J Am Oil Chem Soc (2012) 89:269274 DOI 10.

1007/s11746-011-1913-x

ORIGINAL PAPER

Effect of Ascorbic Acid or Acyl Ascorbate on the Stability of Catechin in Oil-In-Water Emulsion
Yoshiyuki Watanabe Takahiro Suzuki Hirofumi Nakanishi Ayumi Sakuragochi Shuji Adachi

Received: 7 December 2010 / Revised: 2 July 2011 / Accepted: 19 July 2011 / Published online: 3 August 2011 AOCS 2011

Abstract The degradation of (?)-catechin in an oil-inwater emulsion using methyl dodecanate as an oil phase with or without ascorbic acid or acyl ascorbate was kinetically examined at 40 C. The rate constant, k, of the rstorder kinetics for the degradation with ascorbic acid or octanoyl ascorbate depended on the added amount, whereas the k value with hexadecanoyl ascorbate was independent of the amount. The k value for a smaller oil droplet with each ascorbate was lower than that for a larger oil droplet. Catechin did not partition well into the methyl dodecanate phase, but did adsorb slightly onto the interface between the methyl dodecanate and water. The suppressive effect of acyl ascorbate on the catechin degradation in the emulsion was lower than that of hydrophilic ascorbic acid at the low concentration, but the peroxidative ability also was lower. Most of the catechin molecules in the emulsion degraded in the water phase. The catechin degradation in the emulsion with small oil droplets depended on the acyl chain length of the ascorbates more than in large oil droplets. Keywords Acyl ascorbate Catechin Degradation kinetics First-order equation Oil-in-water emulsion

Introduction
L-Ascorbic acid, recognized as vitamin C, is a water-soluble vitamin and widely used as an additive in foods and cosmetics because of its strong reducing ability. The enzymatic synthesis of acyl ascorbate through the condensation of ascorbic and fatty acids using a lipase in an organic solvent has been studied [13]. When compared to a chemical method, the enzymatic synthesis has some advantages, such as the simplicities of the reaction and purication processes and its high regioselectivity. Acyl ascorbate is an amphiphilic antioxidant, because it consists of ascorbic and fatty acids as a hydrophilic antioxidant and lipophilic group, respectively. Acyl ascorbate would be expected to be a useful food additive as it has an antitumor activity and metastasis-inhibitory effect [4, 5]. Many foods are emulsied materials, for example, milk, mayonnaise, coffee creamers, salad dressings, butter, and baby foods [6], and an improvement of the lipid oxidation in emulsions is crucial for the production and storage of food products. Acyl ascorbate has a good emulsifying ability [7], and exhibits an antioxidative activity against microencapsulated lipids prepared by emulsication in a food polymer solution and spray-drying [8]. Many studies on the lipid oxidative stability in emulsions and the effects of the emulsiers and antioxidants on it have been reported [9, 10]. However, there has been no report about the effect of the amphiphilic antioxidant, such as acyl ascorbate, against a water-soluble and unstable compound in the oil-in-water type emulsion. A better understanding of its behavior in the emulsion would be required for its effective use in many emulsied products. Catechins are constituents of green tea with an antioxidative activity [11] and have a suppressive ability against adipocyte differentiation [12]. Most tea drinks containing catechins are kept warm in the vending system during the

Y. Watanabe (&) T. Suzuki H. Nakanishi A. Sakuragochi Department of Biotechnology and Chemistry, Faculty of Engineering, Kinki University, 1 Takayaumenobe, Higashihiroshima 739-2116, Japan e-mail: wysyk@hiro.kindai.ac.jp S. Adachi Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

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cold season, and heat processing at high temperature is needed to sterilize the spores of the thermophilic anaerobes. Therefore, the stability of tea catechins in an aqueous solution has been investigated under various conditions [13, 14]. Furthermore, the degradation has been kinetically examined using rst-order kinetics [15, 16]. We have studied the inuence of ascorbic acid or octanoyl ascorbate, which is one of the most soluble acyl ascorbates in water, on the degradation of (?)-catechin in an aqueous solution, and that of the coexistence of octanoyl ascorbate and citric acid on the catechin stability [17, 18]. However, the effect of acyl ascorbate on the stability of catechins in the emulsion, which is a more complex system, is not clear. In this study, the degradation of (?)-catechin in an oil-inwater emulsion with or without ascorbic acid or acyl ascorbate was kinetically examined. The degradation was expressed by rst-order kinetics, and the kinetic parameters were evaluated. In addition, emulsions with the different droplet size distributions were prepared, and the effect of the diameter of the oil droplets in the emulsion in the presence of acyl ascorbate on the degradation kinetics was also investigated.

