Kor. J. Microbiol. Biotechnol. Vol. 35, No. 3, (2007)

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1 Kor. J. Microbiol. Biotechnol. Vol. 35, No. 3, (2007) q m w³, wy wx y sƒ Á 1 Á yá½ 2 Á«Áy* w tw, 1 ww, 2 Evaluation for the Antimicrobial, Antioxidant and Anti-thrombosis Activity of Korean Traditional Liquors. Ryu, Hee-Young, Eun-Joo Kum 1, Kyung-Hwa Bae, Young Kwan Kim 2, In Sook Kwun, and Ho-Yong Sohn*. Department of Food and Nutrition, Andong National University, Andong , Korea, 1 Institute of Korean Medicine and Biomedical Science, Suwon, , Korea, 2 Gyeongbuk Institute for Bioindustry, Andong , Korea To identify the useful biological activities and the superiority in quality of Korean traditional liquors, we prepared the alcohol-free concentrates from different types of commercial traditional liquors, Takju (T1~T3), Yakju/Cheongju (Y-1~Y-5), Fruit wine (F-1) and Soju (S-1, S-2), respectively. We investigate their compositions as well as antimicrobial, antioxidant, and anti-thrombosis activity. Among the 11 traditional liquors, Y-3, Y-4, Y-5 and F-1 showed higher total-polyphenol, total-flavonoids and reducing sugars than the others. The strong antibacterial and anti-thrombosis activities were identified in Y-3, Y-4, Y-5 and F-1, and a minor antioxidant activity was found in F-1. The antibacterial activity of the Y-3, Y-4, Y-5 and F-1 alcohol-free concentrates showed a broad-spectrum, and growth inhibition was found in gram-positive, gram-negative, and ampicillin-resistant bacteria. The sequential solvent fractionation of Y-3, Y-4, Y-5 and F-1, and following analysis showed that ethyl acetate fractions of Y-3, Y-4, Y-5 and F-1 possess strong antibacterial and anti-thrombosis activity. Especially, the ethyl acetate fractions of Y-3, Y-4 and F-1 showed superior antithrombosis activity compared than that of aspirin. Our results suggest that the useful substances are produced from substrates and edible plant added during the fermentation, and the Korean traditional liquors could be developed as strong antibacterial and anti-thrombosis agents. Key words: Antimicrobial, antioxidant, anti-thrombosis, korean traditional liquors ù šl y k, w w z mw m, w m y, t w ƒ, m, - - w e w. m» ù, 1909 ƒ w w w z»w š w m n», w m» 620 mƒ š, [16]. ù, 1980 z v w e, Ÿ my d *Corresponding author Tel: , Fax: hysohn@andong.ac.kr, ü m yy, w z» w w, m zt w ƒ, m w wì š [8-11]. m w, k z», y zj q zz w [2-4, 12], ƒ yw z» ƒ t y [1, 5, 6] t w [5, 7]ƒ, w ƒ, w m x (,»,,,,,,,, y,, k,,,,,,, e, f, ) ƒwš e, zj š [7, 10, 11, 17-19, 23, 24]. p e ƒ m, e ƒ m angiotensin converting enzyme wy, œ, xw y z š [11, 17, 23,

2 PHYSIOLOGICAL FUNCTIONALITIES OF KOREAN TRADITIONAL LIQUORS ]. ù, x ƒw m w, ü š q m w y w w [8], ½ 2004 ü š w m s sz[15] y z[14]ƒ., s flavonoids, polyphenol, vitamin B peptide»w wy, wšx y, angiotensin I converting enzyme w z y z [21, 22] y y š, w mw,, s š., ü m y mw m yw» w, ü q m yw wš, g w m w³ y, wy y p w wx y sƒw,, ü q m / w w³, w x wy y ùkû yw», ü m t y šw. x x w m tœ k, /, w, 3 k (T-1~T-3), 5 /(Y-1~Y-5), 1 (F-1), 2 (: S-1, S-2) w w. ƒƒ t Table 1 ùkü, q m ƒƒ 300 ml 50 o C 15~20 (Eyela