Food Sci. Technol. Res., +- (,), +0,ῌ+0/, ,**1

Note

E#ects of koji Production and Saccharification Time on the Antioxidant Activity of

ῌ Noriaki SAIGUSA and Riichiro OHBA

Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University; .ῌ,,ῌ+ Ikeda, Kumamoto 20*ῌ**2,,

Received March ,3, ,**0; Accepted December ,1, ,**0

Amazake was prepared using yellow-koji () or white-koji (Aspergillus kawachii). In both cases, when -koji and water were mixed in a ratio of +: ,, saccharification was completed in 0 hand reducing was formed. Changes in antioxidant activity were investigated for each amazake saccharification process. In both amazake preparations, DPPH (+,+-diphenyl-,-picrylhydrazyl) radical- scavenging activity increased for the first , h of the saccharification period, and then gradually decreased. The lipid peroxidation inhibitory activity with b-carotene, however, did not change with saccharification time in either amazake preparation, and the antioxidant activity was roughly constant during saccharifica- tion. Next, both types of amazake were prepared by saccharifying for 0 hat//ῌC, with the respective rice-koji obtained with di#erent koji production times (.,, .2 and /. h). Antioxidant activity was then compared. It was found for both types of rice-koji that longer koji production time yields amazake with higher DPPH radical-scavenging activity and lipid peroxidation inhibitory activity.

Keywords: amazake, DPPH radical-scavenging activity, lipid peroxidation inhibitory activity, rice-koji.

Introduction contained in . Ohta et al. (+33,) reported on anti- Amazake is a sweet beverage made from rice that is oxidative substances with lipid peroxidation inhibitory unique to Japan. It is one of several traditional Japanese activity in sake, and proposed that they were peptides fermented products prepared from polished rice, rice-koji, and ferulic acid produced from rice by koji enzymes. and water. Rice-koji is produced by adding mycelia, a Amazake may contain these antioxidative substances. type of mold, to steamed rice. Amazake is prepared by In this paper, the e#ects of koji production time and adding water to rice-koji and heating the mixture for amazake saccharification time using yellow-koji (normally several hours. The main sweet component in amazake is used for amazake) and white-koji (usually used for shochu) glucose, which is formed from the decomposition of rice on the antioxidant activity of amazake are discussed. starch by the amylase secreted from the mycelia. Oligo- saccharides are also formed during this process. On the Materials and Methods rice surface, there are many proteins, and so large amounts Materials DPPH was purchased from Wako Pure of amino acids and peptides are formed by the proteolytic Chemical Co., Ltd. (Osaka, Japan). b-Carotene was pur- enzymes of the mycelia. Proliferation of mycelia pro- chased from Sigma Chemical Co. (St. Louis, MO). 0- duces vitamins and amino acids and large amounts of Hydroxy- ,,/,1,2 -tetramethylchroman- , -carboxylic acid these dissolve in the amazake. Therefore, amazake is (Trolox) was purchased from Sigma-Aldrich Co. (St. Louis, sometimes referred to as a Japanese-style yogurt, and is MO). Butylated hydroxytoluene (BHT) was purchased currently undergoing consideration as a nutritious health from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). All beverage. In recent years, the beneficial e#ects of ama- other chemicals were of reagent grade. Tane-koji for zake have been widely studied, and blood pressure-lowering making rice-koji were purchased from Kawauchi genichiro e#ects, antiobesity e#ects, liver-protecting e#ects, and shouten Co., Ltd. (Kagoshima, Japan). antiamnesic e#ects have been reported (Ohura, ,**-). Rice-koji production Polished rice (,** g) was soaked One of our aims is to establish production technology to in water for ,* min, and the water was then drained for , make amazake that has beneficial e#ects. Kitagaki et al. h. Following this, the rice was steamed for .* min with a (+333) proposed 1 substances- tannic acid, gallic acid, ferulic steamer, and was allowed to stand until the temperature acid, protocatechuic acid, (ῌ)-epicatechin, -,/-dimethoxy- decreased to .*῍C. Next, *., goftane-koji, either yellow- .-hydroxycinnamic acid, and p-hydroxycinnamic acid- or white-koji, was added to the steamed rice, and the that have the free radical-scavenging ability naturally mixture was stirred until it became uniform. Except for the steaming process, the entire procedure was performed ῌ To whom correspondence should be addressed. under sterile conditions. The steamed rice was then E-mail: [email protected] transferred to a petri dish and packed with a spatula. A Antioxidant activity of amazake 163

