• 산업기술연구
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• 제28권B호
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• pp.53-57
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• 2008

From the extensive screening for small cryptic plasmid among about 23 lactic acid bacteria (LAB), 2.4 kb of cryptic plasmid was isolated from Lactobacillus farciminis strain KCTC 3681 and named as pLF24. The plasmid pLF24 was a circular molecule of 2,396 base-pairs in length with a G+C content of 38%. Two protein-coding sequences could be predicted. ORF1 and ORF2 showed homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and the plus origin, were similar to replication regions of other gram-positive bacteria as shown in plasmids such as pLH2, pLS141-1 and pLC2. The nucleotide sequence of pLF24 was deposited into Genbank data base with an accession number of EU429343. The newly isolated plasmid can be used for construction of shuttle vector in Lactobacillus bacteria.

• Asian-Australasian Journal of Animal Sciences
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• 제30권4호
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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.

• 한국초지조사료학회지
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• 제30권4호
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• pp.333-342
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• 2010

옥수수는 우리나라에서 매우 중요한 사료작물이다. 대부분 사일리지 형태로 이용이 되고 있으며 많은 비율의 곡실을 함유하고 있어 반추가축의 우수한 사료로 이용된다. 본 시험은 옥수수 사일리지용 미생물 첨가제 개발을 위해 수집된 균주의 발효능력을 평가하기 위해 수행되었다. 양질의 옥수수 사일리지에서 선발한 젖산균을 0.02%의 Sodium azide가 들어있는 MRS 배지에 접종을 한 후 균을 분리하여 MRS broth에 접종하여 산 생성 능력을 조사하였다. 총 6종의 젖산균을 선발하였고 동정결과 C3-2, B13-1 및 CC9-1은 Lactobacillus plantarum으로 판명되었고 C11-4는 Lactobacillus fermentum, B14-1은 Lactobacillus paracasei, 그리고 A3-1은 Leuconostoc lactis로 동정되었다. 선발된 5종의 균주와 시판되는 2종 첨가제 그리고 무처리구를 두고 황숙기에 수확된 옥수수에 접종하여 사일리지를 제조하고 2개월 후에 개봉하여 분석한 결과 B13-1 및 CC9-1 처리구의 사일리지가 pH가 낮았고 젖산함량도 높았다. 프리그 점수와 품질등급에 있어서도 B13-1 및 CC9-1 처리구에서 가장 높게 나타났다. 이상의 결과를 종합하여 볼 때 Lactobacillus plantarum B13-1 및 CC9-1는 사일리지 접종제로 사용이 권장되었다.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1386-1392
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• 2010

A bacterial strain designated GR5$^T$, previously isolated from a freshwater culture pond in Taiwan while screening for bacteria for antimicrobial compounds, was characterized using a polyphasic taxonomic approach. Strain GR5$^T$ was found to be Gram-negative, aerobic, greenish-yellow colored, rod-shaped, and motile by means of a single polar flagellum. Growth occurred at $10-40^{\circ}C$ (optimum, $35^{\circ}C$), pH 7.0-8.0 (optimum pH 8.0), and with 0-2.0% NaCl (optimum, 0.5-1.0%). The major fatty acids were $C_{16:1}{\omega}7c$(36.3%), $C_{16:0}$(16.6%), $C_{12:0}$ 3-OH (12.5%), and $C_{18:1}{\omega}7c$(9.1%). The major respiratory quinone was Q-8, and the DNA G+C content of the genomic DNA was 51.9 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GR5$^T$ belongs to the genus Rheinheimera, where its most closely related neighbors are Rheinheimera texasensis A62-14B$^T$ and Rheinheimera tangshanensis JA3-B52$^T$ with sequence similarities of 98.1% and 97.5%, respectively, and the sequence similarities to any other recognized species within Gammaproteobacteria are less than 96.5%. The mean level of DNA-DNA relatedness between strain GR5$^T$ and R. texasensis A62-14B$^T$, the strain most closely related to the isolate, was $26.5{\pm}7.6%$. Therefore, based on the phylogenetic and phenotypic data, strain GR5$^T$ should be classified as a novel species, for which the name Rheinheimera aquatica sp. nov. is proposed. The type strain is GR5$^T$ (=BCRC 80081$^T$=LMG 25379$^T$).