N-1000, Eyela Co., Japan)w g w 30 ml w(alcohol-free concentrates), w ph, brix, y, w, sr v w dw. ƒƒ m w³y, DPPH p w wx y sƒw z, y m, n-hexane, ethyl acetate, butanol w zwš, zw. ƒƒ z w zz dw ùkü, ƒƒ w³y, DPPH p w wx y s ƒw. x 1 n xl w, x z 4 C o 5,000 g 5 w x wš þw k w( x), v w w w. w³y d ³ Escherichia coli KCTC 1682, ³ Streptococcus mutans JC-2 w, q³ ampicillin ü³ Pseudomonas cepacia YAM-10 Pseudomonas rhodesiae YAM-12 ³ [20] w m w³y 1 sƒw. w³ rp yw» w 2 w³y sƒ Bacillus subtilis KCTC 1914, Staphylococcus aureus KCTC 1916, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa KACC 10186, Proteus vulgaris KCTC 2433, Salmonella typhimurium KCTC 1926, Escherichia coli H7:O157 ATCC ³ zz Saccharomyces cerevisiae IF0 0233, e ³ ³ Candida albicans KCTC 1940 q ³ Botrytis cineria KACC w., ³ Nutrient broth (Difco Co., USA) ƒƒ ³ w 37 o C 24 w z, ƒ ³ O.D w Nutrient agar (Difco Co., USA) sww ³ petri dish (90 15 Table. 1. The Korean traditional liquors used in this study. Classification Traditional liquors Ethanol content (%) Remark Takju T white rice 80%, wheat 20% T white rice 80%, wheat 20%, fungal fermentation T white rice 40%, wheat 60% Y white rice 100% Y white rice 100% Yakju/Cheongju Y glutinous rice, medicinal herb Y glutinous rice, edible plant added Y white rice 100%, natural resource added Fruit wine F plant fructus fermentation Soju (distilled liquor) S white rice 100% S white rice 100%

3 240 RYU et al. mm,, w ) 100 µl wš, ƒƒ m z 7 µl ³ disc-paper ( 6.5 mm, Whatman No.2) ƒw, 37 C 24 w o, ³ Sabouraud dextrose w w 37 C 24 o z, y j» dw w³y sƒw[20, 27-29]. w³ ampicillin (Sigma Co., USA) w, y j» ùkù mm dw, 3z d z t ùkü. wx y ƒƒ m z wx y thrombin time dw sƒw. p w y Amelung coagulometer KC-1A (Japan) w x š dw sƒw[20, 25, 26]. 37 C o 0.5 U p(sigma Co., USA) 50 µl 20 mm CaCl 2 50 µl, w 10 µl coagulometer p yw w 2 k z, x 100 µl ƒw z x š ¾ dw, v(sigma Co., USA), DMSO w. DMSO 33 š ùkü, p w wx y 3z w x s³ ùkü. DPPH free radical y ƒ wy y DPPH (1,1-diphenyl-2-picryl hydrazyl) radical œw» w y dw sƒw[20, 26]., w w 20 µl 99.5% k wk M DPPH 380 µl š yww 37 C 30 o k z, 516 nm microplate reader (Asys Hitech, Expert96, Asys Co., Austria) w Ÿ dw. butylhydroxytoluene, vitamin C vitamin E (Sigma Co., USA) w. DPPH free radical ƒ ƒ tw, IC 50 50% ùkü w. DPPH scavenging activity (%) = {1 (Sample O.D.)/(Control O.D.)} 100»k sƒ w y w DNS [20, 25] d w, w Lowry[24] dw, sr w Singleton [25] 400 µl 50 µl Folin-ciocalteau, 100 µl Na 2 CO 3 sy š 1 ew z 725 nm Ÿ dw. t tannic acid (Sigma Co., USA) w. v w Davis[25] dw, ƒƒ 400 µl 90% diethyleneglycol 4 ml ƒwš 1N NaOH 40 µl š 37 o C 1 z 420 nm Ÿ dw. t rutin (Sigma Co., USA) w. Brix d refractometer (Atago N- 1E, Japan) w. š q m m w Table 2 ùkü. k ph 4.25~4.61, /(3.3~4.12) (3.42~3.82) e, (F-1) ph 3.18 ƒ û. Brix /(Y-1~Y-5) (F-1) Table 2. ph, brix and contents of sugar, protein, total polyphenol and flavonoids in the alcohol-free concentrates prepared from different traditional liquors. Traditional liquors ph Brix (%) Reducing sugar (%, w/v) Protein (%, w/v) Total polyphenol (mg %, w/v) Total flavonoids (mg %, w/v) T T T Y Y Y Y Y F S ND ND S ND ND ND: Not Detected.

