JFS:

Food Chemistry and Toxicology

The Effects of Antioxidants on Browning and on Degradation Products Caused by Dehydroascorbic Acid
M. SAWAMURA, T. NAKAGAWA, S. KATSUNO, H. HAMAGUCHI, AND H. UKEDA

Food Chemistry and Toxicology

ABSTRACT: The efficiency of L-cysteine (cysteine) and sodium sulfite as antioxidants was examined in the browning of an aqueous solution of 100 mM dehydroascorbic acid (DHA). The browning was suppressed at 100 mM cysteine and at 40 mM and higher concentrations of sodium sulfite, but it increased in the presence of 10 mM of those agents. These agents did not allow the reduction of DHA to L-ascorbic acid (AA). These results suggest that the suppression or acceleration of browning is likely to be related to some degraded intermediates of DHA. The two colorless intermediates, which during DHA breakdown eventually transform into browning pigments, were discussed with regard to the browning regulation mechanism. Key Words: browning, dehydroascorbic acid, ascorbic acid, antioxidant, sodium sulfite

Introduction

ONENZYMIC

BROWNING HAS BEEN

noted as a food quality issue. Some browning, caused by Strecker degradation and aminocarbonyl reaction, is considered favorable in cooked foods such as roasted meat and baked goods, including cookies and biscuits. Other occurrences of browning are a serious problem for manufacturers of juices and wines. The majority of reports on nonenzymic browning have focused on citrus browning (Clegg 1964; Robertson and Samaniego 1986; Handwerk and Coleman 1988; Kennedy and others 1990; Nagy and others 1990; Lee 1992; Lee and Nagy 1992; DelCastillo 1998). The browning of citrus juices such as yuzu (Citrus junos Tanaka), lemon and grapefruit are more conspicuous than that of orange and mandarin juices, because the natural color of those juices is white as a result of a shortage of carotenoids. The browning of citrus juices is principally caused by the degradation of DHA (Li and others 1989). Citrus fruit is one of the important resources of Vitamin C. Citrus juices contain ascorbic acid (AA) in the range between 30 and 60 mg/100 g. The process of browning proceeds slowly from AA, but once DHA is formed in the juice, the browning is accelerated. The authors have studied browning from DHA in a model solution (Sawamura and others 1991), and identified 2 degraded compounds from DHA: 3,4-dihydroxy-5-methyl-2-5(H)-furanone as a browning pigment; 2-furancarboxylic acid as a colorless product (Sawamura and others 1994). However, several brown pigments and colorless intermediates which are precursors of browning pigments re-

main unidentified. Cysteine and sodium sulfite are known to be effective as antioxidants in food and beverage. Friedman and Molnar-Perl (1990) and Molnar-Perl and Friedman (1990) reported the inhibition of browning by sulfur-containing amino acids including cysteine in amino acid-glucose model system and fruit juices. There has been, however, no report on the effects of these additives on browning caused by DHA. The present study aims to investigate the effects of antioxidants on the browning and the behavior of intermediates of DHA degradation in a model.

Results and Discussion

Effects of cysteine and sodium sulfite on browning
Addition of amino acids to DHA solution accelerated increases in the browning, due to the Maillard reaction as well as DHA degradation. The effects of several amino acids on browning are shown in Fig. 1. L-Aspartic acid and glycine (which are found in citrus juices) and L-lysine increased browning. Cysteine, containing a SH functional group, was expected to prevent browning (Friedman and Molnar-Perl 1990; MolnarPerl and Friedman 1990). The degree of browning that was developed by cysteine, however, surpassed that for other amino acids, reaching nearly double that of a control solution of DHA. It does not seem that an excess of browning caused by cysteine, in comparison to that of other amino acids, resulted from the Maillard reaction, because there was no difference in the degree of browning between both aspartic acid and glycine and lysine, composing a more

reactive -amino group with carbonyl compounds. Friedman and Molnar-Perl (1990) investigated the relationship between browning and the ratio of amino acid to glucose. They examined the browning at various ratios of 1 to 24:1 or 1:1 to 4 of -alanine to glucose. They also mentioned that the SH-containing compounds played the role of nucleophiles and then reduced carbonyls or reacted with carbonyl groups and double bonds in brown products to form colorless materials. In our model system the ratios of amino acids to DHA were 1:1, 1:4, and 1:10. In the case of the ratio of 1:10, where the browning was accelerated by 10 mM cysteine, it is assumed that addition of the SH group because of low proportion of cysteine to DHA would rarely occur. We

Fig. 1Effects of cysteine and several amino acids on browning of DHA. Amino acids of 10 mM in 100 mM DHA were as follows: none (DHA alone) (), L-cysteine (), L-aspartic acid (), glycine () and L-lysine ().

