• Journal of Life Science
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• v.33 no.11
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• pp.897-904
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• 2023

This study was done to develope genetic markers with the unique characteristics of genes according to the genomic information of Bacillus velezensis K10. B. velezensis K10 maintained a total of 4,159,835 bps, which was found to encode 5,136 open reading frames (orfs). B. velezensis K10 was found to have much more gene migration due to external factors overall compared to standard strain B. velezensis JS25R. In order to discover genetic selection markers, orfs on the genome to be easily induced to gene mutation were surveyed such as recombinase, integrase, transposase, and phage-related genes. As a result of the investigation, 9 candidate markers were isolated with high possibility as genetic selection markers. Although a part in the various origin's areas showed specificities in comparison with homology, the selected markers were all existed in phage-related areas because they were relatively lower homologies in phage-related genes. PCR analysis was done on B. licheniformis K12, B. velezensis K10, B. subtilis, and B. cereus to establish them as inter-species candidate selection markers. As a result, it was confirmed that B. velezensis K10-specific PCR products were formed in a total of 6 primer sets such as BV3 and BV5 to 9. On the other hand, analysis at the subspecies level observed the formation of B. velezensis K10-specific PCR products in 4 primer sets such as BV3, 5, 8, and 9. Among them, since BV5 and BV8 were detected by very specific results, we suggest that BV5 and 8 can be used as B. velezensis K10 gene selection markers at the species and sub-species level.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1758-1766
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• 2012

An extensive investigation was carried out to describe the kinetics of cell growth, substrate consumption, and product formation in the batch fermentation using starch as substrate. Evaluation of intrinsic kinetic parameters was carried out using a best-fit unstructured model. A nonlinear regression technique was applied for computational purpose. The Andrew's model showed a comparatively better $R^2$ value among all tested models. The values of specific growth rate (${\mu}_{max}$), saturation constant ($K_S$), inhibition constant ($K_I$), and $Y_{X/S}$ were found to be 0.109 $h^{-1}$, 11.1 g/l, 0.012 g/l, and 1.003, respectively. The Leudeking-Piret model was used to study the product formation kinetics and the process was found to be growth-associated. The growth-associated constant (${\alpha}$) for protease production was sensitive to substrate concentration. Its value was fairly constant up to a substrate concentration of 30.8 g/l, and then decreased.

• Journal of Life Science
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• v.19 no.7
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• pp.994-1002
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• 2009

A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus planiarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5~ 7.0 and a temperature of 37$^\circ$C for 36$\sim$48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25$^\circ$C for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. It's activity was not affected by heat treatment at 100$^\circ$C for 30 min and 50% of activity was retained after heat treatment at 100$^\circ$C for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.

• Fisheries and Aquatic Sciences
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• v.19 no.10
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• pp.42.1-42.10
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• 2016

The objectives of this study were to identify the histamine-forming bacteria and bacteriocin- producing lactic acid bacteria (LAB) isolated from Myeolchi-jeot according to sequence analysis of the 16S rRNA gene, to evaluate the inhibitory effects of the bacteriocin on the growth and histamine accumulation of histamine-forming bacteria, and to assess the physico-chemical properties of the bacteriocin. Based on 16S rRNA gene sequences, histamine-forming bacteria were identified as Bacillus licheniformis MCH01, Serratia marcescens MCH02, Staphylococcus xylosus MCH03, Aeromonas hydrophila MCH04, and Morganella morganii MCH05. The five LAB strains identified as Pediococcus acidilactici MCL11, Leuconostoc mesenteroides MCL12, Enterococcus faecium MCL13, Lactobacillus sakei MCL14, and Lactobacillus acidophilus MCL15 were found to produce an antibacterial compound with inhibitory activity against the tested histamine-producing bacteria. The inhibitory activity of these bacteriocins obtained from the five LAB remained stable after incubation at pH 4.0-8.0 and heating for 10 min at $80^{\circ}C$; however, the bacteriocin activity was destroyed after treatment with papain, pepsin, proteinase K, ${\alpha}$-chymotrypsin, or trypsin. Meanwhile, these bacteriocins produced by the tested LAB strains also exhibited histamine-degradation ability. Therefore, these antimicrobial substances may play a role in inhibiting histamine formation in the fermented fish products and preventing seafood-related food-borne disease caused by bacterially generated histamine.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.30-38
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• 2012

