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Quality Retention of Calcium and Ascorbic Acid Fortified Muscadine Grape Juice

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Title:
Quality Retention of Calcium and Ascorbic Acid Fortified Muscadine Grape Juice
Creator:
Pires, Danielle M. ( Author, Primary )
Copyright Date:
2008

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Calcium ( jstor )
Food ( jstor )
Forts ( jstor )
Grape juice ( jstor )
Grapes ( jstor )
Hydroxybenzoic acids ( jstor )
Juices ( jstor )
pH ( jstor )
Pigments ( jstor )
Standard error ( jstor )

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University of Florida
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University of Florida
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Copyright Danielle M. Pires. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/1/2005
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79904602 ( OCLC )

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QUALITY RETENTION OF CALCIUM AND ASCORBIC ACID FORTIFIED MUSCADINE GRAPE JUICE By DANIELLE M. PIRES 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 2003

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ii ACKNOWLEDGMENTS I thank my major professor, Dr. Stephe n Talcott, for the guidance and patience through my graduate program. I would also like to thank my parents, Ed and Emily, and my brother, Jason, for all the support through everything in my life. I will forever thank my boyfriend, Stephen Downes, who pushed me to keep going throughout all the hard times and supported me in all my decisions. I will always be gratef ul to my lab mates, Joon-hee Lee, Melanie Kemmerer, David Del Pozo-Insfran, and Jennifer Moore; I do not know if I would have made it without them.

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iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. ii LIST OF TABLES...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi ABSTRACT..................................................................................................................... vi ii CHAPTER 1 INTRODUCTION.. 2 LITERATURE REVIEW... Anthocyanins..3 Anthocyanin and Ascorbic Acid Metal Chelation and Ant hocyanin Copigmentation...6 Processing and Storage...8 3 THE EFFECTS OF FORTIFICATION AND PROCESSING PROCEDURE ON PHYTOCHEMICAL AND QUALITY RETENTION OF MUSCADINE GRAPE JUICE.........................................................................................................................9 Introduction................................................................................................................9 Methods and Materials...............................................................................................9 Study I: Color Retention.................................................................................10 Study II: Polyphenolic (hydorxybenzoic acid derivatives) Retention............10 HPLC analysis of polyphenolics in juice..10 Identification of polyphenolics.....11 Study III: Ascorbic Acid Retention................................................................11 Spectural analysis of juice....................................................................12 HPLC analysis of asco rbic acid in juice... Statistical analysis.................................................................................13 Results and Discussion. Study I: Color Retention in Fortified Grape Juice..13 Study II: Polyphenolic Re tention in Fortified Grape Juice............................14 Study III: Ascorbic Acid Retenti on in Fortified Grape Juice.........................17

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iv 4 THE EFFECTS OF FORTIFICATI ON WITH ASCORBIC ACID AND/OR CALCIUM ON ANTHOCYANI N AND NON-ANTHOCYANIN POLYPHENOLICS OF MUSCADINE JU ICE AT VARIOUS ANTHOCYANIN CONCENTRATIONS..............................................................................................20 Introduction......................................................................................................20 Methods and Materials.............................................................................................22 Results and Discussion.............................................................................................23 Anthocyanin Concentration and Color Retention...23 Ascorbic Acid Retention.25 Polyphenolics (ellagic acid derivati ves and flavonoid glycosides)................34 5 CONCLUSION........................................................................................................39 LIST OF REFERENCES...................................................................................................41 BIOGRAPHICAL SKETCH.44

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v LIST OF TABLES Table page 4-1. Means of duplicate samples of anthocya nin concentration (mg/ L) expressed as cyanidin3-glucoside equivalents..26 4-2. Percent Loss of polyphenolics from Day 4 to Day 48...38

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vi LIST OF FIGURES Figure page 2-1. A copigment-metal-anthocyanin coordi nate complex formed by ascorbic acid, copper, and cyanidin (Sarma et al., 1997)..................................................................7 3-1. Total anthocyanin concentration expresse d as cyanidin 3-glucoside equivalents for non-pasteurized fortified juice samples over time. ..................................................14 3-2. Chromatagraph showing polyphenolic standard marking hydroxybenzoic acids and polyphenolic standard overlayed with 60C hot-pressed muscadine juice nonfortifed...........15 3-3. Four hydroxybenzoic acids from ethyl ace tate extracts of muscadine grape juice with no fortification held up to 30 days at 37 C. ....................................................16 3-4. Four hydroxybenzoic acids from ethyl acetate extracts of muscadine grape juice fortified with 500 mg/L ascorbic acid and held up to 30 days at 37 C...................16 3-5. Four hydroxybenzoic acids from ethyl ace tate extracts of mu scadine grape juice fortified with 500 mg/L calcium and held up to 30 days at 37 C...17 3-6. Ascorbic acid recovery (%) due to various fortification and pasteu rization treatments in muscadine juice samples fortified with 10 0 mg/L and 500 mg/L ascorbic acid.19 3-7. Ascorbic acid recovery (%) due to various fortification and pasteurization treatments in muscadine jui ce samples fortified with 100 mg/L and 500 mg/L calcium ascorbate and iron ascorbate..19 4-1. Effects of ascorbic acid on color in co ld-pressed juice compared to non-fortified control..27 4-2. Effects of ascorbic acid on color in hot-pressed juice (50 C) compared to nonfortified control 4-3. Effects of ascorbic acid on color in hot-pressed juice (60 C) compared to nonfortified control 4-4. Effects of ascorbic acid on color in hot-pressed juice (70 C) compared to nonfortified control

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4-5. Effects of calcium on color in cold -pressed juice compared to non-fortified control..29 4-6. Effects of calcium on color in hot-pressed juice (50 C) compared to non-fortified control...29 4-7. Effects of calcium on color in hot-pressed juice (60 C) compared to non-fortified control... 4-8. Effects of calcium on color in hot-pressed juice (70 C) compared to non-fortified control... 4-9. Effects of ascorbic acid plus calcium on color in cold-pressed juice compared to non-fortified control.....31 4-10. Effects of ascorbic acid plus ca lcium on color in hot-pressed juice (50 C) compared to non-fortified control....31 4-11. Effects of ascorbic acid plus ca lcium on color in hot-pressed juice (60 C) compared to non-fortified control....32 4-12. Effects of ascorbic acid plus ca lcium on color in hot-pressed juice (70 C) compared to non-fortified control 4-13. Ascorbic acid recovery of juice fortified with 500 mg/L of ascorbic acid and 500 mg/L ascorbic acid and calcium...33 4-14. Concentration of ellagitannin (averagi ng all treatments) over time at each juice pressing temperature...35 4-15. Concentration of free ellagic (averagi ng all treatments) over time at each juice pressing temperature...36 4-16. Concentration of myricet in (averaging all treatments ) over time at each juice pressing temperature...36 4-17. Concentration of querce tin (averaging all treatments) over time at each juice pressing temperature...37 4-18. Concentration of kaempferol (averagi ng all treatments) over time at each juice pressing temperature...37

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viii 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 QUALITY RETENTION OF CALCIUM AND ASCORBIC ACID FORTIFIED MUSCADINE GRAPE JUICE By Danielle M. Pires May 2003 Chair: Dr. Stephen Talcott Major Department: Food Science and Human Nutrition Muscadine grapes ( Vitis rotundifolia ) have developmental po tential to become an important economic crop for growers in the southeastern US, and grape juice is an important commodity by which muscadine grapes can be introduced to the remainder of US market. However, important quality changes during processing and storage may affect advancement of future markets. Para mount is color degrada tion that occurs under normal storage conditions. Presumably the re sult of oxidation of anthocyanins, the major grape pigments, juice color turns from a deep red or purple to a redbrown color that is visually unappealing. Off color developmen t may also result from oxidation of nonanthocyanin polyphenolics that t ogether could hinder sales of muscadine grape juice. Therefore, it is hypothesized that fortifica tion of muscadine grape juice with various compounds may serve to extend shelf life, co lor, and antioxidant properties. Various fortificants were evaluated including ascorb ic acid, calcium, and various metal chelated forms of ascorbic acid an attempt to cont rol oxidation and stabilize anthocyanins during

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ix processing and storage. Fortified juices were evaluated over time for anthocyanin and color stability, composition and concentration of phenolic acids, and antioxidant capacity compared to non-fortified controls. Results show 70% loss of ant hocyanin concentration in samples containing 100 mg/L ascorbic ac id alone and 100 mg/L ascorbic acid with calcium, and 94% loss when fortified at 500 mg/L level after 14 days of storage. Ascorbic acid retention after 4 days was onl y seen in samples forified with 500 mg/L ascoribic acid and 500 mg/L ascoribic acid a nd calcium. Cold-pressed juice retained about 56% and hot-pressed juice at 70 û C retained about 43%. The polyphenolics were not affected by fortifications and the only differences seen were in various processing methods: Juice hot-pressed at 50 û C gave the highest percent lo ss of flavonoid glycosides. Therefore, there are no additional benefits of fortifying muscadine grape juice with ascorbic acid and/or calcium.

