• KSBB Journal
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• v.15 no.4
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• pp.411-413
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• 2000

A method is described for the rapid and simple isolation of genomic DNA from 3 mL culture of Lactobacillus crispatus KLB46 The isolated DNA using this method was shown to be an excellent substrate for restriction endonclease digestion and PCR. The method is expected to be used in gentic manipulation of L. crispatus KLB46.

• KSBB Journal
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• v.16 no.6
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• pp.626-631
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• 2001

Lactobacilli have been considered to play important roles in the health of human vagina. They secrete inhibitory substances to prevent vaginal infection by pathogenic organisms. In a previous study, we have isolated several lactobacilli from Korean woman and one of them (KLB46) was selected and indentified as Lactobacillu crispatus which showed high antimicrobial activity. In this study. cold adaptation prior to subsequent stresses exposure was examined whether L. crispatus KLB46 maintain the viability better than the non-adapted calls under stresses. For pharmaceutical formulation, the lyophilization process is required where stresses such as freezing/thawing and dehydration are routinely applied. Formulated L. crispatus KLB46 can be used for ecological treatment of bacterial vaginosis. The response of cold-adapted cells to other environmental stresses such as acid, heat, ethanol, NaCl, and H$_2$O$_2$ was also examined. The results showed that cold-adapted cells maintained higher survival rate compared with the non-adapted cells (freezing-thawing. 3-folds; dehydration: 3-folds; acid, 3-folds; heat, 10-folds). However, we did net observe any positive effect of cold adaptation on other stresses such as ethanol, NaCl and H$_2$O$_2$. When chloramphenicol was added during cold adaptation, adaptation effect was abolished. This confirms that de novo protein synthesis is necessary during the adaptation process. Moreover, we have identified cold shock protein homolog that codes for a major cold shock protein by PCR amplification using degenerate primers.

• KSBB Journal
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• v.29 no.2
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• pp.81-86
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• 2014

Lactobacilli, the dominant species of microorganisms in the vaginal flora of healthy women, play important roles to prevent bacterial vaginosis and other sexually transmitted diseases. In this study, we carried out studies on stress adaptation prior to various stress treatment. We found that heat or salt adapted KLB46 showed higher cell viability than non adapted upon heat stress at $60^{\circ}C$ for 20 min. When chloramphenicol was added during the adaptation process, heat tolerance was abolished. This result suggested that de novo protein synthesis was essential during adaptation.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.128-132
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• 2001

Bacterial vaginosis can be treated by restoring the normal vaginal flora using lactobacilli. Lactobacillus crispatus KLB46 that was isolated from the human vagina has a string antimicrobial activity and was grown in a batch and in a continuous fermentor. During batch cultivation, the maximum specific growth rate of L. crispatus KLB 46 was 0.63h(sup)-1 and the highest viable cell count (1.9$\times$10(sup)9 CFU/mL) was obtained at pH 5.5. L. crispatus KLB 46 did not grow well at either pH 3.5 or 7.5. During continuous cultivation, the highest viable cell count (1.53$\times$10(sup)9 CFU/mL) was obtained at a dilution rate of 0.32h(sup)-1, and was 7.33$\times$10(sup)11 CFU L(sup)-1 h(sup)-1, that is approximately 5 times higher than that obtained from batch culture.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.918-922
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• 2001

In a previous study, we have isolated a number of lactobacilli from Korean women, and one of them (KLB46) was identified as Lactobacillus crispatus by 16S rRNA gene sequencing. For the ecological treatment of bacterial vaginosis (BV) cell suspension of L. crispatus KLB46 was instillated into BV patients. L. crispatus KLB46 was found to persist for several days in cell suspension with no nutrients. In this study, in order to assess the influence of starvation on physiological activity, we compared the viability and culturability of KLB46 following suspension in various buffer solutions. A pair of in situ fluorescent dye was used to assess viability (i.e. membrane integrity) and the culturability was examined by plate count assay. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit $(BacLight^{TM})$ was applied to estimate both viable and total counts of bacteria in cell suspension. $BacLight^{TM}$ is composed of two nucleic acid-binding stains ($SYTO\;9^{TM}$ and propidium iodide). $SYTO\;9^{TM}$ penetrates all bacterial membranes and stains the cells green while propidium iodide only penetrates cells with damaged membranes, therefore the combination of the two stains produces red fluorescing cells. Optimal staining conditions for $BacLight^{TM}$ were found to be with 0.0835M $SYTO\;9^{TM}$ and 0.05M propidium iodide for 15 min incubation at room temperature in dark. When cells were microscopically examined during 140 hours of starvation, the culturability decreased markedly while the viability remained relatively constant, which suggests that large fraction of KLB46 cells became viable but non-culturable (VBNC) upon starvation.

• KSBB Journal
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• v.26 no.6
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• pp.529-532
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• 2011

Whey based medium was optimized for the production of viable Lactobacillus crispatus KLB 46 isolated from the vagina of Korean women. Among the various nitrogen sources such as yeast extract, beef extract, and proteose peptone no. 3 supplemented to whey, beef extract showed the highest viable cell production. The addition of Tween 80 to the whey based medium increased viable cell concentration. As beef extract supplementation is not economically attractive, corn steep liquor was added as a supplementary nitrogen sources. When corn steep liquor was supplied with beef extract with the ratio 5 : 1, the viable cell count was $3.11{\times}10^9$ CFU/mL. Also, the addition of mineral salts containing sodium acetate (5 g/L), potassium phosphate dibasic (2 g/L), magnesium sulfate (0.1 g/L) and manganese sulfate (0.05 g/L) to the whey medium increased viable cell count further ($5.00{\times}10^9$ CFU/mL).

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.372-375
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• 2000

Lactobacillus cristatus KLB 46 isolated from Korean woman was grown on supplemented whey medium and medium compositions were optimized for maximum viable cell count. Among the nitrogen sources tested, beef extract yielded the highest viable cell number. When corn steep liquor was applied as an additional nitrogen source, the viable cell number was highest $(3.11{\times}10^9\;CFU/ml)$ in the medium containing 50g/ l corn steep liquor and 10g/ l beef extract. The highest viable cell $count(5.00{\times}10^9\;CFU/ml)$ was obtained from the supplemented whey medium that contains beef extract(10g/ l ), corn steep liquor(50g/ l ), tween 80(0.1%, v/v) and trace amounts of sodium acetate(5g/ l ), dipotassium phosphate(2g/ l ), magnesium sulfate(0.1g/ l ), and manganese sulfate (0.05g/ l ).

• KSBB Journal
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• v.17 no.3
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• pp.311-313
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• 2002

Lactobacillus spry. are Gram-positive bacteria playing important roles in human health. In this study, we successfully isolated the total RNA from the cells broken by glass beads using hot phenol method. Moreover, we were able to omit lysozyme and proteinase K treatment by using glass beads to break cell more efficiently. This method was more rapid and simple when compared to the previous one. Prepared RNA can be used for the transcriptional analysis of Lactobacillus spp.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.312-317
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• 2002

Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.