4 44~85 ùkü k(t-1, T-2) 20, (S- 1, S-2) ƒƒ ùküš, w y d w ùkù F-1 / y w ùkü. w û, F-1 k(t-2), (Y-3, Y-4) 0.02~0.1% w. wr sr v t pw ql ùkù, F-1 (Y-3, Y-4, Y-5) w ùk ü, ƒw l sr v d. PHYSIOLOGICAL FUNCTIONALITIES OF KOREAN TRADITIONAL LIQUORS 241 m w³, wx, wy y sƒ m w³, wx, wy dw Table 3 ùkü. Ampicillin E. coli S. mutans w w³ ùküù Pseudomonas sp. w w³ y ùkù [20], m Y-3, Y-4, Y-5 F-1 w w³ ùkü. w m ³ w ü³ wš. w w³ ùkü / 3 1 w³ rp Table 3. Antimicrobial, anti-thrombosis and antioxidant activity of the alcohol-free concentrates prepared from different traditional liquors. Traditional liquors/chemicals Antimicrobial activity (mm) P. cepacia P. rhodesiae E. coli S. mutans Antithrombin activity thrombin time inhibition (%) DPPH scavenging activity IC 50 (µg/ml) T > 2,700 > 2,000 T > 2,000 T > 2,000 Y > 2,000 Y > 2,000 Y > 2,700 > 2,000 Y > 2,700 > 2,000 Y > 2,700 > 2,000 F > 2,700 1,000 S S ampicillin nd nd aspirin 2 nd nd nd nd 300 nd vitamin C nd nd nd nd nd 25.0 vitamin E nd nd nd nd nd 23.7 BHT 3 nd nd nd nd nd 21.1 P. cepacia: Pseudomonas cepacia, P. rhodesiae: Pseudomonas. rhodesiae, E. coli: Escherichia coli, S. mutans: Streptococcus mutans. -: No activity. nd: not determined. ampicillin 1 : 1 µg/disc, aspirin 2 : 1.5 mg/ml, BHT 3 : butylhydroxytoluene. Table 4. Broad-spectrum of antibacterial activity of the alcohol-free concentrates prepared from Y-3, Y-4, Y-5 and F-1 traditional liquors. Gram (+) bacteria Gram (-) bacteria Fungi Strains Antimicrobial activity (mm) Y-3 Y-4 Y-5 F-1 Bacillus subtilis Staphylococcus aureus Staphylococcus epidermidis Streptococcus mutans Pseudomonas aeruginosa Proteus vulgaris Salmonella thyphimurium Escherichia coli H7:O Escherichia coli Saccharomyces cerevisiae Candida albicans Botrytis cineria

5 242 RYU et al. w. Y-3, Y-4, Y-5 F-1 ³ w w³ y ùkü ù, w³ y ùkù (Table 4). p 4, S. aureus P. vulgaris w w³y ùkü t q ³ w w q. wr m wx y sƒ w, T-1, Y- 3, Y-4, Y-5 F-1 x w, ƒ p k 27 j w w p w y ùkü(table 3). Y-3, Y-4, Y-5 F-1 y wx y wš ƒƒ w z, wp w z dw, T-1 y w y ùkù y w., 20 mg/ml Y-3, Y- 4, Y-5 F-1 2,700% w ùkü, wz Y-4, F-1, Y-5, Y-3 w ùkû(fig. 1). m ƒ p wz q. x wx š v 1.5 mg/ml 300% p wy ùký šw [20, 25, 26], m wx y wš Fig. 1. Thrombin inhibitory activities of the alcohol-free concentrates prepared from Y-3, Y-4, Y-5 and F-1 traditional liquors and aspirin. ù, w y k šw y ƒ v w wx z ƒ ùkú š d. wr DPPH y s ƒ F-1 w y ùkûù, (vitamin C, vitamin E, butylhydroxytoluene) w y (Table 3). q /(Y-3, Y-4, Y-5) (F-1) y z z y sƒ w³ y wx y w Y-3, Y-4, Y-5 F- 1 n-hexane, ethyl acetate, butanol w z w(table 5). 80~95% ƒ w, ethyl acetate z 0.8~3.4% û ùkû. ƒƒ z w w³ y sƒw, 4 ethyl acetate z y ùkû, p B. subtilis, P. aeruginosa S. typhimurium w w wz ùkü( ). Ethyl acetate z 1.8~4.0% y sww, 0.6~0.9% sr, 6.6~11.2% ww š,» w swwš d(table 5). x z y k w, w / w³ y»w y. wr ƒƒ z w wp y sƒw, 4 ethyl acetate z w y y, butanol m w wz ùkü( ).» wx zƒ butanol z ùkù [13, 25, 26], ethyl acetate z wp y v w Fig. 2 ùkü. Y-5 ethyl acetate z w Y-3, Y-4 F-1 z 1.5 mg/ml 2,000% w ùkü, 300% w ùkü v w wz ùkü. p m, ethyl acetate z wp w z Y-3, F-1, Y-4, Y-5 w ùkû. w m y wp Table 5. Sequential organic solvent fractionation of the alcohol-free concentrates of Y-3, Y-4, Y-5 and F-1 traditional liquors and composition of ethyl acetate fraction. Traditional liquors Fraction yield (% w/w) Composition of ethyl acetate fraction (%) Hexane Ethyl Acetate Butanol Water residue Reducing sugar Protein Total polyphenol Y Y Y F

