins and ascorbic acid decreasing in each others presence. Although the exact

mechanism of this degradation is not known, several have been proposed. Several

researchers have proposed a condensation reaction between the two compounds (Poei-

Langston et al.1981, Garzon et al. 2002). Degradation products of L-ascorbic acid and

dehydroascorbic acid such as 2,3-diketogulonic acid, furfurals such as HMF, and other

aldehydes have been shown to increase anthocyanin destruction (Es-Safi et al. 1999,

2002). Previous studies have shown a decrease of anthocyanins with increasing amounts

of ascorbic acid in many fruits including cranberry, strawberry, grapes, and blood orange

juices (Choi et al. 2001). Therefore, this limits the extent to which anthocyanin

containing products, such as fruit juices, can be fortified with vitamin C. It also may limit

the amount of fruit juice blends that can be made with anthocyanin containing fruits.

The kinetics of anthocyanin stability has also been previously studied with total

anthocyanin concentration over time concluded to follow first order kinetics by many

authors (Brenes et al. 2005, Turker et al. 2004, Garzon et al. 2002). First order kinetics,

including rate of degradation and half life (the time necessary for a 50% reduction in

concentration) are determined by changing concentration into a natural log (In) scale,









over storage time. A high correlation of this data represents first order kinetics. First

order kinetics has also explained anthocyanin + L-ascorbic acid, as well as anthocyanin +

polyphenolic (copigmentation) kinetics (Brenes et al. 2005).

Copigmentation is a weak association between an anthocyanin molecule and other

molecules such as other anthocyanins, metals, or polyphenolic compounds, which results

in color enhancement or greater color stability of the matrix during processing and

storage. Copigmentation usually results in an increase in red color intensity

(hyperchromic shift) and a bathochromic shift from reddish to bluish hues (Gutierrez et

al. 2004). Copigmentation can be demonstrated by comparing different varieties of wines.

For example, Gutierrez et al. 2004 conducted a study on the phenolic and anthocyanin

compositions of the wine varieties Cabernet Sauvignon, Cencibel, and Syrah and found

that Cencibel demonstrated the lowest extent of copigmentation with aging that was

attributed to its low flavanol concentration, compared to the other two varieties. This

shows the effect of intermolecular copigmentation, in which an anthocyanin molecule

forms a copigment with a colorless molecule, usually a polyphenolic compound through

weak hydrophobic forces (Mazza et al. 1990). Although copigmentation would most

likely not occur in an anthocyanin juice/tropical juice blend, the added polyphenolics

such as catechin and epicatechin found in guava juice may help to protect the

anthocyanins during storage. Several studies have shown that anthocyanins will condense

with proanthocyanidins such as (+)-catechin and (-)-epicatechin to form polymeric

compounds which in turn are more stable during storage (Dallas 1996, Bishop 1984).

Anthocyanin stability in fruits such as grapes, strawberries, and acai has been

previously studied in model systems (Del Pozo et al. 2004; Brenes et al. 2005). These









studies have looked at the effects of vitamin C fortification, as well as added substances

in hope to form copigments with the anthocyanins. For example Del Pozo et al. (2004)

studied the effects of vitamin C fortification of acai juice on color stability. They

concluded that the added ascorbic acid decreased the anthocyanin stability in the acai

juice. However, no studies exist on the effects on color stability of adding an anthocyanin

extract to a fruit juice with high vitamin C and polyphenolics content, such as guava. As

demonstrated by the wine example (Gutierrezz 2004), polyphenolics may help to stabilize

anthocyanins in a real juice system.














CHAPTER 3
THE EFFECTS OF THERMAL PROCESSING AND STORAGE ON THE
PHYTOCHEMICALS AND ANTIOXIDANTS IN GUAVA NECTAR

Antioxidant phytochemicals are steadily gaining the interests of American

consumers, expanding markets and increasing sales of fruits, vegetables, botanicals,

dietary supplements, and numerous food and related products containing these

compounds. Likewise, tropical fruits are gaining popularity in the United States due to

their exotic flavors and potentially unique phytonutrient composition. Of these tropicals,

guava has yet to reach the popularity of other tropicals such as banana, mango, and

papaya yet products that contain processed forms of the fruit are gaining in popularity.

Fresh guavas suffer from a relatively short shelf life, which effectually prevent US

imports and limited domestic fruit production. However, due to its diverse phytochemical

composition and unique flavor, guava has the potential for market expansion in the area

of juices, purees, and other processed products.

Investigations into the thermal stability and shelf life properties of antioxidant

phytochemicals present in guava puree are limited despite the fact that thermally

processed purees, juices, and jellies are the most predominant guava-based products in

US markets (Jimenez-Escrig et al. 2001). Many of these products are shelf stable while

others are lightly pasteurized and held refrigerated creating conditions for significant

phytonutrient degradation during storage. Guava is known to contain the second highest

concentration of ascorbic acid among all fruits, with only acerola cherry containing more

on average. Ascorbic acid is well known for its oxidative protection of other









phytochemicals including carotenoids, folic acid, tocopherols, and polyphenolic

compounds and serves to retard oxidation and stabilize phytochemical compounds during

thermal processing and storage (Dewanto et al. 2003, Gregory 2000). The objectives of

this study were to determine the effects of two thermal pasteurization time and

temperature combinations and two storage temperatures on the stability of vitamin C,

lycopene, polyphenolics, and antioxidant capacity of guava puree.

Materials And Methods

Materials And Processing

Mature guava fruit were obtained from Sardinia Farms in Homestead, Florida and

transported to the Food Science and Human Nutrition Department in Gainesville, Florida.

The whole guava fruit were mechanically pressed into puree and immediately frozen at -

20 C. On the day of processing, the puree was thawed under cold running water and

diluted 50% with a 0.5M citric acid buffer at pH 3.8. This dilution served to thin the

puree for ease of pasteurization and to mimic a commercial puree. The nectar was then

divided into two equal portions for processing at 820C for 20 seconds (Process 1) or 87C

for 90 seconds (Process 2) in accordance with commercial processors in Hawaii (Dr.

Alvin Huang, University of Hawaii).

The nectar was pumped by a peristaltic pump (Cole Parmer, Chicago, IL) through a

stainless steel tube into a temperature controlled water bath (Hart Scientific, American

Fork, UT) were it was held at the time and temperatures mentioned above. The juice was

then passed through a cooling tube and chilled to 10 C where it was collected into sterile

glass containers. Following pasteurization, sodium azide (50 mg/L) was added to the

nectars to insure inhibition of microbial growth during storage. The pasteurized samples

were then individually filled in triplicate into 30 ml test tubes and stored at 4 and 250 C in









the dark for 0, 14, 28, and 42 days. A non-pasteurized nectar that also contained sodium

azide was retained and stored until identical conditions as a control for the experiment.

Aliquots of the nectar were removed for extraction of lycopene and the remaining nectar

was treated with 0.1% v/w of pectinase (100,000 AJDU/ml) and incubated at 320 C for 30

minutes. The nectar was then clarified by centrifugation to obtain a clear serum that was

used for the remaining chemical analyses.

Physicochemical Analysis

The clarified nectars were assayed for vitamin C, antioxidant capacity, total

soluble phenolics, individual phenolic acids, and the original nectar for lycopene with all

assays conducted within 48 hrs of their respective processing and storage times.

Vitamin C

Vitamin C was determined in all samples as previously described by Talcott et al.

(2003) by extracting in a 3% aqueous citric acid solution, filtering through a 0.45 .im

Whatman syringe filter, and injecting into a Waters Alliance 2695 HPLC separation unit

(Waters Corp. Miflord, MA) with an isocratic flow at a rate of 1 mL/min with 0.2M

K2H2PO4 as the mobile phase. Separations were made on a Supelcosil LC-18 column

(Supelco, Bellefonte, PA) with detection at 280 nm with a Waters 996 PDA detector.

Vitamin C characterized based on retention time and spectroscopic determinations

against an authentic standard (Sigma Chemical Co., St. Louis, MO).

Total soluble phenolics

Total soluble phenolics were analyzed using the Folin-Ciocalteu metal reduction

assay, previously described by Talcott et al. 2000, using gallic acid as a standard. For

analysis, 100 pL of clarified nectar was reacted with a commercial preparation of 0.25N

Folin-Ciocalteu reagent and after a 3 min reaction was neutralized with IN sodium









carbonate. After 7 minutes, water was added to bring total volume to 10 mL and the

sample mixed using a vortex. Approximately 30 minutes later, the samples and standard

curve was pipetted in to a microtiter plate and the absorbance at 726 nm read using a

Spectra Max 190 spectrophotometer (Molecular devices, Sunnyville, CA). Total soluble

phenolics were reported in mg/L equivalents of gallic acid.

