• Korean Journal of Food Science and Technology
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• v.38 no.1
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• pp.82-87
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• 2006

Twenty one strains strongly producing protease were isolated from Korean traditional Cheonggukjang. Eight strains selected by sensory evaluation on Cheonggukjang prepared with isolated strains were identified with based on biochemical properties a and 16S rDNA sequencing. Identified strains were Bacillus subtilis MB4, and Bacillus amyloliquefaciens A1, A2, B1, MC1, SB2, SC1, and SD1. Protease activities, important strain selection factor, were higher in Cheongjukjang prepared with B. subtilis MB4 (179.6 Unit) and B. amyloliquefaciens SB2 (201.9 Unit) than commercial traditional Cheonggukjang (97.9 Unit). Sensory evaluation revealed Cheonggukjang prepared with B. subtilis MB4 had flavor very similar to commercial traditional Cheonggukjang. Cheonggukjang prepared with B. suhtilis MB4 (0.0006 Pa s) and commercial traditional Cheonggukjang (0.0002 Pa s) revealed lower viscosities than those of Cheonggukjang prepared with B. amyloliquefaciens SB2, MC1, B1, A1, SD1, A2, and SC1 (0.006 to 0.008 Pa s at 1001/s. Results show Cheonggukjang could be prepared using single strain of B. subtilis MB4, maintaining high protease activity and very similar sensory and viscosity qualities with those of commercial traditional Cheonggukjang.

• Food Science and Preservation
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• v.13 no.1
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• pp.77-82
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• 2006

This study was carried out to investigate the Cheongbukjang fermentation characteristics and to select the strain showing a fibrinolytic activity. Fibrinolytic activity of 5 strains isolated from the commercial Chungkukjang was tested N2 strain showed the highest activity $(41.7\%)$ while N3 and N5 had similar activity $(27.8\%)$ compared to plasmin 1 unit/mL The selected N2 strain was determined as B. subtilis with $90.1\%$ homology by API kit analysis. Quality characteristics of Cheongbukjang fermented by 6 kinds of strains were tested Among 3 strains cultured, B. subtilis (KCTC 3014) showed the highest viscous substance, fibrinolytic activity and amino type nitrogen content. After isolated, B. subtilis N2 showed the highest viscous substance, fibrinolytic activity and amino type nitrogen content. Optimum steam-time for Cheongbukjang fermented by B. subtilis (KCTC 3014) and B. subtilis N2 was 45 min while optimum fermentation-time was 20 hr.

• Microbiology and Biotechnology Letters
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• v.44 no.2
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• pp.185-193
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• 2016

A novel proteolytic bacterium was isolated from soil at Yeungnam University, South Korea. The strain, named FBL-1, was rod-shaped with a smooth surface. Biolog and API 50CHB test results revealed that strain FBL-1 was a Bacillus species. Based on 16S rDNA sequencing and chemotaxonomic characterization, the strain was identified as Bacillus subtilis because it had the highest homology with Bacillus subtilis subsp. subtilis NCIB 3610 (99.5%). In liquid culture at 37℃ with shaking at 200 rpm, fructose and yeast extract were found to be the best carbon and nitrogen sources, respectively, for cell growth and protease production. The highest protease activity (451.640 U/ml) was obtained when the strain was cultured in medium containing 20 g/l of fructose and 5 g/l of yeast extract. Although further studies are needed to characterize the protease and enhance its activity, the newly isolated protein-degrading B. subtilis FBL-1 can be applicable for the production of peptides and for the degradation of proteins in various industries.

• Journal of Food Hygiene and Safety
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• v.38 no.5
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• pp.356-360
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• 2023

We analyzed the heat resistance of non-pathogenic Bacillus atrophaeus, Bacillus subtilis, and Geobacillus stearothermophilus spores which exhibit strong heat resistance and evaluated the possibility of using them to determine direct sterilization when manufacturing retort foods. The D₁₂₁-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 2.9±0.1 min, 4.3±0.1 min, and 3.7±0.1 min, respectively. The Z-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were 43.0±1.4℃, 25.0±1.6℃, and 35.8±1.4℃, respectively. The D₁₂₁-values of B. subtilis, G. stearothermophilus, and B. atrophaeus spores were all higher than that of Clostridium botulinum spores used to confirm retort food sterilization. Considering these results, B. subtilis, G. stearothermophilus, and B. atrophaeus spores can be used instead of the pathogenic spore-forming bacteria C. botulinum when sterilizing retort food. In addition, sterilization can be confirmed in 2 to 3 days, a shorter time than the 13 days required for existing bacterial growth experiments based on the Korean food code.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.131-142
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• 2019

