CHEMISTRY/BIOCHEMISTRY

JOURNAL OF FOOD SCIENCE

Black Currant Nectar: Effect of Processing and Storage on Anthocyanin and Ascorbic Acid Content
C. K. Iversen

ABSTRACT The degradation of anthocyanins and ascorbic acid in black currant nectar was studied during processing and storage. A continuous production process was used where the nectar was bottled within 4.0h; 20 % of both components were lost in the main stream process. The choice of enzyme mainly affected the recovery of ascorbic acid. Pasteurization resulted in a loss of 26 % of the ascorbic acid content. About 50% of the original content of monomeric anthocyanins remained after 6 months storage at 208C. The stabilities of delphinidins and cyanidins during storage were very similar. Deaeration of the nectar before bottling had no effect on anthocyanin content. Key Words: juice, stability, anthocyanins, currant, HPLC methods

INTRODUCTION
ANTHOCYANINS ARE RESPONSIBLE FOR THE ATTRACTIVE COLOR of black currant nectar and they have been hypothesized as important antioxidants (Rice-Evans et al., 1996). However anthocyanins disappear as monomeric compounds and are transformed into polymeric forms (Markakis, 1982; Baranac et al., 1996; Francia-Aricha et al., 1997; Johnston and Morris, 1997). This transformation results in a color change to a more brownish shade (Skrede et al., 1992). The stability of anthocyanin pigments is affected by oxygen, pH, temperature, light, metal ions, enzymes and ascorbic acid; and may also be affected by the actual anthocyanin concentration (Mazza and Miniati, 1993). Anthocyanins, as well as other phenolics, can act as antioxidants by donating hydrogen to highly reactive radicals, thereby preventing further radical formation (Rice-Evans et al., 1996). Ascorbic acid, being closely related to the phenolic compounds, may also act as an antioxidant, but surprisingly, many reports have indicated that ascorbic acid tended to accelerate anthocyanin degradation rates (Starr and Francis, 1968, Skrede et al., 1992). In early studies it was reported that ascorbic acid was more stable in black currant juice than in other juices, and this was confirmed (Miller and Rice-Evans, 1997). Model systems resembling black currant juice have been used to study the degradation rate of anthocyanin and ascorbic acid but results have been contradictory. Some phenolic compounds in black currants appeared to protect ascorbic acid (Clegg and Morton, 1968) while anthocyanins, in model systems, seemed to accelerate the degradation of ascorbic acid (Harper et al., 1969). The cell walls of fruits consist of a continuous phase of pectin (galacturonic acid joined together by -1,4; -1,3 and -1,6 linkages) and short chain heteropolymers of up to 6 sugar components joined by

-1,2; -1,3; -1,4 and -1,6 linkages (Boyce, 1986; Englyst et al., 1988). Degradation of plant cell walls involves a variety of enzyme activities. Plant cell wall degrading enzymes are often derived from Aspergillus species. After fermentation the desired cell wall degrading enzymes are recovered and purified, but the final enzyme preparation may have several other activities, among them -glucosidase (CanalLlauberes, 1993). The -glucosidase activities can decompose anthocyanins by hydrolyzing the glycosidic substituent, producing an unstable aglycon (Wrolstad et al., 1994; Wightman and Wrolstad, 1995). Pectolytic enzymes are normally used during juice processing to improve juice yield and anthocyanin extraction. When recommended dosage and times are followed, it should be possible to avoid degradation of anthocyanins to their unstable aglycon forms (Wightman and Wrolstad, 1996). Black currant berries contain very high levels of both anthocyanins and ascorbic acid. Under Danish growing conditions, the cultivar Ben Lomond contains on average 410 mg anthocyanins and 180 mg ascorbic acid /100g of berries (Andersen and Vang-Pedersen, 1993). Aroma, color and ascorbic acid content are the most important quality parameters of black currant nectar. Aroma changes during black currant nectar processing have been described (Iversen et al., 1998). Our current objective was to determine how processing and storage conditions affect the degradation of total anthocyanins, individual anthocyanins and ascorbic acid in cloudy black currant nectar.