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1 CHAPTER 1 INTRODUCTION The muscadine grape ( Vitis rotundifolia ) is native to the southe astern United States. Its range extends along the Atlantic coast from Virginia to central Florida, and west along the Gulf coast to eastern Texas. Muscadine grapes are used for fresh fruit consumption, preservatives, wine, and ju ice production. Quality defects plague the muscadine grape industry through the formation of insolubl e sediments during storage and poor color stability during processing and storage of the fr uit juice. A high quality red grape juice is defined by it organoleptic qua lities including a deep red-pu rple color; however with prolonged storage muscadine grape juice slowly transforms into a brownish-red color that is visually unappealing and considered a quali ty defect to consumers. Since color plays an important role in food acceptability and choices for consumers, a juice manufacturer must be able to prevent color loss during processing and storage. Metal chelation has been show to form stable anthocyanin-metal-copigment complexes when a flavonoid contains an ortho-dihydroxy group. Cyanidin was shown to rapidly chelate with aluminum causing a 2025 nm bathochromic shift in the visible spectra while subsequent addition of ascorbic acid gave an additional 10-15 nm shift indicating that ascorbic acid may complex with a metal-chelated anthocyanin (Sarma et al., 1997). A similiar principle will be evaluate d in muscadine grape juice using calcium in place of aluminum, due to potentially harmful e ffects of aluminum on human health, to access an in vivo response that may serve to improve processing and storage stability.

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2 The objectives of these studies were 1. To determine if fortifying with ascorbic ac id and calcium affect color deterioration. 2. To evaluate the effects of various concen trations of ascorbic acid, calcium, and anthocyanin in a model system on colo r loss and polyphenolic retention. 3. To determine if there are any other lo sses in non-anthocyanin polyphenolics. 4. Finally, to show the effects of storag e on the anthocyanin and non-anthocyanin polyphenolics with fortification

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3 CHAPTER 2 LITERATURE REVIEW Anthocyanins Anthocyanins comprise the largest group of water-soluble pigments in the plant kingdom (Bridle and Timberlake, 1996). In food plants, anthocyanins are widespread and occur in at least 27 families, 73 genera and a multitude of species (Bridle and Timberlake, 1996). More than 240 different anthocyanins have been identified and are distinguished in their degree of hydroxyl ation, methoxylation, glycosylation, and acylation, which has a direct effect on th eir color, expressed general stability characteristics (Wagner, 1982; Strack and Wray, 1989). Anthocyanins are best known for their brilliant red and purple colors and as polyphenolic co mpounds their antioxidant and antiradical capacity has been firmly esta blished (Heinonen et al ., 1998; Abuja et al., 1998; Frankel et al., 1998; Ghiselli et al., 1998 ; Lapidot et al., 1999; Espín et al., 2000). Anthocyanins exist in muscadine grap es primarily as 3,5-diglucosides of delphinidin, cyanidin, petunidi n, peonidin, and malvidin in non-acylated forms (Flora, 1978, Goldy et al., 1986) and are the most a bundant flavonoids present. Acylation is known to confer additional stability to an anthocyanin by preventing it from hydration, making it more stable than the corresponding nonacylated pigm ent (Saito et al., 1995). The stability of the anthocyani ns is influenced by several f actors, including the chemical structure of the pigment. Hydroxyl, methoxyl, sugar, and acylated sugar substitute groups have pronounced effects on th e stability of the anthoc yanin (Markakis, 1982).

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4 Anthocyanin 3,5-diglucosides were reported as less stab le to oxidation and heat compared to corresponding 3-glucosides (Mar kakis, 1982) and may result in rapid color loss during wine or juice storag e. The 3,5-diglucosides report ed to be most unstable in muscadine grape juice were delphinidin a nd petunidin (Flora, 1978, Goldy et al., 1986) and their oxidation during storage were correl ated to decrease radical scavenging activity (Talcott and Lee, 2001). Both delphinidin and petunidin in, along with cyanidin, contain at least one o -dihydroxy group, making them more sus ceptible to oxidation than the other anthocyanin forms. Flora (1978) reported large reductions in delphinidin, cyanidin, and petunidin-3,5-diglucosides in muscadine gr apes after severe heat treatments when analyzed by thin-layer chromatography. Malv idin 3,5-diglucoside was found to be less stable than acylated forms of malvidin pres ent in red cabbage (Hr azdina et al., 1970), but in model systems the stability of malvidin 3,5-diglucoside was grea ter than malvidin 3glusoside both with and without added asco rbic acid (Hrazdina et al., 1970; GarciaViguera and Bridle, 1999). The relative stabil ity of muscadine anthocyanins is likely a function of a complex chemical matrix, struct ural features, and the combined effects of processing and storage. Anthocyanins and Ascorbic Acid Anthocyanins and ascorbic acid have been shown to be mutually destructive in the presence of oxygen, limiting fortification efforts in foods containing these natural pigments. Oxidation of either anthocyanins or ascorbic acid results in decreased radical scavenging properties of muscadine grape ju ice, and their combination may actually promote quality degradation during processing and storage. However, Garcia-Viguera and Bridle (1999) showed anthocyanins offer some degree of stabilization towards ascorbic acid, and ascorbic acid degradat ion is slower according to the degree of

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5 substitution of the anthocyanin, presumably due to a higher stability of 3,5-diglucosides compared to 3-glucosides. Fortification with as corbic acid can severly alter the color in a juice system containing anthocyanin, howev er, anthocyanins might help retain the fortified ascorbic acid in the juice. Landbo and Meyer (2001) reported that as corbic acid added to red and white Vitis vinifera grape juice enhanced antioxidant pr operties as long as polyphenolics were present in the system. Degradation of anthocya nins in the presence of ascorbic acid and oxygen seems to be commodity (or anthocyanin) specific and are not affected in a similar manner during processing or storage. Kaack and Austed (1998) reported a protective effect on anthocyanins containing ascorbic acid or when sparged with nitrogen in elderberry juice, while ascorbic acid alone was responsible for anthocyanin decreases in Concord grape juice (Calvi and Francis, 1978) . In red wine, Datzbe rger et al. (1992) found that the addition of ascorbic acid was detrimental to anthocyanin color but at a level unperceivable to the naked eye, therefore not affecting overall quality characteristics. In model systems, Poei-L angston and Wrolstad (1981) demonstrated the role of ascorbic acid as harmful to anthocyanins under bot h aerobic and anaerobic conditions, and proposed that degradation occurred through a direct condensation reaction between the pigment and ascorbic acid. It was also determined that ascorbic acid was influential in causing browning reacti ons involving catechin, further implicating condensation reactions i nvolving ascorbic acid. Differences in model and in vivo systems seem to implicate a complex phytochemical ma trix in altering co-oxidative reactions between ascorbic acid and anthocyanins.

