• Asian-Australasian Journal of Animal Sciences
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• v.30 no.4
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• pp.576-584
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• 2017

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ${\beta}-defensin-2$ (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.8
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• pp.1126-1131
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• 2010

The antiobesity effect of soymilks fermented with Bacillus subtilis KC-3 (KCCM 42923) from cheonggukjang was compared with other sources of B. subtilis KCCM 11316 and B. subtilis MYCO. The antiobesity effect was investigated by measuring the release of leptin, Oil red O staining, glycerol secretions and adipogenic transcription factor by reverse transcription-polymerase chain reaction (RT-PCR) in the 3T3-L1 adipocytes. Fermented soymilk with B. subtilis KC-3 (F-KC) led to decrease levels of leptin secretion and increase levels of glycerol secretion in the cells. In addition, F-KC reduced contents of Oil red O dye in the 3T3-L1 adipocytes. Also, mRNA expression levels of both SREBP-1c (sterol regulatory element-binding protein 1-c) and PPAR-$\gamma$ (peroxisome proliferator-activated receptor-$\gamma$), which are adipogenic transcription factor, in cells treated with F-KC were markedly down regulated. These results demonstrate that the Bacillus subtillis fermented soymilk (F-KC) decreased lipid content in 3T3-L1 adipocytes by inhibiting lipogenesis. All B. subtilis fermented soymilks had shown antiobesity activities, however, F-KC exhibited the strongest antiobesity effect in the 3T3-L1 adipocytes. Our study suggests that especially F-KC increased the potential of antiobesity effects.

• Korean Journal of Food Science and Technology
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• v.41 no.4
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• pp.399-404
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• 2009

The goal of this study was to improve the quality of cheonggukjang by the optimization of the inoculation methods of the Bacillus subtilis (B. subtilis) and Lactobacillus plantarum (L. plantarum) strains. In order to optimize the mixed cultivation of B. subtilis and L. plantarum, the B. subtilis strain was inoculated into steamed soybeans after cultivation of L. plantarum. Inoculation size of B. subtilis was changed to the simultaneous inoculation method in order to stimulate the growth of the L. plantarum in cheonggukjang. The viable cell count of L. plantarum increased from $2{\times}10^7$ CFU/g to $2-6{\times}10^8$ CFU/g and B. subtilis grew to $9{\times}10^8$ CFU/g. These results showed that 2 strains were successfully able to grow in the steamed soybean for good quality of cheonggukjang by optimization of the inoculation methods. The sensory evaluation indicated that a favorable aroma and overall acceptance of cheonggukjang by the optimized mixed cultivation of B. subtilis and L. plantarum, which was relatively higher than those of cheonggukjang by single strain inoculation of B. subtilis.

• Journal of Chitin and Chitosan
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• v.23 no.4
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• pp.228-233
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• 2018

We isolated microorganisms from soil, which is sampled at forest, Kyeonbuk, Korea, as cellulolytic microorganisms. The isolated strains were identified by analysis of 16S rRNA gene from the starins. The result, four kinds of Bacillus subtilis, one kind of Bacillus amyloliquefaciens, and one kind of Bacillus cereus were identified. Among these strains, Bacillus subtilis was selected due to its high cellulase activity and this strain was named as Bacillus subtilis CNS. The optimum pH and temperature of the cellulase from Bacillus subtilis CNS was pH 5.0 and $40^{\circ}C$, respectively. In the investigation of pH and temperature stability, the cellulase from Bacillus subtilis NSC stabled pH 4.0~6.0 range and until $40^{\circ}C$ for 30 min perfectly. In the enzyme activity for various cellulosic substrate, cellulase from Bacillus subtilis CNS showed the highest activity for CM-cellulose. And, the enzyme activities for alkali swollen cellulose, Alpha-cellulose, Sigmacell-cellulose, and Avicel were approximately 31%, 8%, 8% and 4% of activity for CM-cellulose, respectively. In the degradation of CM-cellulose, the 0.26 U/ml and 0.52 U/ml of cellulase showed 0.43 and 0.76 U/ml activity for CM-cellulose after the reaction of 120 min, respectively.