Antioxidant activity

Antioxidant capacity of hydrophilic compounds was determined by the oxygen

radical absorbance capacity (ORAC) assay as previously described by Prior and others

(2001). This assay measures the peroxyl radical scavenging properties of guava

phytochemicals generated by 2,2'-azobis (2-amidinopropane) dihydrochloride in the

presence of fluorescein, the fluorescent probe used as an indicator of oxidation.

Antioxidant capacity was calculated by integrating the area under the fluorescence decay

curve in the presence of guava phytochemicals and calibrated using a standard curve of

Trolox, a water soluble vitamin E analogue with data reported in [iM Trolox

equivalents/mL of guava nectar.

Lycopene

Lycopene was extracted from the initial guava nectar using an equal mixture of

hexane and acetone. Upon extraction, water was added to the solvent mixture to partition

lycopene into the hexane layer, which was collected and filtered for for HPLC analysis.

Lycopene was separated by HPLC using a YMC C30 Carotenoid TM column (250 X 4.6

mm) with detection at 470 nm. Sample concentration was determined from a standard

curve of lycopene calculated based its molar extinction coefficient in hexane.









Statistical Analysis

This study consisted of a 2 x 2 x 4 x 3 full factorial design where the factors

included 2 processing time/temperatures, 2 storage temperatures, and 4 storage times

following pasteurization with analysis for each treatment conducted in triplicate.

Regression analyses, Pearson correlation, and analysis of variance were conducted using

JMP5 software (SAS Institute 2002), with mean separation performed by the least

significant difference (LSD) test (P < 0.05).

Results And Discussion

Effects On L-Ascorbic Acid

Guava is well documented as having one of the highest concentrations of vitamin

C among all fruits, typically containing between 200-300 mg of vitamin C/100 g.

Previous studies on the effects of storage time on freeze dried guava demonstrated an

inverse relationship between vitamin C concentration and storage time and temperature

(Uddin et al. 2002). No other studies exist on thermal pasteurization or storage of guava

nectar. However, L-ascorbic acids low thermal and storage stability has been widely

studied in other fruits. Talcott et al. (2003) found that pasteurization (85C for 30 min) of

passion fruit juice resulted in a 25% loss of L-ascorbic acid and all L-ascorbic acid was

lost after 14 days of storage at 37C. Likewise, this study demonstrated similar results for

changes in L-ascorbic acid over time whereby it decreased as a function of storage

time/temperature and processing conditions (82C for 20 seconds and 890C for 90

seconds).

Both L-ascorbic acid and dehydroascorbic acid are functional as vitamin C in

vivo, yet a goal of this study was to focus on those factors influencing antioxidant

properties and phytochemical retention. It is likely that L-ascorbic acid was initially









converted to dehydroascorbic acid, which has no antioxidant capacity, and then into other

degradation products that have negative implications for nectar quality such as off-colors

and flavors. Immediately after thermal processing, no significant differences in L-

ascorbic acid were observed between Process 1 and Process 2 and the non-pasteurized

nectars (Figure 3-1). This indicated that the conditions of processing did not create an

environment suitable for ascorbic acid oxidation or degradation. However, during storage

ascorbic acid concentration rapidly decreased in response to the pasteurization conditions

temperature of storage. Guava nectar pasteurized with Process 1, lower temperature and

shorter time, retained 26% more ascorbic acid during storage at 40C than Process 2 and

Process 1 nectar stored at 250C retained 35% more L-ascorbic acid than the Process 2

nectar, which lost all L-ascorbic acid after just 14 days. The results of this study suggest

that thermolabile, oxidative or other degradation products formed during thermal

processing that did not originate from ascorbic acid itself affected the stability of the L-

ascorbic acid during storage.

The greatest effect on the retention of ascorbic acid during storage was storage

temperature, irregardless of pasteurization conditions, with nectars at 40C retaining L-

ascorbic acid for a longer period of time than nectars stored at 250C. This effect of

storage temperature can be seen previous studies on citrus fruit. Burdurlu et al. (2005)

studied ascorbic acid degradation in oranges, tangerines, lemons, and grapefruits and

found that ascorbic acid retention after storage at 28, 37, and 45C was about 54.5-83.7%,

23.6-27%, 15.1-20%, respectively. L-Ascorbic acid was completely destroyed in Process

2 after 14 days of storage at 250 C whereas nectars stored at 40 C still contained 43% of










the initial concentration. Likewise complete loss was observed for Process 1 after 28 days

at 25 C but still retained 26% at 40C.

Factors that influence the oxidation of ascorbic acid include pH, oxygen, water

activity, and the presence of certain metal ions such as iron and copper and is generally




350

-*-- Processed: 820C/20 sec, stored: 40C
300 -
-0- Processed: 820C/20 sec, stored: 250C
-V- Processed: 870C/90 sec, stored: 40C
o 250 --- Processed: 870C/90 sec, stored: 250C
o \ unprocessed control
200
0

J 150 -
-J

E 100 -


50 -


0
day 0 day 14 day 28 day 42

Figure 3-1. Effects of processing and storage on L-ascorbic acid. Bars represent the
standard error of the mean, n=3.

exacerbated with light and elevated temperatures (Fennema 2000). Although the guava

nectars were stored in the dark, minimizing their exposure to light, the effect of the

storage temperature differential was significant indicating the major cause for oxidation

of L-ascorbic acid. Another potential factor contributing to L-ascorbic acid degradation

was the presence of oxygen. No steps to control atmospheric oxygen in the headspace or

dissolved in the nectar were made, and likely was causative towards oxidation. Despite

no observable change in L-ascorbic acid following pasteurization, the higher temperature









and longer processing time of Process 2 along with storage at 250C for both processes

created an environment for rapid oxidation of L-ascorbic acid.

This temperature dependence is important since many guava products, such as

nectars, purees, and juice blends are currently shelf stable products and therefore stored at

room temperature. In conclusion, a recommendation to guava processors would be to

optimize pasteurization conditions to avoid excessive heat exposure followed by

refrigerated storage in effort to retain the greatest amount of L-ascorbic acid.

Effects On Lycopene

The main distinguishing characteristic for pink guavas is the presence of the red

carotenoid lycopene. Lycopene concentrations have been well documented in other

commodities such as tomatoes, watermelon, grapefruit, and papaya but guava is an often

over-looked source for this important phytochemical. All trans-lycopene was measured

by HPLC before and after processing with a 40% decrease observed for Process 2, yet no

difference was observed between Process 1 and the unpasteurized control (Figure 3-2).

After the initial loss due to pasteurization, Process 2 did not result in subsequent loss in

lycopene with storage at either 4 or 250C. However, Process 1 exhibited a large decrease

during storage and by Day 14 a 34 and 28% decrease occurred when stored at 4 and 250

C, respectively. Through 28 days of storage, lycopene concentration was mainly a

function of the processing parameters rather than storage temperature and by Day 42

there were no differences among the nectars (Figure 3-2).

Carotenoids, including lycopene are sensitive to oxygen, temperature, light,

peroxides, and storage (Maini and Sudhakar 1994). The appreciable loss of lycopene after

Process 2 may be explained by the conversion of trans-lycopene into cis-lycopene

through heat and oxidation. Cis-lycopene is more easily oxidized than trans-lycopene










into degradation products which in turn do not act as antioxidants (Bohmet al. 2004). Cis-

lycopene isomers may also re-isomerize to trans-lycopene




30

28 Processed: 820C/20 sec, stored: 40C
-0- Processed: 820C/20 sec, stored: 250C
26 --- Processed: 870C/90 sec, stored: 40C
--V- Processed: 870C/90 sec, stored: 250C
24 unprocessed control

22
-j
j 20-

E 18


16 -

14

12
day 0 day 14 day 28 day 42

Figure 3-2. Effects of processing and storage on lycopene. Bars represent the standard
error of the mean, n=3.

throughout storage (Lovric et al. 1970, MacDougall 2002), which may explain the

increase in trans-lycopene observed at Day 28 for Process 2 stored at 40 C. Previous

studies (Giovanelli and Paradiso 2002, Lovric et al. 1970) with tomatoes have also

demonstrated re-isomerization from cis- to trans- during processing and storage. Lovric

et al. concluded that all-trans-lycopene was more stable at 200 C compared to -10, 2 and

37 C and attributed this to the fact that re-isomerization is favored at this temperature.

Lycopene stability in products such as guava nectar is also a function of light, water

activity, oxygen, pH, temperature, and the presence of pro-oxidants or antioxidants (Chou

& Breene 1972; Minguez-Mosavera 1995; Yanislieva et al.1998). Among the









antioxidants available in guava, ascorbic acid, despite the disparity in polarity, is known

to stabilize lycopene (Mortensen et al. 2001). Although this protecting effect was not

observed during pasteurization, it may help explain the stability of lycopene during

storage.