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6 Metal Chelation and Anthocyanin Copigmentation Augmenting anthocyanin color characteristics by chelation of metal ions or binding with colorless polyphenolics is a common occurrence in natu re. Associations of this nature are known as “copigmen tation” reactions and are impo rtant features affecting the color and oxidative stability of natural pigm ents. Often, various copigments are added to fruit/vegetable extracts or juices containing anthocyanins to help improve color and stability features, adding value to the food or food application. Copigment reactions are generally weak chemical associations w ith anthocyanins (possibly hydrogen bonding) and can occur with various polyphenolics or by chelation with various monoand polyvalent metal ions (Osawa, 1982). These co mplexes serve to confer enhanced color characteristics to anthocyanins and may be in fluential in altering th eir stability under the conditions of processing. It was previously observed that addition of flavonoids to an anthocyanin-ascorbicmetal ion model system could prevent degrad ation of the anthocyanins (Shrikhande and Francis, 1979), results that we re attributed to the antioxi dant properties of flavonoids. Other authors have also observed higher stab ility for anthocyanins in foods high in flavonols (Clegg and Morton, 1968; Poei-Langs ton and Wrolstad, 1981), but the effects of metal ions in a similar role have not b een determined. Lee et al. (1996) showed that certain metal ions and ascorbic acid in the presence of oxygen we re destructive to anthocyanins isolated from purple sweet potatoes. Conversely, Sarma et al (1997) demonstrated that oxidation of ascorbic acid by prooxidant metal ions could be prevented by the addition of an anthocyanin, demonstrat ing that not only can anthocyanins chelate metal ions but also form an ascorbate-me tal ion-anthocyanin complex (Figure 2-1) preventing their mutual destru ction under oxidizing conditions.

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7 The unique phytochemical composition of mus cadine grapes makes attempts at this mechanism worthy of the effort. However, cal cium was chosen to replace copper because it has the same valency state as copper and could be used for commercial purposes. With the exception of a few hybrid grape cultivars, most Vitis vinifera species do not contain anthocyanin 3,5-diglucosides (which act as more efficient copigment acceptors) and none are known to contain ellagic acid. Additionally, the use of ascorbic acid (which also has metal and anthocyanin binding properties) may significantly augment functional properties of muscadine juice, providing addi tional shelf stability and increasing overall quality parameters. Figure 2-1. A copigment-metal-an thocyanin coordinate complex formed by ascorbic acid, copper, and cyanidin (Sarma et al., 1997)

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8 Processing and Storage Storage temperature has a great affect on the browning of the pigments. In a ninemonth storage study, muscadine wine was held at three different te mperatures (20,30, and 40°C). Higher storage temperatures resulted in a larger loss of free anthocyanins most likely due to the destruction of anthocya nins, as well as, polymerization at high temperatures (Sims and Morris, 1984). Due to the proven detrimental effect of heat on anthocyanins, samples were kept at room temperature to decrease this risk. The pH of a system has an effect on the anthocyanin pigment over time. The lower the pH the more stable the anthoc yanin. In a storage study over 90 days, the anthocyanin pigment retained most in the system had the lowest pH. The system with the pH of 3.95 had almost no pigment levels remaining, pH 3.45 had about 9% remaining, and pH 2.95 had 44% of pigments remaining (Wesche-Ebeling, et al., 1996). The pH of the juice system is important because of the sensitivity of anthocyanins in various pH environments. When fortifying juice samples w ith calcium and ascorbic acid the pH was adjusted back to original jui ce pH to ensure pH was not a factor in analysis of color.

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9 CHAPTER 3 THE EFFECTS OF FORTIFICATION AND PROCESSING PROCEDURE ON PHYTOCHEMICAL AND QUALITY RETENTION OF MUSCADINE GRAPE JUICE Introduction Investigations into fortifying muscadine gr ape juice with ascorbic acid and calcium have not been investigated, although products with these agents ar e currently in the market with V. vinifera grapes. The inherent instability of quality attributes of muscadine grape products make them a good model fo r evaluation of phytochemical changes associated with processing and storage. The studies here were designed to evaluate the overall effects of fortificants to muscadine gr ape juice as an initial basis for bulk changes in a typical juice. Specifically, changes asso ciated with fortification and pasteurization were evaluated for ascorbic acid retenti on and individual phenolic compounds quantified by HPLC (i.e. hydroxybenzoic acids). The results of these studies were then applied to a more comprehensive evaluation on the effects of muscadine grape ju ice fortification. Methods and Materials Red muscadine grapes (cv. Noble) were obtained from a local vineyard and processed in the University of Florida, Food Science and Human Nutrition pilot plant. Grapes were heated to 70°C for 15 min and hot -pressed into juice. Juice was then frozen for up to 6 months and used as needed in study I and study II. Study III was conducted using frozen grapes that were hot-pressed the day of use.

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10 Study I: Color Retention Color retention of fortified muscadine grape juice was assessed in the presence of un-pasteurized grape juice in the presence of 500 mg/L ascorb ic acid, 500 mg/L calcium chloride, and a mixture of 500 mg/L ascorb ic acid and 500 mg/L calcium chloride compared to control and a control contai ning 500 mg/L EDTA as a metal chelator. Approximately 50 mg/L sodium azide was added as a bacterial inhibitor. Treated juices were filled into individual screw-cap vials (i n duplicates) and stored at 20°C for 0, 6, 25, and 34 days whereby samples were removed for spectrophotometric color determination at 514 nm following appropriate dilution. Study II: Polyphenolic (Hydroxybenzo ic Acid Derivatives) Retention Polyphenolics (primarily hydroxybenzoic acid derivatives) composition were evaluated following fortification with 500 mg /L ascorbic acid or 500 mg/L calcium chloride compared to a non-fortified control in the same frozen grape juice evaluated in Study I. Following fortificati on, each treatment was sealed into individual screw-cap vials and pasteurized at 90°C for 15 min to inactive residual enzymes and for sterility. Samples were stored at 37°C for 0, 15, and 30 days and analyzed for polyphenolics by HPLC. HPLC analysis of polyphenolics in juice HPLC analysis was performed with a Wa ters Alliance 2690 system equipped with a Nova-Pak C18 column (150 mm × 3.9 mm, Waters, Milford, MA) and a Waters 996 photodiode array detector. Millennium32® was used for data ac quisition and analysis. Chromatographic conditions were as follo ws: flow-rate, 0.8 ml/min; injection volume, 100 µl; detection, 280 nm. Mobile phase c ontained 98 % water and 2 % acetic acid (Fisher, Fair Lawn, NJ), and mobile pha se B contained 68% water, 30% acetonitrile

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11 (Fisher, Fair Lawn, NJ), and 2% acetic acid. Gradient elution program was: start with 100 % A, to 70 % A in 20 min, to 0 % A in 30 min, stay at 0%A for 20 min, return to initial conditions in 2 minutes. The UV spectra were recorded between 210 and 400 nm. A standard was run containing ga llic, protocatechuic, p-OH-benz oic, catechin, epicatechin, vanillic, caffeic, resveratrol, p-coumaric, a nd ellagic acid (all Sigm a, St. Louis, MO) for quantification of phenolic acids (R2=0.998). Identification of polyphenolics Identification and separation of phenolic acids were accomplished by HPLC analysis using ethyl acetate extracts. Ethyl acetate extrac ts contain all non-anthocyanin polyphenolics and was prepared by adding two vol umes of ethyl acetate to two volume of juice (Salagoity-Auguste et al., 1986). The uppe r, ethyl acetate phase was collected and evaporated to dryness with a rotary evaporator (Buchler Instrument, NJ). The dry residue was dissolved in 50% methanol acidified w ith 0.01% HCl back to original volume of juice. Polyphenolics were quantified without further manipulation by filtering through at 0.45 µ m filter and injecting directly into th e HPLC. Polyphenolics were identified by identical retention time and UV spectra with an authentic standard. Study III: Ascorbic Acid Retention Ascorbic acid retention in fortified muscadine grape juice was evaluated as a function of ascorbic acid c oncentration (100 or 500 mg/L ) and processing conditions using fresh Noble grapes. Grapes were obtaine d from a local grower in central Florida and held frozen for ca. 2 months prior to extr action. Frozen grapes were then placed into an open steam kettle without crushing and slow ly heated to 60 °C and held for 30 min prior to pressing in order to keep enzyme s, like polyphenol oxidase active. The juice obtained was divided into thr ee equal portions for additional processing and fortification