6 PHYSIOLOGICAL FUNCTIONALITIES OF KOREAN TRADITIONAL LIQUORS 243 ƒw wš. REFERENCES Fig. 2. Thrombin inhibitory activities of the ethyl acetate fractions prepared from alcohol-free concentrates of Korean traditional liquors (Y-3, Y-4, Y-5 and F-1) and aspirin. z w[13, 25, 26], butanolz ethyl acetate z y, m z,»w d. wz, y w ml y ƒw q. ü m t y yw» w, q m yw wš, g w m w³ y, wy y p w wx y sƒw. ü q k (T1~T3), /(Y-1~Y-5), (F-1) (S-1, S-2), z» ƒ ùküù, Y-3, Y-4, Y-5 F-1 w y, sr, v w ùkü. w y y sƒw. Y-3, Y-4, Y-5 F-1 w w³ y wx y ùkü, DPPH F-1 w y. p w³ y» w ü ùkü ³ w w y ùkü, ³ wy ù kü. Y-3, Y-4, Y-5 F-1 m» w yz, w³ wx y ethyl acetate z y, p wx y Y-3, Y-4 F-1 aspirin w p w ùkü. m z ƒw z» l w, y œw ù kw, ü ml w y 1. Bae, I. Y., E. J. Yoon, J. M. Woo, J. S. Kim, H. G. Lee, and C. B. Yang The development of Korean traditional wine using the fruits of Opuntia ficus-indica var. saboten-i. characteristics of mashes and sojues. J. Kor. Soc. Appl. Biol. Chem. 45: Jeong, E. J., Y. S. Kim, D. Y. Jeong, and D. H. Shin Yeast selection and comparison of sterilization method for making strawberry wine and changes of physicochemical characteristics during its fermentation. Kor. J. Food. Sci. Technol. 38: Kim, H. R., S. H. Baek, M. J. Seo, and B. H. Ahn Feasibility of cheonghju brewing with wild type yeast strains from nuruks. Kor. J. Microbiol. Biotechnol. 26: Kim, H. S., J. S. Hyun, J. Kim, H. P. Ha, and T. S. Yu Enzymological characteristics and identification of useful fungi isolated from traditional Korean nuruk. Kor. J. Microbiol. Biotechnol. 26: Kim, I. H., W. S. Park., and Y. J. Koo Comparison of fermentation characteristics of Korean traditional alcoholic beverages prepared by different brewing methods and their quality changes after aging. Kor. J. Dietary Culture. 11: Kim, I. H., W. S. Park., and Y. J. Koo Comparison of fermentation characteristics of Korean traditional alcoholic beverages with different input sep and treatment of rice and nuruk (Korean-style bran koji). Kor. J. Dietary Culture. 11: Kim J. H. D. H. Lee, S. H. Lee, S. Y. Choi, and J. S. Lee Effect of Ganoderma lucidum on the quality and functionality of Korean traditional rice wine, Yakju. J. Biosci. Biotechnol. 97: Kim, J. H., D. H. Lee, S. Y. Choi, and J. S. Lee Characterization of physiological functionalities in Korean traditional liquors. Kor. J. Food Sci. Technol. 34: Kim, J. H., S. C. Cheng, N. M. Kim, and J. S. Lee Effect of indian millet koji and legumes on the quality and angiotensin I-converting enzyme inhibitory activity of Korean traditional rice wine. Kor. J. Food Sci. Technol. 35: Kim, J. H., S. H. Lee, N. M. Kim, S. Y. Choi, J. Y. Yoo, and J. S. Lee Manufacture and physiological functionality of Korean traditional liquor by using dandelion (Taraxacum platycarpum). Kor. J. Appl. Microbiol. Biotechnol. 28: Kim, J. S., E. J. Kwak, and Y. S. Lee Effect on the quality characteristics of Korean traditional wines with the addition of rosemary (Rosemarinus officinalis L.). Kor. J. Food Cookery Sci. 22: Kim, J. Y. and J. S. Go Screening of brewing yeasts and saccharifying molds for foxtail millet-wine making. J.

