Radical Scavenging Activity of the Japanese Traditional Food, Amazake
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Food Sci. Technol. Res., 17 (3), 209–218, 2011 Radical Scavenging Activity of the Japanese Traditional Food, Amazake 1 1 2 3 3* Shinpei Yamamoto , Yu naKaSHima , Junpei YoSHiKawa , Naoki wada and Seiichi matSugo 1 Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan 2 Department of Chemistry and Chemical Engineering, Faculty of Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan 3 School of Natural System, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan Received August 3, 2010; Accepted January 14, 2011 The traditional Japanese beverage amazake was evaluated as a possible treatment for oxidative stress. For this purpose, the radical scavenging activities of four kinds of amazake were investigated by determin- ing diphenylpicrylhydrazyl radical, galvinoxyl radical and superoxide anion radical quenching activities using a spectrophotometric method and an electron spin resonance (ESR) technique. ESR measurements were used to assess the superoxide anion radical scavenging activity by employing dimethylpyrroline N- oxide as a spin trapping reagent. The ferricyanide-reducing activity and chelating activity were evaluated spectroscopically. The results indicated that amazake made from brown rice had stronger radical scaveng- ing, metal chelating and metal reducing activities than amazake produced from white rice and sake cake (by-product of sake). Keywords: amazake, radical-scavenging activity, metal-chelating activity, metal-reducing activity, fermentation Introduction manufactured using sake cake, which is obtained as a by- Amazake, a sweet beverage made from fermented rice, product of sake. This kind of amazake is simply made by has been consumed in Japan for more than a thousand years mixing the sake cake and sugar in hot water. Generally, this (Yamamoto and Matsugo, 2008). Steamed rice, rice-koji and type of amazake is called instant amazake. Sake cake is water are used as the raw materials for making traditional the condensate of rice protein and contains alcohol (Yama- amazake (Shinmura, 1998; Murakami, 1986; Nakano, 1967). moto and Matsugo, 2008; Kagawa, 2009). Recently, instant These three components are mixed together and kept at 55 amazake has become popular because of its low price. − 60℃ for 15 − 18 h. During this period, rice starch is sac- In spite of a long history of traditional amazake, the charized by the enzymes present in rice-koji, with traditional characteristics of amazake as a functional food are not well amazake containing approximately 20% glucose (Yamashita, examined. It has become apparent that foods fermented by 2009). Overall, the saccharification process is the same as koji have various physiological functions including antioxi- that of sake (an alcoholic beverage originating in Japan); dative activities. The physiological functions of soy sauce, however, the alcoholic fermentation process is not used in miso, sake and mirin (alcoholic seasoning in Japan) are well amazake production. The high temperature used to make studied (Soy Sauce Information Center, 2004; Japan Miso amazake is the main factor that prevents yeast propagation; Promotion Board, 1999; Ohta et al., 1992; Ishizaki et al., therefore, traditional amazake contains no alcohol. 2006); however, no comprehensive study exists comparing Recently, drinking of traditional amazake has increased the characteristics of traditional and instant amazake. because of the tendency for consumers to prefer natural Many different functional nutrients are produced from foods. Although the normal raw material of traditional raw materials during the fermentation process. We have cho- amazake is polished rice, brown rice is sometimes used. sen to examine the functionality of amazake by focusing on Since the 1970s, a different kind of amazake has been its antioxidative activity. Antioxidative activities are gener- ally evaluated based on the radical scavenging activity of the *To whom correspondence should be addressed. compound of interest. Several methods to evaluate radical E-mail: [email protected] scavenging activity have been developed; however, the re- 210 S. Yamamoto et al. sults are sometimes dependent on the radical species exam- tics of these amazakes, which are labeled according to the Ja- ined (Yamada and Matsuda, 2009; Kikuchi et al., 2004; Park pan Agricultural Standards Act and the Food Sanitation Act, et al., 2002). Four kinds of amazake found in the Japanese are shown in Table 1. The nutritional composition of these market were used in this