Studies on the Degradation of Ascorbic Acid by Saskatoon Berry Juice

Studies on the Degradation of Ascorbic Acid by Saskatoon Berry Juice Marguerite Panther and Fred H. W olfe* Department of Food Science University of Alberta Edmonton, Alberta

Abstract Ascorbic Acid (Vitamin C) has been shown to be absent although dehydroascorbic acid has been found in several varieties of Saskatoon berries currently being cultivated at the Canada Department of Agriculture Research Station at Beaverlodge, Alberta. Synthetic L-ascorbic acid is rapidly degraded after addition to expressed juice of the berries. Evidence of an ascorbic acid oxidizing system has been revealed, and the effects of pigmentation and approaching maturity on the enzyme system have been examined. The effects of various pigments, polyphenols and oopper ions on the oxidation have also been studied, and the implications of these results are discussed.

Resume Les effets de differentes temperatures de congelation sur des echantillons stabilises et non stabilises de differents gras furent etudies en vue d'en etablir le rapport solide-liquide. Les analyses furent faites sur un appareil it resonance magnetique nucleaire Newport it large bande du type MKI. Les echantillons furent congeles it 0 0 et it _60 0 ainsi qu'en combinaison de ces temperatures. Les resultats presentent des differences qui sont d'autant plus marquees que les echantillons qui avaient ete stabilises. La reproductibilite des epreuves se maintient au niveau de 1% de la phase solide peu importe le type de gras ou la temperature etudies.

Introduction The laboratories of the Department of Food Science at the University ,of Alberta have been conducting routine analyses since Hl67 on a number of selections of Saskatoon berries (Amelanchier alnitolia Nutt.) propagated, cultivated and harvested by Dr. Robert E. Harris of the Canada Department of Agriculture Research Station, Beaverlodge, Alberta. The object of these studies has been to evaluate processing parameters, and the potential for more effective utilization of this native fruit in the food processing industry. Wolfe and Wood (1971) studied organic acid and reducing sugar content of Saskatoon berries, and the changes in these compounds during ripening. Early studies of Tuba et al. (1944) on sources of ascorbic acid (Vitamin C) in native Alberta fruits indicated that Saskatoon berries might be a useful source of this important vitamin. These workers, using the classical 2,6-Dichlorophenolindophenol titration for Vitamin C estimation, reported detection of ascorbic acid in Saskatoon berries in the range of 5-38 mg/100 g fresh fruit. The method they used is subject to limi· tations since Saskatoons contain purple anthocyanin pigments, which obscure the endpoint of the titration. Subsequently, tests at the Department of Food Science revealed that the varieties of Saskatoons from the Beaverlodge Research Station had a negligible ascorbic acid content, and that ascorbic acid added to the juice Can. Inst. Food SeL Teehnol. J. Vol. 5, No. 2, 1972

expressed from these berries was rapidly degraded. Ascorbic acid oxidizing enzymes have been identified in a variety of plants (Bonner, 1957; Vines and Oberbacher, 1963; Clark et al., 1966), however, solutions of ascorbic acid are subject to spontaneous oxidation by atmospheric oxygen in the absence of any enzyme, and the rate of the nonenzymic reaction can be greatly altered by catalytic amounts of the transition metal ions (Kahn and Martel, 1967a and 1967b; Hamilton et al., 1969). As well, the interaction of trace amounts of copper ions with a variety of pure flavonoids and other phenolic compounds, such as those generally found in plant tissue, has marked effects on the rates of degradation in this system (Harper, 1969). This study outlines the results of experiments dealing with the loss of ascorbic acid in Saskatoon berry juice, and examines both the enzymatic and nonenzymatic phases of the degradation.

