Kinetics of Disappearance and Discoloration of L-Ascorbic Acid 2-Glucoside Powders with Different Water Contents

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1 Japan Journal of Food Engineering, Vol. 9, No. 3, pp , Sep. 28 Kinetics of Disappearance and Discoloration of L-Ascorbic Acid 2-Glucoside Powders with Different Water Contents Lan-hsin HUNG, Yukitaka KIMURA and Shuji ADACHI Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto , Japan The disappearance and discoloration of L-ascorbic acid 2- glucoside powders with water contents of 2 to 12% (w/w) during storage at a temperature from 7 to 9 were investigated. The disappearance of L-ascorbic acid 2-glucoside with the higher water content proceeded faster at the higher temperature. The disappearance process was expressed by the Weibull equation. The change in the L-ascorbic acid was analyzed by assuming that both the hydrolysis and decomposition of L-ascorbic acid were expressed by first-order kinetics. A kinetic comparison of the overall disappearance by the hydrolysis indicated that the hydrolysis was not the sole route for the disappearance. The discoloration kinetics was also analyzed on the basis of the modified Weibull equation. It was shown that the enthalpy-entropy compensation held for all the processes involved in the overall disappearance of L-ascorbic acid 2-glucoside, its hydrolysis, the decomposition of L-ascorbic acid and the discoloration of L-ascorbic acid 2-glucoside. It was also demonstrated that the discoloration was suppressed by the glucosylation of L-ascorbic acid, but that L-ascorbic acid 2-glucoside was more easily discolored than L-ascorbyl 6-palmitate. Key words: L-Ascorbic acid 2-glucoside, disappearance kinetics, discoloration kinetics, water content 1 Introduction L-Ascorbic acid, known as vitamin C, is widely used in food systems because it is an essential nutrient for humans and also a natural antioxidant. However, L-ascorbic acid easily decomposes depending on many factors, e.g., oxygen partial pressure, ph, temperature, and the presence of heavy metal ions [1]. In addition to its decomposition, L-ascorbic acid also easily discolors during the storage of food products. The discoloration of L-ascorbic acid in the solid state has already been investigated [2-4]. The susceptibility of L-ascorbic acid to discoloration has led to the interest in derivatives with an increased stability. For example, derivatives of L-ascorbic acid, which were acylated with long-chain fatty acids, were synthesized [5,6]. The acylation altered the highly hydrophilic behavior of L-ascorbic acid that improved its solubility and miscibility in a hydrophobic environment; for example, L-ascorbyl 6-palmitate is increasingly used as a preservative in edible oils or as an antioxidant in other foods. Austria et al. [7] reported that L-ascorbyl 6-palmitate was (Received 17 Apr. 28: accepted 16 Jun. 28) Fax: , adachi@kais.kyoto-u.ac.jp more stable than L-ascorbic acid in their solutions. We also investigated the discoloration kinetics of L-ascorbyl 6-palmitate in the solid state with a water content of 2 to 1% (w/w) during storage at a temperature of 6 to 9 by dissolving it in water and monitoring the absorbance of the solution at 5 nm [8]. It was found that the acylation seemed to suppress the discoloration of the L-ascorbyl moiety. The effect of the hydroxyl group modification at the C-2 position of the L-ascorbic acid on its discoloration is also of interest. For example, L-ascorbic acid 2-glucoside has been synthesized by enzymatic transglucosylation, and it showed a high stability in a neutral solution [9]. L-Ascorbic acid 2-glucoside also showed a higher stability toward oxidative stress and UV irradiation than L-ascorbic acid [1,11]. L-Ascorbic acid 2-glucoside is widely used in skin care products and as a food additive [12,13]. Kubota et al. [12] reported that L-ascorbic acid 2-glucoside can effectively inhibit the discoloration and/or odor of a composition and thus improving the storage stability of the composition. However, the discoloration of the L-ascorbic acid 2-glucoside in its solid state has not been reported. Therefore, we measured the discoloration of the L-ascorbic acid 2-glucoside samples with various water contents to examine

