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Enhanced production of bacteriocin by Staphylococcus sp. NPSI 38 (NPSI 38) using koji extract medium was investigated. The bacteriocin produced by NPSI 38 in MRS medium was found to be effective for inhibiting the growth of Lactobacillus hilgardii NBRC 15886 T , one of the representative hiochi-bacteria. In cultivations using test tubes without pH control, meat extract and Polypepton were effective for bacteriocin production by NPSI 38 with 10%(v/v) koji extract medium. When the koji extract medium supplemented with hydrolyzate of a rice protein preparation was used instead of meat extract and Polypepton, NPSI 38 produced 160 U/ml of bacteriocin in the cultivation with pH control, which was almost as high as that (156 U/ml) observed in cultivation using MRS medium with pH control. When the cells of L. hilgardii NBRC 15886T collected at logarithmic growth and stationary phases were inoculated into fresh modified MRS medium with 11 U/ml of the bacteriocin, negligible cell growth was observed, irrespective of different growth phases of cells inoculated. Little or no increase in cell concentrations in the media containing bacteriocin showed that the action of the bacteriocin produced by NPSI 38 was bacteriostatic against the hiochi-bacterium.Keywords: bacteriocin, hiochi-bacteria, Lactobacillus hilgardii, koji extract medium, rice protein *To whom correspondence should be addressed. Email: mtanig@eng.niigata-u.ac.jp IntroductionHiochi-bacteria are recognized as major potential spoilage microorganisms of sake. Hiochi-bacteria have been identified as several Lactobacillus species (Lactobacillus fructivorans, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum and Lactobacillus hilgardii) by molecular biology and biochemistry methods (Goto-Yamamoto et al., 1994, Niidome et al., 1995. Hiochi caused by outbreaks of the bacteria results in lowering of the pH, occurrence of turbidity, and formation of off-flavors such as diacetyl. The spoilaging phenomena seriously deteriorate the quality of sake. To prevent hiochi from emerging in the sake brewing industry, pasteurization (hiire) at around 65℃ for a short duration of 2-3 min or microfiltration for removing hiochibacteria has been carried out in the sake brewing process.However, the spoilage of sake caused by hiochi-bacteria is not completely repressed at present.A large number of bacteriocins, antimicrobial peptides, are produced by many lactic acid bacteria (LAB) (Onda et al., 2002, Soriano et al., 2004, Sparo et al., 2006. Since bacteriocins are isolated from foods, such as meat and dairy products, which normally contain LAB, they have unknowingly been consumed for centuries by humans (Goktepe, 2006). Thus, bacteriocins from LAB have been shown to be safe, and have the potential to be effective as natural food preservatives (Ennahar et al., 2000, Cleveland et al., 2001. Many bacteriocins are small proteins, stable at high temperature, digested by protease (Ray, 1992, Heng et al., 2007, and only effective against a limited narrow spectrum of bacteria, typic...

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The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of Lactobacillus hilgardii, a hiochi bacterium, at the mizu-koji stage in the sake brewing process. The bacteriocins produced by C101910 and NBRC 12007 were slowly inactivated in the koji extract by proteases, depending on the pH and temperature of the koji extract. However, the activities of bacteriocins from C101910 and NBRC 12007 remained about 50% and 70% of the initial values, respectively, even after incubation at pH 3 and 10 for 12 h. By adding the bacteriocin solutions, prepared according to the method reported previously, from C101910 and NBRC 12007 to koji extract (pH 3 and 10) at a volume ratio of 5% (v/v) and 1% (v/v), respectively, the number of viable L. hilgardii cells decreased to below the detection limit (1.0 10 2 cells/ml) from the initial concentration (3.2 10 5 cells/ml) within 12 h. In mizu-koji consisting of rice koji and lactic acid solution at pH 3 and 10 , the growth-inhibitory effect of bacteriocins from C101910 and NBRC 12007 on L. hilgardii decreased as compared with that in koji extract. However, the number of viable L. hilgardii cells decreased by more than two orders of magnitude as compared with the initial value (1.0 10 5 cells/ml) by adding the bacteriocin solution at a volume ratio of 10% (v/v).