filter paper was inserted inside the cover, and the mixture Results and Discussion was incubated at -*῎C. The rice-koji was stirred every +, When amazake was prepared with yellow- or white-koji h, and three types of rice-koji (culture time: .,, .2,or/. h) obtained with a koji production time of .2 h, the amount were prepared. of reducing sugar gradually increased for 0 h of saccha- Preparation of amazake Amazake was prepared by rification at //῎C in both cases. Sugar formation was not adding +** ml of deionized water to /* g of either yellow- seen beyond 0 h (Fig. +). or white-koji, followed by saccharification for 0 hina Although both yellow- and white-koji were prepared constant-temperature bath at //῎C. Total reducing sugar under the same conditions, more reducing sugar was content was determined using the method of Somogyi- formed in amazake prepared from white-koji. Under these Nelson (Somogyi, +3/,; Nelson, +3..). conditions, changes in antioxidant activity during the 0-h Measurement of DPPH radical-scavenging activity saccharification process were investigated for each amazake Amazake was added to the same volume of deionized (Fig. ,). water, and the mixture was centrifuged (-,*** rpm, +* min). DPPH radical-scavenging activity significantly increased The resulting supernatant was then mixed with the same with saccharification in yellow-koji amazake (p῍*.*+)and volume of ethanol, and the mixture was again centrifuged. white-koji amazake (p῍*.*+) for the first , h of sacchari- The obtained supernatant was thus .-fold diluted amazake fication, after which it gradually decreased. Furthermore, containing /*ῌ ethanol. This amazake was further diluted .-fold with /*ῌ ethanol, and this final +0-fold diluted amazake containing /*ῌ ethanol was used for analysis. The radical-scavenging activity was measured based on the method of Suda et al. (,***) and Wang et al. (,**.). DPPH radical-scavenging activity was evaluated in terms of Trolox equivalent (mM). Measurement of lipid peroxidation inhibitory activity Lipid peroxidation inhibitory activity was measured based on the method of Kudo et al. (,***) using b-carotene. For analysis, +0-fold diluted amazake containing /*ῌ eth- anol was prepared in the same way as analysis of DPPH radical-scavenging activity. Lipid peroxidation inhibitory activity was evaluated in terms of BHT equivalent (mM). Assay of glucoamylase activity Glucoamylase activi- ty was assayed using the o$cial analytical method of the National Tax Administration Agency of Japan (+33-). One unit of glucoamylase activity was defined as the Fig. +. Change in reducing sugar during amazake making. amount of enzyme that produced + mg of glucose in the Symbols: ῏, white-koji; ῐ, yellow-koji. rice-koji in 0* min.

Fig. ,. E#ects of saccharification time on antioxidant activity of amazake. Antioxidant activity was evaluated by DPPH radical-scavenging activity (left) and inhibition of lipid peroxidation (right). Each value is the meanῌstandard deviation of three replicates. Statistical di#erence was analyzed by a multiple comparison post test using Tukey’s method. Values in columns followed by di#erent superscript letters in each amazake (a-b, a-c, a-cd, and a-e) are significantly di#erent (p῍*.*+). Also, each amazake (a-d, c-d, and b-e) is significantly di#erent (p῍*.*/) as well. 164 N. SAIGUSA &R.OHBA