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should consider not only reductivity of the antioxidants, but also the ability of addition to carbonyl compounds as intermediates from DHA degradation. Sodium sulfite is known as a popular and strong agent for antioxidation and bleaching. Figure 2 shows the change of browning in the presence of sodium sulfite during storage. The tendency for browning of a 10 mM sodium sulfite solution in DHA was the same as that of a 10 mM cysteine solution (Fig. 1). Though the browning was accelerated in 10 mM sodium sulfite, it was suppressed by the addition of 40 mM sodium sulfite, and did not occur in a solution of 100 mM sodium sulfite over a 28-d period. We examined the effect of a mixture of cysteine and sodium sulfite at 10 mM or 100 mM on browning (data are not shown). Those results were similar to those described above. It does not seem that there is a synergistic effect of these 2 antioxidants. Cysteine and sodium sulfite have been widely used as food additives aimed at antioxidation, and the prevention of browning. It was, however, demonstrated that these additives did not always prevent browning, but could also promote browning, depending on their concentration. Knowledge of the precise behavior of these antioxidants is scant.

amount of AA after 28 d. The retention of AA with cysteine and sodium sulfite (10 mM) showed almost the same behavior as the AA control, in spite of some difference in the degree of browning (Fig. 3, top). No conversion of DHA to AA occurred in either the absence or presence of cysteine and sodium sulfite at 100 mM (data are not shown). We observed that addition of 100 mM cysteine or sodium sulfite to a specific citrus (yuzu) juice suppressed browning. According to Japan Food Hygienic Law, however, the tolerance of cysteine and sodium sulfite in juice is zero and less than 0.15 g/kg as SO2, respectively. An aqueous solution of 100 mM sodium sulfite accounts for 7 g/kg. We must consider, of course, the safety of these additives, when we practically apply it to juice products. We do not think the concentration of 100 mM sodium sulfite used in this experiment is too high, if we consider the model solution of 100 mM AA or DHA. It is not our goal to prevent browning of citrus juice products by use of these additives, but to elucidate the mechanism of browning and find more safe, effective, and natural inactivators of brown-

Effects of cysteine on the degradation products of DHA

The 3 colorless products and 4 brown pigments degraded from DHA alone, named arbitrarily here C-I, C-II, and C-III, and B-I, B-II, B-III, and B-IV, respectively, are formed in the process of browning (Sawamura and others 1991). The authors have already identified C-III and B-III as 2furancarboxylic acid and 3,4-dihydroxy-5methyl-2-5(H)-furanone, respectively. The formations of these colorless products were examined in the presence of 10 mM cysteine in 100 mM DHA (Fig. 4). Three amino acids L-aspartic acid, glycine, and Llysine were simultaneously examined and compared with cysteine. It has been observed that C-II is obtained in the form of a white powder after lyophilization, but it immediately changes into a brown pigment, B-IV, upon returning from vacuum to normal atmospheric pressure. C-III can be obtained as white crystalline substance and is one of the final and most stable products of DHA degradation. As shown in Fig. 4, these amino acids solutions in DHA showed a similar behavior to the control of DHA in the formation of C-II and C-III. It was suggested that the formation of C-II and C-III was not affected by amino acids. When C-I was formed, the cysteine sample gave a different behavior from the control and other amino acids. It is supposed that the decrease of C-I caused by cysteine may bear some relation to the acceleration of browning indicated in Fig. 1.