Effects On Polyphenolics

Total soluble phenolics were measured by the Folin-Ciocalteu assay, which

measures the ability of phytochemical compounds to reduce an oxidized metal ion.

Although the target compounds for this assay were polyphenolics, compounds such as

ascorbic acid, certain soluble proteins, melonoidins, and reducing sugars give a

measurable interference in the assay. Results of the assay revealed that following

pasteurization with both conditions the total soluble phenolics significantly increased

from 806 mg/L gallic acid equivalents (GAE) to 1046 mg/L (30% increase) and 1108

mg/L (37% increase) for Process 1 and 2, respectively (Figure 3-3). These increases

following thermal processing may be explained by several factors, including the

conditions of the assay itself. Due to the broad range of compounds detected by the assay,

changes in these compounds following pasteurization can easily influence the ability to

reduce metal ions in solution. Additionally the use of guava puree to create the nectar

likely contained intact plant cells whereby the conditions of heating helped to release

cell-wall or vacuole-bound compounds with metal reducing capabilities. Any of these

compounds present or their effect on the assay following processing could account for the

increase. Dewanto et al. (2002) observed similar results in sweet corn. In this study, total

soluble phenolics in corn, as measured by the Folin-Ciocalteu assay increased 24, 32, and

36 % when heated at 1150C for 10, 25, and 50 minutes, respectively.









Additional evidence for the influence of heat on the breakdown or release of

cellular constituents was obtained by adding pectinase to 100% guava puree and

incubating at 350 C for 45 minutes and centrifuged to obtain a clear serum (Figure 3-4).

The residual solids were then extracted a second time with water, centrifuged, and a

second serum obtained. This process was repeated 5 times with the final sample placed in

boiling water for 5 min to determine the release of cellular-bound compounds with metal

reducing capabilities. Results indicated that significant amounts of compounds with metal

reducing capabilities persisted with a 35% increase observed after heating. Results may

indicate that increased total soluble phenolics after processing may be related to the

rupturing of cells and resulted in better extractability. Findings also indicate that that the

use of clarified guava is likely underestimating the total concentration of phytochemicals

present. It may be necessary in the future to explore other extraction techniques such as

alcoholic solvents to insure complete phytochemical solubilization.

During storage, the residual concentration of total soluble phenolics was mainly a

function of storage temperature, whereas nectars stored at 40 C retained higher

concentrations than samples stored at 250 C for both pasteurization parameters and each

decreased continuously at a similar rate over time. Throughout the 42 days of storage at

4 C, there were no differences between the samples processed under the two different

conditions, whereas only small differences were observed for nectars stored at 250 C. For

Process 1 and Process 2 samples stored at 250 C there was a 28% and 17% decrease in

total soluble phenolics after 42 days, respectively.

Effects On Antioxidant Activity

Antioxidants are important in foods in terms of their potential health benefits as

well as their radical scavenging abilities, which can prevent oxidation in foods.










Therefore, it is important to have an overall measurement of the antioxidant ability of

fruits and vegetables, which are the leading source of antioxidants in the American diet.

Many antioxidant capacity assays are currently used in the food industry,



2000

10 -*- Processed: 820C/20 sec, stored: 4C
1800 -0- Processed: 820C/20 sec, stored: 250 C
o -- Processed: 870C/90 sec, stored: 4C
S1600 -V- Processed: 870C/90 sec, stored: 250 C
unprocessed control

o 1400
t-
w 1200
0
w 1000
0
J 800
E
600
day 0 day 14 day 28 day 42

Figure 3-3. Effects of processing and storage on total soluble phenolics. Bars represent
the standard error of the mean, n=3.

including the total radical trapping antioxidant parameter (TRAP), Trolox equivalence

antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), and the

oxygen radical absorbance capacity (ORAC) method (Huang et al. 2005).

Antioxidant activity was measured in this study, using the ORAC assay for

hydrophilic antioxidants, which excludes compounds such as lycopene, tocopherol, or

other carotenoids that may be present in the nectars. However, a study on the antioxidant

capacity of lycopene found low radical scavenging activities for lycopene (Bangalore et

al. 2005), therefore the hydrophilic compounds likely represented the majority of

antioxidant compounds in guava such as ascorbic acid and polyphenolics.












100

S I Total Soluble Phenolics
80


S60-


40 -


20 -
20


0 --- -- -- -- -- -- -- -- -- ----
J2 J3 J4 J5 J6

Figure 3-4. Percent initial of total soluble phenolics after water extraction and dilutions.
J2: 100% guava juice, J3: puree from J2, diluted 1:1 with water, J4: puree
from J3, diluted 1:1 with water, J5: puree from J4, diluted 1:1 with water, J6:
puree from J4, heated for 1 minute at 100C. Bars represent the standard error
of the mean, n=3.

Antioxidant capacity of the guava nectars was unchanged immediately after

pasteurization in all samples when compared to the unpasteurized control (Figure 3-5).

Overall, loss of antioxidant capacity was a function of storage temperature rather than

processing conditions, with nectars stored at 4 oC retaining a higher antioxidant capacity

than nectars stored at 25 o C as previously observed for ascorbic acid and total soluble

phenolics. For those nectars stored at 25 o C, there was a significant decrease (22%) in

antioxidant capacity after 14 days, while the samples stored at 4 o C remained unchanged

throughout the 42 day study. Nectars pasteurized with Process 1 and 2 and stored at 25 o

C lost 47 and 39% of the antioxidant capacity after 42 days, respectively and followed a

similar decrease over time.










Table 3-1 shows correlations between ORAC and L-ascorbic acid, total soluble

phenolics, and lycopene. Antioxidant capacity in guava puree was attributed mainly to

the hydrophilic antioxidants L-ascorbic acid and the various polyphenolic compounds.

Higher correlations were seen between ORAC vs. L-ascorbic acid, followed by total

soluble phenolics, while ORAC vs. lycopene was poorly correlated. Overall, ORAC was

better correlated to all phytochemicals during 25C storage, due to increased stability of

and fluctuations observed in the samples stored at 4C. Leong and Shui (2002)

contributed 50% of guava's antioxidant activity to ascorbic acid, which is similar to the

results of this study.






-- Processed: 820 C/20 sec, stored: 40 C
-0-- Processed: 820 C/20 sec, stored: 250 C
10 -
E --- Processed: 870C/90 sec, stored: 40 C
u -v-- Processed: 870C/90 sec, stored: 250 C
S, unprocessed control


=.



8 6
x




4

0 10 20 30 40


Figure 3-5. Effects of processing and storage on antioxidant activity. Bars represent the
standard error of the mean, n=3.










Table 3-1. Correlations (R2) of ORAC vs. L-ascorbic acid, total soluble phenolics, and
lycopene during storage for 28 days at 4 and 25C
Total soluble
Sample Set L-ascorbic acid phenolics Lycopene
Process 1, 40 C 0.26 0.22 0.20
Process 1,250 C 0.73 0.40 0.45
Process 2, 40 C 0.18 0.16 0.01
Process 2, 250 C 0.45 0.49 0.00


Conclusions

Overall, the stability of antioxidants in guava nectar was a function of storage

temperature and time with the conditions of processing a less important factor following

pasteurization. Storage temperature was the major factor in stability of L-ascorbic acid,

total soluble phenolics, and total antioxidant capacity, while processing time/temperature

was the major factor in stability for lycopene. Guava nectar can be thermally processed

and stored without affecting the majority of its antioxidants as long as strict temperature

control is maintained during storage. All L-ascorbic acid was lost and total antioxidant

capacity was significantly decreased while polyphenolic compounds were the most stable

compounds during processing and storage for 42 days at both 4 and 25 C. A nitrogen

purge may be a sufficient way to increase the stability of L-ascorbic acid and therefore

total antioxidant capacity in guava puree.