MATERIALS & METHODS

BLACK CURRANTS, RIBES NIGRUM, CULTIVAR BEN LOMOND, WERE grown at Fyn, Denmark in 1995. Berries were harvested mechanically, stored at 5C overnight and then frozen and stored at -24C.
Nectar processing

Author Iversen is affiliated with the Dept. of Fruit, Vegetable & Food Sciences, Danish Institute of Agricultural Sciences, Kirstinebjergvej 6, 5792 Aarslev, Denmark.

The black currant nectar was prepared from berries thawed at 20C overnight. Berries (25 kg) were homogenized with 10 kg of water at 0C in a model ML-150 (FRYMA-Maschinen AG, Rheinfelden) colloid mill at a flow of 350 kg/h. The mash was enzyme treated at 50C for 2h after the addition of 0.0057% v/v (dosage recommended by Novo Nordisk Ferment for mash treatment) of either PectinexTM Ultra SP (enzyme A), 8800 PDU/mL, or PectinexTM AR (enzyme B), 3000 PDU/mL; both from Novo Nordisk Ferment (Dittingen, Switzerland). Enzyme-treated mash was centrifuged at 50C. Centrifuging was performed by use of a screw-strain separator Siebtechnik (Germany), model Konturbex H 200, with 20 strain, with slits 0.18 mm 3 mm, at 2635 rpm. In the separator, the mash was separated into raw juice and pulp. The raw juice was left 1h at 35C before further processing. Before pasteurization 20 kg of raw juice was mixed with 24 kg of water; another 4 kg water was heated to 80 C and 5.00 kg sugar and 1.62 kg honey were added. After solubilization the two samples were mixed. The final nectar was 16.0 Brix, pH was 3.0. The nectar was deaerated at -925 mbar in a Fryma vacuum deaeration unit type LVE/D. The nectar was pasteurized at 80C for 27s in a APV Pasilac plate heat exchanger, model 1090, at flow rate 180 l/h and filled directly into 250 mL

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Black Currant Nectar Processing . . .

glass bottles. Total processing time was 4h. Glass bottles were stored at 20C either in daylight or in darkness. Nectar processing and storage were replicated twice. For each assay (ascorbic acid or anthocyanin) two bottles were sampled/ treatment. In a separate storage experiment, black currant mash was treated with 0.0057% v/v Enzyme A, separated and sweetened as described. Nectar was then bottled with or without deaeration.
Anthocyanin analysis

tration was measured in the bottles immediately after uncapping. Two bottles were analyzed per treatment.
Calculation of kinetics

HPLC was performed on a system which consisted of two ShimadzuTM LC-6A pumps, a system controller, type SCL-6B, an auto injector, type SIL-6B, an UV-detector, type SPD-10AV and an integrator, type C-R7A. The analytical column was a Merck LichrospherTM RP-18 (125-4) and the preparative column was a Merck LichrocartTM 250-10. Solvents were HPLC grade. Two mobile phases were generally used: (A) water-acetonitrile-formic acid (82:8:10, v:v:v) and (B) water-acetonitrile-formic acid (40:50:10, v:v:v). Sample preparation: raw juice and nectar samples were diluted 1:1 with water-acetonitrile-formic acid (78:12:10, v:v:v) and filtered through a 0.25 m filter before injection into the HPLC. Berries, mash and pulp (from separator) were extracted with the solvent water-acetonitrile-formic acid (84:6:10, v:v:v) and centrifuged, and the solid residue was reextracted three times. The separation was carried out with solvent A by isocratic elution at a flow rate of 1 mL/min at 35C. After elution of anthocyanins the column was rinsed with 100% of eluent B for 5 min, and equilibrated with solvent A for 5 min before the next injection. This was a modification of the method of Goiffon et al. (1991).
Preparation of anthocyanin standards

If the degradation during storage was a first order reaction a straight line relation would occur between ln (concentration of anthocyanin) and storage time. The slope of the linear regression line would indicate the reaction constant. The total anthocyanin concentration was calculated as the sum of dpd-rut, dpd-glu, cya-rut and cya-glu divided by the weighted mean molecular weight (603 dalton), concentration in mmol/kg. The molecular weight of ascorbic acid is 176.1 dalton. Ln concentration was plotted against time, the slope of the best linear regression line equals the reaction constant. A t-test was performed assuming that the two lines had the same slope; if this hypothesis was rejected at P0.05 we then concluded that the reaction constants were different (Zar, 1984).