7 244 RYU et al. Kor. Soc. Appl. Biol. Chem. 47: Kim, K. W., J. K. Baek, Y. W. Jang, E. J. Kum, Y. S. Kwon, H. J. Kim, and H. Y. Sohn Screening of antibacterial agent against Streptococcus mutans from natural and medicinal plants. J. Life Sci. 15: Kim, S. J., J. Y. Baek, C. K. Park, and G. W. Kim Gastroprotective effect of Korean rice-wine (Yakju). Kor. J. Food Sci. Technol. 36: Kim, S. J., S. H. Ko, W. Y. Lee, and G. W. Kim Cytotoxic effects of Korean rice-wine (Yakju) on cancer cells. Kor. J. Food Sci. Technol. 36: Kim, Y, J. and Y. S. Han The use of Korean traditional liquors and plan for encouraging it. Kor. J. Food Culture. 21: Lee. D. H., J. H. Kim, N. M. Kim, and J. S. Lee Manufacture and physiological functionality of Korean traditional liquor by using chamomile (Matricaria chamomile). Kor. J. Food Sci. Technol. 34: Lee. D. H., W. J. Park, B. C. Lee, J. C. Lee, and J. S. Lee Manufacture and physiological functionality of Korean traditional liquor by using gugija (Lycii fructus). Kor. J. Food Sci. Technol. 37: Min, Y. K. and H. S. Jeong Manufacture of some Korean medicinal herb liquors by soaking. Kor. J. Food Sci. Technol. 27: Ryu, H. Y., K. H. Bae, E. J. Kum, S. J. Park, B. H. Lee, and H. Y. Sohn Evaluation for the antimicrobial, antioxidant and antithrombosis activity of natural spices for fresh-cut yam. J. Life Sci. 17: Saito Y., K. Wanezaki, A, Kawato, and S. Imayasu Antihypertensive effects of peptide in sake and its by-products on spontaneously hypertensive rats. Biosci. Biotechnol. Biochem. 58: Saito Y., K. Wanezaki, A, Kawato, and S. Imayasu Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees. Biosci. Biotechnol. Biochem. 58: Seo, S. B., J. H. Kim, N. M. Kim, S. Y. Choi, and J. S. Lee Effect of acasia (Robinia pseudoacasia) flower on the physiological functionality of Korean traditional rice wine. Kor. J. Microbiol. Biotechnol. 30: Seo, S. B., S.. M. Han, J. H. Kim, N. M. Kim, and J. S. Lee Manufacture and physiological functionality of wines and liquors by using plum (Prunus salicina). Kor. J. Biotechnol. Bioeng. 16: Sohn, H. Y., C. S. Kwon, K. H. Son, G. S. Kwon, H. Y. Ryu, and E. J. Kum Antithrombin and thrombosis prevention activity of buckwheat seed, Fagopyrum esculentum Moench. J. Kor. Soc. Food Sci. Nutr. 35: Sohn, H. Y., C. S. Kwon, K. H. Son, G. S. Kwon, Y. S. Kwon, H. Y. Ryu, and E. J. Kum Antithrombosis and antioxidant activity of methanol extract from different brands of rice. J. Kor. Soc. Food Sci. Nutr. 34: Sohn, H.-Y., K. H. Son, C.-S. Kwon, G.-S. Kwon, and S. S. Kang Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai. Phytomedicine 11: Sohn, H. Y., Y. S. Kim, E. J. Kum, Y. S. Kwon, and K. H. Son Screening of anti-acne activity of natural products against Propionibacterium acnes. Kor. J. Microbiol. Biotechnol. 34: Tabrizizadeh M., and M. Mohammadi In vitro evaluation of antibacterial activities of root canal sealers. J. Clin. Dent. 16: (Received June 11, 2007/Accepted July 28, 2007)