CHAPTER 4
THE EFFECTS OF ANTHOCYANIN-CONTAINING JUICE BLENDS ON THE
PHYSIOCHEMICAL AND ANTIOXIDANT CAPACITY OF GUAVA JUICE

Introduction

Due to limited availability of fresh guava, most fruit destined for US markets are

processed into juice, puree, jams, jellies, and syrup (Jimenez-Escrig and others 2001),

with a majority utilized in juice blends. Numerous fruit blends that incorporate guava and

other tropical fruits exist in retail markets, but few of these blends contain anthocyanin-

containing juices and may even be colored with artificial colorants to give an impression

of red or pink colors. Anthocyanins are polyphenolic compounds that give fruit their

blue, purple, and red colors and contribute to the health promoting properties and the

appearance of the fruit and juice. Anthocyanins are commonly extracted from natural

sources such as grapes, red radish, black carrots, purple sweet potatoes, and red cabbage

and may be added to juices, yogurts, and ice cream among other products to add

nutritional benefits and as a natural color substitute for artificial pigments. However,

issues surrounding natural color fortification or blending juices containing anthocyanins

is their inherent instability during processing and storage. Anthocyanin stability is

dependent on pH, temperature, light, and oxygen which tend to adversely impact

retention as well as the presence of other polyphenolic compounds that act as cofactors

and serve to enhance overall retention. A diversity of polyphenolic compounds such as

catechin and rosmarinic acid were shown to serve as cofactors to anthocyanins which

help to increase their stability. Anthocyanins are also affected by the presence of ascorbic









acid which has shown to degrade anthocyanins in a mutually destructive process in fruit

juices such as grapes, strawberries, and acai (Choi et al 2001, Del Pozo et al. 2004;

Brenes et al. 2005). Despite the high concentration of naturally occurring polyphenolics

present in guava that may serve as cofactors to anthocyanins, the unusually high

concentration of ascorbic acid present in guava may be a primary reason why

anthocyanin concentrates and juices naturally containing anthocyanins are not commonly

blended with guava juice.

Two cyanidin based anthocyanin sources, acai and black current, were chosen for

this study. Cyanidin-based anthocyanin pigments were chosen due to their abundance in

natural products including blackberry, elderberry, black carrot, sweet potato, red cabbage,

black currant, and acai (Malien-Aubert et al. 2000, Stintzing et al. 2002, Del Pozo et al.

2004). Cyanidin 3-glucoside, which is the predominant anthocyanin in black currant and

agai (Del Pozo et al. 2004, Nielsen et al. 2003) is the most commonly occurring

anthocyanin in nature (Mazza et al. 1993). Acai and black currant juices vary mostly in

their non-anthocyanin polyphenolic composition. Acai pulp has been previously reported

to contain predominantly ferulic acid, epicatechin, p-hydroxy benzoicv c acid, gallic acid,

protocatechuic acid, and (+)-catechin, (Del Pozo et al. 2004) while black currant has been

reported to contain predominantly m-coumaric acid, p-courmaric acid, salicylic acid,

ferulic acid, and caffeic acid (Zademowski 2005).

Preliminary studies were performed on acai juice and C18 isolates under

accelerated conditions (37C). Total anthocyanins were measured in samples every 30

minutes for a total of 4 hours and results indicated acai juice anthocyanins to be more

stable than acai C18 bound anthocyanins in the presence of L-ascorbic acid. This









suggested that an unknown compound or compounds in the acai juice matrix has a

protecting effect on the anthocyanins and therefore was the justification for further

investigations of the difference between the stability of anthocyanin containing juices and

their C18 bound isolates. This study will also answer the question to whether clarified

guava juice, which is naturally yellow in color, can be color fortified with natural

anthocyanin pigments for better marketability, and if clarified guava juice, which is often

used in juice blends, can be blended with anthocyanin containing juice, despite the high

ascorbic acid content.

This study was designed to explore the effects of anthocyanin-containing juices

(black current and acai) and anthocyanin concentrates blended with clarified guava juice,

which is high in both ascorbic acid and polyphenolic compounds. The juices and

anthocyanin isolates were mixed into a pH buffered solution and evaluated with and

without the addition of ascorbic acid. Juices were pasteurized and stored at 6 and 250 C

for a total of 4 weeks to determine degradation kinetics of the antioxidant and

phytochemical constituents.

Materials And Methods

Materials And Processing

Clarified guava juice was prepared as previously described in Chapter 3 and

adjusted to pH 3.5 with citric acid. Looza brand black currant juice (30% juice, 150

Brix) was purchased from a local market and adjusted to pH 3.5. Frozen acai puree was

obtained from Bolthouse Farms (Bakersfield, CA) and clarified by centrifugation and

filtered though a bed of diatomaceous earth. The resultant juice was adjusted to 150 Brix

with the addition of sucrose and diluted to 30% juice using pH 3.5 buffer. Anthocyanin

isolates were prepared from the black current and acai juices by partitioning from









Waters C-18 5g Sep Pak Vac cartridges, collecting anthocyanins and non-anthocyanin

polyphenolics with acidified methanol. The acidified methanol was then evaporated and

re-dissolved in pH 3.5 citric acid buffer. Isolation in this manner removed sugars, organic

acids, and other small, polar molecules leaving behind a C18 bound isolate.

Juices and reconstituted C18 isolates were blended 1:1 with guava juice with

respective controls processed by blending each treatment 1:1 with pH 3.5 buffer, with

and without L-ascorbic acid fortified at the concentration present in the diluted guava

juice (400 mg/L). The juices were placed into 50 mL glass containers and pasteurized in a

hot water bath to 80 C then immersed in an ice water bath until the samples reached

250C. The juices were then placed into labeled test tubes, purged with nitrogen, capped

and stored at either 6 or 250C in the dark. Pre and post pasteurized juices and controls

were immediately analyzed for ascorbic acid, antioxidant capacity, and total anthocyanins

as previously described in Chapter 3 while analysis samples for individual polyphenolics

by HPLC were stored at -20 C until time for analysis (<30 days). Total anthocyanins, L-

ascorbic acid, antioxidant activity, and polyphenolics will be reported as percent of initial

concentration (Day 0, post-pasteurization).

Chemical Analysis

L-ascorbic acid

L-ascorbic acid was assayed as described in Chapter 3.

Phenolics by HPLC

Predominant flavonoids and phenolic acids were analyzed by HPLC according to

conditions of Talcott et al. (2002), using a Waters 2695 Alliance HPLC system with a

Waters Spherisorb 5 [m ODS2 column (250 X 4.6 mm) with detection at 280 nm using

a Waters 996 PDA detector. Compounds were characterized or identified by









spectroscopic interpretation, retention time, and comparison to authentic standards

(Sigma Chemical Co., St. Louis, MO).

Total anthocyanins

Total anthocyanins were measured using a pH shift method as previously

described by Talcott et al. 2002. Samples were diluted 10x with a hydrochloric acid

buffer at pH 1.0. An aliquot of the buffer was pipetted into a 96 well microplate and

absorbance read using a Spectra Max 190 spectrophotometer at 520 nm with absorbance

values adjusted to a 1 cm light path. An extinction coefficient of 29,600 was used to

quantify total anthocyanins in mg/L equivalents of cyanidin 3-glucoside.

Percent monomeric and polymeric anthocyaninins

The proportion of monomeric/polymeric anthocyanins was determined as

previously described by Brenes et al. 2005, based on color retention at 520nm at pH 3.5

in the presence of sodium bisulfite, which bleaches monomeric anthocyanins with

remaining color attributable to polymeric forms. Samples were read against a blank that

consisted of sample diluted with buffer at pH 3.5 and read on a Spectra Max 190

spectrophotometer.

Statistical Analysis

This study consisted of a 4 x 2 x 5 x 3 full factorial design. The factors studied

were 4 guava juice blends, 2 storage temperatures, and 4 storage times, with each sample

run in triplicate. Regression analyses, Pearson correlation coefficients, and analysis of

variance were conducted using JMP software Version 5 (SAS Institute 2002), with mean

separation performed by the least significant difference (LSD) test (P < 0.05).









Results And Discussion

Effects On Anthocyanins

Guava juice blends

Pasteurization had a small, but statistically significant effect on total anthocyanin

concentration in all guava juice blends. The acai juice + guava juice anthocyanins were

the most stable during pasteurization with only a 4% decrease in their total anthocyanins

(Figure 4-1). The other guava juice blends, black currant juice + guava, black currant C18

+ guava, and acai C18 + guava showed no differences from each other due to processing

with an average decrease of 6% of their total anthocyanins Figure 4-1 and 4-2).

Pasteurization also increased the concentration of polymeric anthocyanins in the juice

blends. When comparing the unpasteurized juices, all juice blends containing guava juice

had slightly higher polymeric anthocyanins when compared to the juices without guava

juice, with acai C18 + guava juice containing the highest amount of polymeric

anthocyanins before pasteurization (71.2%). The high proportion of polymeric

anthocyanins in all of the C18 bound juices most likely resulted from the extraction

process which involved slight heating (< 40 C) during evaporation. The guava juice

blend that started with the least amount of polymeric anthocyanins was black currant

juice + guava at 39.9%. Pasteurization significantly increased the polymeric anthocyanins

6% in acai juice while having no significant effect on acai C18 + guava juice blends.

Pasteurization also significantly increased the polymeric anthocyanins by 8% in both

black currant juice and C18 blends. This again demonstrates the stability of the acai C18

+ guava juice blend.