0.001, 0.01, 0.1, 1.3, 2.5 or 3.8 mmol/L. The mixture was emulsied using a rotor/stator homogenizer (Ultra-Turrax T25, IKA Japan, Nara, Japan) for 5 min at 1 9 104 rpm in the test tube immersed in ice water to produce the coarse oil-in-water emulsion. After the pre-emulsication, the coarse emulsion was circulated through a membrane lter with a pore size of 0.8 or 3.0 lm using a peristaltic pump at 2.0 mL/min for 30 min to reduce and monodisperse the diameter of the oil droplets. The prepared emulsion was put into an amber glass vial with a screw-cap and stored in the dark at 40 C with magnetic stirring at 100 rpm. Stability of Catechin At appropriate intervals, 200 lL of the emulsion was removed from the vial and mixed with 400 lL of a mixture of chloroform and methanol (2:1 by vol.). The mixture was centrifuged for 5 min at 1.5 9 104 rpm. Fifty microliters of the water phase was mixed with 50 lL of 1.3 mmol/L caffeine solution and 900 lL of the eluent, which was a mixture of methanol, water and phosphoric acid (20:80:0.5 by vol.), for the HPLC analysis. Twenty microliters of the mixture was applied to a Chemcosorb300-5C18 column (4.6 mm/ 9 250 mm; Chemco Scientic, Osaka) and eluted at 1.0 mL/min using a Shimadzu LC-10AT pump (Kyoto). The elution proles of the catechin and caffeine were monitored using a Shimadzu SPD-10A UV detector at 280 nm. The amount of remaining catechin, which was recovered in the water phase, in the emulsion was measured in duplicate, and the mean value was calculated. As below mentioned, the partition coefcient of catechin between methyl dodecanate and water was very low or zero. Therefore, the remaining catechin in the emulsion was judged to be in the aqueous phase after centrifugation. Oil-Droplet Size Distribution in the Oil-In-Water Emulsion Twenty microliters of the emulsion were periodically removed from the vial to measure the oil-droplet size distribution using a centrifugal particle size analyzer (SA-CP3L, Shimadzu, Kyoto). The sample was diluted 100400 times with distilled water, followed by the droplet size analysis. The droplet size distribution in the emulsion was measured in triplicate, and the mean value was calculated from the median diameter, d, of the oil droplet as an index of the emulsion stability. Partition Coefcient of Catechin Between Methyl Dodecanate and Water The partition coefcient, P, of catechin between methyl dodecanate and distilled water was measured, according to

Experimental Procedures Materials (?)-Catechin (purity [ 98%) was purchased from Sigma, St. Louis, MO, USA. The immobilized lipase from Candida antarctica was obtained from Roche Molecular Biochemicals, Mannheim, Germany. L(?)-Ascorbic acid was purchased from Nacalai Tesque, Kyoto, Japan. The octanoic and hexadecanoic acids, and methyl dodecanate were purchased from Wako Pure Chemical Industries, Osaka, Japan. The hydrophilic surfactant, SY-Glyster ML-750 (decaglycerol monododecanate), was supplied by Sakamoto Yakuhin Kogyo, Osaka. Caffeine, which was used as the internal standard for the determination of catechin by HPLC, was purchased from Kishida Chemical, Osaka. All other chemicals of analytical grade were purchased from either Wako or Yoneyama Chemical, Osaka. 6-O-Octanoyl and hexadecanoyl L-ascorbate were synthesized by the condensation of ascorbic and octanoic or hexadecanoic acids in acetone using the immobilized lipase and then puried according to previous methods [3]. Preparation of Oil-In-Water Emulsion Five milliliters of a 1.0 mmol/L catechin aqueous solution with 10 g/L SY-Glyster ML-750 as the water phase and the same volume of methyl dodecanate as the oil phase were added to a test tube. Ascorbic acid or acyl ascorbate was added to the water phase at the initial concentration of