The primary objective of this study was to determine the content of biogenic amines in Korean traditional fermented soybean pastes (doenjang) and to isolate potential probiotic Bacillus sp. with the ability to inhibit biogenic amines accumulation. There were significant differences in the bacterial cell counts, pH value, titratable acidity, salinity, and biogenic amine content between the samples. Among Bacillus strains isolated from doenjang, Bacillus (B.) licheniformis DB102, B. subtilis DB203, B. stearothermophilus DB206, Bacillus sp. DB209, Bacillus sp. DB310, B. coagulans DB311, B. cereus DB313, B. amyloliquefaciens DB714, Bacillus sp. DB917, B. cereus DB 915, B. subtilis DB1020, and Bacillus sp. DB1022 were found to be able to produce biogenic amines. On the other hand, biogenic amine-degrading strains were identified as Bacillus sp. DB403, Bacillus sp. DB407, B. subtilis DB517, B. licheniformis DB612, and B. subtilis DB821. In particular, Bacillus sp. DB407 and B. subtilis DB821 showed probiotic properties including tolerance to artificial digestive juices, adherence to intestinal epithelial cells, resistance to antibiotics, and antibacterial activity against biogenic amine-producing strains. In conclusion, the two probiotic Bacillus strains may be considered as the suitable starter for manufacture of fermented soybean foods with low biogenic amines content.

• Microbiology and Biotechnology Letters
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• v.28 no.1
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• pp.14-20
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• 2000

The transformation of Bacillus subtilis K-54 and J-10 was carried out with constructed vectors containing structure and enhancer genes of aprN and prtR, to increase their fibrinolytic enzyme activity. Bands for the aprN and prtR genes were identified from B. subtilis J-10 by PCR that was carried out with the constructed primers for the genes. In addition, the gene fragments contained promoter site based on the results of analysing their nucleotide sequence. The two gene fragments, aprN and prtR, obtained by the PCR, were, then, inserted to vector such as T-vector and E.coli/Bacillus shuttle vector. The constructed vector were designated as pAPR2 (aprN), pENC2 (prtR) and pFLA1 (aprN and prtR), respectively. The constructed vector was used for transformation of the strains of B.subtilis J-10 and B. subtilis K-54 and the fribrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and the fibrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and pFLA1, resulted in the increase of fibrinolyitic enzyme activity in B. subtilis J-10 by 27.3% and 16%, respectively. However, the introduction of pENC2 to B. subtilis J-10 did not seem to induce increase of the enzyme activity. The strain of B.subtilis K-54 transformed with pENC2 showed an increased fibrinolytic activity by 5 folds compared with that of the original strain of B. subtilis K-54.

• 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.

• Food Science and Preservation
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• v.22 no.4
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• pp.545-552
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• 2015

This study investigated the quality characteristics of popped rice Doenjang prepared with different Bacillus strains (Bacillus subtilis KACC 15935, and Bacillus subtilis HJ18-9). The changes in the enzyme activity (protease, cellulase, and ${\alpha}$-amylase), amino-type nitrogen and ammonia-type nitrogen contents, and the reducing sugar were investigated during the fermentation period. Enzymes such as protease, cellulase, and a-amylase plays an important role in the changes in composition of nutrients, and in flavor and taste of popped rice Doenjang. Protease activities of the popped rice deonjang fermented with different Bacillus strains (control, B. subtilis KACC 15935, and B. subtilis HJ18-9) was in the range of 171.77-185.97 unit/g at the beginning of fermentation, and there were no significant differences among the samples. On the other hand, the protease activity in popped rice Doenjang fermented with B. subtilis HJ18-9 increased significantly up to $248.77{\pm}4.53unit/g$ at the end of fermentation (p<0.05). Cellulase activity and a-amylase activity of popped rice Doenjang in HJ18-9 was higher than these of other samples. After 56 days of fermentation, amino-type nitrogen in popped rice deonjang fermented with control, B. subtilis KACC 15935, and B. subtilis HJ18-9 increased significantly up to $174.99{\pm}3.70$, $166.59{\pm}1.40$, $225.39{\pm}3.70mg%$, respectively (p<0.05). These results suggested that B. subtilis HJ18-9 was a suitable starter for the preparation of soybean paste.

• 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 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.