Fig. - E#ects of koji production time on antioxidant activity of amazake. Antioxidant activity was evaluated by DPPH radical-scavenging activity (left) and inhibition of lipid peroxidation (right). Each value is the meanῌstandard deviation of three replicates. Statistical di#erence was analyzed by a multiple comparison post test using Tukey’s method. Bars topped by di#erent letters are significantly di#erent. the DPPH-radical-scavenging activity of amazake was .**ῌ white-koji with koji production times of .,, .2,or/. hand 0** mM-Trolox eq., which is the same as the fresh weight saccharification of 0 h. The antioxidant activities of the of Nigaure, Papaya and Hechima (Maeda et al., ,**0). On obtained amazake were compared (Fig. -). The DPPH the other hand, Kudo et al. (,**+) reported that the pep- radical-scavenging activity of amazake prepared from tides produced from casein during lactic acid fermenta- either yellow-koji (p῍*.*+) or white-koji (p῍*.*/) signifi- tion have lipid peroxidation inhibitory activity. In addi- cantly increased with koji production time (.,, .2 hand.,, tion, they suggested that it is important to produce pep- /. h). However, there was almost no di#erence between tides with specific structures to induce lipid peroxidation .2 hand/. h. Similarly, the lipid peroxidation inhibitory inhibitory activity, and that lipid peroxidation inhibitory activity of amazake prepared from both yellow- and activity may decrease following decomposition of the white-koji significantly increased (p῍*.*+) with koji pro- peptides by proteolytic enzymes. duction time (.,, .2 hand.,, /. h). However, unlike DPPH The change in lipid peroxidation inhibitory activity radical-scavenging activity, a significant di#erence (p῍ during lactic acid fermentation was very similar to the *.*+) was observed between koji production times of .2 h change in DPPH radical-scavenging activity during the and /. hinamazake prepared using only white-koji. The saccharification of amazake. From these findings, it was relationship between the antioxidant activity of six types conjectured that antioxidant substances with some spe- of amazake and the glucoamylase activity of their respec- cific structure were released in amazake during the first , tive rice-koji were investigated (Fig. .). h of saccharification; after that, the structures changed. A positive correlation was observed between the DPPH Therefore, the radical-scavenging activity of amazake in- radical-scavenging activity of amazake and the gluco- creased during the first , h, after which it gradually amylase activity of rice-koji. However, no correlation was decreased. On the other hand, lipid peroxidation in- observed between the lipid peroxidation inhibitory activ- hibitory activity remained largely unchanged during the ity of amazake and the glucoamylase activity of rice-koji. saccharification process for amazake prepared from either Recently, it was reported that ferulic acid with antioxi- yellow- or white-koji. It was inferred from these results dant activity binds arabinose, which is the side chain of that the lipid peroxidation inhibitory activity of amazake xylan, the structural material of hemicellulose in plant is due to materials formed during the koji production cell walls, and that ferulic acid was released from rice on process. These materials were probably not a#ected by addition of takadiastase, originating from Aspergillus the enzymes in rice-koji during the saccharification oryzae (Koseki et al., +33.). Furthermore, Koseki et al. period. Furthermore, the lipid peroxidation inhibitory (+330) reported that the ferulic acid released from rice was activity of amazake was ,/*ῌ/** mM, which was /ῌ/* times changed to .-vinyl guaiacol by acid and heating during higher than that of the sweet potato yogurt reported by the shochu process. From these findings, we Kudo et al. (,**+). When the radical-scavenging and lipid conjectured that the release of ferulic acid by the increase peroxidation inhibitory activity of amazake prepared of glucoamylase activity might cause a increase in DPPH from yellow- and white-koji was compared, amazake pre- radical-scavenging activity of amazake with increasing pared from white-koji exhibited more activity at all time koji production time. Furthermore, we hypothesize that points. Next, amazake was prepared from yellow- or ferulic acid is oxidized during saccharification and its Antioxidant activity of amazake 165