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a previous report [17]. One mililiter of each 0.1, 1, or 10 mmol/L catechin solution, Vaq, and each 5, 10 or 20 mL of methyl dodecanate, Vorg, were mixed in an amber vial. The vial was immersed in a water bath at 40 C. After shaking at 100 revolutions per h for 1 h, the vial was left for about 15 min for phase separation. The concentration of catechin in the water phase at the partition equilibrium, Caq, was measured by the above-mentioned HPLC analysis, proceeded by sampling 500 lL of the water phase. The measurement of the partition coefcient was done in triplicate, and the mean value was estimated. Statistical Analysis The effect of the concentration of the ascorbates on the rate constant for the degradation of catechin in the emulsion was determined by ANOVA. Signicant differences were determined by t tests at P \ 0.05. For the average values of the median diameters of the oil droplets of 5.3 lm, the degree of freedom was 4 for no ascorbates. The degrees with ascorbic acid were 2 at 0.001 mmol/L, 4 at 0.01 mmol/L, 8 at 0.1 mmol/L, 6 at 1.3 mmol/L, 5 at 2.5 mmol/L, and 4 at 3.8 mmol/L, respectively. The degrees with octanoyl ascorbate were 3 at 0.01 mmol/L, 5 at 0.1 mmol/L, 4 at 1.3 mmol/L, 8 at 2.5 mmol/L, and 4 at 3.8 mmol/L, respectively. The degrees with hexadecanoyl ascorbate were 4 at 0.001 mmol/L, 5 at 0.01 mmol/L, 5 at 0.1 mmol/L, and 5 at 1.3 mmol/L, respectively. At 9.9 lm of the average median diameters, the degrees were 4 for no ascorbates, 5 for 0.01 mmol/L ascorbic acid, 4 for 0.01 mmol/L octanoyl ascorbate, and 4 for 0.01 mmol/L hexadecanoyl ascorbate, respectively.

Results and Discussion Effect of the Amount of Ascorbic Acid or Acyl Ascorbate on the Stability of Catechin in the Oil-In-Water Emulsion Figure 1 shows the degradation processes of catechin in the oil-in-water emulsions at 40 C with no ascorbates, ascorbic acid, octanoyl ascorbate, and hexadecanoyl ascorbate. The average values of the median diameters of the oil droplets were 5.3 and 9.9 lm, and the standard deviations were 0.23 and 0.95. C/C0 represents the fraction of remaining catechin in the emulsion, where C and C0 are the catechin concentrations at any time t and t = 0, respectively. The transient changes in the median diameters of the oil droplets in the oil-in-water emulsion containing catechin and no ascorbates at 40 C are present in Fig. 2. The data indicates that the emulsions were stable during the measurements for the catechin degradation regardless of initial mean diameter. The degradation kinetics of catechin was assumed to be expressed by rstorder kinetics [18]: C expkt C0 1

where k is the rate constant. The rate constant of the rstorder kinetics was calculated to best-t the experimental results by the regression function of Microsoft Excel 2007 as shown by the solid curves in Fig. 1. From previous results, each residual area per molecule, a, evaluated from the surface tension for 6-O-hexanoyl, octanoyl, decanoyl and dodecanoyl ascorbates was ca. 0.30 nm2 [19]. Using

1.0

(a)

(b)

(c)

(d)

0.8

C/C0

0.6

0.4

0.2

100

200 0

100

200 0

100

200 0

100

200

Time [h]
Fig. 1 Degradation processes of catechin in the oil-in-water emulsion at 40 C with a no ascorbates, b ascorbic acid, c octanoyl ascorbate and d hexadecanoyl ascorbate at the concentrations of 0.001 (open circles),0.01 (open diamonds), 0.1 (open triangles),1.3 (inverted open triangles),2.5 (right pointed open triangles) and 3.8 (left pointed open triangles) mmol/L. C and C0 are the catechin concentrations at time t and initial one, respectively. The squares and other symbols represent the average values of the median diameters of the oil droplet in the emulsion of 9.9 and 5.3 lm, respectively. The closed symbols represent the emulsion without ascorbic acid and acyl ascorbates. The solid curves were calculated using the estimated rate constant of rst-order kinetics

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10-2

10

d [m]

k [h-1]
10-3 10-4

10-3

10-2

10-1

100

50

100

150

200

10

Time [h]
Fig. 2 Transient changes in the median diameters of the oil droplets in the oil-in-water emulsion with catechin and no ascorbates at 40 C. The circles and squares represent the mean values of the diameters of 5.3 and 9.9 lm, respectively. The bars show the standard deviation (n = 3)

mA/m
Fig. 3 Dependence of the rate constants, k, of the rst-order kinetics for the degradation of catechin in the oil-in-water emulsion with the mean diameter of 5.3 lm at 40 C on the amount of ascorbic acid (open circles), octanoyl (open squares) and hexadecanoyl (open triangles) ascorbate. The bars represent 95% CI. The solid curves were empirically drawn. The broken curve represents the k value for the degradation without any ascorbates

the a value, the total number of oil droplets, n, and interfacial area per oil droplet, A, the maximum number of adsorbable ascorbate molecules on the overall interface, m, was estimated: n V 6V v pd 3 nA 6V a da 2 3 4