RESULTS & DISCUSSION

THE PROCESS EQUIPMENT WE USED RESEMBLED THAT OF A COMmercial nectar processing plant, so data from our pilot plant trials would be directly applicable to large scale production. The enzyme levels were as recommended by the enzyme supplier for large scale juice production. Enzyme A caused higher ascorbic acid degradation than Enzyme B (Table 1). During pasteurization 2% of the ascorbic acid was lost in the Enzyme B treated nectar and 6% from the Enzyme A treated nectar. Loss of 2-6% vitamin content was assumed to be quite reasonable since ascorbic acid is very heat-sensitive (Viberg et al., 1997). Anthocyanins are present only in the skin of black currant berries. During the mash treatment, cell walls of the skin were disintegrated by action of pectolytic mash enzymes, and anthocyanins were released into the raw juice either in soluble form or in small suspended fruit particles. There was a small increase in total anthocyanin content (Table 2) in pasteurized nectar compared with raw juice. This was probably because a small amount of anthocyanins was released from fruit particles during pasteurization (as this was a cloudy nectar). The two

The four main anthocyanins of black currants were isolated in our laboratory. They were not commercially available, and we had previously isolated them (Nrbaek et al. 1996). Each of the four anthocyanins was isolated after extraction from freeze dried black currant pulp (3 g extracted with 10 mL water-acetonitrile-formic acid ( 84:6:10, v:v:v)) . This extract (300L) was injected onto a preparative column and the 4 anthocyanins were collected. This procedure was repeated several times. Standard curves were developed for delphinidin-3-glucoside (dpd-glu) (Mw 465), delphinidin-3-rutinoside (dpd-rut)(Mw 611), cyanidin-3-glucoside (cya-glu)[Mw 446 (theoretically 449)] and cyanidin-3-rutinoside (cya-rut)(Mw 595). Concentrations of the anthocyanins were calculated from absorbance measurement at 525 nm (in a spectrophotometer) using the molar absorption coefficient for cyanidin-3-glucoside = 29 600 (1% HCl, pH 2) (Wrolstad, 1976) and the molecular weights.
Ascorbic acid analysis

Table 1Ascorbic acid concentrations during processing

Enzyme A mg/100g berries masha raw juice pasteurized nectara
in Materials & Methods. b,cDifferent at P 0.01.

Enzyme B mg/100g 1645 116c3 41.5c1.3 % of expected 100 99 97

% of expected 100 91 87 82

1645 1062 102b1 35.1b1.0

The analysis was performed on the same HPLC system described for anthocyanin analysis with a Merck LichrosorbTM RP-18 (125-4) column and UV-detection at 250 nm. Samples were extracted with 0.1% oxalic acid and diluted to 20 mg AA /100g. The sample was filtered through a 0.45 m filter and flushed with nitrogen; the samples were analyzed within 2h. Oxalic acid extraction and nitrogen flushing was used to stabilize the ascorbic acid. The ascorbic acid analysis was a modification of the HPLC method described by Gensler et al. (1995). The separation was carried out by isocratic elution with solvent C (0.01M NaH2PO4 ) with detection at 250 nm. After elution of ascorbic acid the column was rinsed with solvent D (methanol:water (80:20, v:v)) for 10 min to remove impurities, and equilibrated with solvent C for 7 min. A standard equation was developed for ascorbic acid in the interval 10-60 mg AA /100g.
Oxygen analysis

aWater was added to the berries before mash treatment and also to the raw juice as described

Table 2Total anthocyanin concentration a during processing

Enzyme A mg/100g berries mashb raw juice pasteurized nectarb 36327 25215 22516 86.74.7. mg/100g pulpc 2556 % of expected 100 97 87 91 % lost 21.2 Enzyme B mg/100g 36480 25213 23717 87.42.8. mg/100g 25414 % of expected 100 97 91 92 % lost 20.8

aTotal anthocyanin calculated as the sum of delphinidin-3-glucoside, delphinidin-3-rutinoside,

Oxygen concentration in the nectar was measured by an oxygen sensor model WTW OximeterTM Oxi 538 with electrode StirroxTM G, both from WTW, D-82362 Weilheim, Germany. The oxygen concenJOURNAL OF FOOD SCIENCEVolume 64, No. 1, 1999

cyanidin-3-glucoside and cyanidin-3-rutinoside. bWater was added to the berries before mash treatment and also to the raw juice as described in Materials & Methods. Percentage of expected when the effect of dilution is normalized. cPercentage lost, when loss of anthocyanin in the pulp is considered, calculation based on a mass balance for anthocyanin: mash raw juice pulp.