Methods and Materials Ascorbic acid was quantitated by the polarographic method of Brezina-Zuman (1956) as modified by Wasa et al. (1961). An acetate buffer, pH 5.5, with 1% formaldehyde added, was saturated with sodium oxalate and filtered through a Whatman #1 filter paper. The filtrate is used as the supporting electrolyte, and in this medium the half-wave potential of ascorbic acid is about +0.07 volts. Under these conditions there was a linear relationship between ascorbic acid present in standard solutions (in the concentration range from to 75 fLg/ml) and wave height as measured in the polarographic trace. Five grams of Saskatoon berries were homogenized at O°C. in 20 ml of 4% trichloroacetic acid and 2% meta-phosphoric acid, pH 2.4, and after filtration through Whatman #1 filter paper to remove cell debris, brought to a volume of 20 ml with extractant. Ten ml of this solution was added to an equal volume of supporting electrolyte, and the resulting solution was purged with nitrogen for four minutes, after which the solution was polarographed from +0.1 V to -0.1 V in the Sargent Model XV Polarograph. All results represent the average of duplicate measurements. Dehydroascorbic acid was measured as ascorbic acid following reductions with either 2,3 dimercaptopro~anol (Gero and Candido, 1969) or sodium sulfide (Baczyk and Karlik, 1970). The degradation of ascorbic acid by diluted Saskatoon juice centrifugates was followed polarographically, in each case 15 g of berries were. homogenized in a Waring Blendor at O°C. for two mmutes, and the juice obtained by passing the mulch through

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a centrifugal juice maker was made to 50 ml with deionized water. This diluted juice was than added to an aqueous solution of L-ascorbic acid (Worthington Biochemicals) to a final volume of 500 ml and a final ascorbic acid concentration of 90 ftg/ml and maintained at 30°C. Ten ml portions of this mixture were then assayed for ascorbic acid polarographically as above, at regular intervals up to two hours. Blanks were run on 90 ftg/ml ascorbic acid solutions at 30°C. and all results corrected for these observed autooxidative losses. Proteins were precipitated from the Saskatoon juice by the ammonium sulfate fractionation method of Dawson and Magee (1955), and the effect of the proteins on ascorbic acid deg-radation determined polarographically as above. Phenolic compounds were purchased from Fluka AG, Switzerland, and all other reag-ents used were at least analytical reag-ent Q"rade. Copper concentrations were determined in Unicarn SP-90 Atomic Absorption Spectrophotometer. The Saskatoon berries used in this study were of the Smoky (pigmented) and Paleface (non-pigmented) varieties cultivated and g-enerously supplied by Dr. Harris of Beaverlodge. The berries were hand picked and immediately frozen on the specified dates, and air shipped directly to the University without thawing. The samples were stored at -20°C. and assayed immediately after thawing, usually within a few days of receipt.

Results and Discussion Both the titrimetric methods of ascorbic acid assay inv,olving the use of 2,6-Dichlorophenolindophenol and the more rapid and sensitive polarographic assay yielded negative results for the Smoky and Paleface Saskatoon juice. Tuba, Hunter and Kennedy (1944) have produced contradictory results which must be Ql1estioned in view of the difficulty in detecting any distinct endnoint in the titrimetric method. Comparin~ the two methods for the estimation of ascorbic acid, the advantal!es of polarography far outweigh the titration methons. The decay of ascorbic acid is rapid ann it is logical to assume that the more accurate results ilre obtained by the faster method. Estimations are carried out in an atmosphere of nitrollen, so that the atmospheric oxidation of the vitilmin is at a minimum. The unmodified method 'of Tillmans (1927) was of no use because the endpoint of the titration was the same color as the Smoky juice. When Paleface berries were used in the analyses, the endpoint was more accurate but it was difficult to ensure that the intensity of color at the end point was always the same. When the 2,6-Dichlorophenolindophenol xylene extraction method of Robertson and Stob; (1945) was used, the color of the "permanent" end point was not stable. Therefore, the uRefulness of this method for ascorbic acid assay in Saskatoons is doubted. The finding that ascorbic acid is absent from the berries is perhaps surprising, especially when fruits similar in appearance do contain the vitamin. However, the Saskatoon is closely related to the apple, pear, and 94