A pd2 m

where V is the total volume of oil, v is the volume per oil droplet, and d is the median diameter of an oil droplet. The relationship between the k value for the emulsion with the mean diameter of 5.3 lm and the ratio of added ascorbate molecules, mA, to m is shown in Fig. 3. Ascorbic acid suppressed the degradation of catechin at the low concentrations. Mochizuki et al. [20] suggested that the rst step for the autoxidation of catechin in an aqueous solution was the one-electron oxidation of the B-ring of the catechins by molecular oxygen. Superoxide anion and a semiquinone radical resulted from one-electron oxidation which in turn catalyze the autoxidation through radical chain reactions. The function of ascorbic acid due to the enediol-lactone resonant system in the molecule would contribute to suppressing the radical chain reactions. However, the suppressive effect gradually decreased with increase in ascorbic acid molecules, and the k value became higher than that with no ascorbate at all. Ascorbic acid is known to exhibit a peroxidative action [21, 22]. Octanoyl ascorbate also showed a similar behavior, but the anti/peroxidative effect was weaker than that of ascorbic acid. This would be attributed to its

amphiphilic property. It is suggested that molecules such as octanoyl ascorbate were difcult to disperse in the water phase in the presence of catechin, in comparison with ascorbic acid, due to sorption onto the interface, the participation in the emulsifying, or partition into the oil phase. At mA/m [ 1, the k value rapidly increased in the presence of octanoyl ascorbate. A similar tendency was observed for the non-amphiphilic ascorbic acid. On the other hand, the k value for the degradation of catechin in the emulsion with hexadecanoyl ascorbate was independent of the concentration of the ascorbate and almost equal to the control. Most of hexadecanoyl ascorbate molecules, with high lipophilicity, appear to be in the oil phase and not involved with the catechin degradation that occurred in the water phase. Effect of Acyl Chain Length of Acyl Ascorbate on the Stability of Catechin in the Oil-In-Water Emulsion with the Different Median Diameters of the Oil Droplets Emulsions with smaller oil droplets show a higher rate of lipid oxidation due to the higher interfacial area [23]. In this study, the degradation of catechin, which was a watersoluble compound, in the oil-in-water emulsion was examined. The stability of catechin in the emulsion with the oil-droplet diameter of 5.3 lm was higher than those with a 9.9 lm diameter, as shown in Fig. 1a for the emulsion in the absence of the ascorbates. Decaglycerol monododecanate, used as an emulsier, on the interface might provide a partial barrier to the radical chain reactions

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10-2

273
10-1

10-2

Cint [mol/m2]

10-3

(C aq,0 Caq) / CaqA [m-2]

k [h-1]

300 200 100 0 8000 12000 16000

10-4

Vorg / VaqA [m-2]

10-3

10-5 10-5
0 4 8 12 16

10-4

10-3

10-2

10-1

100

10

Caq [mol/L]

Acyl chain length

Fig. 4 Relationship between the rate constants, k, of the rst-order kinetics for the degradation of catechin in the oil-in-water emulsion with 0.01 mmol/L ascorbates at 40 C and acyl chain length of the ascorbates. The symbols are the same as in Fig. 2. The bars represent 95% CI. The solid curves were empirically drawn

for the degradation of catechin. Figure 4 shows the effect of the acyl chain length of the acyl ascorbate on the rate constant for the degradation of catechin in the oil-in-water emulsion with the two median diameters of the oil droplets. Each concentration of ascorbic acid or acyl ascorbate in the emulsion was 0.01 mmol/L. For each ascorbate at this concentration, the k value for the smaller oil droplets was lower than that for the larger ones. Furthermore, both k values for the two different droplet diameters were slightly higher for the longer acyl chain length. This would indicate that the inuence of the oil droplet size on the stability of the catechin was stronger than that of the ascorbate type, and that the ascorbate with a shorter acyl chain length was more efcient. Location of Catechin in Methyl Dodecanate and Water System The P values of catechin between the methyl dodecanate and water, which were estimated assuming no adsorption onto the interface, increased with the increasing initial catechin concentration and also depended on the volume ratio of the organic phase to the water phase. Generally, the P value should be independent of the initial concentration and the volume ratio of the two phases. Therefore, we postulated that catechin molecules are adsorbed onto the interface between the methyl dodecanate and water at the surface concentration of Cint. Based on this assumption, the mass balance for catechin is given by the following equation: Caq;0 Vaq Caq Vaq Corg Vorg Cint A 5