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.102.2-103
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• 2003

This studies were investigated the occurrence of Sclerotinia rot by Sclerotinia Sclerotiorum at the lettuce field in Uiryeong-Gun, Gyeongsangnam-Do and were isolated the most effective microorganism for the biological control to the pathogen, S. sclerotiorum YR-1 strain from diseased soil and lettuce leaves. For the pathogenicity test, the most suitable inoculmn density of YR-1 strain was selected as the mycelial suspension of 40m1 showing disease incidence of 80％, and the symptom showed as same as at the fields, the leaves and stem had rotten and developed white downy mycelial at the diseased lesion on the leaves and stems, and produced black and irregular sclerotinia. On the PDA dual test, about 300 isolates were examined the antifungal activity to the pathogen, YR-1 strain, and among them, A-2, A-7, and RH-4 strain were selected most effective antagonistic bacteria. At pots test, the control value of A-7 strain showed the highest value as 85％ which was more effective than that of others in a growth chamber. For the promotion of control effect, the selected 3 isolates were spayed on the lettuce leaves as a sole and/or mixed treatments in a growth chamber, the mixed treatment of A-7 and RH-4 strain showing the control value of 90％ was most effective than that of sole treatment with A-7 or RH-4 strain showing the control value of 80％, respectively and mixed treatment with A-2 and A-7 strain and A-2, A-7 and RH-4 strain. In addition, 3 bacteria re-isolated from diseased soils, and all of the selected 6 isolates investigated the control effect at pots in a growth chamber, According to the results, A-7 and Pro-EB 15 strain showed the control value of 91.0％ and 90.1％ respectively, and they were selected most effectual antagonistic bacteria to control lettuce sclerotinia rot and identified as the Bacillus mojanuinensis by 16s RNA analysis. This is the first report on the biological control using by B. mojanvinensis to the lettuce Sclerotinia rot.

• Journal of Microbiology and Biotechnology
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• 제12권4호
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• pp.610-616
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• 2002

The growth-inhibiting effects of Pinus densiflpora leaf-derived materials on nine human intestinal bacteria were investigated using the impregnated paper disk method, and their activities were compared with those of 13 commercially available terpenes. The biologically active constituent of the extract of P densiflora leaf was characterized as the monoterpene (1R)-(+)-$\alpha$-pinene by various spectroscopic analyses. Responses varied according to bacterial strain, chemicals, and dose. At 10 mg/disk, limonene and (1R)-(+)-$\alpha$-pinene strongly inhibited the growth of Clostridium perfringens, Staphylococcus aureus, and Escherichia coli, without adverse effects on the growth of five lactic acid-bacteria (Bifidobacterium adolescentis, B. bifidum, B. longum, Lactobacillus acidophilus, and L. casei). Little or no inhibition against seven bacteria was observed with anethole, borneol, camphor, caryophyllene, 1,8-cineole, estragole, linalool, and $\alpha$-terpineol. Structure-activity relationship revealed that (1R)-(+)-$\alpha$-pinene had more growth-inhibiting activity against C. perfringens than (1R)-(+)-$\beta$-, (1S-(-)-$\alpha$-, and (1S-(-)-$\beta$-pinenes. Furthermore, the growth-inhibition against L. casei was much more pronounced in (1R)-(+)-$\beta$- and (In-(-)-$\beta$-pinenes than (1R)-(+)-$\alpha$- and (1S)-(-)-$\alpha$-pinenes. These results indicate that the (+)-$\alpha$ form seems to be required against C. perfringens and $\beta$ form against L. casei for growth-inhibiting activity. Morphologically, most strains of C. perfringens were damaged and disappeared at 5 and 2 mg/disk of (1R)-(+)-$\alpha$-pinene. Morphological study revealed that (1R)-(+)-$\alpha$-pinene had more growth-inhibiting activity against C. perfringens than (1R)-(+)-$\beta$-, (1S)-(-)-$\alpha$-, and (1S)-(-)-$\beta$-pinenes. As naturally occurring growth-inhibiting agents, the Pinus leaf-derived materials described above could be useful preventive agents against diseases caused by harmful intestinal bacteria such as clostridia.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• 미생물학회지
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• 제49권3호
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• pp.253-261
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• 2013