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quince and the term "berry" is a misnomer, as the fruit is in fact a pome. Typically, these fruits contain very low quantities of ascorbic acid, ranging from 0 to 10 mg/100 g of fruit. Although no ascorbic acid could be detected, the reduction methods with both 2,3 dimercaptopropanol and sodium sulfide yielded positive results for dehydroascorbic acid. Polarographic analysis after the reduction treatments of solutions of Saskatoon extracts revealed 23.9 mg/100 g of berries of dehydroascorbic acid in the former case and 21.9 mg/100 g of berries in the latter case. These results indicate that considerable oxidation of ascorbic acid to the dehydro form has occurred. Ascorbic acid was not found at any of the examined stages of growth, so it is conceivable that the acid is degraded as rapidly as it is formed. Figure 1 depicts the effect of added Saskatoon berry extract on the degradation of ascorbic acid with time. These results have been corrected for the autooxidation of ascorbic acid by adding the amount of ascorbic acid lost in standard solutions polarographed at equivalent concentrations after the same time periods. The general shape of this curve is characteristic of all of the decay curves obtained. The loss of ascorbic acid was greatly inhibited by boiling the juice prior to the addition of the synthetic Vitamin C, which implies the existence of an enzyme system involved in the degradation process. In order to ascertain the role of possible enzymes, an ammonium sulfate precipitation scheme (Dawson and Mag-ee, 195fi), was uspd to prepare a crude protein fraction from the Smoky juice. Figure 2 depicts the rate of decay of added ascorbic acid to the salt precipitated material from the equivalent of 100 g of berries, which had been dialysed for 36 hours against deionized water to remove pigments and small molecules. The close similarity with the rate of decav of the ascorbic acid added to whole diluted juice (Fig. 1) clearly establishes the role of enzymes J. Inst. Can. Sel. Teehnol. Aliment. VoJ. 5, No 2. 1972

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The copper content of the Saskatoons was found to be around 9 parts per million. Increasing the copper ion content of juice extracts to 19, 34 and 59 ppm led to the interesting effect -of apparently inhibiting the rate of degradation, as the amount of ascorbic acid remaining after one hour was increased to 72, 77 and 84 ttg/ml respectively, as opposed to about 68 ttg/ml for these late harvest Smoky varieties without added copper ions. The apparent stability which is conferred to the Vitamin C by the added oopper may be the Can. Inst. Food Sel. Technol. J. Vol. 5, No. 2, 1972

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in the ascorbate degradation. No ascorbic acid was found at the various stages of maturity examined, but the activity of the enzyme system involved in the degradation was found to show interesting variations with the onset of maturity. Figure 3 presents the results of studies on the loss of ascorbic acid added to diluted filtered juice of both Smoky and Paleface Saskatoon berries harvested at five stages of maturity. The July 8 berries were the first formed, hard green fruit; those from July 17 were larger and slightly pink; the July 24 berries were full size and quite pink; those from July 30 were considered optimally ripe. The August 6 harvest contained many overripe fruits. In order to simplify the comparison of the pigmented and non-pigmented varieties the results of measurements of ascorbic acid remaining after addition to diluted juice are presented as percent of Vitamin C remaining one hour after addition to the juice, for each harvest date. It is apparent from these data that the decay of ascorbic acid at different harvest dates, related to the ascorbic acid oxidase activity, is approximately the same for both varieties, although the degradative activity of the Smoky juice decreases more rapidly with the onset of maturity than the Paleface variety. This may be the result of a protective effect on ascorbic acid exerted by the plant pigments.

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result of the formation of a copper-ascorbate complex (illustrated below), such as suggested by Kahn and Martel (1967a), possibly interfering with the interaction of the enzyme and vitamin.

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Since Clegg and Morton (1968) found that flavonols and other plant pigments including anthocyanins can affect the rate of oxidation of ascorbic acid, a series of experiments was conducted with a variety of phenolic compounds to learn the effect of added phenols on the rate of decay of the vitamin added to the Saskatoon juice system. The phenols and pigments were added at levels of 1 X 10-4 NI:, a value chosen after the work of Harper et al. (1969). The results of these experiments are presented in Figure 4, expressed as % ascorbic acid remaining one hour after the addition of synthetic ascorbic acid to the juice of both Smoky and Paleface berries. It is interesting to note that the anthocyanin pigments peonin and malvin, and the anthocyanidin, delphinidin, had only minor effects on the stability of the vitamin in the already pigmented Smoky juice, but had important stabilizing effects in the juice of the non-pigmented Paleface berries. This observation may indeed reflect differences in the ascorbic acid oxidizing system from these two berries. The other polyphenolic compounds exert a varying sparing effect on the ascorbate, probably due to their relative ease of oxidation by the enzymes present, or possibly due to chelation of needed cofactors. 95

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The authors gratefully acknowledge the financial assistance of the .A.lberta Agricultural Research Trust (AR #55-08276), the National Research Council (A-5751) and the generous assistance of Dr. R. E . Harris, CDA Research Station, Beaverlodge, Alberta.