Fig. 5 Effect of the concentration, Caq, of catechin in the water phase at the volume ratio of 5 (open circles), 10 (open squares) and 20 (open triangles) of the organic phase to water phase on the adsorbed amount, Cint, of catechin onto the interface between methyl dodecanate and water at 40 C. The solid curves were empirically drawn. The inset shows the relationship between (Caq,0 - Caq)/CaqA and Vorg/VaqA at the initial concentration, Caq,0, of catechin in the water phase of 0.1 (lled circles), 1 (lled squares) and 10 (lled triangles) mmol/L. The broken curves in the inset indicate Cint/CaqA at the partition coefcient of zero, P, of catechin between two phases

where Caq,0, is the initial concentration in the water phase, Corg, is the concentration in the organic phase at the partition equilibrium, A is the interfacial area, and Vaq and Vorg are the volumes of the water and organic phases, respectively. As the P value is dened as Corg/Caq, Eq. 5 is converted to Eq. 6 Caq;0 Caq Vorg Cint P : Caq A Vaq A Caq Vaq 6

The inset in Fig. 5 shows the relationship between (Caq,0 - Caq)/CaqA and Vorg/VaqA. The plots at any initial catechin concentration lay on a horizontal line, indicating that the P value is very low or practically zero. Assuming that P = 0, Cint was estimated from the intercept of the line. Figure 5 shows the dependence of Cint on Caq. For each volume ratio, Cint proportionally increased with the change in Caq. This would indicate that catechin molecules do not partition well into the methyl dodecanate phase and a slight adsorption of catechin occurred on the interface. In this study, a 1 mmol/L catechin aqueous solution was consistently used for the measurements of the degradation processes. The P value at the initial concentration of 1 mmol/L was very low or zero. Therefore, the partition of catechin to the oil phase in the emulsion would not occur well, though the composition in the emulsion containing decaglycerol monododecanate as an emulsier was different from that of the mixture for the partition experiment. On the other hand, the slight adsorption onto the interface