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12 evaluations. The first portion was sub-divi ded and fortified with 0, 100, and 500 mg/L ascorbic acid, and then pasteurized at 95 °C for 15 min, while the second portion was initially pasteurized and then fortified. These treatments were compared to a control juice that was fortified at each ascorbic acid con centration but not pasteurized. All juices were equilibrated to 23 °C and held for 2 hrs af ter which isolates were obtained for HPLC analysis. Spectral analysis of juice Juice treatments at each st orage time were diluted with buffer solutions at pH 1.0 and pH 4.5. Buffer at pH 1.0 was prepared by adding 62.5 mL of concentrated HCl (Fisher, Fair Lawn, NJ) to 687.5 mL water, and dissolving 3.72g KCl (Fisher, Fair Lawn, NJ) in 250 mL water, both solutions were then combined and the pH was adjusted to 1.0 using 1M HCl. Buffer at pH 4.5 was prep ared by adding 236.4 mL of 1M HCl to 54.44 g of sodium acetate (trihydrate) (Fisher, Fair Lawn, NJ) and adjusting to pH 4.5 with 1M HCl or sodium acetate. Cold-pressed sample and hot-pressed sample at 50 û C were diluted 10X and hot-pressed juice at 60 û C and 70 û C were diluted 20X. Samples were read on a Beckman DU® 640 Spectrophotometer at fixe d wavelength of 514 nm. The absorbance readings were back calculated to account for dilutions and the absorb ance readings at pH 4.5 was subtracted from the ab sorbance readings at pH 1.0 giving the total anthocyanin concentration (Wrolstad, 1976). HPLC analysis of asco rbic acid in juice Anthocyanins were binded to an ACCUBOND® ODS filter. Samples were then filtered through a 0.45 µ m filter and injecting directly in to the HPLC. HPLC analysis was performed with a Waters Alliance 2690 system equi pped with a Nova-Pak C18 column (150 mm × 3.9 mm, Waters, Milford, MA) and a Waters 996 photodiode array detector.

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13 Millennium32® was used for data acquisition and an alysis. Chromatographic conditions were as follows: flow-rate, 0.8 ml/min; in jection volume, 20 µl; detection, 254 nm. Mobile phase A contained 0.2M KH2PO4 in water mobile phase was adjusted to pH 2.4 with o -phosphoric acid (Bartolomé et al., 1993; Ba rtolomé et al., 1996). Isocratic elution program was 100% A. The UV spectra were recorded between 193 and 400 nm. A calibration curve of ascorbic acid (Sigma , St. Louis, MO) at 254 nm was used for quantification of ascorbic acid (R2= 0.9998). Statistical analysis All data represent the mean and standa rd deviation from duplicate analyses. Analysis of variance and mean separation was conducted using Duncan’s multiple-range test (P<0.05; SAS Institute, 1999). LSD te st (P<0.05) was employed to determine treatment effect on individual analyzed values by using JPM software (SAS Institute, 1996). Results and Discussion Study I: Color Retention in Fortified Grape Juice The effect of fortification with 500 mg/L of ascorbic acid, calcium, ascorbic acid and calcium, and EDTA on the total anthocya nin concentration of the non-pasteurized juice samples was evaluated. Th e juice samples used for this study were from frozen noble muscadine hot-pressed juice that was not pasteurized. Samples were evaluated spectrally at 514 nm by the pH differential met hod outlined in the material and methods. Results seen in Figure 3-1 show at the samp le time of 25 days a significant difference (p=0.05) between the control and calcium fortified samples and the rest of the samples. Day 34 samples showed a significant differen ce between the control and the rest of the samples; however, the mix of ascorbic acid and calcium was signifi cantly different from

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14 the rest of the samples. This suggest s a small protective effect by calcium on anthocyanins from ascorbic acid; however, the difference seen is minimal. It is clear that from these samples at days 25 and 34 that ascorbic acid has a de structive effect on anthocyanin color in muscadine juice. Thes e samples were not pa steurized therefore enzymes, such as polyphenol oxidase, may be de structive against anthoc yanins as well as the treatments. Time (Days) 010203040 Total anthocyanin concentration (mg/L) 0 50 100 150 200 250 Control AA 500 mg/L Ca 500 mg/L AA+Ca 500 mg/L EDTA 500 mg/L Figure 3.1. Total anthocyanin concentrati on expressed as cyanidin 3-glucoside equivalents for non-pasteurized fortifie d juice samples over time. (n=2; error bars represent standard error of mean; average sta ndard error = 3.467) Study II: Polyphenolic Retention in Fortified Grape Juice Knowing that fortifying muscadine juice w ith ascorbic acid decreases anthocyanins in muscadine juice, a study was conducte d to evaluate if the hydroxbenzoic acid polyphenolics decrease as well over time. Past eurized muscadine juice samples were fortified with 500 mg/L of ascorbic acid a nd 500 mg/L of calcium and were stored at

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15 37 û C for 30 days. Samples were evaluated at 0, 15, and 30 days for phenolic acids using HPLC by an ethyl acetate ex traction outlined in the methods section. Hydroxybenzoic acids (gallic acid, protocatachuic acid, p -OH-benzoic acid and vani llic acid) (figure 3-2) increased over time regardless of treatmen ts (Figures 3-3, 3-4, 3-5). These compounds are cell wall polyphenolics and most likely incr ease due to the breakdown of the cell wall during storage. Concluding that the destructive effect seen on muscadine anthocyanins when fortified with ascorbic acid does not seem to the have the same effect on the nonanthocyanin polyphenolics. Figure 3-2. A) Polyphenolic standard s howing hydroxybenzoic acids B) Polyphenolic standard (solid line) and 60 û C hot-pressed muscadine juice non-fortifed (dotted-line).

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16 Time (Days) 01530 mg/L 0 50 100 150 200 250 Gallic Acid Protocatachuic Acid p-OH-benzoic Acid Vanillic Acid Figure 3-3. Four hydroxybenzoic acids from et hyl acetate extracts of muscadine grape juice with no fortification held up to 30 days at 37 °C. (n=2; error bars indicate standard error of the mean) Time (Days) 01530 mg/L 0 50 100 150 200 250 Gallic Acid Protocatachuic Acid p-OH-benzoic Acid Vanillic Acid Figure 3-4. Four hydroxybenzoic acids from et hyl acetate extracts of muscadine grape juice fortified with 500 mg/L ascorbic acid and held up to 30 days at 37 °C. (n=2; error bars indicate standard error of the mean)

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17 Time (Days) 01530 mg/L 0 50 100 150 200 250 Gallic Acid Protocatachuic Acid p-OH-benzoic Acid Vanillic Acid Figure 3-5. Four hydroxybenzoic acids from et hyl acetate extracts of muscadine grape juice fortified with 500 mg/L calcium and held up to 30 days at 37 °C. (n=2; error bars indicate standa rd error of the mean) Study III: Ascorbic acid Retent ion in Fortified Grape Juice These studies were conducted to determine the best time to fortify muscadine juice; before or after pasteurization. Juice sample s were put under three different processing treatments, pasteurized then fortified, for tified and not pasteurize d, and fortified then pasteurized. Pasteurization was take n place in a hot water bath of 90 û C for 15 minutes. Ascorbic acid was evaluated using the HPLC method outlined in methods and materials. Two separate studies were conducted; one looki ng at the effect of fo rtifying with 100 and 500 mg/L of ascorbic acid and the other fo rtifying with 100 and 500 mg/L of calcium ascorbate and iron ascorbate (Fluka BioC hemika, Switzerland). Pasteurization to denature residual enzymes followed by fortifica tion gave >68% retention of ascorbic acid that reflected non-enzymatic, concentration-inde pendent destruction of ascorbic acid in the presence of anthocyanins. In non-past eurized juices, enzymes such as, polyphenol

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18 oxidase (PPO) was suspected in co-oxidative reactions that caused 73 and 43% ascorbic acid loss at 100 and 500 mg/L ascorbic acid fortifications, respectively (Figure 3-6). Greater retention at higher as corbic acid concentrations was an indication of enzyme suppression by keeping substrates in a redu ced form. Additionally, the combination of enzyme action prior to pasteurization and degradation due to thermal pasteurization resulted in ascorbic acid losses exceeding 88% when fortified at 100 mg/L. These results show the effect of an enzyme that causes as corbic acid degradation being inhibited during pasteurization. Results of this study illustrate the most effective pasteurization technique would be to pasteurize then fo rtify, therefore, in the rest of the studies conducted, juice was pasteurized before any fortifications were taken place. Another aspect looked at was to see if fo rtifying samples with ascorbic acid already bound to a metal would have a hi gher retention of ascorbic acid. The theory would be that ascorbic acid already bound would not have a chance to react with the anthocyanin to degrade it. However, fortifyi ng with calcium ascorbate and iron ascorbate showed lower retention of ascorbic acid than samples fortif ied with just ascorbic acid alone (Figure 37). Due to these results, and that the use of th ese fortificants would not be practical on an industry level, calcium ascorbate and iron as corbate were not eval uated again throughout the remainder of the research. These extreme ascorbic acid losses highlight the importance of heating prior to ascorbic acid fortification of muscadine grape juice for nutrient and quality re tention. Processing and treatment parameters that allow for ascorbic acid fortification without phytoc hemical and quality deterioration will be a critical factor impacting future.