Storage temperature, anthocyanin source, and the anthocyanin-based food matrix

all attributed differences in total anthocyanin concentration over a total storage period of
















100



40 -
80

0

< 60
--


40

control 1 7 14 21 28
Days

Acai Juice + Buffer, 25 OC Storage
~ Acai Juice + Buffer + AA, 25 OC Storage
Acai C18 + Buffer, 25 OC Storage
~ Acai C18 + Buffer + AA, 25 OC Storage
Acai Juice + Guava Juice, 25 OC Storage
Acai C18 + Guava Juice, 25 OC Storage



Figure 4-1. Percent initial of total anthocyanins stored at 25 o C for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3.

28 days. When comparing only the juice blends with added guava juice, all were

significantly different in their total anthocyanin concentration at 28 days of storage at

both 6 and 25 o C except for acai C18 + guava juice and acai juice + guava juice which

showed no differences between each other at 6 C storage after 28 days. The juices in

order of decreasing anthocyanin retention at 6 oC storage were acai C18 + guava juice

(83%), acai juice + guava juice (80%), black currant juice + guava juice (72%), and the

least stable being black currant C18 + guava juice (64%). The juices stored at 25 0 C in

order of decreasing percent of initial of total anthocyanin concentration were acai C18 +

guava juice (74%), acai juice + guava juice (47%), black currant C18 + guava juice













120


S110

0
100

E
U) 90
C--

o 80
e--


70
I-

60 -
control 1 7 14 21 28

Days

--- Acai Juice + Buffer, 60 C Storage
-0- Acai Juice + Buffer + AA, 60 C Storage
-v- Acai C18 + Buffer, 60 C Storage
-v- Acai C18 + Buffer + AA, 60 C Storage
-U- Acai Juice + Guava, 60 C Storage
--- Acai C18 + Guava, 60 C Storage


Figure 4-2. Percent initial of total anthocyanins stored at 60C for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3.

(42%), and black currant juice + guava juice (39%). This demonstrates a large storage

temperature dependence, with those stored at 40C retaining significantly more total

anthocyanins than those stored at 250C. These results also indicate that acai anthocyanins

were are more stable in the presence of guava than black currant anthocyanins.

The proportion of monomeric anthocyanins decreased over time in each juice


treatment and as a result of storage temperature. Turker et al. (2004) observed a similar

trend in percent monomeric/polymeric anthocyanins during storage time in a fermented

black carrot beverage, showing that the % monomeric anthocyanins decreasedl7.8, 49.2,













120



c 100 -









80 --------------------------
4--
80

0-
80

0
o 60



o 40
I-



20 ,
control 1 Zays 14 21 28

--- Black Currant Juice + Buffer, 25 C Storage
--- Black Currant Juice + Buffer + AA, 25 OC Storage
--- Black Currant C18 + Buffer, 25 OC Storage
-V- Black Currant C18 + Buffer + AA, 25 OC Storage
--- Black Currant Juice + Guava Juice, 25 OC Storage
-- Black Currant C18 + Guava Juice, 25 OC Storage



Figure 4-3. Percent initial of total anthocyanins stored at 25oC for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3.

and 93.3% in the carrot beverages stored for 90 days at 4, 25, and 40 C, respectively,


with a respective increase in % polymeric for the carrot beverages over the 90 day period.


An increase in polymeric anthocyanins has been extensively investigated for red wine


aging, where polymeric anthocyanins are a desired quality aspect (Vidal et al. 2002,

Arnous et al. 2001). Likewise, storage studies with blood orange juice found that


anthocyanins could polyermize with hydroxycinnamic acids to form larger


pyranoanthocyanins (Hillebrand et al. 2004) similar to a model system where malvidin


3,5-diglucoside condensed with catechin to form a colorless condensation product







41





120


S110


S100

-J
90 -
E
U)
C 80-

0
7 70


o 60 -
F--

50 .
control 1 7 14 21 28

Days

--- Black Currant Juice + Buffer, 6 C Storage
-0- Black Currant Juice + Buffer + AA, 6 OC Storage
--- Black Currant C18 + Buffer, 6 C Storage
-v- Black Currant C18 + Buffer + AA, 6 C Storage
--- Black Currant Juice + Guava Juice, 6 C Storage
-D- Black Currant C18 + Guava Juice, 6 C Storage


Figure 4-4. Percent initial of total anthocyanins stored at 6oC for 28 days. Control is the
unpasteurized juice. Bars represent standard error of the mean, n=3.

(Bishop and Nagel 1984). After 28 days of storage at 6 oC Acai C18 + Guava and Acai

C18 + Buffer + L-Ascorbic Acid had the highest polymeric anthocyanins within the juice

blends stored at this temperature, at 77.8 and 76.2 % polymeric anthocyanins,

respectively. This is a slight increase from the pre-processed control, which started with

71% polymeric. The Black Currant C18 + Guava had the second highest amount of

polymeric anthocyanins out of all guava containing samples (53.6% compared to its L-

ascorbic acid control at 45.5%). Black Currant Juice + Guava had the lowest amount of

polymeric anthocyanins compared to the other guava juice blends, and consisted of 41.5

% after 28 days at 4 o C. At 25 C storage, the Acai C18 + Guava and the Black Currant









C18 + Guava had the most polymeric anthocyanins (83.8 and 79.7 % respectively.

Similarly, the Acai Juice + Guava and Black Currant Juice + Guava were not different

from each other (68.0 and 69.2 % respectively). These results show a strong indication

that the concentration of polymeric anthocyanins were instrumental in preventing the loss

of total anthocyanins, which is based on a colorimetric assay. Since consumers tend to

make purchasing decisions based on visual perception of anthocyanin-based color, this

stability is important in consumers' perception of fruit juices. Anthocyanins, as found in

fresh fruit and juices are in their monomeric form. Over storage time, the monomeric

anthocyanins in fruit juices may undergo a condensation reaction to form polymeric

pigments as previously discussed (Bishop and Nagel 1984, Hillebrand et al. 2004, Wang

2003). Several mechanisms have been theorized for this condensation, reaction which

include: a reaction between proanthocyanidin and anthocyanin, which results in colorless

or orange xanthylium salts, or an acetaldehyde, glyoxylic acid, furfural, or HMF related

condensation between anthocyanin and proanthocyanidin resulting in the formation of

purple pigments (Bishop et al 1984, Dallas et al. 1996, Es-Safi et al. 2000). Despite the

mechanism of reaction, polymeric anthocyanins are more stable than their monomeric

counterparts. This may explain the high stability of the acai C18 samples, which started

with the highest degree of anthocyanin polymerization.

Synthetic L-ascorbic acid models

Pasteurization also had a small, but statistically significant effect on total

anthocyanin concentration in the model juices containing L-ascorbic acid, resulting in a

decrease in total anthocyanins in all juices. Acai juice and C18 anthocyanins decreased 8

and 4%, respectively while black currant juice and C18 anthocyanins decreased 5 and

9%, respectively. Synthetic ascorbic acid juice models had a lower increase of polymeric









anthocyanins overall than the guava juice blends, suggesting that guava polyphenolics

may be polymerizing with the anthocyanins while in the juice matrix. Also, the

concentration of polymeric anthocyanins in the juice matrix was higher than those in the

C18 isolate, which may be attributed to the higher concentration of HMF in the juice

system. As previously explained, one suggested mechanism for the formation of

polymeric anthocyanins is a condensation of non-anthocyanin polyphenolics, such as

condensed tannins (catechin and epicatechin) with the anthocyanin pigments in the

presence of HMF. The polymeric anthocyanins in the Acai Juice + L-Ascorbic Acid

blend increased 3% post pasteurization; compared to the Acai Juice + Guava blend which

increased 6%. The polymeric anthocyanins in Acai C18 + L-Ascorbic Acid did not

significantly change, as in the Acai C18 + Guava blend. The polymeric anthocyanins in

the Black Currant Juice + L-Ascorbic Acid increased 3% compared to the Black Currant

Juice + Guava which increased 8%, while the Black Currant C18 + L-Ascorbic Acid

increased 10%, which was not significantly different than the 8% increase in the guava

blend.

In order to determine if the guava matrix had an effect on the anthocyanin

containing juices during storage, juices consisting of anthocyanin source, buffer, and L-

ascorbic acid were compared to those juices containing the anthocyanin source and guava

juice. Guava juice showed a protecting effect on several of the juice blends, which is

attributed to the guava polyphenolics forming more stable polymeric compounds with the

anthocyanins. Acai C18 + Guava and Black Currant C18 + Guava, compared to their

counter parts with equal amounts of L-ascorbic acid however lacking the guava juice, had















Unprocessed Control
I Post-Processing, Day 0
Day 7 at 250 C Storage
I Day 28 at 250 C Storage















Acai Juice Acai Juice Acai Juice AcaiC18 Acai C18
+ Buffer + Buffer + AA + Guava + Buffer + Buffer + AA


Acai C18
+ Guava


Figure 4-5. Percent polymeric anthocyanins of acai based samples stored at 250C. Bars
represent standard error of the mean, n=3.