2 136 Lan-hsin HUNG, Yukitaka KIMURA and Shuji ADACHI whether or not the hydrophilic glucose moiety affects the discoloration. Because the hydrolysis of the glucoside would occur and affect the discoloration of the L-ascorbyl moiety, the degree of hydrolysis was also measured. The discoloration and hydrolysis were then kinetically analyzed. 2 Materials and Methods 2.1 Materials L-Ascorbic acid 2-glucoside (purity, >99.9%) was purchased from Hayashibara Biochemical Laboratories (Okayama, Japan). Tetrabutylammonium hydroxide (4% (w/w) in water) was purchased from Aldrich Chemical Company, Inc. (USA). All other chemicals of analytical grade were purchased from Wako Pure Chemical Industries (Osaka, Japan). the dilution was applied to the column, which was kept at 3, and eluted with the eluent at the flow rate of.8 ml/ min. The absorbance of the effluent was measured at 26 nm and then recorded. 3 Results and Discussion 3.1 Disappearance of L-ascorbic acid 2-glucoside Figures 1 (a), (b) and (c) show the changes with time in the fraction of the remaining L-ascorbic acid 2-glucoside which had the water contents of 2 to 12% (w/w) and stored at 9, 8 and 7, respectively. L-Ascorbic acid 2-glucoside with the higher water content more rapidly disappeared. The disappearance also proceeded faster at the higher temperatures. Because the disappearance of the 2.2 Sample preparation The stability of the L-ascorbic acid 2-glucoside powders was investigated with water contents from 2 to 12% (w/w). The L-ascorbic acid 2-glucoside powders (5 mg) were placed in a vial. Distilled water was pipetted over the powders in the vial to produce a water content of 2, 4, 6, 8, 1 or 12% (w/w), and the vial was then screw-capped. The samples were then stored at a temperature of 6, 7, 8 or 9. A sample (27 mg) was periodically removed from the vial and analyzed. 2.3 Analysis The discoloration of the L-ascorbic acid 2-glucoside powders was monitored by dissolving 27 mg of the powders in 3 ml of distilled water and measuring the absorbance of the solution using a Shimadzu UV-16 UV-visible spectrophotometer (Kyoto, Japan). The wavelength range from 3 to 6 nm was scanned at a sampling pitch of 2. nm. The optical path length was 1 cm. The sample was also analyzed to determine the fraction of unchanged L-ascorbic acid 2-glucoside using a Shimadzu LC-1AT liquid chromatograph (Kyoto, Japan) equipped with a Shimadzu SPD-6A UV spectrophotometer and Shim-pack VP-ODS column (4.6 mm 25 mm; Shimadzu). The eluent was prepared as follows: 1.4 g of KH 2 PO 4 and 6 ml of tetrabutylammonium hydroxide were dissolved in 9 ml of distilled water. The ph of the solution was adjusted to 5.1 by adding phosphoric acid. The volume of the solution was adjusted to 1. L by adding distilled water. The solution was mixed with acetonitrile at a ratio of 9/1 (v/v), and then degassed by sonication. The sample solution was diluted 1-times and 1 L of Fraction of remaining ascorbyl glucoside 1..5 (a) (b) (c) Time [d] Fig. 1 Changes with time in the fraction of the remaining L-ascorbic acid 2-glucoside with various water contents and stored at (a) 9, (b) 8 and (c) 7. The water contents were ( ) 2%, ( ) 4%, ( ) 6%, ( ) 8%, ( ) 1% and ( ) 12% (w/w).

3 Disappearance and Discoloration of L-Ascorbic Acid 2-Glucoside 137 L-ascorbic acid 2-glucoside could not be expressed by the first-order kinetics for all the samples stored at any of the temperatures, we adopted the Weibull equation, which has the potential for describing many deterioration and degradation processes [14]: m AG / m AG, exp [ (k d t) nd ] (1) where m AG and m AG, are the amounts of L-ascorbic acid 2-glucoside at any time t and the initial one, respectively. k d and n d are the rate and shape constants for the disappearance, respectively. The parameters, k d and n d, were evaluated by best-fitting the experimental data using the Solver of Microsoft Excel. The solid curves in Fig. 1 were drawn based on Eq. (1) using the estimated parameters. Because the extent of the disappearance for the sample with the water content of 2% (w/w) was low, the parameters of Eq. (1) were not estimated for the sample. The experimental results were smoothly connected by the broken curves. The water-content dependencies of the rate and shape constants, k d and n d, are shown in Figs. 2 (a) and (b), respectively. Both the k d and n d were larger at the higher water content and higher temperature. The higher k d value at the higher water content indicated that water promoted the disappearance of L-ascorbic acid 2-glucoside although the mechanism is unknown. The disappearance of the L-ascorbic acid 2-glucoside would consist of at least two processes: one is its hydrolysis to the constituent L-ascorbic acid and glucose and the other is its direct change into other compounds. The latter would include discoloration. L-Ascorbic acid 2-glucoside should be easily hydrolyzed at the higher water contents. This seems to be the reason for the higher k d value for the L-ascorbic acid 2-glucoside with higher water content. The amount of L-ascorbic acid produced by the hydrolysis of L-ascorbic acid 2-glucoside was determined for the L-ascorbic acid 2-glucoside samples with various water contents at 7, 8 and 9. Figure 3 shows the changes with time of the L-ascorbic