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19 Pasteurized, Fortified Non-pasteurized, fortified Pasteurized, fortified Ascorbic Acid Retention (%) 0 20 40 60 80 AsA (100 mg/L) AsA (500 mg/L) Figure 3-6. Ascorbic acid recovery (%) due to various fortifica tion and pasteurization treatments in muscadine juice sample s fortified with 100 mg/L and 500 mg/L ascorbic acid. (error bars represent stand error of the mean) Pasteurized, fortified Non-pasteurized, fortified Fortified, pasteurized Ascorbic Acid Retention (%) 0 10 20 30 40 50 60 Ca/AA 100 mg/L Ca/AA 500 mg/L Fe/AA 100mg/L Fe/AA 500 mg/L Figure 3-7. Ascorbic acid recovery (%) due to various fortifica tion and pasteurization treatments in muscadine juice sample s fortified with 100 mg/L and 500 mg/L calcium ascorbate and iron ascorbate (erro r bars represent st andard error of the mean)

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20 CHAPTER 4 THE EFFECTS OF FORTIFICATION WITH ASCORBIC ACID AND/OR CALCIUM ON ANTHOCYANIN AND NON-ANTHO CYANIN POLYPHENOLICS OF MUSCADINE JUICE AT VARIOUS ANTHOCYANIN CONCENTRATIONS. Introduction Color of food plays an important role in consumer acceptance. Consumers often relate “off-color” of food systems to dete rioration of food qual ity. Anthocyanins are primarily responsible for the color of red gr apes. There are over 300 different naturally occurring anthocyanins in nature , ranging from red to blue. Muscadine grapes ( V. rotundifolia ) contain nonacylated 3,5-diglucosides of malvidin, peonidin, petunidin, cyanidin, and delphinidin. Nonacylated anthocyanins are less stable at higher temperatures than the acylated anthocyanins, therefore, poorer color quality (Robinson et al., 1966). It is well known that anthocya nins and ascorbic acid in the presence of oxygen are mutua lly destructive. However, st udies with anthocyanins and ascorbic acid have also been ambiguous since Kaack and Austed (1998) reported a protective effect on anthocyanins containing ascorbic acid or when sparged with nitrogen in elderberry juice, while ascorbic acid alon e was responsible for anthocyanin decrease in Concord grape juice (Calvi and Francis, 1978). Color degradation of blood orange juice, which naturally contains cyanidin-3-glucoside and ascorbic acid, was found to correlate with ascorbic acid concentrations and resulted in juice discoloration and loss of fortified ascorbic acid (Choi et al., 2002). Therefore, de gradation reactions of this nature seem to be commodity specific depending on the phyt ochemical or anthocyanin composition.

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21 Ascorbic acid is commonly added to fruit juices to provide additional sources of vitamin C. Vitamin C is known for its anti oxidant activity and recognized that human consumption of dietary antioxidants affords pr otection against some pathological events. The destructive effect of ascorbic acid on anthocyanins, resulting in color of a model juice system has been researched, as we ll as, a protective effect on ascorbic acid degradation by anthocyanins (Poei-Langstr on and Wrolstad, 1981; Garcia-Viguera and Bridle, 1999). Marti et al. (2002) showed fortifying pomegranat e juice (containing 3glucosides and 3,5-diglucosides of delphinidin, cyanidin, and pelargonidin) with 330mgl-1 of ascorbic acid has no additional benefits. Ascorbic acid degraded very rapidly, and after 4 days of storage at 25 û C and 5 û C, ascorbic acid was completely degraded, leaving the samples with a slight orange hue. An important objective of this study was to determine if any additional benefits arise fr om fortifying with ascorbic acid despite the color loss, for example better re tention of polyphenolics. Interest in phenolic acids have been on the rise due to positive associations between consumption rates and beneficial he alth factors such as anticarcinogenic and radical scavenging activ ity (Meyer et al., 1998). Studies have been conducted on storage effects on non-fortified juices. Talcott and L ee (2002) showed losses of ellagic acid and flavonols (myrecetin, quercetin, and kaempferol) were obser ved following storage at 37 û C of Noble hot pressed muscadine juice. They showed decreases in flavonols during storage were due to autoxidative reactions, wi th losses of myrecetin greater than those of quercetin or kaempferol. This study evaluate s if these losses seen by Talcott and Lee (2002) can be slowed down or even prevented with fortification.

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22 With increasing numbers of fruit juice pr oducts being fortified with vitamins and minerals, muscadine juice should also be evalua ted to observe the eff ects of fortification with calcium and/or ascorbic acid in hope s of a future market. This study evaluates fortifying muscadine juice afte r pasteurization with ascorbic acid, calcium, and a mixture of ascorbic acid and calcium on the anthocyani n concentration. The last objective of this study was to determine if fortification with ascorbic acid and calcium would alter the polyphenolic content of hot pressed and cold pressed muscadine juice Methods and Materials Red muscadine grapes (cv. Noble) were divided into four equal parts and processed into juice following either cold pr essing at room temperature, or three hot pressing treatments that included 50 û C for 10 minutes, 60 û C for 10 minutes, and 70 û C for 10 minutes yielding a concentra tion gradient of soluble phytochemicals. Each juice was pasteurized for 15 minutes at 90 û C and cooled to room temperature. Juices were then filtered using a GAST® vacuum pump and Whatman® 4 filter paper (Whatman International, England) to remove insoluble sediments. Sodium azide (5 mg/L) (Fisher, Fair Lawn, NJ) was added to retard microbiol ogical growth during stor age of juice. Each juice was then fortified with 100 mg/L and 500 mg/L of L-as corbic acid (Fisher, Fair Lawn, NJ), 100mg/L and 500mg/L of calcium chloride (Sigma, St. Louis, MO), and 100mg/L and 500mg/L mixture of both Lascorbic acid and calcium chloride. Fortifications all took place on a stock sample and replications were taken from the stock samples. Fortification solutions were all adjusted to the orig inal juice pH of 3.4 using 1M HCl or 1M NaOH. The samples were then evenly distributed into test tubes and stored in the dark at room temperature (20 û C). Non-fortified controls were kept in the same conditions as the fortified samples. Samp les were analyzed spectrally for total

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23 anthocyanin, and ascorbic acid and polyphenol ics were evaluated by HPLC, as described in Chapter 3. Results and Discussion Anthocyanin Concentration and Color Retention Color loss due to fortification treatments in fortified juices was obtained by evaluating the absorbance values of anthoc yanins at 514 nm and comparing against a non-fortified control. Juice samples were dilu ted (with citric acid buffer at pH 3.4) 10X for cold press and hot pressed at 50 û C, and 20X for hot pressed juices at 60 û C and 70 û C. Anthocyanin concentrations are shown in Table 4-1, as cyanidin 3-glucoside equivalents, and demonstrate the destructive e ffect of fortification of with 500 mg/L of ascorbic acid alone or in the presence of calcium over time on muscadine anthocyanins. The percent loss from day 4 to day 14 of anthocyanins are highest for samples that contained 500 mg/L ascorbic acid, and the loss es are also greater for the cold-press juice than the juice hot-pressed at 70 û C. Therefore, less the anthoc yanins present in the juice system faster the loss of anthocyanins. Evaluating the color of these samples can further support anthocyanin concentration results. Samples fortified w ith 100 mg/L and 500 mg/L ascorbic acid for cold-pressed juice, hot-pressed juice at 60 û C, and 70 û C (figures 4-1, 4-3, 4-4) showed each treatment was significantly different fr om each other and the non-fortified control (p=0.05). Hot-pressed juice at 50 û C (figure 4-2) showed juice fortifed with 500 mg/L ascorbic acid was significantly different (p =0.05) from the non-for tified control and 100 mg/L ascorbic acid fortified sample, however , samples fortified with 100 mg/L ascorbic acid were not significantly different than th e non-fortified control. Thus, illustrating degradative effects to color when juice is fo rtified with ascorbic acid compaired to non-