• Journal of Microbiology and Biotechnology
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• v.4 no.1
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• pp.68-74
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• 1994

A bacterial strain YB-70 which has powerful biocontrol activity against Fusarium solani causing plant root-rot resulting in considerable losses of many economical crops was isolated and selected from over 500 isolates from a ginseng rhizosphere in suppressive soil, and identified as a strain of Bacillus subtilis. In several biochemical and in vitro antibiosis tests on F. solani with culture filterates from B. subtilis YB-70, our data strongly indicated metabolites which mediated inhibition of the fungal growth were presumed to be heat-stable, micromolecular, and ethyl alcohol solutable antifungal substances. Suppression of root-rot by B. subtilis YB-70 was demonstrated in pot trials with eggplant (Solanum melongena L) seedlings. Treatment of the seedling with the bacterial suspension (1.7~1.9$\times$$10^5$ CFU/g) in F. solani-infested soil significantly reduced disease incidences by 68 to 76% after 25 to 30 days. The results supported that B. subtilis YB-70 have excellent potentials as a biocontrol agent.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.227-231
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• 1991

A gene coding for a $\beta$-galactosidase of Bacillus subtilis HP-4 was cloned in E. coli JM109 by inserting HindIII digested fragment of B. subtilis HP-4 chromosomal DNA into the site of pBR322 and selecting recombinant transformant showing blue color on X-gal plate. The recombinant plasmid, named pBG109, was found to contain the 1.4 Kbp HindIII fragment originated from B. subtilis HP-4 chromosomal DNA by Southern hybridization. The cloned gene was stably maintained and expressed in E. coli JM109 and the pBG109 encoded $\beta$-galactosidase had the same enzymatic properties as those of $\beta$-galactosidase produced by B. subtilis HP-4.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.327-331
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• 2000

The application of LFH-PCR(long flanking homology region-PCR) for Bacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved from B. subtilis genome data base, kanamycin resistance gene was inserted into two genes of B. subtilis involved in sporulation, spoIIIE and spoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene of B. subtilis or other prokaryote in the genomic era.

• Microbiology and Biotechnology Letters
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• v.40 no.3
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• pp.186-189
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• 2012

A gene coding for mannanase (manA) from Bacillus subtilis was introduced into a shuttle vector pGK12 between Escherichia coli, B. subtilis and Lactobacillus paracasei. As a result of transferring the resultant plasmid, designated pGK12M3, into three different strains, the manA gene was found to be expressed in L. paracasei as well as in B. subtilis and E. coli. In a 4 L fermentor culture, the production of mannanase by recombinant L. paracasei (pGK12M3) reached a maximum level of 5.4 units/ml in an MRS medium with a fixed pH 6.5. Based on the zymogram of mannanase, it is assumed that mannanase produced by recombinant L. paracasei is not maintained stably with proteolytic degradation. The optimal temperature and thermostability of mannanase produced by recombinant L. paracasei were also found to be different from those of enzymes produced by B. subtilis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.5
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• pp.846-851
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• 1998

Characteristics of chunggugjang fermented by Bacillus subtilis DC-2, a pigment producing bacterium, were investigated. More water soluble browning materials were produced with fermentation time. The pH was gradually alkalized. The contents of amino nitrogen were extraordinarily increased with fermentation time. Both strength and hardness were gradually decreased during fermentation. Total 30 volatile compounds were identified in the chunggugjang fermented by B. subtilis DC-2. The pyrazines were detected more than any other compounds. The good aroma of the chunggujang fermented by B. subtilis DC-2 was considered to be contributed by tetramethylpyrazine, trimethylpyrazine, 1-octen-3-ol, 2, 5-dimethylpyrazine and guaiacol.

• Journal of Microbiology
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• v.42 no.4
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.