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enzymes gave similar anthocyanin extractions. Anthocyanins were lost for two main reasons which have to be considered separately. In the main stream process, anthocyanins were destroyed as a result of enzymatic break-down and heat treatment; in addition, some anthocyanins were removed with the pulp. Degradation of anthocyanins during processing at the applied dosage level of enzymes was limited. After 2h mash treatment at 50C, 97% of the original anthocyanin content in the berries was intact. The mash was separated in a screw-strain centrifuge into raw juice and pulp. After separation the raw juice was left for 1h at 35C, then mixed with sugar, honey and water and pasteurized. More anthocyanin was lost during this standing period than during the mash treatment. This confirmed results of Wightman and Wrolstad (1995), who reported that the anthocyanin destroying activity of mash enzymes was much higher after pulp removal. A much higher loss of anthocyanin and ascorbic acid has occurred in industrial practice. The content of monomeric anthocyanins and ascorbic acid in Ribena black currant drink concentrate has been reported (Miller and Rice-Evans, 1997). Concentrations of anthocyanins were 0.035 mM dpd-glu, 0.12 mM dpd-rut, 0.014 mM cyd-glu and 0.09 mM cyd-rut (calculated as concentrate). Compared with our data (Table 3) it is seen that anthocyanin concentration in the black currant nectar was about 5 times higher than in the undiluted black currant drink concentrate. Concentration of ascorbic acid in Ribena drink concentrate was 0.22 mM and in black currant nectar 2.0-2.4 mmol/kg. During production of clarified black currant nectars or juice drinks it is common practice after pressing to leave the raw juice for hours or days in storage tanks for stabilization before final clarification and pasteurization. This is probably a very critical point because the mash enzymes are highly active at that step, and major losses of anthocyanins and ascorbic acid may result.
Nectar storage

Table 3Anthocyanins in Ben Lomond berries and the corresponding nectar

dpd-glu berries mg kg-1 mmol kg-1 % mg kg-1 mmol kg-1 % 3197 0.690.02 12.7 794 0.170.01 9.2 dpd-rut 168990 2.760.15 50.7 4566 0.750.01 53.2 cyd-glu cyd-rut 1195 102437 0.270.01 1.720.06 5.0 31.6 312 2917 0.070.01 0.490.01 3.6 34.0

nectar

Table 4Kinetic constants for ascorbic acid and total anthocyanin contenta
Compound Ascorbic acid Total anthocyanin Conditions light darkness light darkness k10-3 (days) 1.0 1.1 4.2 3.2 t (days) 693 630 165 216 r2 0.83 0.77 0.95 0.94

aCalculations refer to Fig. 2 and 3.

The four main anthocyanins: delphinidin-3-glucoside (dpd-glu), delphinidin-3-rutinoside (dpd-rut), cyanidin-3-glucoside (cyd-glu) and cyanidin-3-rutinoside (cyd-rut), were quantified. Their distribution in cloudy nectar produced from black currants (cv Ben Lomond) is shown (Table 3). The distribution of anthocyanins depends very much on the cultivar and Ben Lomond has a rather high delphinidin/cyanidin ratio.

The nectar was pasteurized at 80C for 27s and stored at 20C in daylight; degradation of individual anthocyanins under these conditions was followed (Fig. 1). The concentration of each anthocyanin on the day of production corresponded to 100%. The degradation rates of the four main anthocyanins were not significantly different. The degradation of total anthocyanin and ascorbic acid in light (Fig. 2) and dark storage (Fig.3) was compared. Anthocyanin destruction was much greater than for ascorbic acid both under conditions of natural lighting and in the dark. The kinetic parameters are shown (Table 4). The degradation of anthocyanin and ascorbic acid was described as a first order reaction, although the correlation coefficient for ascorbic acid was lower than for the anthocyanins. The slopes of the regression lines (equivalent to reaction constants) were compared by statistical methods. The reaction constants of anthocyanin degradation in light (k 4.2 103) and in darkness (k 3.2 103) were different (P0.05). The reaction constants for ascorbic acid in

Fig. 1Degradation of individual anthocyanins in black currant nectar stored in bottles at 20C in daylight. The concentration of each anthocyanin on the day of production corresponds to 100 %. dpd-glu: delphinidin-3-glucoside; dpd-rut: delphinidin-3rutinoside; cya-glu: cyanidin-3-glucoside; cya-rut: cyanidin-3-rutinoside. LSD = 7.0.