acid amount. The ordinate is expressed by the ratio of the amount m AA of L-ascorbic acid at any time to the initial amount m AG, of the L-ascorbic acid 2-glucoside. The amount of L-ascorbic acid increased during the early stage of storage and then declined after a prolonged storage. These facts indicated that the L-ascorbic acid 2-glucoside was hydrolyzed to produce L-ascorbic acid and the acid was further decomposed. These processes were analyzed by assuming the following consecutive reactions and that each step was expressed by first-order kinetics: k d 1 1 L-Ascorbic acid 2-glucoside H 2 O k 1 (2) L-ascorbic acid glucose n d 1..5 m AA /m AG, Water content [% (w/w)] Time [d] Fig. 2 Water-content dependencies of (a) the rate constant k d, and (b) the shape constant n d for the overall disappearance of the L-ascorbic acid 2-glucoside powders at ( ) 7, ( ) 8 and ( ) 9. The curves were drawn empirically except for the points for 2% water content because of only slight progress of the disappearance at the water content. Fig. 3 Transient changes in the amount of L-ascorbic acid, which was normalized by the initial amount of L-ascorbic acid 2-glucoside, m AA /m AG, for the glucoside powders with different water contents and stored at 9. The symbols are the same as those in Fig. 1.

4 138 Lan-hsin HUNG, Yukitaka KIMURA and Shuji ADACHI L-Ascorbic acid k 2 other compounds (3) where k 1 and k 2 are the rate constants for the hydrolysis of L-ascorbic acid 2-glucoside and the further decomposition of L-ascorbic acid, respectively. The reaction order of each step was unknown. Because the shape constant n d was in the range of.3 to 1.3 for the disappearance of L-ascorbic acid 2-glucoside (Fig. 2(b)), the reaction order for the step was roughly assumed to be one. Therefore, we roughly adopted the first-order kinetics for both the hydrolysis of L-ascorbic acid 2-glucoside and the decomposition of L-ascorbic acid. Based on these assumptions, the transient changes in m AA /m AG, are given by Eq. (4) [15]: m AG 1 m (e -k 2 t e -k1t ) (4) AG, 1 where k 2 /k 1. The rate constants k 1 and k 2 were evaluated by best-fitting the experimental data using the Solver of Microsoft Excel except for the L-ascorbic acid 2-glucoside with the water content of 2% (w/w), for which the formation of L-ascorbic acid was too low to estimate the constants. The solid curves in Fig. 3 were drawn based on Eq. (4) using the estimated k 1 and k 2 values. The broken curve for the sample with the initial water content of 2% (w/w) was drawn by smoothly connecting the experimental points. Figure 4 shows the water-content dependencies of k 1 and k 2 for the hydrolysis of the L-ascorbic acid 2-glucoside and decomposition of the resultant L-ascorbic acid, respectively. The k 1 value at any temperature was in linear proportion to the water content on the semi-logarithmic scale, indicating that the k 1 became exponentially large value. This would be reasonable because the present of more water facilitated the hydrolysis of L-ascorbic acid 2-glucoside. On the other hand, the k 2 values became smaller at the higher water content. This fact suggests that L-ascorbic acid was more stable at the higher water content. A comparison of the initial rate for the overall disappearance to that for the hydrolysis of L-ascorbic acid 2-glucoside to L-ascorbic acid and glucose would suggest whether or not the hydrolysis is the predominant route for the disappearance. However, the differential coefficient of Eq. (1) in terms of the time t at t is always zero, and we cannot know the initial rate for the overall disappearance of L-ascorbic acid 2-glucoside. Therefore, the rate constant, k d, app, was approximately estimated by applying the first-order kinetics to the first three points for overall disappearance under each condition. The rate constant for the hydrolysis of L-ascorbic acid 2-glucoside, k 1, is plotted versus the k d, app in Fig. 5. When the hydrolysis is the only route for the disappearance of the L-ascorbic acid 2-glucoside, the plots should be on or near a diagonal line. However, most of the plots lie under the line, indicating that the hydrolysis is not the only route for the L-ascorbic acid 2-glucoside, but there are other routes such as the discoloration of the L-ascorbic acid 2-glucoside itself k 1, k 2 1. k Water content [% (w/w)] k d,app Fig. 4 Water-content dependencies of the rate constants (closed symbols) k 1 and (open symbols) k 2 for the hydrolysis of L-ascorbic acid 2-glucoside and the decomposition of L-ascorbic acid, respectively at (, ) 7, (, ) 8 and (, ) 9. Fig. 5 Relationship between the approximated rate constant k d, app for the overall disappearance of L-ascorbic acid 2-glucoside and the rate constant k 1 for the hydrolysis of the glucoside.

