Production of Bacteriocin by Staphylococcus sp. NPSI 38 in Koji Extract Medium with Rice Protein Hydrolyzate and Its Growth-inhibitory Activity against Hiochi-bacteria

Abstract

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...

Growth-Inhibition of <i>Lactobacillus hilgardii</i>, a Bacterium Related to Hiochi, in the Mizu-Koji Process by Bacteriocins from Lactic Acid Bacteria

“…Bacteriocin production was carried out in a medium bottle using 10% (v/v) koji extract medium supplemented with RPH. The koji extract solution used as the medium for bacteriocin production and RPH (Shimada Kagaku, Nagaoka) were prepared as described previously [7]. Bacteriocin-producing lactic acid bacteria were cultured statically at 30 for 24 h at an initial turbidity of 0.1-0.2 at 660 nm.…”

“…The antimicrobial activity of bacteriocin was determined by the agar diffusion method using a stainless cup according to the procedure reported previously [7]. The agar concentration of each medium was adjusted to 1%.…”

“…To prevent hiochi from occurring in sake brewing, the exploitation of bacteriocins with antimicrobial activity against the strains of Lactobacillus described above was considered [6]. Recently, it was reported that some lactic acid bacteria produce bacteriocin in koji Growth-Inhibition of Lactobacillus hilgardii, a Bacterium Related to Hiochi, in the Mizu-Koji Process by Bacteriocins from Lactic Acid Bacteria extract medium supplemented with rice protein hydrolyzate (RPH), which can be legally used in the sake brewing process [7], and that the culture supernatant containing the bacteriocin was found to be effective in inhibiting the growth of L. hilgardii in MRS medium [5].…”

Analysis of Free Fatty Acids inSakeby an Enzymatic Method and Its Application for Estimating Ethyl Caproate and Selecting Yeast with High Productivity of the Ester

“…8), produced a greater amount of either ethyl caproate or FFAs than the parental or standard sake yeast strains (nos. 1-7), the correlation between FFAs (x) and ethyl caproate (y) produced in the koji extract medium 12) showing a regression line with a high correlation coefficient (r ¼ 0:9824, y ¼ 14:6661x À 0:0798, p < 0:01). Furthermore, we confirmed the high productivity of FFAs/ethyl caproate by these strains (nos.…”