Browning from AA
An aqueous solution of 100 mM AA was browned slowly, as shown in Fig. 3 (top). Its degree of browning after 28-d storage was a quarter of that for the DHA control in Fig. 1 and 2. Addition of cysteine and sodium sulfite to DHA solutions further decreased browning. There was no increase in browning in an AA solution containing 100 mM sodium sulfite during a 28-d period. Figure 3 (bottom) shows that AA with 100 mM sodium sulfite retained 95% of the initial

Effects of sodium sulfite on the degradation products of DHA

The levels of C-I, C-II, and C-III decreased when the concentration of sodium sulfite increased. (Fig. 5). This suggests that sodium sulfite affects the overall mechanism of the degradation or browning of DHA. There was no difference in the formation of C-II and C-III between the DHA control and the solution of 10 mM sodium

Fig. 2Effects of sodium sulfite on browning of DHA. Sodium sulfite of different concentrations in 100 mM DHA were as follows: none (DHA alone) (), 10 mM (), 40 mM ( ) and 100 mM ().

Fig. 3Effects of cysteine and sodium sulfite on browning from AA (top) and retention of AA (bottom). The materials were added to 100 mM AA as follows: none (AA alone) (), 10 mM L-cysteine ( ), 10 mM sodium sulfite () and 100 mM sodium sulfite ( ).

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Food Chemistry and Toxicology

ing. Yuan and Chen (1998) reported that a predominant degradation product of AA was furfural at pH 1. The pH of DHA aqueous solutions, in addition to the pH of the model solutions to which several amino acids and sodium sulfite were added at various concentrations, was about the same (pH 1). Thus, the present experiments were carried out under the same pH conditions by using MacIlvaine buffer (pH 1.2). The browning of citrus juices results primarily from the oxidation of AA to DHA, where the role of furfural is not so great in browning (Li and others 1989). That is the motivation for investigating the degradation and browning caused by DHA in a model (Sawamura and others 1991, 1994).

Prevention of Nonenzymatic Browning . . .

sulfite during a 7-d period. C-I seems to be formed quickly from the beginning of the reaction, reaching a maximum within 7 d, then decreasing gradually. C-I and C-II have been considered to be precursors of the brown pigments. Therefore, the suppression of these products at 40 mM and 100 mM sodium sulfite is associated with decrease of the browning activity as described in Fig. 2. However, from the beginning of storage, the formation of C-I at 10 mM sodium sulfite was as low as that at 10 mM cysteine (Fig. 4). It is suggested that CI is reactive with cysteine and sodium sulfite or that these agents are involved in the suppression of C-I formation. solution. The browning of DHA solution (control) increased continuously for 14 d. The browning of 10 mM cysteine in DHA increased twice as much as that of the control in a 14-d period, and further increased in a 100 mM cysteine solution. The level of C-I, in contrast, decreased, reaching 0 at 100 mM cysteine. From these results, we hypothesize a mechanism in which another browning pigment would be readily formed from the reductant of C-I reduced

Addition of cysteine or sodium sulfite to the browning media

We can observe the behavior of C-I and C-II and the relationship between browning and those intermediates by adding cysteine or sodium sulfite to previously browned DHA solutions, as shown in Fig. 6. Browning activity of C-I and C-II during 14-d storage was significantly different (P < 0.05) by t-test between the DHA alone and other treatments except for the browning activity of 10 mM sodium sulfite

Food Chemistry and Toxicology

Fig. 4Effects of cysteine and several amino acids on the formation of the degradation products of DHA. Amino acids of 10 mM in 100 mM DHA were as follows: none (DHA alone) (), L-cysteine (), L-aspartic acid (), glycine () and L-lysine ().

Fig. 5Effects of sodium sulfite on the formation of the degradation products of DHA. Sodium sulfite of different concentrations in 100 mM DHA were as follows: none (DHA alone) (), 10 mM (), 40 mM () and 100 mM ().

Fig. 6Effects of cysteine and sodium sulfite added to 7-d stored DHA solutions on the browning and C-I and C-II degradation products. The materials were added to 100 mM DHA stored previously for 7 d, then further stored for another 7 d as follows: none (DHA alone) (a), 10 mM L-cysteine (b), 100 mM Lcysteine (c), 10 mM sodium sulfite (d), 40 mM sodium sulfite (e) and 100 mM sodium sulfite (f). ND: Not detected.

