• Journal of Microbiology
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• 제40권1호
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• pp.26-32
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• 2002

From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The pretense is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10 and at $45^{\circ}C$, although it is unstable at $60^{\circ}C$.

• Journal of Microbiology
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• 제44권6호
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• pp.694-698
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• 2006

Several marine bacterial strains were isolated from Undaria pinnatifida (Miyok in Korean). Sixty-six strains were isolated on R2A agar media at $10^{\circ}C$ and identified by a phylogenetic analysis of the 16S rRNA gene sequences. They were grouped into 10 different sequence types based on the initial sequence analysis of the 5' domain of the gene (approximately 500 bp). Full sequences of 16S rRNA gene, were obtained from one strain in each sequence type and the species-affiliation was determined using phylogenetic and sequence similarity analyses. The results of the analyses indicated that they were closely related to Psychrobacter aquimaris, P. celer, P. nivimaris, P. pulmonis, Psychromonas arctica or Bacillus psychrodurans. These bacteria are marine or psychrotrophic bacteria. Because the sporophytes of U. pinnatifida are cultured on the costal area during winter, the U. pinnatifida-associated bacteria appeared to grow at low temperatures. U. pinnatifida sporophytes can be a good source for the isolation of psychrotrophic bacteria.

• Journal of Dairy Science and Biotechnology
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• 제18권1호
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• pp.38-46
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• 2000

Since generalization of cold storage of raw and processed milk, psychrotrophic bacteria has become more important. The number present in raw milk is related to sanitary conditions during pro-duction and to length and temperature of storage before pasteurization. Growth of psychrotrophs In raw milk often reduces the quality of pasteurized products. Recently, some pathogenic bacteria like Listeria monocytogenes, Yersinia enterocolitica, Bacillus cereus are reported to grow at low temperature and cause food poisoning. The presence of gram positive psychrotrophic bacteria which can survive pasteurization can limit the shelf life of pasteurized milk during extended storage and the survival of heat stable proteases and lipases produced by gram negative psychrotrophic bacteria often brings about proteolytic damage to milk protein in the products. Therefore, in order to prevent the deteorioration of milk and milk products by the growth of psychrotrophs, it is necessary to cool down the temperature of raw milk as soon as possible after milking and to keep the temperature below 5t during storage at farm. As psychrotrophic bacteria become readily predominant in raw milk under refregeration, it can be considered to change the traditional incubating temperature for SPC from 30${\sim}$32$^{\circ}C$ to 25${\sim}$27$^{\circ}C$ at which the psychrotrophs prefer to grow. The psychrotrophic bacterial count(PBC) is of limited use in dairy industry, because of the 10 days incubation period. Although estimates of psychrotrophic bacteria may provide an acceptable shelf-life prediction, there is no single, generally acceptable rapid method for replacing the PBC at the moment. Consequently, faster method for esmating psychrotrophic bacteria has to be developed.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.213.1-213
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• 2005

• 한국축산식품학회지
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• 제31권2호
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• pp.311-315
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• 2011

The objective of this study was to evaluate the antimicrobial effects of various natural flavonoids against growth of psychotropic Bacillus cereus strains, which cause dairy food outbreaks. Flavonoids were first screened for their ability to inhibit growth of B. cereus strains using the paper-disc diffusion test. Second, the growth inhibitory effect of selected flavonoids was evaluated in tryptic soy broth supplemented with 0.6% yeast extract, and the bactericidal effect of the flavonoids was measured in 0.8% (w/v) NaCl solution. Based on the paper-disc diffusion test, kaempferol was effectively active against B. cereus P14 and B. cereus KCCM 40935. Kaempferol had an antimicrobial effect at concentrations greater than 100 ${\mu}M$, and the numbers of B. cereus P14 and B. cereus KCCM 40935 decreased by 3.55 and 1.5 log cycles, respectively. The cell numbers of B. cereus P14 and B. cereus KCCM 40935 treated with 50 ${\mu}M$ kaempferol were reduced by 4.18 and 2.84 log cycles during a 24 h incubation to test the bactericidal effect of kaempferol (p<0.05). The results indicate that kaempferol had the greatest antimicrobial effect among the psychotropic B. cereus strains and the natural flavonoids tested.

• Journal of Microbiology
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• 제41권1호
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• 미생물학회지
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• 제39권4호
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• pp.230-234
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• 2003

Bacillus amyloliquefaciens psychrotrophic strain이 분비하는 세포 외 단백질 가수분해효소를 정제하여, endopeptidase 활성에 관한 특성을 분석하였다. Protease SE910로 명명된 효소는 단백질 내부의 leucine에 연결된 peptide 결합만을 가수분해하는 endopeptidase로 작용한다. 효소를 특이한 아미노산 잔기에 작용하는 화학수식제와 반응하였을 때 효소의 활성부위에 관여하는 아미노산 잔기가 수식되었을 때는 효소활성이 저해를 받는다. 본 효소는 serine을 수식하는 PMSF에의해 endopeptidase 활성이 완전히 저해되었으며, 카르복실 기능기를 수식하는 화학수식제에 의해 저해되었고, lysine을 화학수식하는 PLP에 의해서는 큰 영향을 받지 않았다. 이것은 본 효소의 endopeptidase 활성에 serine과 aspartic acid 잔기가 관여하는 것을 의미한다. 구조적으로 leucine을 포함하는 유도체인 bestatin은 효소의 endopeptidase 활성을 경쟁적으로 저해하였다.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.861-869
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• 1999

An extracellular protease was purified to homogeneity from the culture supernatant of psychrotrophic bacteria Bacillus sp. JH 108 using procedures including ammonium sulfate fractionation, anion exchange chromatography, gel filtration chromatography, and cation exchange chromatography. The enzyme exhibited a molecular weight of 36 kDa, an optimum pH of 8 to 9, and optimum temperature of $60^{\circ}C$. The enzyme preferentially hydrolyzed leucine at the N-terminus of peptides and thus can be classified as an aminopeptidase. It was strongly inhibited by metal chelating agents such as EDTA and l, l0-phenanthroline. The activity lost by EDTA was restored with $Zn^{2+}{\;}or{\;}Co^{2+}$. These divalent cations also stimulated the native enzyme. This suggests that the enzyme is a metalloprotease acting as a leucine aminopeptidase.

• Food Science and Biotechnology
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• 제17권1호
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• pp.166-171
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• 2008

Abstracts Soybean sprouts which are a popular vegetable in Korea, are produced using the techniques of sous vide. The purpose of this study was to determine the effect of nisin and storage temperature on the microbiological and physicochemical qualities of sous vide processed soybean sprouts during storage in order to improve shelf-life and industrial applications. During storage of the cook-chilled soybean sprouts at $3^{\circ}C$, no development of mesophilic microorganisms was observed. However, at $10^{\circ}C$ storage without nisin, the number of mesophilic microorganisms increased markedly, whereas sprouts stored at the same temperature with nisin showed no observed increase. Psychrotrophic microorganisms, anaerobic microorganisms, and Bacillus cereus all showed similar trends. The ascorbic acid content, following the sequential heat processing of soybean sprouts through blanching and pasteurization decreased markedly during early storage, and stabilized thereafter. During storage, no major changes in the color or ascorbic acid content of samples at either temperature were observed. With regard to microbial and physicochemical qualities, the presences of nisin and storage temperature are important factors for extending shelf-life of soybean sprout.

• Journal of Microbiology
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• 제43권3호
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• pp.237-243
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• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.