study. Three of them are catego- amazakes was examined according to the conventional pro- rized as traditional amazake and the other is instant amazake. cedure. The data summarized in Table 2 were obtained with We evaluated the antioxidative activities of these amazakes technical help from Ishikawa Health Service Association and against several radical species including 1,1-diphenyl-2- Industrial Research Institute of Ishikawa. Free amino acids picrylhydrazyl (DPPH) radical, galvinoxyl radical and super- were analyzed using the L-8900 analyzer (Hitachi, Tokyo, •− oxide anion radical (O2 ). Furthermore, we also evaluated Japan) after dilution with distilled water. The results shown the reducing and chelating activities of these amazakes by in Table 3 were obtained with the help of Industrial Research spectroscopic analyses using ferric-ferrous metal complexes. Institute of Ishikawa. Materials DPPH radical, xanthine oxidase, phenol Materials and Methods reagent (Folin reagent), and 5,5-dimethyl-1-pyrroline-N- Samples Two traditional amazakes made from brown oxide (DMPO) were purchased from Nacalai Tesque (Kyoto, rice (BRA and BRA2) were used in this study. The other tra- Japan). Disodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- ditional amazake (WRA) was made from white rice. Instant (4-disulfophenyl)-2H-tetrazolium (WST-1) and sodium amazake (SCA) was made from sake cake. The characteris- 3-(2-pyridyl)-5,6-diphenyl- 1,2,4-triazine-4′,4″-disulfate Table 1. Raw materials and manufacturer of amazake samples used in this study. Abbreviations Raw Materials Manufacturer BRA brown rice, koji made from brown rice Ayumasamune-shuzo BRA2 brown rice, koji made from white rice Yamato-soysauce-miso WRA white rice, koji made from white rice Shinozaki SCA sugar, sake cake, koji made from white rice, Morinaga(seller) sodium chloride, acidifier Table 2. Nutrition components in amazakes. /100g BRA BRA2 WRA SCA Water (g) 75.80 71.10 74.70 84.40 Energy (kcal) 95.00 113.00 112.00 66.00 Protein (g) 1.80 2.00 1.70 0.80 Lipid (g) 0.10 0.20 0.50 0.40 Carbohydrate (g) 21.80 25.80 25.20 14.50 Reducing sugar (g) 13.00 20.00 17.20 4.60 Mineral Na (mg) 35.00 8.00 2.00 63.00 K (mg) 54.00 20.00 3.00 5.00 Mg (mg) 29.00 18.00 1.00 1.00 P (mg) 73.00 23.00 6.00 7.00 Vitamin B1 (mg) 0.06 0.07 0.01 0.00 B2 (mg) 0.03 0.06 0.04 0.02 pH 5.98 5.96 5.66 4.40 Radical Scavenging Activity of the Japanese Traditional Food, Amazake 211 Table 3. Free amino acid composition of amazakes (mg/100 g). AminoAcid BRA BRA2 WRA SCA Total 316.6 464.0 310.0 120.6 Asp 20.8 36.8* 17.9 6.3 Thr 13.1* 17.9 11.5 3.7 Ser 18.0 29.3 17.6 5.2 Asn 16.2** 6.9 12.0** 10.2** Glu 22.1 42.7* 20.5 7.7 Gln 3.6 22.3* 37.5** 1.5 Gly 9.7 17.2 11.0 4.4 Ala 25.5 35.7 21.1 14.8** Val 20.8 29.1 17.4 6.3 Cys 1.7 2.3 3.1 1.8* Met 4.8 7.6 4.3 1.4 Ile 16.2 21.4 13.1 4.9 Leu 31.4 43.6 25.8 9.2 Tyr 20.3 31.5 20.0 6.7 Phe 23.5* 25.8 15.5 6.5 Trp 9.6** 8.3 5.4 4.3 Lys 14.6* 16.5 16.7** 7.9* His 5.4 7.4 6.1* 2.5* Arg 20.5 45.0* 19.2 9.7 Pro 7.0 11.5 7.7 2.3 4-amino butanoic acid 11.9** 5.0 7.3* 3.3* ** remarkably high value by comparison with the proportion of the four amazakes * slightly high value by comparison with the proportion of the four amazakes (ferrozine) were purchased from Wako Pure Chemical Indus- to the DPPH radical was measured using a JASCO V-550 tries (Osaka, Japan). Gallic acid was purchased from Kanto spectrometer (JASCO Corporation, Tokyo, Japan). The ex- Chemical (Tokyo, Japan) and galvinoxyl radical was pur- periment was repeated three times. DPPH radical scavenging chased from Tokyo Chemical Industry (Tokyo, Japan). activity was calculated using the following equation: Determination of DPPH radical scavenging activ- DPPH radical scavenging activity (%) = ity Amazake samples BRA, BRA2, WRA and SCA were {1 – (A1 – A3)/(A2 – A3)} × 100 (1) centrifuged at 15℃ at 6700 × g for 20 min. The supernatant A1 = absorbance at 527 nm in the presence of amazake was filtered by microfiltration (6 μm pore size) and stored at A2 = absorbance at 527 nm in the absence of amazake –20℃ until use. Just before use, the stock sample was de- A3 = absorbance at 527 nm in the presence of 2 mM frosted at 35℃ for 30 min and centrifuged at 6700 × g for 10 ascorbic acid (blank test) min. The supernatant (C0) was diluted with ultrapure water to obtain the required concentration (C). An aliquot of the Electron spin resonance (ESR) spectra of DPPH radical diluted solution (200 µl) was mixed with ultrapure water (1.8 were measured using a JEOL JES-FR30EX Free Radical ml) and ethanol (2.5 ml) and then a solution of DPPH radical Monitor (JEOL Ltd., Tokyo, Japan).