Fig. . Relationship between glucoamylase activity of rice-koji and antioxidant activity of amazake. Amazake was prepared from yellow-koji or white-koji obtained using koji production times of .,, .2 or /. h (saccharification time: 0 h). structure might be changed; accordingly, radical-scavenging in the digests of a soybean protein. J. Agric. Chem., .0, .3ῌ/-. activity might be decreased. In addition, we believe that Kitagaki, T., Iwatsuki, Y. and Tsugawa, M. (+333). Sensory evalu- the substances with lipid peroxidation inhibitory activity tion of a model sake endowed with free radical scavenging ability. J. Brew. Soc. Japan., 3., /*,ῌ/*0. in amazake might be formed in mycelia during the koji Koseki, T., Ito, Y., Ito, K., Iwano, K. and Tadenuma, M. (+33.). production process, and might increase with the growth Phenolic compounds found in . J. Brew. Soc. Japan., and proliferation of mycelia. These substances may not 23, .*2ῌ.++ (in Japanese). be a#ected by the enzymes in rice-koji, or by oxidization Koseki, T. and Iwano, K. (+330). Flavor compounds in Shochu during saccharification. Chen et al. (+332) reported that generated from raw material and the enzyme from filametous peptides with lipid peroxidation inhibitory activity did fungus Aspergillus contributing to hydrolysis. Nippon Nogei- 1* 02.ῌ020 not have DPPH radical-scavenging activity. It is also kagaku Kaishi, , (in Japanese). Kudo, Y. and Matsuda, S. (,***). E#ect of lactic acid bacteria on possible that the antioxidant substances in amazake need antioxidative activity of sweet potato yogurt. Nippon # to be identified, as they may be di erent substances. Shokuhin Kagaku Kogaku Kaishi, .1, 1,1ῌ1-* (in Japanese). In this study, amazake was prepared from two types of Kudo, Y., Matsuda, S., Igoshi, K. and Oki, T. (,**+). Antioxidative rice-koji: yellow- and white-koji. Antioxidant activity was peptides from fermentation with Lactobacillus delbrueckii higher for amazake made from white-koji than from yellow- subsp. Bulgaricus IFO+-3/-. Nippon Shokuhin Kagaku Kogaku koji. While the same koji production conditions were Kaishi, .2, ..ῌ/* (in Japanese). used in this study; optimal conditions were not used. Maeda, G., Takara, K., Wada, K., Oki, T., Masuda, M., Ichiba, T., Chuda, Y., Ono, H. and Suda, I. (,**0). Evaluation of antioxidant The antioxidant activity of amazake may be a#ected by activity of vegetables from and determi- enzyme activity. Thus, the conditions for mycelial pro- nation of some antioxidative compounds. Food Sci. Technol. liferation and enzyme production for white-koji may have Res., +,, 2ῌ+.. been closer to optimal than those for yellow-koji. In Nelson, N. (+3..). A photometric adaptation of the Somogyi conclusion, it was confirmed that antioxidant activity is method for the determination of glucose. J. Biol. Chem., +/-, a#ected by koji production conditions, saccharification -1/ῌ-2*. $ conditions, and tane-koji species. Therefore, the antioxi- O cial Method of Analysis of the National Tax Administration Agency of Japan, In .th ed. (+33-). The Society of dant activity of amazake can be controlled by altering Japan, ,+-ῌ,+. (in Japanese). production conditions. In order to prepare highly func- Ohura, S. (,**-). The functionality of sake and by-products. tional amazake, future investigations will aim to deter- Seibutsu-kogaku Kaishi, 2+, /+.ῌ/+0 (in Japanese). mine the components involved in the antioxidant activity Ohta, T., Takashita, K., Todoroki, K., Iwano, K. and Ohba, T. of amazake, and to establish technology that can produce (+33,). Antioxidative substances in sake. J. Brew. Soc. Japan, 21, su$cient amounts of these components. 3,,ῌ3,0 (in Japanese). Somogyi, M. (+3/,). Notes on sugar determination. J. Biol. Chem., +3/ +3ῌ,- Acknowledgements The authors are grateful to Ms. Seiko Taneoka , . Suda, I. (,***). Antioxidant activity In “The Methods of Food for assistance in conducting this research. Functions Analysis,” ed. by Shinohara, K., Suzuki, T. and Kaminogawa, S., Korin, Japan, ,+2ῌ,,* (in Japanese). References Wang, L., Li, L., Fan, J., Saito, M. and Tatsumi, E. (,**.). Radical- Chen, H.-M., Muramoto, K., Yamauchi, F., Fujimoto, K. and scavenging activity and isoflavone content of sufu (fermented +332 Nokihara, K. ( ). Antioxidative properties of histidine- ) extracts from various region in China. Food Sci. Technol. containing peptides designed from peptide fragments Found Res., +*, -,.ῌ-,1.