• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1462-1470
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• 2022

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70℃ and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

• KSBB Journal
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• v.22 no.3
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• pp.162-167
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• 2007

Ammonia gas is one of the major pollutants which cause environmental pollution and damage to the human and the livestock. The objective of this study was to investigate the important parameters for the development of efficient removal of ammonia gas by Bacillius subtilis IB101 and to optimize the medium composition for the mass production of B. subtilis IB101. The ammonia gas removal efficiency was evaluated at different growth phases and by changing culture conditions (temperature, pH). The effect of $(NH_4)_2SO_4$ concentration in preculture medium was examined. Medium optimization for the mass production of B. subtilis IB101 was performed by using Plackett-Burman design and one factor at a time method. The removal of ammonia gas was more efficient at exponential phase by 20% than at stationary phase. The ammonia gas removal was the highest at pH 4 and 30 $^{\circ}C$. There was not any significant influence of concentration of $(NH_4)_2SO_4$ on the removal of ammonia gas. The components of optimized medium for the production of viable Bacillus subtilis IB101 was yeast extract 10 g/l, soluble starch 2.5 g/l, $MgSO_4$ 6 g/l, $CaCl_2$ 1.55 g/l, $(NH_4)_2SO_4$ 5 g/l, $KH_2PO_4$ 0.75 g/l, soy bean meal 8 g/l.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.84-92
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• 2010

Antibacterial compound from Bacillus subtilis MJP1 was purified using C18 Sep-Pak cartridge, ion exchange chromatography, and gel filtration chromatography. The purified antibacterial compound showed antibacterial activity against Listeria monocytogenes, Bacillus subtilis, Staphylococcus aureus subsp. aureus, and Enterococcus faecalis. The purified antibacterial compound was found to be stable at $100^{\circ}C$ for 5 min and in the pH range of 3.0~9.0, but it was unstable at pH 10.0. It was inactivated by proteinase K and pronase E, and heat treatment at $121^{\circ}C$ for 15 min, but it was stable with lipase and $\alpha$-amylase treatment, which indicated its proteineous nature. Ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry analysis were used to identify the purified antibacterial compound and confirmed the existence of two peptides (3356.54 Da, 3400.5244 Da).

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.131-137
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• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.

• Microbiology and Biotechnology Letters
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• v.42 no.4
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• pp.393-401
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• 2014

A bacterium producing antimicrobial substance was isolated from cheonggukjang. The bacterium was identified as a strain of Bacillus subtilis by 16S rDNA sequencing and designated as Bacillus subtilis HH28. The antimicrobial substance produced from Bacillus subtilis HH28 was purified by 0-80% ammonium sulfate precipitation, DEAE-sepharose FF column chromatography, and Sephacryl S-200 HR gel chromatography. The molecular weight of the purified antimicrobial substance was estimated to be approximately 3,500 Da using Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and direct detection analysis. Antimicrobial substance from B. subtilis HH28 not only inhibited B. cereus, but also Listeria monocytogenes and Vibrio parahaemolyticus. The purified antimicrobial substance was stable at $40-80^{\circ}C$, and between pH 2 and 8. Antimicrobial activity of the purified substance was completely destroyed by treatment of protease, proteinase K, and pronase E, indicating that it is proteinaceous.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.671-674
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• 2001

Cyclodextrin glucanotransferase(CGTase) derived from recombinant Bacillus subtilis was partial purified and concentrated by ultrafiltration. The prepared CGTase were immobilized on various matrices by ionic interaction or covalent bond. CGTase covalently bound on CNBr-activated sepharose 4B were identified to be the highest immobilization activity among various immobilization methods. The optimum conditions for CGTase immobilization were determined; $30^{\circ}C$, 6Orpm, using O.2g CNBr-activated sepharose 4B in pH 6.0 phosphate buffer and 9hr immobilization.