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by antioxidants. While the solution of 10 mM sodium sulfite was not significantly different from the control in browning, it still only showed half of the browning of that of cysteine. We expected that the browning tendency of sodium sulfite at 10 mM would be similar to that for cysteine, considering the data in Fig. 1 and 2. The fact that the amount of browning in 10 mM sodium sulfite was smaller can be explained by the level of C-I, which was not so different from the control after 14 days. However, it is not clear why C-I was not reduced as much with sodium sulfite, whose reducing power is stronger than that of cysteine. Antioxidants were added to 7-d DHA solutions, as shown in Fig. 6. This status is quite different from the experiments represented in Fig. 1 and 2, where there would be many intermediates degraded from DHA in 7-d old stored solutions. Sodium sulfite would have been consumed to reduce most of these intermediates, resulting in a small degree of browning. If antioxidants were added from the onset of storage, as seen in Fig. 1 and 2, C-I would be more predominantly reduced among the few intermediates in the solutions. Thus, it seems that there is no contradiction between the behaviors in the 2 experiments for 10 mM cysteine and sodium sulfite and those described in Fig. 1 and 2.

In the case of 40 mM and 100 mM sodium sulfite in Fig. 6, the degree of browning of the solutions was decreased. In 10 mM sodium sulfite, the level of C-I was less than the control, although there was a small increase of the browning activity. The level of C-I decreased considerably with a decrease of browning in 40 mM sodium sulfite, and disappeared in 100 mM of sodium sulfite. The reduced form of C-I seems to be blocked from proceeding in a new browning mechanism in the presence of sodium sulfite. In these solutions sodium sulfite also affects suppression of oxidation in the overall mechanism of DHA degradation to browning. The authors presume that C-II is probably a precursor of B-IV, because the deeply browned pigment derived from C-II gives the same retention time on a chromatogram as that of B-IV. All the levels of cysteine and sodium sulfite except 10 mM, cysteine reduced the formation of C-II. The C-II level in 100 mM sodium sulfite was equal to that of the control for the first seven days. This result suggests that the conversion of C-II to B-IV was effectively blocked by 100 mM sodium sulfite. In conclusion, AA is not reconverted from DHA even in the presence of cysteine and sodium sulfite. Therefore, DHA is directly broken down to form the degradation products via 2,3-DKG and L-xylosone

without conversion of DHA to AA. Sodium sulfite blocks the conversion of C-II to B-IV in a DHA aqueous solution, while C-I shifts rapidly to formation of browning pigments. It is supposed that cysteine would reduce C-I to form the reductant of C-I, but the reducing power of cysteine is not strong enough to advance the reductant of C-I to form a new browning mechanism. Sodium sulfite reduces C-I to its reductant, and can suppress the oxidation of the reductant, resulting in browning due to its strong antioxidative property. There is also a possibility of the addition of cysteine or sodium sulfite to carbonyl compounds. If browning is suppressed by the formation of adducts with either sulfite or cysteine and carbonyls, we cannot explain the increase of browning at 10 mM sulfite or cysteine without further study on the interaction between the reductivity and addition reactivity of sodium sulfite and cysteine. We suggest that the overall system of DHA breakdown is perfectly suppressed in the presence of 100 mM sodium sulfite. There is an obvious need for further studies of the identification of intermediates and browning pigments, as are studies of the acceleration and suppression of browning with sodium sulfite and cysteine. This knowledge would be useful for feedback to browning suppression of citrus juices.

Materials and Methods

Preparation of DHA
An aqueous DHA solution was prepared according to the method described in Rose and Nahrwold (1981), in which a solution of AA is oxidized into DHA with bromine. After completion of AA to DHA, the remaining bromine in the solution was substituted with nitrogen. A 10-mL solution of DHA with a given concentration of sodium sulfite or Lcysteine (free from HCl) in MacIlvaine buffer (pH 1.2) was stored in a 10-mL vial sealed tightly with a gum stopper and an aluminum ring at 37 0.1 C for

28 d in the dark. The headspace gas in the vial was 3.7 mL atmosphere containing 0.74 mL oxygen. Thus, the oxygen level in a vial was about 0.03 mmoles. All the measurement was triplicated and the results were shown in mean of triplicate. The standard deviation was designated with bars in figures, when necessary.