본 연구에서는 숙성된 된장으로부터 분리된 유산균에 의한 aflatoxin $B_1$의 결합 정도를 배양조건에 따라 측정하였고, 물리화학적 처리조건이 aflatoxin $B_1$에 대한 유산균 세포의 결합력에 미치는 영향을 살펴보았다. Enterococcus faecium DJ22, Lactobacillus fermentum DJ35, Lactobacillus rhamnosus DJ42 및 Lactobacillus pentosus DJ47는 19.3-52.1% 정도의 aflatoxin $B_1$ 결합 효과를 나타내어 균종에 따라 결합력에 차이가 있었다. 하지만 E. faecalis DJ14, Lactobacillus panis DJ29 및 Pediococcus halophilus DJ50 균주는 aflatoxin $B_1$에 대한 결합력을 나타내지 않았다. Aflatoxin $B_1$에 대한 유산균의 결합력과 결합속도는 독소의 농도, 반응시간 및 온도와 초기 세포수 등의 배양 조건에 따라 유의한 차이가 있었다. Aflatoxin $B_1$의 결합력은 세척 횟수에 따라 현저하게 감소하였고, 감소율은 살아있는 세포와 가열 처리한 세포에서 비슷하게 나타났다. 가열, 산성 pH, ${\alpha}$-amylase, protease, lysozyme 혹은 sodium metaperiodate의 처리에 의해 결합력이 유의하게 감소된 것으로 보아 주로 세포벽에 존재하는 당이나 단백질에 aflatoxin $B_1$이 결합되며, urea의 처리에 의해 결합력에 낮아지는 것은 이들 사이에는 소수성 결합이 작용하는 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제23권1호
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• pp.40-46
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• 2013

A variety of nuruk were collected from various provinces in Korea, and their microflora profiles were analyzed at the species level. A total of 42 nuruk samples were collected and when the viable cell numbers in these nuruk were enumerated, the average cell numbers of bacteria, fungi, yeast, and lactic acid bacteria from all nuruk were 7.21, 7.91, 3.49, and 4.88 log CFU/10 g, respectively. There were no significant differences in viable cell numbers of bacteria or fungi according to regions collected. Bacillus amyloliquefaciens and B. subtilis were the predominant bacterial strains in most samples. A significant portion, 13 out of 42 nuruk, contained foodborne pathogens such as B. cereus or Cronobacter sakazakii. There were various species of lactic acid bacteria such as Enterococcus faecium and Pediococcus pentosaceus in nuruk. It was unexpectedly found that only 13 among the 42 nuruk samples contained Aspergillus oryzae, the representative saccharifying fungi in makgeolli, whereas a fungi Lichtheimia corymbifera was widely distributed in nuruk. It was also found that Pichia jadinii was the predominant yeast strain in most nuruk, but the representative alcohol fermentation strain, Saccharomyces cerevisiae, was isolated from only 18 out of the 42 nuruk. These results suggested that a variety of species of fungi and yeast were distributed in nuruk and involved in the fermentation of makgeolli. In this study, a total of 64 bacterial species, 39 fugal species, and 15 yeast species were identified from nuruk. Among these strains, 37 bacterial species, 20 fungal species, and 8 yeast species were distributed less than 0.1%.

• 생약학회지
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• 제34권2호통권133호
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• pp.142-144
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• 2003

Acetone extracts of 301 strains of marine-derived fungus were tested for antimicrobial activity against three strains of bacteria. The bacteria consisted of three pathogens, Staphylococcus aureus, methicillin-resistant S. Aureus, and multidrug-resistant S. aureus. The acetone extracts of 10 strains (MFA117, MFA130, MFA134, MFA206, MFA217, MFA268, MFA277, MFA291, MFA292, MFA301) showed strong activity, inhibiting 100% of the bacterial growth. These antimicrobial active strains were cultlued in SWS medium on a 1 L scale and the resulting broth and mycelium were extracted to afford mycelium extract (000M) and broth extract (000B), respectively. Antimicrobial activity for all extracts has been tested as the results, the mycelium extract of one strain (217M) and the broth extracts of 9 strains (117B,130B, 134B, 206B, 268B, 277B, 291B, 292B, 301B) exhibited relatively high levels of activity at minimal inhibitory concentration (MIC) values of $500-125\;{\mu}g/mL$ range. Among them, the extracts, 277B, 291B, 292B and 301B showed the most significant antimicrobial activity with $IC_{50}$ values of $125\;{\mu}g/mL$.