Fig. 2Degradation of total anthocyanin and vitamin C content in black currant nectar stored at 20C in daylight. LSD vitamin C = 0.110. LSDanthocyanin = 0.065.

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light (k 1.1 103) and in darkness (k 1.0 103) were not different (P0.05). The degradation rate of total anthocyanin appeared to be four times faster than ascorbic acid degradation in storage in daylight (Table 4), and about three times faster in dark storage. The degradation rate of anthocyanins in black currant juice concentrate stored in the dark was reported by Eder (1996), who concluded that delphinidins and cyanidins broke down at very similar rates. The reaction kinetics fitted well to a first order reaction and the reaction constants, calculated for each of the anthocyanins, were between 6.3 and 7.5 103 (days) with storage at 17C. Taylor (1989) studied the degradation rate of the same four anthocyanins in juices from 40 different black currant cultivars. In that study the juice was pasteurized by immersing the whole bottle in hot water at 75C for 15 min. Delphinidin-3-rutinoside was concluded to be more stable than cyanidin-3-rutinoside when the bottles were stored at room temperature under conditions of natural lighting. In that study the degradation rate seemed to be of mixed orders. The degradation of ascorbic acid in a model system consisting of ascorbic acid and glucose or sucrose was also reported as a first order reaction (Rojas and Gerschenson, 1997), but under these conditions the reaction constants were approximately 5 times higher than in black currant nectar. Several reports on ascorbic acid suggest that the decomposition of this substance in stored juice systems can also be zero-order (Davies et al., 1991). This indicates that the kinetics of degradation of both ascorbic acid and anthocyanins depends very strongly on the nature of the total system, i.e. the presence of anthocyanins, ascorbic acid and other antioxidants. Declines in ascorbic acid content have been compared in apple, orange and black currant juice/drink (Miller and Rice-Evans, 1997). It was shown that ascorbic acid was considerably more stable in the black currant drink when the beverages were subjected to conditions of mild oxidation. The antioxidant activity of several phenolic compounds was measured. However, ascorbic acid, and not the monomeric anthocyanins in black currant drinks, was found to be the main source of antioxidant activity. A large part of the antioxidant activity in black currant drink was unaccounted for, but was ascribed to some unidentified phenolics (Miller and Rice-Evans, 1997). It seems reasonable to suggest that such unidentified compounds might well be anthocyanin polymers, as the main part of the original anthocyanins in Ribena were probably transformed into their polymeric forms (Francia-Aricha et al., 1997; Roux and Ferreira, 1985). Both ascorbic acid and anthocyanins can form Table 5Oxygen concentration (ppm) in the nectar during storage.
Day Deaerated Not deaerated
a,bDifferent at P 0.01.

0 1.60a0.07 5.10b0.14

1 1.050.07 1.070.07

35 0.080.01 0.070.01

75 0.050.01 0.050.01

stable radicals. The vitamin C protecting effect of anthocyanins in black currant drink results in losses of the anthocyanins. Ascorbic acid radicals can be regenerated to ascorbic acid (Davies et al., 1991) by oxidizing one molecule of anthocyanin to its well stabilized radical form. We carried out another storage experiment with black currant nectar that had been either deaerated or not deaerated before filling into bottles. Oxygen concentration was measured in both nectars during the first period of storage, as can be seen (Table 5) oxygen concentration decreased so rapidly during storage that there was no difference between deaerated nectar and not deaerated nectar after one day. From these data it can be concluded that deaeration of nectar was of no value for increasing anthocyanin or ascorbic acid stability, and this was confirmed (Fig. 4). When the slopes of the two regression lines for anthocyanin were compared no difference was seen, and the same hold for ascorbic acid.