5 Disappearance and Discoloration of L-Ascorbic Acid 2-Glucoside Discoloration of the L-ascorbic acid 2-glucoside powders The discoloration of the L-ascorbic acid 2-glucoside powders with a water content of 2 to 12% (w/w) during storage at a temperature of 6 to 9 was monitored by the absorbance at 5 nm of their aqueous solutions. Figure 6 shows the changes in the absorbance at 5 nm for the L-ascorbic acid 2-glucosides stored at various water contents and at 6 to 9. The absorbance at 5 nm increased with the storage time at a higher rate for the higher storage temperature and also for the sample with the higher water content. For most of the samples, except for those with the higher water contents of 1 or 12% (w/ w) which became too stiff to be sampled in the late stage of the storage period, the absorbance at 5 nm reached a maximum, which depended on both the water content and the storage temperature. Therefore, the change with time of the discoloration of the L-ascorbic acid 2-glucoside powders was analyzed in the same way as described in our previous study [4]. The experimental data were analyzed based on the modified Weibull distribution of the following equation: (A max A)/(A max A ) exp[ (k c t) nc ] (5) where A is the absorbance at time t, A max is the maximum absorbance, A is the initial absorbance, which is equal to zero in this study, k c is the rate constant of discoloration, and n c is the shape constant. The kinetic parameters, A max, k c, and n c, were also evaluated by best-fitting the experimental data using the Solver of Microsoft Excel. The solid curves in Fig. 6 were drawn based on Eq. (5) using the estimated parameters. Except for the samples with water contents of 1 and 12% (w/w) and being stored at 6 and 8, which became too stiff to be sampled after 28- and 8-days storage, respectively, the maximum absorbance A max increased with both the water content and the storage temperature. Most of the rate constants k c increased with the storage temperature and decreased with the water content, which showed the same tendency as those of the L-ascorbic acid [4] and L-ascorbyl 6-palmitate [8]. Figures 7 (a), (b) and (c) show the water-content dependencies of the A max, k c and n c, respectively, for the 1 1 A max.1 3 (a) (c) Absorbance at 5 nm 1 2 (b) (d) k c n c Storage period [d] 5 1 Water content [% (w/w)] Fig. 6 Changes in absorbance at 5 nm of the solutions of L-ascorbic acid 2-glucoside samples with various water contents stored at (a) 6, (b) 7, (c) 8, and (d) 9. The symbols are the same as in Fig. 1 (a). Fig. 7 Water-content dependencies of (a) A max, (b) k c, and (c) n c for the discoloration of the L-ascorbic acid 2-glucoside powders at ( ) 7, ( ) 8 and ( ) 9.