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24 fortified controls. Cold-press juice, hot-pressed juice at 50 û C, 60 û C and 70 û C fortified with 100 mg/L and 500 mg/L calcium (figures 4-5, 4-6, 4-7, 4-8) showed juice samples fortified with 100 mg/L calcium were not si gnificantly different (p=0.05) from the nonfortifed control, however, 500 mg/L calcium fo rtifed samples were significantly different from the non-fortifed contro l and 100 mg/L calcium for tified samples. Therefore, fortifying with 500 mg/L calcium significanly alters the color of muscadine juice, however, fortifying with 100 mg/L calcium does not significantly change color compaired to non-fortified controls. Samples fo rtified with ascorbic acid in conjunciton with calcium followed the same trend as samples fortifed with ascorbic acid alone. Coldpressed juice, hot-pressed juice at 60 û C, and 70 û C fortifed with 100 mg/L and 500 mg/L ascoribic acid and calcium were all signifi cant (p=0.05) from each other and from the non-fortified control (figure 4-9, 411, 4-12). Hot-pressed juice at 50 û C fortified with 100 mg/L and 500 mg/L acorbic acid plus calcuim (figure 4-10) showed non-fortified control and 100 mg/L fortified samples were not si gnificantly different (p=0.05), but samples fortified with 500 mg/L ascorbic plus calci um showed a significant change in color compaired to the control and the samples fortified with 100mg/L ascorbic acid plus calcium. These results are in concurrence with other researcher s (Poei-Langston and Wrolstad, 1981; Marti et.al., 2002) where the fortification with as corbic acid showed decreases in anthocyanin concentration compar ed to non-fortified controls. Since samples fortified with ascorbic acid and calcium demonstrate similar trends as samples fortified with ascorbic acid alone, the schematic propos ed by Sarma et al. (1997) where ascorbic acid acts likes a copigment and directly in teracts with a metal-chelated anthocyanin forming a stable complex is not observed with muscadine anthocyanins when calcium is

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25 used as the metal. In conclusion, fortifying sa mples with high levels of ascorbic acid by itself or in the presence of calcium prove to deteriorate the color significantly. Ascorbic Acid Retention Many factors play a role in the degradation of ascorbic acid in fruit juice systems. Anthocyanins such as malvidin 3-glucosid e and malvidin 3,5-diglucoside have shown a protective effect against asco rbic acid degradation (Gar cia-Viguera and Bridle, 1999; Marti, 2002). However, malvidin is one of th e most stable anthocyanins due to its two methyl groups, and muscadine grape juice c ontains only about 14% malvidin. The main anthocyanins found in muscadine juice are delphinidin and pet unidin, which are less stable due to being an ortho-diphenolic st ructure that can easily undergo oxidation Ascorbic acid, after four days, was only r ecovered in samples that were fortified with 500mg/L ascorbic acid and 500mg/L ascorb ic acid and calcium; samples that were fortified with 100mg/L ascorbic acid were degraded. All samples were degraded by day 14. Overall percent recovery was higher in sample fortified only with ascorbic acid. This effect could be due to the pr oxidative effect of calcium at high levels. Cold-press juice, having the least amount of anthocyanins, had the highest percent recovery (56% for AA sample and 47% for AA+Ca sample) of ascorbic acid in both fortified samples (Figure 413). Therefore, the protection effect of asco rbic acid degradation by anthocyanins, as previously reported, is not seen with mus cadine juice. Pomegranate juice containing mainly 3-glucosides and 3,5-diglucosides of delphinidin, cyanidi n, and pelargonidin, showed similar ascorbic aci d degradation results.

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26 Table 4-1. Means of duplicate samples of an thocyanin concentration (mg/L) expressed as cyanidin3-glucoside equivalents Juice Pressing Conditions Treatment Day 4 Day 14Day 48 Percent Loss from Da y 4 to da y 14 Control 1713 a 1401a* 728 a* 58 AA 100mg/L 1630 b 1232 b* 489 b* 70 AA 500 1423 c 716 c* 92 c* 94 Ca 100 1643 d 1359 d* 731 d* 56 Ca 500 1521 e 1229 e* 626 e* 59 AA+Ca 100 1659 f 1272 f* 522 f* 69 CP AA+Ca 500 1359 g 683 g* 87 g* 94 Control 2970 a 2853 a* 1277 a* 57 AA 100 3311 b 2691 b* 1178 b* 64 AA 500 2978 c 1568 c* 292 c* 90 Ca 100 3304 d 2698 d* 1279 d* 61 Ca 500 3100 e 2374 e* 1066 e* 66 AA+Ca 100 3392 f 2641 f* 1224 f* 64 50°C AA+Ca 500 2984 g 1626 g* 303 g* 90 Control 6238 a 5281 a* 3001 a* 52 AA 100 5930 b 4730 b* 2424 b* 59 AA 500 5790 c 2841 c* 694 c* 88 Ca 100 6127 d 5173 d* 3108 d* 49 Ca 500 5196 e 4382 e* 2666 e* 49 AA+Ca 100 6178 f 4762 f* 2798 f* 55 60 û C AA+Ca 500 5592 g 2971 g* 728 g* 87 Control 7812 a 6662 a* 4059 a* 48 AA 100 7868 b 6323 b* 3670 b* 53 AA 500 6900 c 4152 c* 1171 c* 83 Ca 100 7725 d 6294 d* 4074 d* 47 Ca 500 7065 e 5862 e* 3701 e* 48 AA+Ca 100 7699 f 6030 f* 3802 f* 51 70 û C AA+Ca 500 6862 g 4182 g* 1235 g* 82 * represents the anthocyanin concentration significant (p=0.05) decreasing at each time from the time before and letters repres ent the differences (p=0.05) between each treatment at each procssing temperature from the control.

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27 Time (Days) 0102030405060 mg/L 0 2 4 6 8 10 12 Control AA 100 mg/L AA 500 mg/L Figure 4-1. Effects of ascorbic acid on colo r in cold-pressed juice compared to nonfortified control (n=2; aver age standard error =0.089) Time (Days) 0102030405060 mg/L 0 5 10 15 20 25 Control AA 100 mg/L AA 500 mg/L Figure 4-2. Effects of ascorbic acid on color in hot-pressed juice (50 C) compared to nonfortified control (n=2; aver age standard error =0.5255) a b c a a b

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28 Time (Days) 0102030405060 mg/L 0 10 20 30 40 50 Control AA 100 mg/L AA 500 mg/L Figure 4-3. Effects of ascorbic acid on color in hot-pressed juice (60 û C) compared to nonfortified control (n=2; aver age standard error =0.3732) Time (Days) 0102030405060 mg/L 0 10 20 30 40 50 60 Control AA 100 mg/L AA 500 mg/L Figure 4-4. Effects of ascorbic acid on color in hot-pressed juice (70 û C) compared to nonfortified control (n=2; aver age standard error =0.7980) a b c a b c

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29 Time (Days) 0102030405060 mg/L 2 4 6 8 10 12 Control Ca 100 mg/L Ca 500 mg/L Figure 4-5. Effects of calcium on color in co ld-pressed juice compared to non-fortified control (n=2; average standard error =0.2248) Time (Days) 0102030405060 mg/L 6 8 10 12 14 16 18 20 22 24 Control Ca 100 mg/L Ca 500 mg/L Figure 4-6. Effects of calcium on color in hot-pressed juice (50 û C) compared to nonfortified control (n=2; aver age standard error =0.5180) a,a b a,a b

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30 Time (Days) 0102030405060 mg/L 15 20 25 30 35 40 45 Control Ca 100 mg/L Ca 500 mg/L Figure 4-7. Effects of calcium on color in hot-pressed juice (60 û C) compared to nonfortified control (n=2; aver age standard error =0.5370) Time (Days) 0102030405060 mg/L 20 25 30 35 40 45 50 55 Control Ca 100 mg/L Ca 500 mg/L Figure 4-8. Effects of calcium on color in hot-pressed juice (70 û C) compared to nonfortified control (n=2; aver age standard error =0.4160) a,a b a,a b