CONCLUSIONS
RETENTION OF ANTHOCYANINS AND VITAMIN C DURING NECTAR production depends on the process as well as nature of the raw material. We used a continuous production process where the final pasteurized nectar was produced within 4.0h. Under these conditions the main stream process loss of both anthocyanins and ascorbic acid was limited. Anthocyanin destroying activity of added enzymes was limited during mash treatment when the recommended dosage levels were used. However, it was much higher in the raw juice after pulp removal. After bottling the nectar, a considerable loss of total anthocyanins occurred during storage. Only half the original content of monomeric anthocyanins was left after 6 months. The degradation of the four main anthocyanins, two delphinidin-glucosides and two cyanidinglucosides indicates no differences between delphinidins and cyanidins. Deaeration of the nectar before tapping did not increase anthocyanin stability.

Fig. 3Degradation of total anthocyanin and vitamin C content in black currant nectar stored at 20C in the dark. LSDvitamin C = 0.061. LSD anthocyanin = 0.101.

Fig. 4Degradation of total anthocyanin (AC) and vitamin C content in black currant nectar stored at 20C in daylight. LSD vitamin C = 0.097. LSD antocyanin = 0.102.

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Mazza, G. and Miniati, E. 1993. Introduction. Ch. 1 in Anthocyanins in Fruits, Vegetables, and Grains, G. Mazza (Ed.). CRC Press, Boca Raton, FL. Miller, N.J. and Rice-Evans, A.C. 1997. The relative contributions of ascorbic acid and phenolic antioxidants to the total antioxidant activity of orange and apple fruit juices and black currant drink. Food Chem. 60: 331-337. Nrbaek, R., Christensen, L.P., Bojesen, G., and Brandt, K. 1996. Anthocyanins in Chilean species of Alstromeria. Phytochemistry 42: 97-100. Rice-Evans, A.C., Miller, N.J., and Paganga, G. 1996. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology & Medicine 20: 933-956. Rojas, A.M. and Gerschenson, L.N. 1997. Ascorbic acid destruction in sweet aqueous model systems. Lebensm.-Wiss. und Technol. 30: 567-572. Roux, D.G. and Ferreira, D. 1985. Part 12. Rationalization of divergent condensation sequences in flavonoid oligomerization. In Annual proceedings of the phytochemical society of Europe, vol 25, The biochemistry of plant phenolics, C.F. Van Sumere C.F. and P.J. Lea (Ed.). Clarendon Press, Oxford, UK. Skrede, G., Wrolstad, R.E., Lea, P., and Enersen, G. 1992. Color stability of strawberry and black currant syrups. J. Food Sci. 57: 172-177. Starr, M.S. and Francis, F.J. 1968. Oxygen and ascorbic acid effect on the relative stability of four anthocyanin pigments in cranberry juice. Food Technol. 22: 12931295. Taylor, J. 1989. Colour stability of black currant (Ribes nigrum) juice. J. Sci. Food Agric. 49: 487-491. Viberg, U., Ekstrm, G., Fredlund, K., ste, R., and Sjholm, I. 1997. A study of some important vitamins and antioxidants in a black currant jam with low sugar content and without additives. Int. J. Food Sci. & Nutr. 48: 57-66. Wightman, J.D. and Wrolstad, R.E. 1995. Anthocyanin analysis as a measure of glycosidase activity in enzymes for juice processing. J. Food Sci. 60: 862-867. Wightman, J.D. and Wrolstad, R.E. 1996. -glucosidase activity in juice-processing enzyme based on anthocyanin analysis. J. Food Sci. 61: 544-547. Wrolstad, R.E. 1976. Color and pigment analyses in fruit products. Station Bulletin 624. Oregon State Univ. Reprinted May 1993. Wrolstad, R.E., Wightman, J.D., and Durst R.W. 1994. Glycosidase activity of enzyme preparations used in fruit juice processing. Food Technol. 48(11): 90-98. Zar, J.H. 1984. Comparing simple linear regression equations, Ch. 18 in Biostatistical analysis, J.H. Zar (Ed.). Prentice-Hall Inc.: Englewood Cliffs, NJ. Ms received 2/20/98; revised 7/2/98; accepted 7/31/98.
This work was supported by the Danish Directorate for Development; Ministry for Food, Agriculture & Fisheries.

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