HPLC operations
The stored solution was filtered through a cellulose nitrate membrane (FM-22) (Fuji Photo Film Co. Ltd., Tokyo, Japan) to be analyzed by HPLC, monitored at 250 nm and 290 nm. Browning

and AA content were measured by absorbance at 420 nm and 210 nm, respectively. A Toyo Soda HPLC composed of a CCPE pump, a UV-8000 absorbance detector and a Rheodyne Model 7125 injector with a 25 l-loop was used. A reversephase column, Wakosil 5C18 (4.6 mm i.d. 150 mm, Wako Pure Chemical Industries, Ltd., Osaka, Japan), was used. The column temperature was 40 C and 0.2 % metaphosphoric acid was used isocratically as the mobile phase at 1.0 mL/min. All the chemical reagents used were of the highest purity of Wako Pure Chemical Industries (Osaka, Japan). Milli-Q water was used in the experiment.

References
Clegg KM. Nonenzymic browning of lemon juice. 1964. J. Sci. Food Agric. 15:878-885. DelCastillo MD, Corzo N, Polo, MC Pueyo E, and Olano A. 1998. Changes in the amino acid composition of dehydrated orange juice during accelerated nonenzymatic browning. J. Agric. Food Chem. 46:277-280. Friedman M, Molnar-Perl. 1990. Inhibition of browning by sulfur amino acids. 1. Heated amino acid-glucose systems. J. Agric. Food Chem. 38:1642-1647. Handwerk RL, Coleman RL. 1988. Approaches to the citrus browning problem. A review. J. Agric. Food Chem. 36:231236. Kennedy JF, Rivera ZS, Lloyd LL, Warner FP, Jumel K. 1990. Studies on non-enzymic browning in orange juice using a model system based on freshly squeezed orange juice. J. Sci. Food Chem. 52: 85-95. Lee HS. 1992. Antioxidative activity of browning reaction products isolated from storage-aged orange juice. J. Agric. Food Chem. 40:550-552.

Lee HS, Nagy S. 1992. Quality changes and nonenzymic browning intermediates in grapefruit juice during storage. J. Food Sci. 53:168-172. Li ZF, Sawamura M, Yano H. 1989. Effect of oxygen on the browning and formation of furfural in yuzu juice. Agric. Biol. Chem. 53:1979-1981. Molnar-Perl, Friedman M. 1990. Inhibition of browning by sulfur amino acids. 2. Fruit juices and protein-containing foods. J. Agric. Food Chem. 38:1648-1651. Nagy S, Lee H, Rouseff RL, Lin JCC. 1990. Nonenzymic browning of commercially canned and bottled grapefruit. J. Agric. Food Chem. 38:343-346. Robertson GL, Samaniego CML. 1986. Effect of initial dissolved oxygen levels on the degradation of ascorbic acid and the browning of lemon juice during storage. J. Food Sci. 51:184-192. Rose RC, Nahrwold DL. 1981. Quantitative analysis of ascorbic acid and dehydroascorbic acid by high-performance liquid chromatography. Anal. Biochem. 114:140-145. Sawamura M, Takemoto K, and Li ZF. 1991. 14C studies on browning of dehydroascorbic acid in an aqueous solu-

tion. J. Agric. Food Chem. 39:1735-1737. Sawamura M, Takemoto K, Matsuzaki K, Ukeda H, Kusunose H. 1994. Identification of two degradation products from aqueous dehydroascorbic acid.J. Agric. Food Chem. 42:1200-1203. Yuan JP, Chen F. 1998. Degradation of ascorbic acid in aqueous solution. J. Agric. Food Chem. 46:5078-5082. MS 19990318 received 3/10/99; revised 10/15/99; accepted 12/2/99.

Authors Sawamura, Hamaguchi, and Ukeda are with Dept. of Bioresources Science, Faculty of Agriculture, Kochi University, B-200 Monobe, Nankoku, Kochi 783-8502, Japan. Authors Nakagawa and Katsuno, are now with Zen-Noh Chokuhan Co. Ltd., 700-1 Kohya, Tomisatomachi, Imba-gun, Chiba 286-0216, Japan. Address inquiries to Dr. Masayoshi Sawamura (Email: sawamura@cc.kochi-u.ac.jp).

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