• Journal of Life Science
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• v.11 no.3
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• pp.235-241
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• 2001

To select the sntagonistic bacteria against B. cinerea, isolates were screened from the eggplant leaves and rhizosphere soils in the eggplnat fields in the greenhouse. W1 and P99 isolates were selected by the inhibition of mycelial growth of B. cinerea E12 in vitro test. These isolates, W1 and P99, were identified as Bacillus subtilis and Pseudomonas putida, respectively, by the Bergeys manual and API systems, For the formulation of the antagonistic bacteria, the media for the mass production were prepared with biji(soybean curd residues) or soybean flour. B. subtilis W1 or P. putida P99 was mass cultured in biji broth or soybean flour extrect broth and then soybean flour, corn starch flour, rice glutinous flour and biji flour as high molecular substrates were added. These mixtures were dried, grinded and formulated as brofungicides of wettable powder type. The assess the control effect of biofungicides against the infection of B. cinerea, six types of formulations were assayed at the pot culturing with eggplant in the greenhouse. According to the results, there were no significant differences among the formulation methods. However, P99S or PppB formulated with P. putida P99 showed the highest control values as 90.4% and 96.1%, respectively. Then. BSB or BSD formulated whit B. subtilis W1 were 80.8% and 83.0%, respectively. There afforementioned values were more effective than that of chemical fungicide. Ipro W.P which showed as 72.6%.

• Journal of Veterinary Clinics
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• v.30 no.6
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• pp.459-463
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• 2013

A study investigated the effects of a mixture of Lactobacillus plantarum (L. plantarum) and Bacillus subtilis (B. subtilis) on diarrhea incidence, immune response, and fecal microflora counts in post-weaning piglets. One hundred 28-day-old piglets were randomly assigned to five treatment groups: negative control (NC), free of antibiotics; positive control (PC), 0.03% chlortetracycline; LB 1, a mixture of L. plantarum and B. subtilis (LB) 0.5 kg/ton feed; LB 2, LB 1.0 kg/ton feed; and LB 3, LB 2.0 kg/ton feed. Diarrhea scores for LB 2 and LB 3 from the 2nd week were significantly reduced compared to NC (P<0.05). In terms of the level of IgG and IFN-${\gamma}$, all treatment groups were significantly higher than NC (P<0.05), and the IgG level of LB 3 was significantly higher than that of PC (P<0.05). Furthermore, fecal lactic acid bacteria (LAB) counts for LB 2 and LB 3 were significantly higher than those of NC and PC (P<0.05). In addition, fecal Enterobacteriaceae (ENT) counts for PC, LB 2 and LB 3 were significantly lower than those of NC (P<0.05). Based on the results from this study, it was concluded that a combination of L. plantarum and B. subtilis strains could be used as potential alternatives to antibiotics to prevent diarrhea incidence in piglets.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.3
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• pp.395-402
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• 2001

A fusant FG-21 was selected on the basis of higher ${\gamma}-GTP$ activity following fusion process between SM-2 and SM-10 of Bacillus subtilis mutants. ${\gamma}-GTP$ activity of the mutant FG-21 was increased up to 612 U/mL when grown for 36 hr at $37^{\circ}C$ in culture media containing 1% glycerol 1% glycerol, 1% peptone, 0.1% citric acid, 5 mM $K_2HPO_4$, 1 mM $FeCl_3$, 1 mM $MgCl_2$, 1 mM $NH_4Cl$, pH 7.0. In fusnat FG-21, the ratio of protein to total sugar contents for biopolymer A was 38 to 59. for biopolymer B from parental strains it was 19 to 78. Fructose contents determined by HPLC were $573.7\;\mu\textrm{g}/mg\;and\;764.4\;\mu\textrm{g}/mg$ for biopolymer A and B, respectively. And glutamic acid content were $163.7\;\mu\textrm{g}/mg\;and\;94.6\;\mu\textrm{g}/mg$ for biopolymer A and B, respectively. In fusant FG-21, the ratio of fructose to glutamic acid contents for biopolymer A was 78 to 22. For biopolymer B from parental strains it was 89 to 11.