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The degradation of ascorbic acid by the juice from two varieties of Saskatoon berries in the presence of 1 X 10-4 M concentrations of select plant pigments and polyphenols. L-ascorbate added to juice at levels of !:l0 j.tg/ml, and the degradation measured after a one hour incubation at 30°C. Data corrected for autooxidation eHects.

Conclusions From these results it has been ascertained that ascorbic acid is not present in Saskatoon berries at any stage of maturity in detectable quantities. While the oxidized form, dehydroascorbic acid, is present in the order of 22 to 24 mg/IOO g fresh weight, the low efficiency of the dehydro form as an antiscorbutic factor precludes the usefulness of Saskatoon berries as good dietary sources of Vitamin C. The rapid degradation of Vitamin C added to Saskatoon juice extracts was attributable to an as· corbic acid oxidizing enzyme system, and this enzyme

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system was precipitable from juice by saturation with ammonium sulfate. Increasing levels of copper ions or certain phenolic compounds exerted a protective effect on the ascorbic acid, suggesting that in Saskatoon products fortified with synthetic Vitamin C, the vitamin potency, might be extended by the addition of natural pigments.

Baczyk, S. and B. Karlik. 1970. "Colorimetrische Bestimmung von Dehydro-L-Ascorbinsaure in Pflanzlichem Material." Z. Unters. Lebensmittel. 143 :184. Bonner. W. D. Jr. 1957. "Soluble oxidases and their functions." Ann. Rev. Plant Physiol. 8:427. Brezina-Zuman. 1956. "Die Polarographie in der Medizin." pp 370. Biochemie und Pharmazie Academie Verlag, Leipzig. Clark, E. E., W. N. Poillon and C. R. Dawson. 1966. "Ascorbate oxidase, I. Oxidase activity." Biochim. Biophys. Acta. 118 :72. Clegg, K. M. and A. D. Morton. 1968. "The phenolic compounds of black-currant Juice and their protective effect on ascorbic acid, 11." J. Food Technol. 3:277. Dawson, C. R. and R J. Magee. 1955. "Ascorbic acid oxidase." In: Methods in Enzymology, Colowick, S. P. and N. O. Kaplan (Ed.l. Volume 11, pp 831. Academic Press, New York. Gero, E. and A. Candido 1969. "Une technique chimique de dosage de l'acide ascorbique total par une reaction d'oxydo-rMuction." Internat. J. Vit. Res. 39:252. Hamilton, G. E. 1969. "Mechanism of two and four electron oxidations catalysed by some metalloenzymes." Adv. Enzymol. 32:55. Harper, K. A. 1969. "Copper-flavonoid complexes in acidic solutions." J. Food Technol. 4:405. Harper, K. A., A. D. Morton anrl E. J. Ro1fe. l o6Q. "'1'he ""°'101ic compounds of blackcurrant Juice and their protective effect on ascorbic acid. Ill." J. Food Technol. 4 :255. Kahn, M. M T. and A. E. Martell. 1967a. "Metal ion and metal chelate catalvsed oxidation of ascorbic cid by molecular oxygen. I." J. Am. Chem. Soc. 89:4176. Kahn, M. M. T. and A. E. Martell. 1967b. "Metal ion and metal chelate catalysed oxidation of ascorbic Acid by molecular oxygen. 11." J. Am. Chem. Soc. 89:7104. Robinson, W. B. and E. Stotz. 1945. "The indophenol-xylene extraction method for ascorbic acid and modifications for interfering substances." J. BioI Chem. 160:217. Tillmans, J. 1927. "Bestimmung der Elektrode Reduktion Oxydation Potentialen und ihre Anwenrlung in der Lebensmittelchemie." Z. Unters. Lebensmittel. 54 :33. Tuba, J., G. Hunter and L. L. Kennedv. 1944. "On sources of Vitamin C. 11. Alberta native fruits." Can. J. Research. 22C :33. Vines, H. M. and M. F. Oberbacher. 1963. "Citrus fruit enzymes." Plant Physiol. 38 :333. Wasa, T., M. Taka~i and S. Ono. 1961. "Polaro~raphic investigations of Vitamin C." Chem. Soc. Japan Bull. 34:518. Wolfe, F. H. and F. W. Wood. 1971. "Non-volatile organic acid and sugar ('omp,,~jtion of Sa$kl\toon berries during ripening." Can. Inst. Food Technol. J. 4:29. Received Sep. 27, 1971.

J. Inst. Can. Scl. Technol. Aliment. Vol. 5, No 2, 1972