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31 Time (Days) 0102030405060 mg/L 0 2 4 6 8 10 12 Control AA+Ca 100 mg/L AA+Ca 500 mg/L Figure 4-9. Effects of ascorbic acid plus calcium on color in cold-pressed juice compared to non-fortified control (n=2; average standard error =0.0868) Time (Days) 0102030405060 mg/L 0 5 10 15 20 25 Control AA+Ca 100 mg/L AA+Ca 500 mg/L Figure 4-10. Effects of ascorbic acid plus calcium on color in hot-pressed juice (50 û C) compared to non-fortified control (n =2; average standard error =0.5205) a b c a,a b

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32 Time (Days) 0102030405060 mg/L 0 10 20 30 40 50 Control AA+Ca 100 mg/L AA+Ca 500 mg/L Figure 4-11. Effects of ascorbic acid plus calcium on color in hot-pressed juice (60 û C) compared to non-fortified control (n =2; average standard error =0.1477) Time (Days) 0102030405060 mg/L 0 10 20 30 40 50 60 Control AA+Ca 100 mg/L AA+Ca 500 mg/L Figure 4-12. Effects of ascorbic acid plus calcium on color in hot-pressed juice (70 û C) compared to non-fortified control (n =2; average standard error =0.2390) a b c a b c

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33 Pomegranate juice fortified with 330 mgl-1 ascorbic acid showed complete degradation in samples stored at 5 û C and 25 û C after 4 days (Marti et al., 2002). In conclusion, ascorbic acid degradation in mus cadine juice is a very rapid reaction when fortified in low levels, especially in the pr esence of abundant anthoc yanins. Fortifying at high levels of ascorbic acid alters the color si gnificantly as stated in chapter 3. Therefore, there does not seem to be significant evidence from these results that fortfiying muscadine juice with ascorbic acid has a ny additional benefits due to the rapid degradation of ascorbic acid. Model System CP506070 Ascorbic Acid Retention (%) 0 10 20 30 40 50 60 70 AA 500 mg/L AA+Ca 500 mg/L Figure 4-13. Ascorbic acid recovery of juice fortified with 500 mg/L of ascorbic acid and 500 mg/L ascorbic acid and calcium. Thes e results are for da y four.(error bars represent standard error of the mean)

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34 Polyphenolics (Ellagic Acid Deri vatives and Flavonoid Glycosides) The polyphenolics can be divided into three categories: hydroxybenzoic acid derivatives (protocatechuic and vanillic acids), ellagic acid derivatives (ellagitannin and free ellagic acid) and flavonoi d glycosides (myricetin, quercetin, kaempferol). Hydroxybenzoic acid derivatives, protocatechui c and vanillic acids, show the same trend regardless of treatment, in occurance with result from chapter three. Statistical analysis (p=0.05) shows that no significant difference between treatments and controls when fortified at 100 mg/L and 500 mg /L ascorbic acid and/or calciu m, therefore, results will be discussed basied on averaging all treatmen ts at each of the four juice concentration levels. Ellagitannin concentr ation decreased over time in a ll four processing treatments, on day 48 there was no significant difference (p=0.05) from cold-p ress juice and juice processed at 50 C, and no significant difference between 60 C and 70 C processed juice (figure 4-14). The percent loss over time (table 4-2) show e llagitannin had the greatest losses in hot-pressed juice at 50 C. Free ellagic acid was stable over time in 50 C and 60 C processed juice, increased by 18% in cold pressed juice, and decreased 34% in 70 C hot-pressed juice (table 4-2) , however, by day 48 there was no significance (p=0.05) between 60 C and 70 C, and there was significa nt difference between 50 C and coldpressed juice (figure 4-15). Flavonoid glycos ides (myricetin, querc etin, and kaempferol) all exhibit the same trends in percent loss; they showed the greates t loss in hot-pressed juice at 50 C and the best retention in hot-pressed juice at 60 C (table 4-2). Overall after 48 days quercetin and kaempferol showed no significant difference (p=0.05) in coldpressed and hot pressed juice at 50 C and no significant difference in 60 C and 70 C hotpressed juice (figures 4-17, 4-18). Myricetin on day 48 showed no significant difference

PAGE 44

35 (p=0.05) between 60 û C and 70 û C hot-pressed juice, however, there was significant difference between 50 û C hot-pressed and cold presse d juice (figure 4-16). Decreases in flavonids were also evalua ted by Talcott and Lee (2002) in Noble hotpressed juice. They showed a decrease in a ll three flavonoids after storage of 60 days at room temperature. Cold-pressed juice showed a decrease in quercetin and kaempferol, myricetin showed no difference after storag e at room temperature at 60 days. In conclusion, the flavonoid glycos ides were the only pheonolic acids to show significant signs of decrease with an d without fortification. Time (Days) 0102030405060 mg/L 0 2 4 6 8 10 12 14 16 18 CP 50oC 60oC 70oC Figure 4-14. Concentration of ellagitannin (averaging all treatments) over time at each juice pressing temperature (n=12; average standard error =0.7667) b,b a a

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36 Time (Days) 0102030405060 mg/L 0 10 20 30 40 50 60 CP 50oC 60oC 70oC Figure 4-15. Concentration of free ellagic (averaging all treatments) over time at each juice pressing temperature (n=12; average standard error =2.095) Time (Days) 0102030405060 mg/L 0 20 40 60 80 100 CP 50oC 60oC 70oC Figure 4-16. Concentration of myricetin (averaging all treatments) over time at each juice pressing temperature (n=12; average standard error =3.593) a b c,c a b c,c

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37 Time (Days) 0102030405060 mg/L 0 5 10 15 20 25 CP 50oC 60oC 70oC Figure 4-17. Concentration of quercetin (ave raging all treatments) over time at each juice pressing temperature (n=12; av erage standard error =0.9391) Time (Days) 0102030405060 mg/L 2 4 6 8 10 12 CP 50oC 60oC 70oC Figure 4-18. Concentration of kaempferol (averaging all treatments) over time at each juice pressing temperature (n=12; average standard error =0.4432) a a b,b a,a b,b

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38 Table 4-2. Percent Loss (%) of Polyphenolics from Day 4 to Day 48 Ellagitannin Free EllagicMyricetin Quercetin Kaempferol CP 41 0* 60 44 48 50C 64 0 61 51 51 60C 42 0 39 34 33 70C 52 34 50 44 47 * Free Ellagic increased by 18% in cold pressed juice from day 4 to day 48.

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39 CHAPTER 5 CONCLUSIONS These studies were conducted to better understand pastuerization and fortification with ascorbic acid and calcium on red muscad ine juice (cv. Noble). When fortifying red muscadine juice with ascorbic acid, it is best to pasteurize the juice then fortify. The less anthocyanins in the system the better the re tention of ascorbic acid. However, ascorbic acid degrades very rapidly, even fortifying samples with 500 mg/L, ascorbic acid was almost completely degraded in 14 days. Mus cadine juice can be fo rtified with 100 mg/L of ascorbic acid, calcium, and ascorbic aci d in combination with calcium without any detrimental loss of color. However, fortifying th e juice with 500 mg/L of ascorbic acid or ascorbic acid and calcium show detrimental loss of color in the juice. The addition of calcium showed no added benefits, with or without the addition of ascorbic acid. Polyphenolics analyzed showed no difference be tween treatments and controls. They are stable when fortified with ascorbic acid and/or calcium at the 100 and 500 mg/L levels, and best retained in hot-pressed juice at 60 û C. The overall conclusion is that fortificati on of muscadine juice with ascorbic acid and/or calcium does not prove to improve the color dete rioration displayed by red muscadine juice. Ascorbic acid alone or in the presence of calcium at 100 mg/L degrades rapidly in red muscadine juice and ascoribic ac id alone or in the presence of calcium at 500 mg/L degrades the color of the juice. Ther efore, there are no addi tional benefits of fortifiying with ascoribic acid alone or in the presence of calcium. Muscadine juice can

PAGE 49

40 be fortified with calcium wit hout any adverse affects to juic e color or phenolics, although this addition would only be for the health benefits of calcium