Acai Juice Acai Juice + AcaiJuice AcaiC18 Acai C18+ AcaiC18
+ Buffer Buffer + AA + Guava + Buffer Buffer + AA + Guava


Figure 4-6. Percent polymeric anthocyanins of acai based samples stored at 60C. Bars
represent standard error of the mean, n=3.
































20 I I l II I
Black Currant Black Currant Black Currant Black Currant Black Currant Black Currant
Juice + Buffer Juice + Buffer Juice + Guava C18 + Buffer C18 + Buffer C18 + Guava
+AA +AA


Figure 4-7. Percent polymeric anthocyanins of black currant based samples stored at
25C. Bars represent standard error of the mean, n=3.


Black Currant Black Currant Black Currant Black Currant Black Currant Black Currant
Juice + Buffer Juice + Buffer Juice + Guava C18 + Buffer C18 + Buffer C18 + Guava
+AA +AA


Figure 4-8. Percent polymeric anthocyanins of black currant based samples stored at 60C.
Bars represent standard error of the mean, n=3.









a greater percent of initial total anthocyanins after 28 days at 4 o C of storage. The Black

Currant Juice + Guava however, had a lower final concentration compared to the black

currant juice fortified synthetically, while the Acai Juice + Guava showed no difference

compared to the acai juice fortified synthetically. This demonstrated that the C18 fraction

was more stable then their respective juices and that these C18 isolates, together with

guava juice, may be more stable as well. This aspect will be further discussed in the

effects on polyphenolics section.

Polymeric anthocyanins significantly increased in all synthetically fortified

ascorbic acid blends. However, after 28 days of storage at both 6 and 25C, guava juice

blends had a greater concentration of polymeric anthocyanins than the synthetically

fortified L-ascorbic acid blends. After 28 days of storage at 250C storage, Acai Juice + L-

Ascorbic Acid had 72% polymeric anthocyanins (9% less than Acai Juice + Guava), Acai

C18 + L-Ascorbic Acid had 91% polymeric anthocyanins (no significant difference than

Agai C18 + Guava), Black Currant Juice + L-Ascorbic Acid had 75% polymeric

anthocyanins (8% less than Black Currant Juice + Guava) and Black Currant C18 + L-

Ascorbic Acid had 86% polymeric anthocyanins (4% less than Black Currant C18 +

Guava). After 28 days at 60C storage, Acai Juice + L-Ascorbic Acid had 49% polymeric

anthocyanins (6% less than Acai Juice + Guava), Acai C18 + L-Ascorbic Acid had 78%

polymeric anthocyanins (no significant difference than Acai C18 + Guava), Black

Currant Juice + L-Ascorbic Acid had 46% polymeric anthocyanins (9% less than Black

Currant Juice + Guava), and Black Currant C18 + L-Ascorbic Acid had 67% polymeric

anthocyanins (no significant difference than Black Currant C18 + Guava).









Anthocyanin controls

For those samples lacking L-ascorbic acid, only the black currant juice and C18

bound black currant juice decreased (5 and 7 %, respectively) as a result of thermal

processing. The acai juice and C18 bound acai juice without added L-ascorbic acid were

the most stable during thermal processing. Under similar processing conditions, Brenes et

al. (2005) reported a 12% decrease in total anthocyanins in a grape juice (Vitis vinifera)

model system with and without added ascorbic acid, which is similar to the results of this

study. Overall, the effect of pasteurization on total anthocyanin concentration was

minimal (<5 or 7%) in all samples. Pasteurization had no significant effect polymeric

anthocyanins on all controls expect black currant juice + buffer, which increased 5%.

This demonstrates the stability of the anthocyanins in a system lacking ascorbic acid.

As expected, anthocyanin/buffer control model juice anthocyanins were the most

stable throughout the 28 days of storage. At 25C storage, Acai Juice + Buffer and Acai

C18 + Buffer retained 76 and 90% of their total anthocyanins, respectively. Black

Currant Juice + Buffer and Black Currant C18 + Buffer retained 60 and 64% of their total

anthocyanins, respectively. At 6C storage, Acai Juice + Buffer and Acai C18 + Buffer

retained 93 and 94%, respectively and Black Currant Juice + Buffer and Black Currant

C18 + Buffer retained 88 and 85%, respectively. This demonstrates an anthocyanin

source (acai versus black currant) dependence and for the acai model juices, and

anthocyanin matrix dependence (juice versus C18 isolate). The anthocyanin/buffer

controls also retained the most monomeric anthocyanins, and therefore had the least

amount of polymeric anthocyanins, after 28 days of storage, containing in the range of

54-70% polymeric anthocyanins when stored at 250C and 42-65% when stored at 60C.









Anthocyanin Degradation Kinetics

Regression analysis was performed on the total anthocyanin concentration in a

natural log (In) scale versus time in order to determine the appropriate kinetics model.

The total anthocyanin stability during storage followed first order kinetics as previously

concluded by several other researchers (Brenes et al. 2005, Turker et al. 2004, Garzon et

al. 2002). From first order kinetic calculations, the half life was determined for total

anthocyanins in the guava juice containing samples. Storage temperature had the greatest

effect on anthocyanin half life, with the samples stored at 6 C having significantly

greater half lives than those stored at 25 C for 28 days (Table 4-1.)


Table 4-1. Effects of storage temperature and L-ascorbic acid on first order regression,
rate, and half life of total anthocyanins. Different letters represent differences
between sample types with or without L-ascorbic acid.
6 C Storage 250 C Storage
Sample R2 k t1/2 R2 k t1/2
Acai Juice + Buffer + AA 0.91 -0.0063 112.6 a,b 0.96 -0.0291 23.85 c
Acai Juice + Guava 0.89 -0.0074 97.73 a,b 0.98 -0.0271 25.58 b,c
Acai C18 + Buffer + AA 0.82 -0.0097 71.99 b 0.92 -0.0226 30.65 b
Acai C18 + Guava 0.76 -0.0055 131.9 a 0.76 -0.0111 62.78 a
Black Currant Juice + Buffer + AA 0.68 -0.0068 110.2 a 0.98 -0.0352 19.69 b.c
Black Currant Juice + Guava 0.85 -0.0107 65.48 b 0.87 -0.0342 21.36 a,b
Black Currant C18 + Buffer + AA 0.89 -0.0178 39.43 b 0.92 -0.0374 18.55 c
Black Currant C18 + Guava 0.93 -0.0148 46.98 b 0.82 -0.0298 23.38 a


The influence of storage temperature on anthocyanin stability was generally in

agreement with previously published data on the subject (Garzon et al. 2002). The juices

lacking L-ascorbic acid however, showed high stability of total anthocyanins during the

28 day storage period, especially those held at 6 C, and therefore kinetics were not run

due to the lack of fit of kinetic models (zero, first, or second order).

ANOVA analyses on total anthocyanin half lives were performed on sample sets

consisting of anthocyanin juice/C 18 + guava, anthocyanin juice/C 18 + buffer, and









anthocyanin juice/C18 + buffer + L-ascorbic acid. Within the acai set stored at 6 oC, Acai

C18 + Guava had the highest half life, 131.9 days (P<0.05), which was significantly

greater than its L-ascorbic acid control, which had a half life of 72 days. However, the

agai C18 samples showed no differences at 6 C when compared to the acai juice

samples. The Acai Juice + Guava and Acai Juice + L-Ascorbic Acid also showed no

statistical differences between each other. The acai samples stored at 25 C showed

similar results, with the Acai C18 + Guava again having a greater half life (62.8 days)

compared to the others, with its L-ascorbic acid control having a half life of 30.7 days.

The half lives of the Acai Juice + L-Ascorbic Acid and the Acai Juice + Guava samples

were 23.9 and 25.6 days, respectively, which were not statistically different from one

another. Therefore, when comparing acai samples, the acai C18 samples had the highest

half life and the guava juice had a protecting effect on the acai C18 samples. Black

currant samples did not show this protecting effect. When comparing the black currant

samples stored at 6 C, Black Currant Juice + L-Ascorbic Acid at the greatest half life

(110 days compared to the juice + guava sample at 65 days) and was significantly greater

than all black currant juice blends stored at 6 C The black currant samples with guava

juice demonstrated slightly greater stability than the black currant juice synthetically

fortified with L-ascorbic acid when stored at 25 C with Black Currant C18 + Guava

having the greatest half life of 23 days. The Black Currant Juice + Guava half life was not

different than the black currant C18 + guava juices nor the Black Currant Juice + L-

Ascorbic Acid juices. These results again indicate that storage temperature plays the

greatest role in the protection of the guava juice blend stability.