6 14 Lan-hsin HUNG, Yukitaka KIMURA and Shuji ADACHI discoloration (changes in the absorbance at 5 nm) of the solutions of the L-ascorbic acid 2-glucoside powders stored at 7 to 9. There was a tendency that A max scarcely depended on the temperature, but that it was higher for the samples with the higher water contents. The shape constant n c was also independent of the temperature and it was roughly 2..5 for all the samples. The k c value depended on both the temperature and water content, and it was higher for the samples with the lower water content and at higher temperature. 3.3 Comparison in the discoloration of the L-ascorbic acid 2-glucoside powders with the L-ascorbic acid and L-ascorbyl 6-palmitate powders Figure 8 shows the absorbance at 5 nm for the L-ascorbic acid 2-glucoside stored at 7, together with the absorbance for the L-ascorbic acid [4] and L-ascorbyl 6-palmitate [8] with similar molar ratio water contents. Discoloration of the L-ascorbic acid 2-glucoside proceeded slower and faster than the unmodified L-ascorbic acid and L-ascorbyl 6-palmitate, respectively. Therefore, the glucosylation of L-ascorbic acid was effective for suppressing the discoloration, but the effect was weaker than the acylation of L-ascorbic acid. This would be ascribed to the high solubility of the L-ascorbic acid 2-glucoside in water because L-ascorbic acid, which is produced through the hydrolysis of the L-ascorbic acid derivatives, easily undergoes the discoloration. Absorbance at 5 nm Time [d] Fig. 8 Changes in the absorbance at 5 nm of L-ascorbic acid 2-glucoside ( ), L-ascorbic acid ( ) [4] and L-ascorbyl 6-palmitate ( ) [8] at 7. The samples had similar water contents on a molar basis, and the molar ratio water contents were 1.13,.98 and.92 for the glucoside, L-ascorbic acid and palmitate, respectively. References [1] H. D. Belitz, W. Grosch; Vitamins. In: M. M. Burghagen, D. Hadziyev, P. Hessel, S. Jordan, C. Sprinz ed. Food Chemistry. 2nd ed. Berlin: Springer. 1999, pp [2] X. Y. He, G. K. Yin, B. Z. Ma; A study on the decomposition kinetics of vitamin C powder. Acta Pharm. Sinica, 25, (199). [3] A. B. Shephard, S. C. Nichols, A. Braithwaite: Moisture induced solid phase degradation of L-ascorbic acid. Part 1: A kinetic study using tristimulus colorimetry and a quantitative HPLC assay. Talanta, 48, (1999). [4] L. H. Hung, Y. Kimura, S. Adachi: Kinetics of moistureinduced-discoloration of L-ascorbic acid powders. Jpn. J. Food Eng., 6, (25). [5] K. Enomoto, T. Miyamori, A. Sakimae, R. Numazuwa: Production of organic acid ester. Japan Kokai Tokkyo Koho H (1991). [6] T. Uragaki, S. Miyamoto, K. Sakashita, A. Sakimae: Production of organic acid ester. Japan Kokai Tokkyo Koho H (1993). [7] R. Austria, A. Semenzato, A. Bettero: Stability of vitamin C derivatives in solution and topical formulations. J. Pharm. Biomed. Anal., 15, (1997). [8] L. H. Hung, Y. Kimura, S. Adachi: Discoloration kinetics of L-ascorbyl 6-palmitate powders with various water contents. Food Sci. Technol. Res., 13, 7-12 (27). [9] I. Yamamoto, N. Muto, E. Nagata, T. Nakamura, Y. Suzuki: Formation of stable L-ascorbic acid -glucoside by mammalian -glucosidase-catalyzed transglucosylation. Biochim. Biophys. Acta, 135, 44-5 (199). [1] V. Křen, L. Martínková: Glycosides in medicine: The role of glycosidic residue in biological activity. Cur. Med. Chem., 8, (21). [11] S. B. Lee, K. C. Nam, S. J. Lee, J. H. Lee, K. Inouye, K. H. Park: 24. Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles production in cooked chicken meat. Biosci. Biotechnol. Biochem., 68, (24). [12] M. Kubota, H. Fukushima, T. Miyake: Browning inhibitor containing ascorbic acid 2-glucoside as the active ingredient and method for inhibiting browning with the same. PCT International Application WO (26). [13] H. Watanabe, A. Inoue, T. Kimura: Storage-stable gel compositions containing ascorbic acid 2-glucoside, and creams containing the gel compositions in outer phases. Japan Kokai Tokkyo Koho (25). [14] L. M. Cunha, F. A. R. Oliveria, J. C. Oliveria: Optimal experimental design for estimating the kinetic parameters of processes described by Weibull probability distribution function. J. Food Eng., 37, (1998). [15] O. Levenspiel: Design for Multiple Reactions. In Chemical Reaction Engineering. 2nd Ed. 1972, pp

7 , Vol. 9, No. 3, p. 141, Sep. 28 L- 2- L- C L- 6 9 [4] Weibull Arrhenius L- 6-O- -L- [8].8 Weibull L- Weibull Arrhenius L- 6-O- -L- L- L- L- 2- L- 2- Weibull k d n d L- 2- L- L- L- L- 2- k 1 L- 2- k d L- 2- L- 2- L- 2-5 nm L- 6-O- -L- A max Weibull A max n c 2..5 k c L- 6-O- -L- L L- 2- L- 6-O- -L- L- L- 2- L Fax: , adachi@kais.kyoto-u.ac.jp