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41 LIST OF REFERENCES Abuja, P.M., Murkovic, M., and Pf annhauser, W. 1998. Antioxidant and prooxidant activities of elderberry ( Sambucus nigra ) extract in low-density lipoprotein oxidation. J. Ag ric. Food Chem. 46:4091-4096. Bartoleme, B., Bengoeshea, M.L., Galvez, M.C., Perez-Ilzarbe, F.J., and Hernandez, T. 1993. Photodiode array detection for elucid ation of the structure of phenolic compounds. J. Chrom. 655:119-125. Bartoleme, B., Hernandez, T., Bengoeshea, M.L., Quesada, C., Gomez-Cordoves, C., and Estrella, I. 1996. Determinati on of some structural feat ures of procyanidins and related compounds by photodiode-array detection. J. Chrom. 723:19-26. Bridle, P. and Timberlake, C.F. 1996. Anthocya nins as natural food colours-selected aspects. Food Chem. 58:103-109. Calvi, J.P. and Francis, F.J. 1978. Stability of Concord grape ( V. labrusca ) anthocyanins in model systems. J. Food Sci. 43:1448-1456. Choi, M.H., Kim, G.H., and Lee, H.S. 2002. Eff ects of ascorbic acid retention on juice color and pigment stability in blood or ange (Citrus sinensis) juice during refrigerated storage. Fo od Res. Intl. 35:735-759. Clegg, M.K. and Morton, A.D. 1968. Phenolic compounds of black currant juice and their protective effect on asco rbic acid. J. Food Tech. 3:227. Datzberger, K., Steiner, I., Washuettl, J., and Kroyer, G. 1992. The influence of wine additives on colour and colour qualit y of young red wine. Zeitschrift fuer Lebensmittel Untersuchung und Forschung 194:524-526. Espín, J.C., Soler-Rivas, C., Wichers, H.J ., and García-Viguera, C. 2000. Anthocyaninbased natural colorants: A new source of antiradical activity for foodstuff. J. Agric. Food Chem. 48:1588-1592. Flora, L.F. 1978. Influence of heat, cultivar, and maturity on the anthocyanidin-3,5diglucosides of muscadine gr apes. J. Food Sci. 43:1819-1821.

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42 Frankel, E.N., Bosanek, C.A., Meyer, A.S., Silliman, K., and Kirk, L.L. 1998. Commercial grape juices inhibit the in vitro oxidation of human low-density lipoproteins. J. Agric. Food Chem. 46:834-838. Garcia-Viguera, C. and Bridle, P. 1999. Influe nce of structure on colour stability of anthocyanins and flavylium salts wi th ascorbic acid. Food Chem. 64:21-26. Ghiselli, A., Nardini, M., Baldi, A., and Scaccini, C. 1998. Antioxidant activity of different phenolic fractions separated fr om an Italian Red wine. J. Agric. Food Chem. 46:361-367. Goldy, R.G., Ballinger, W.E., and Maness, E.P. 1986. Fruit anthocyanin content of some Euvitis X Vitis rotundifolia hybrids. J. Amer. Soc. Hort. Sci. 111:955-960. Hrazdina, G., Borzell, A.J., and Robinson, W.B. 1970. Studies on the stability of the anthocyanin-3,5-diglucosides. Am. J. Enol. Vitic. 21:201. Heinonen, I.M., Meyer, A.S., and Frankel, E.N. 1998. Antioxidant activity of berry phenolics on human low-density lipoprote in and liposome oxidation. J. Agric. Food Chem. 46:4107-4112. Kaack, K., and Austed, T. 1998. Interaction of vitamin C and flavonoids in elderberry ( Sambucus nigra L .) during juice processing. Plant Foods Hum. Nutr. 52:187-198. Landbo, A. K. and Meyer, A.S. 2001. Ascorbic acid improves the antioxidant activity of European grape juices by improving the jui ces' ability to inhibit lipid peroxidation of human LDL in vitro. Int’l J. Food Sci. Technol. 36:727-735. Lapidot, T., Harel, S., Akiri, B., Granit, R., and Kanner, J. 1999. pH-dependent forms of red wine anthocyanins as antioxida nts. J. Agric. Food Chem. 47:67-70. Lee, S.K., Rhim, J.W., Kim, S.J., and Chung, B.C. 1996. Study on the stability of anthocyanin pigment extracted from pur ple sweet potato. Korean J. Food Sci. Technol. 28:352-359. Markakis, P. 1982. Stability of anthocyanins in foods In Anthocyanins as Food Colors . Markakis. P., Ed., Academic Press, New York. 163. Marti, N., Perez-Vicente, A., and Garcia-V iguera, C. 2002. Influence of storage and ascorbic acid addition on pomegranate juice. J. Sci. Food and Agric. 82:217-221. Osawa, Y. 1982. Copigments of anthocyanins. In Anthocyanins as Food Colors . Markakis, P., Ed. Academic Press, Inc. New York, New York. 58-60. Poei-Langston, M.S. and Wrolstad R.E. 1981. Color degradation in ascorbic acidanthocyanin-flavanol model system. J.Food Sci. 46:1218-1222.

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43 Robinson, W.B., Weirs, L.D., Bertino, J.J., a nd Mattick, L.R. 1966. The stability of wine anthocyanin composition to color stability of New York State wines. Am. J. Enol. Vitic. 17:147. Saito, N., Tatsuzawa, F., Yoda, K., Yokoi, M., Kasahara, K., Iida, S., Shigihara, A., and Honda, T. 1995. Acylated cyanidin glycos ides in the violet-blue flowers of Ipomoea purpurea. Phytochem. 40:1283-1289. Salagoity-Auguste, M.H., Tricard, C., Marsal, F. and Sudraud, P. 1986. Preliminary investigation for the differentiation of enological tannins according to botanical origin: Determination of gallic acid and its derivatives. Am. J. Enol. Vitic. 37: 301-303. Sarma, A. D., Sreelakshmi, Y., and Sh arma, R. 1997. Antioxidant ability of anthocyanins against ascorbic ac id oxidation. Phytochem. 45:671-674. SAS Institute, Inc. 1996. JMP Start Statistics , SAS Institute Inc. Cary, NC. SAS Institute, Inc. 1999. Program: SAS Institute Inc., SAS Campus Drive Cary, NC. Sims, C.A. and Morris, J.R. 1984. Effects of pH, sulfur dioxide, storage time, and temperature on the color and stability of red muscadine grape wine. Am. J. Enol. Vitic. 35:35-39. Shrinkhande, A.J. and Francis, F.J. 1979. E ffect of flavonols on ascorbic acid and anthocyanin stability in model systems. J. Food Sci. 39:904-906. Strack, D. and Wray, V. 1989. Anthocyanin in Methods in Plant Biochemistry , Vol. 1, Plant Phenolics , ed. P.M. Dey & J.B. Harborne . Academic Press, New York, 325. Talcott, S.T., Brenes, C.H., Pires, D.M., and Del Pozo-Insfran, D. Phytochemical stability and color retention of copigmen ted and processed muscadine grape juice. J. Agric. Food Chem. 51:957-963. Talcott, S.T. and Lee, J.H. 2002. Ellagic ac id and flavonoid antioxidant content of muscadine wine and juice. J. Agric. Food Chem. 50:3186-3192. Wagner, G.J. 1982. Cellular and subcelluar lo calization in plant metabolism. Rec. Adv. Phytochem. 16:1. Wesche-Ebeling, P., Argaiz-Jamet, A., Herna ndez-Porras, L.G., and Lopez-Malo, A. 1996. Preservation factors and processing effects on anthocyanin pigments in plums. Food Chem. 57:399-403.

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44 BIOGRAPHICAL SKETCH Danielle Michelle Pires was born in Boston, Massachusetts, in March of 1979, and moved to Longwood, Florida, in 1986, wh ere she attended Woodlands Elementary School until 1990. She attended Greenwood Lakes Middle School from 1990-1993, and finished her college preparatory education at Lake Mary High School, graduating in 1997. She was accepted into the University of Florida and started her undergraduate degree in August of 1997, in the College of Agricultural and Life Sciences. She completed her B.S. degree in food science and human nutrition in May of 2001, and was accepted into graduate school in the College of Agricultural and Life Sciences. She graduated in May 2003, with her Master of Science degree in food science.