We investigated the effects on soil microbial diversity and the growth promotion of red pepper resulting from inoculation with a microbial agent composed of Bacillus subtilis AH18, B. licheniformis K11 and Pseudomonas fluorescens 2112 in a red pepper farming field. Photosynthetic bacteria, Trichoderma spp., Azotobacter spp., Actinomycetes, nitrate oxidizing bacteria, nitrite oxidizing bacteria, nitrogen fixing bacteria, denitrifying bacteria, phosphate solubilizing bacteria, cellulase producing bacteria, and urease producing bacteria are all indicator microbes of healthy soil microbial diversity. The microbial diversity of the consortium microbial agent treated soil was seen to be 1.1 to 14 times greater than soils where other commercial agent treatments were used, the latter being the commercial agent AC-1, and chemical fertilizer. The yield of red pepper in the field with the treated consortium microbial agent was increased by more than 15% when compared to the other treatments. Overall, the microbial diversity of the red pepper farming field soil was improved by the consortium microbial agent, and the promotion of growth and subsequent yield of red pepper was higher than soils where the other treatments were utilized.

• Journal of Dairy Science and Biotechnology
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• v.28 no.2
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• pp.7-11
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• 2010

In recent times, antimicrobial resistance has been a concern because of its relation to national health and food safety. In this study, we reviewed the management of antibiotics and antimicrobial-resistant microorganisms in domestic and foreign countries and analyzed microorganisms and antimicrobial resistance in dairy farms and raw milk. The isolates from dairy farms were Micrococcus spp., Staphylococcus chromogens, Escherichia coli, Bacillus licheniformis, Escherichia coli O157:H7, Pseudomonas plecoglossicida, Enterobacter spp., and Kluyvera intermedia. Rummeliibacillus stabekisii, Paenibacillus badius, Pantoea agglomerans, B. oleronius, B. fusiformis, and B. badius were isolated from feed E. coli and Kurthia gibsonii, from feces and S. pasteuri, S. aureus, S. chromogenes, and Salmonella spp., from raw milk. Pathogens isolated from dairy farms and raw milk were tested for susceptibility to 20 types of antibiotics. E. coli (EAEC) and E. coli O157:H7 (EHEC) isolated from dairy farms, E. coli (EAEC) isolated from feces, and Salmonella spp. isolated from raw milk showed resistance to multiple antibiotics. These results show that antimicrobial-resistant microorganisms should be more effectively managed to improve the safety of dairy farms.

• Food Science of Animal Resources
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• v.38 no.4
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• pp.749-758
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• 2018

The objective of this study was to provide preliminary data for food industry by investigating the distribution of microorganisms in raw materials and sausage examining the effect of heating temperature on sausage quality. Total microbes in sausage ranged 2.21-3.11 Log CFU/g. Bacillus pumilus, B. licheniformis, Staphylococcus saprophyticus, and Enterococcus faecalis were detected on sausage. Total microbes in raw materials was 1.59-7.16 Log CFU/g. Different types of microorganisms were found depending on raw materials, with B. pumilus and B. subtilis were being detected in both raw materials and sausage. Total microbes in sausage after heating was in the range of 1.10-2.22 Log CFU/g, showing the trend of decrease in total microbe with increasing heating temperature, although the decrease was not significant. With increasing heating temperature, pH and hardness were also increased. The yield of sausage manufactured at $85^{\circ}C$ was 95.42% while that manufactured at $65^{\circ}C$ was 96.67%. Therefore, decreasing heating temperature during sausage production might increase yield and save energy without microbiological effect.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.48 no.1
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• pp.127-133
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• 2016

This study is performed that furniture and interior materials of MDF's (Medium Density Fiberboard) biodegradation properties, and the goal of this study is investigation of possibility of waste-MDF's composting after landfilling. To investigate biodegradation, this study was performed according to KS M ISO 14855-1, and there were two different soil conditions including a compost condition and an activated vermiculite condition as artificial soil. This experiment was tested for 40 days. The measurement of carbon-dioxide generation was processed every 24 hour in 1-2 week, and every 48 hour after 3 week. In the same days, MDF showed 24.4% of biodegradation in compost condition, and 6.2% in activated vermiculite. Also, the reference material of TLC (thin-layer chromatography) grade cellulose showed 26.4%, 11.4% in compost and activated vermiculite respectively. The dilution plate method was performed for biological analysis in the study. This experiment was used for investigation of inoculum's (Bacillus licheniformis) activity. As the result of bioassay, compost has more other germs include inoculum than activated vermiculite in the first week. Especially in the 2nd week, the reference material under the compost condition showed the most germ's activity, and also the biodegradation was the highest. Consequentially, compost condition was able to reduce a performing period of biodegradation testing than activated vermiculite. However, activated vermiculite could be stabilizing errors between repetition.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1999-2009
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• 2017

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three Bacillus licheniformis alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.