Effect On Naturally Occurring And Fortified L-Ascorbic Acid

As previously discussed in chapter 3, guava contains very high concentrations of

L-ascorbic acid and when combined with anthocyanins in solution has shown to be

mutually destructive in a variety of food products (Brenes et al. 2005, Choi et al. 2001).

This destruction has been linked to the formation of dehydroascorbic acid breakdown

products, but the exact mechanism for their adverse interaction has not been completely

elucidated. This study demonstrated similar results with juices containing anthocyanins

losing L-ascorbic acid at a much greater rate than the guava juice control which contained

no anthocyanins. All L-ascorbic acid containing samples in this study started with similar

amounts of L-ascorbic acid, containing between 350-400 mg/L. The clarified guava juice

used in the study was assayed to contain 700 mg/L L-ascorbic acid which is in the range

of previously published data on guava ascorbic acid (USDA nutrient handbook 2005,

Uddin 2002). Unlike the results of Chapter 3 where guava nectar was found to be

impervious to losses during pasteurization, several juice treatments lost L-ascorbic acid

as a result of pasteurization. The L-ascorbic acid in the Acai C18 + Guava, Black Currant

Juice + Guava, and Black Currant C18 + Guava all significantly decreased as a result of

pasteurization while none of the fortified treatments on anthocyanin control treatments

significantly decreased. Therefore, it is concluded that guava juice, which naturally

contains L-ascorbic acid, had a negative effect on the L-ascorbic acid stability of

anthocyanin containing juices compared to those fortified with L-ascorbic acid.

In agreement with previous studies (Brenes 2005, Garzon 2002), the L-ascorbic

acid in this study followed first-order kinetics and half life and rate were calculated as

previously described. Storage temperature played the major role in L-ascorbic acid

stability during storage time, with samples stored at 6 C having much greater half lives









than those stored at 250 C. The L-ascorbic acid source (natural versus fortified) was not

significant in terms of L-ascorbic acid stability over storage time. This is an important

result, and demonstrates that guava juice had no protecting effect on the L-ascorbic acid.

The average L-ascorbic acid half life for anthocyanin containing samples stored at 6 and

25 C were 29 and 2 days, respectively. By 28 days, all juices stored at 25 C contained

no detectable L-ascorbic acid. The guava juice control was the most stable at both storage

temperatures compared to all other samples with half lives of 67 and 5 days at 6 and 250

C, respectively. This demonstrates the mutual destruction of ascorbic acid in the presence

of anthocyanins.

This is an important finding for the food industry and suggests that juices must be

kept at refrigerated temperatures (4-7C) in order to protect the L-ascorbic acid. L-

ascorbic acid is extremely important in fruit due to its antioxidant properties, as well as

its protecting effect on other phytochemicals such as phenolic acids and carotenoids. Its

stability is especially important in anthocyanin containing juice blends since it is evident

that the degradation products of L-ascorbic acid degrade anthocyanins as well (Es-Safi et

al. 1999, 2002; Brenes et al. 2005). L-ascorbic acid is therefore often used as an indicator

of fruit juice quality and actions taken to improve its stability may also improve the

overall juice shelf life in terms of nutrition and quality.

Effects On Antioxidant Activity

Hydrophilic antioxidant capacity was measured using the oxygen radical

absorbance capacity assay as described in Chapter 3, primarily measuring contributions

from anthocyanins, non-anthocyanin polyphenolics, and ascorbic acid. This section will

discuss the antioxidant activity of guava juice blends. Prior to pasteurization, the samples












Table 4-2. Effects of storage temperature and anthocyanins on first order regression, rate,
and half life of L-ascorbic acid. Different letters represent differences between
sample types with or without L-ascorbic acid. N/A = not applicable due to
high stability or fluctuation in the data.
6 C Storage 250 C Storage
Sample R2 k t1/2 R2 k t1/2
Acai Juice + Buffer + AA 0.86 -0.0211 32.94 b 0.89 -0.2951 2.36 b,c
Acai Juice + Guava N/A N/A N/A 0.82 -0.2652 2.69 b
Acai C18 + Buffer + AA 0.84 -0.0388 24.06 b 0.88 -0.3000 2.36 b,c
Acai C18 + Guava N/A N/A N/A 0.96 -0.3725 1.86 c
Guava Juice 0.65 -0.0118 66.79 a 0.67 -0.1453 4.77 a
Black Currant Juice + Buffer + AA 0.65 -0.0411 31.31b 0.83 -0.2963 2.44 b
Black Currant Juice + Guava N/A N/A N/A 0.96 -0.3725 1.86 b
Black Currant C18 + Buffer + AA 0.76 -0.0258 28.67 b 0.97 -0.2863 2.45 b
Black Currant C18 + Guava 0.78 -0.0545 14.87 b 0.99 -0.3533 1.96 b
Guava Juice 0.65 -0.0118 66.79 a 0.67 -0.1453 4.77 a


160

140

120

100

80

60

40


Control Day 1 Day 7 Day 17 Day 21 Day 28


Figure 4-9. Percent initial of L-ascorbic acid in acai based samples stored at 250C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n=3.


-*- Acai Juice + Buffer + AA, 25 C Storage
-0- Acai C18 + Buffer +AA, 25 C Storage
-V- Acai Juice + Guava Juice, 25 C Storage
-- Acai C18 + Guava Juice, 25 C Storage
- Guava Juice, 25 C Storage





















0
4-

.0
-o
<


53






160


140


120 -


100


80


60


o
0
(I)
40 -
-j


20 -

n


Control Day 1 Day 7 Day 14 Day 21 Day 28





Figure 4-10. Percent initial of L-ascorbic acid in acai based samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n=3.







160 Black Currant Juice + Buffer + AA, 25 OC Storage
-0- Black Currant C18 + Buffer + AA, 25 OC Storage
140 Black Currant Juice + Guava Juice, 25 OC Storage
S- Black Currant C18 + Guava Juice, 25 OC Storage
S120 Guava Juice, 25 OC Storage
0
o 100

< 80
o
60
)



20
40 -



Control Day 1 Day 7 Day 14 Day 21 Day 28





Figure 4-11. Percent initial of L-ascorbic acid in black currant based samples stored at

250C. Control is the unpasteurized juice. Bars represent standard error of the
mean, n=3.


- Acai Juice + Buffer + AA, 6 OC Storage
-0- Acai C18 + Buffer + AA, 6 OC Storage
-- Acai Juice + Guava Juice, 6 OC Storage
-V- Acai C18 + Guava Juice, 6 OC Storage
- Guava Juice, 6 OC Storage







54





180
-*- Black Currant Juice + Buffer + AA, 6 C Storage
160 -0- Black Currant C18 + Buffer + AA, 6 C Storage
-v- Black Currant Juice + Guava Juice, 6 C Storage
S140 -v- Black Currant C18 + Guava Juice, 6 C Storage
0 -- Guava Juice, 6 oC Storage
120
.-0
100 -

80-
0
< 60 -

40

20
Control Day 1 Day 7 Day 14 Day 21 Day 28



Figure 4-12. Percent initial of L-ascorbic acid in black currant based samples stored at
6C. Control is the unpasteurized juice. Bars represent standard error of the
mean, n=3.

had an antioxidant capacity between the ranges of 9-14 uM Trolox eq/mL, with the black

currant and agai C18 + guava samples having the lowest antioxidant activity (9.17 and

10.11 uM Trolox eq/mL respectively). Agai pulp has previously been reported to have an

antioxidant activity of 48.6 uM TE/mL (Del-Pozo Insfran et al. 2004) and black currant

berries between 54.4 and 101.4, depending on cultivar (Wu et al. 2004), while no

published data exists on guava antioxidant activity as measured by the ORAC assay. The

lower antioxidant activity of the C18 samples was attributed to the lower concentration of

total anthocyanins and the higher degree of polymeric anthocyanins which occurred as a

result of the isolation process. Tsai and Huang (2003) reported the antioxidant capacity in

the roselle flower, as measured by the FRAP, TEAC, and DPPH scavenging effect, to

decrease with increasing polymerization, similar to the results of this study. Each










treatment acted independently of each other as a result of pasteurization. Guava juice,

Agai C18 + Guava Juice, and Black Currant C18 + Guava Juice all had increasing

antioxidant values after processing, while the Acai Juice + Guava Juice ORAC value

decreased and the Black Currant Juice + Guava Juice ORAC value did not change.