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J Am Oil Chem Soc (2012) 89:269274 4. Miwa N, Yamazaki H (1986) Potentiated susceptibility of ascites tumor to acyl derivatives of ascorbate caused by balanced hydrophobicity in the molecule. Exp Cell Biol 54:245249 5. Nagao N, Matsubara H, Yamanaka T, Etoh T, Miwa N, Iwagaki H, Saiki I, Yamaoka S, Itoh S, Kokata E (1996) Functions of vitamin C for cells (V) Mechanism for tumor inltration- and metastasis-inhibitory effects of L-ascorbic acid (in Japanese). Vitamins 70:199200 6. Ponginebbi L, Nawar WW, Chinachoti P (1999) Oxidation of linoleic acid in emulsions: effect of substrate, emulsier, and sugar concentration. J Am Oil Chem Soc 76:131138 7. Kuwabara K, Watanabe Y, Adachi S, Nakanishi K, Matsuno R (2003) Emulsier properties of saturated acyl L-ascorbates for preparation of O/W emulsions. Food Chem 82:191194 8. Watanabe Y, Fang X, Minemoto Y, Adachi S, Matsuno R (2002) Suppressive effect of saturated acyl L-ascorbate on the oxidation of linoleic acid encapsulated with maltodextrin or gum arabic by spray-drying. J Agric Food Chem 50:39843987 9. Coupland JN, McClements DJ (1996) Lipid oxidation in food emulsions. Trends Food Sci Technol 7:8391 10. Frankel EN (2001) Interfacial lipid oxidation and antioxidation. J Oleo Sci 50:387391 11. Huang SW, Frankel EN (1997) Antioxidant activity of tea catechins in different lipid systems. J Agric Food Chem 45:30333038 12. Furuyashiki T, Nagayasu H, Aoki Y, Bessho H, Hashimoto T, Kanazawa K, Ashida H (2004) Tea catechin suppresses adipocyte differentiation accompanied by down-regulation of PPARc2 and C/EBPa in 3T3L1 cells. Biosci Biotechnol Biochem 68: 23532359 13. Chen ZY, Zhu QY, Wong YF, Zhang Z, Chung HY (1998) Stabilizing effect of ascorbic acid on green tea catechins. J Agric Food Chem 46:25122516 rgens HS (2000) Effect of pH on the stability of 14. Friedman M, Ju plant phenolic compounds. J Agric Food Chem 48:21012110 15. Komatsu Y, Suematsu S, Hisanobu Y, Saigo H, Matsuda R, Hara K (1993) Effects of pH and temperature on reaction kinetics of catechins in green tea infusion. Biosci Biotechnol Biochem 57:907910 16. Wang R, Zhou W, Wen RH (2006) Kinetic study of the thermal stability of tea catechins in aqueous systems using a microwave reactor. J Agric Food Chem 54:59245932 17. Watanabe Y, Okayasu T, Idenoue K, Adachi S (2009) Degradation kinetics of catechin in aqueous solution in the presence of ascorbic acid or octanoyl ascorbate. Jpn J Food Eng 10:117124 18. Watanabe Y, Idenoue K, Nagai M, Adachi S (2010) Stability of catechin in aqueous solution with coexistent ascorbic acid or octanoyl ascorbate and organic acid. Food Sci Technol Res 16:111114 19. Watanabe Y, Adachi S, Fujii T, Nakanishi K, Matsuno R (2001) Surface activity of 6-O-hexanoyl, octanoyl, decanoyl and dodecanoyl ascorbates. Jpn J Food Eng 2:7375 20. Mochizuki M, Yamazaki S, Kano K, Ikeda T (2002) Kinetic analysis and mechanistic aspects of autoxidation of catechins. Biochim Biophys Acta 1569:3544 21. Podmore ID, Grifths HR, Herbert KE, Mistry N, Mistry P, Lunce J (1998) Vitamin C exhibits pro-oxidant properties. Nature 392:559 22. Nielsen JH, Kristiansen GH, Andersen HJ (2000) Ascorbic acid and ascorbic acid 6-palmitate induced oxidation in egg yolk dispersions. J Agric Food Chem 48:15641568 23. Gohtani S, Sirendi M, Yamamoto N, Kajikawa K, Yamano Y (1999) Effect of droplet size on oxidation of docosahexaenoic acid in emulsion system. J Dispers Sci Technol 20:13191325

between the oil and water phases could occur. In the emulsion with the small oil-droplets, catechin molecules adsorbed onto the interface would increase due to the large interfacial area. This may contribute to the high stability of catechin in the small oil-droplets emulsion, as shown in Fig. 1a. In the presence of acyl ascorbate, some catechin on the interface might be replaced by the adsorbed ascorbate. Catechin liberated by the replacement is prone to degradation in the water phase. This is affected by the added amount of the ascorbate and the droplet size in the emulsion, that is, the interfacial area. This is only a hypothesis, and more research needs to be done to conrm this speculation.

Conclusions Most of the catechin molecules in the emulsion must exist and degrade in the water phase. Hydrophilic ascorbic acid effectively suppressed the oxidative degradation of catechin. Amphiphilic octanoyl ascorbate also exhibited an antioxidative ability against the catechin degradation in the emulsion, but the effectiveness was low due to the adsorption onto the interface between the water and oil phases. Hexadecanoyl ascorbate, with a high lipophilicity, did not act as an antioxidant against catechin in the emulsion indicating that the ascorbate was not in the water phase. It would support these suggestions that the dependence of the degradation in the emulsion with small oil droplets on the acyl chain length of the ascorbates was stronger than that with the large oil droplets. As these ascorbates act not only as an antioxidant, but also as a proxidant, their consumed quantity should be focused on. The appropriate quantity is different among acyl ascorbates and the applied processing food systems. Thus, further studies are needed for effective application.
Acknowledgments This work was supported by a Grant-in-Aid for Young Scientists (B) 19780106 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

References
1. Humeau C, Girardin M, Coulon D, Miclo A (1995) Synthesis of 6-O-palmitoyl L-ascorbic acid catalyzed by Candida antarctica lipase. Biotechnol Lett 17:10911094 2. Watanabe Y, Adachi S, Nakanishi K, Matsuno R (1999) Condensation of L-ascorbic acid and medium-chain fatty acids by immobilized lipase in acetonitrile with low water content. Food Sci Technol Res 5:188192 3. Yan Y, Bornscheuer UT, Schmid RD (1999) Lipase-catalyzed synthesis of vitamin C fatty acid esters. Biotechnol Lett 21: 10511054

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