Similar to the previous phytochemicals discussed, the antioxidant activity in the

juices was temperature dependent, with the samples stored at 6 C retaining more

antioxidant activity than the samples stored at 25 C. When comparing antioxidant

activities after 28 days of storage to the initial post-processing values, differences were

only seen in C18 samples at both storage temperatures. After 28 days of storage at 6 oC,

the acai C18 + guava and black currant C18 + guava retained 88.55 and 90.96 % of their

initial antioxidant activity and when stored at 25 C, retained 87.16 and 83.98 % of their

antioxidant activity, respectively.



130

E 120

o 110
I--
S100 -

9- 90

80 -
--- Acai Juice + Guava Juice, 25 C Storage
S70 -O- Acai C18 + Guava Juice, 25 C Storage
-Y- Guava Juice, 25 C Storage
60 -.
control day 0 day 7 day 14 day 21 day 28



Figure 4-13. Percent initial of antioxidant activity in acai based samples stored at 250C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n=3.
















130 -


120


110


100


90 -


80 -


control day 0 day 7 day 14 day 21 day 28


Figure 4-14. Percent initial of antioxidant activity in acai based samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n=3.


control day 0 day 7 day 14 day 21 day 28


Figure 4-15. Percent initial of antioxidant activity in black currant samples stored at
250C. Control is the unpasteurized juice. Bars represent standard error of the
mean, n=3.







57




130


120


2 110 -
H--

100-


90 -

S-* Black Currant Juice + Guava Juice, 6 C Storage
E 80 -0- Black Currant Juice + Guava C18, 6 C Storage
-<- Guava Juice, 6 C Storage

70 1
control day 0 day 7 day 14 day 21 day 28



Figure 4-16. Percent initial of antioxidant activity in black currant samples stored at 60C.
Control is the unpasteurized juice. Bars represent standard error of the mean,
n=3.

Effects On Polyphenolics

This discussion will identify the non-anthocyanin polyphenolics in guava

containing samples, determine their storage stability, and determine their effects on

anthocyanin stability. Major polyphenolic compounds were evaluated in pure guava

juice. Figure (4-17) shows a typical polyphenolic chromatogram of guava juice at 280

nm, separated by HPLC conditions as previously outlined. Due to the numerous

compounds separated, only the compounds found in the greatest concentration and/or the

resolved compounds were evaluated. Few studies exist that identify and quantify the

polyphenolics present in guava, but have included ellagic acid and condensed tannins

(Misra and Seshadri, 1967), and gallic acid, catechin, epicatechin, and chlorogenic acid

(Kondo et al., 2005). Based on retention times, spectral properties, and comparison to

authentic standards, gallic acid, catechin, and ellagic acid were positively identified in











guava juice in this study (Table 4-3), while several compounds were tentatively identified

and classified into general groups, such as benzoic acid esters, procyanidins, and ellagic

acid derivatives. These compounds, listed in Table 4-3, were evaluated in all guava

containing samples in terms of their storage stabilities at 6 and 25 C (Table 4-4).




0.40-
0.35
0.30-

0.25-

AU 0.207
0.15-




0.00-
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
Minutes


Figure 4-17. HPLC chromatogram (at 280nm) of polyphenolic compounds found in
guava juice. 1) gallic acid 2) (+)-catechin 3) ellagic acid 4) ellagic acid
derivative 5) unknown characteristic guava polyphenolic.




Table 4-3. Tentative identification of guava juice polyphenolics, measured by
HPLC/PDA.
Retention Time Spectral Properties
Peak No. (min) (nm) Tentative Identification
1 17.06 215, 271 gallic acid
2 31.28 281 catechin
3 49.89 252, 369 ellagic acid
4 51.38 252, 365 ellagic acid derivative
5 54.36 219, 276 unknown



Individual peaks (1-5) were summed for each sample after four weeks of storage at

both 6 and 25 o C and reported as percent initial of day 0 concentrations (Table 4-4).

ANOVA analysis was performed, comparing the different samples at their respective











Table 4-4. Percent of initial polyphenolics after four weeks of storage at 6 and 25 C.
Letters represent statistical differences (P<0.05, n=3).
Sample 6 oC 25 C
Guava 80.8 b 73.0 a
Acai Juice + Guava 98.6 a 74.6 a
Acai C18 + Guava 72.4 c 51.1 c
Black Currant Juice + Guava 93.3 a 61.2 b
Black Currant C18 + Guava 94.4 a 65.2 b


storage temperatures. At 6 oC of storage, Acai Juice + Guava, Black Currant Juice +

Guava and Black Currant C18 + Guava retained the most polyphenolics (98.6, 93.3, and

94.4 %, respectively). Guava retained the second greatest amount (80.8%), while Acai

C18 + Guava retained the least amount of polyphenolics (72.4%). Polyphenolic retention

at 25 o C was lower than those stored at 6 o C. Acai C18 + Guava and the pure Guava

sample retained 74.6 and 73 % of initial, respectively, at 25 o C storage. Both black

currant samples, Black Currant Juice + Guava and Black Currant C18 + Guava retained

61.2 and 65.2 percent while the Acai C18 + Guava again retained the least amount of the

polyphenolic compounds measured (51.1%). These results are unexpected since the Acai

C18 + Guava had the highest anthocyanin stability compared to the other samples.

However, this may be a result of the some of the polyphenolic compounds, such as

procyanidins (catechin) polymerizing with the anthocyanins in the presence of furfurals

such as HMF, as seen in blood orange juice (Hillebrand 2004) and black carrot juice (Wu

2004). HMF was detected in the juice blends with a chromatographic peak at retention

time 8.6 minutes at 285 nm.

Conclusions

Only slight decreases in total anthocyanins, L-ascorbic acid, and antioxidant

activity were seen as a result of pasteurization in the juice blends. Phytochemical and









antioxidant stability in guava juice and guava juice/anthocyanin juice and C18 mixtures

were storage temperature dependent. It is recommended that juice manufactures keep

strict temperature control under refrigerated conditions for anthocyanin and L-ascorbic

acid containing juices. Further investigations on acai C18 + guava juice mixtures are

needed in order to determine exact reasons for its stability. Possible studies include

HPLC/MS determinations of exact anthocyanins and other polyphenolics that may form

as a result of polymerization. However the results of this study indicate that the acai C18

isolate may be polymerizing with the guava juice therefore increasing the stability of the

anthocyanins. This is evident from the high degree of polymeric anthocyanins, the

increased stability of total anthocyanins compared to the synthetic ascorbic acid fortified

blend, and the overall decrease of non-anthocyanin polyphenolics.














CHAPTER 5
SUMMARY AND CONCLUSIONS

Guava products such as juice, nectar, purees, and jams are steadily increasing in

the United States market. Few studies are available pertaining to guava juices in relation

to pasteurization and storage, especially those relating to the stability of its health and

quality aspects such as the color and antioxidant phytochemicals. To provide an

overview, these studies consisted of two pasteurization conditions on guava nectar with

two subsequent storage temperatures. This study concluded that storage temperature is

the biggest factor relating to increasing the stability of L-ascorbic acid, lycopene,

polyphenolics, and antioxidant activity, with the nectar stored at 40C better retaining L-

ascorbic acid, polyphenolics and antioxidant activity, and the nectar stored at 250C better

retaining lycopene. The second study was conducted in order to determine the effects of

blending clarified guava juice with anthocyanin containing juices and concentrates, for

the purpose of juice blending and color fortification, respectively. The L-ascorbic acid

found in guava juice was found to be mutually destructive in the presence of the added

anthocyanins. This study demonstrated temperature dependence as well, with the juices

stored at 60C retaining more color overall (anthocyanins) and L-ascorbic acid, than those

stored at 250C. Guava polyphenolics were also found to have a protecting effect on acai

and black currant C18 concentrates. This suggests possible interactions between guava

phenolics (catechin) and anthocyanins, due to antioxidant protection and formation of

more stable polymers. Overall conclusions and suggestions to industry are to keep juice






62


blends at refrigerated temperatures (4-70C) in order to protect overall quality and

antioxidant phytochemicals
















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BIOGRAPHICAL SKETCH

Lanier Elaine Fender was born May 15, 1982 in Dothan, Alabama. She then moved

to Ocala, Florida, where she was raised. After completion of high school in 2000, Lanier

attended Central Florida Community College in Ocala, Florida, where she completed her

Associate of Arts degree in May 2002. Lanier received her Bachelor of Science degree in

food science and human nutrition in April 2004 from the University of Florida. She

continued at the University of Florida, where she completed her Master of Science

degree, specializing in food science, in December 2005.