• 한국미생물·생명공학회지
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• 제19권6호
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• pp.619-623
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• 1991

Lactococcus lactis ssp. lactis ML8(L.lactis ML8)의 nisin 생산과 저항 특성을 구명하기 위하여 배지의 종류 및 pH가 nisin의 역가에 미치는 영향, 균체의 생육에 따른 nisin의 생산특성, nisin이 균체생육에 미치는 영향 및 $Ca^[2+}$ 이온의 존재가 균주의 nisin 저항성에 미치는 영향을 조사하였다. Nisin의 역가를 Micrococcus flacus에 대하여 항생효과를 나타내는 성질을 이용하여 agar diffusion법으로 측정하였을 때, M.flavus 생육에 대한 저해직경은 nisin 농도 (0.5`20 unit/ml)의 log치에 비례하였다.

• 한국미생물·생명공학회지
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• 제21권3호
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• pp.200-206
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• 1993

The effect of pH (7, 5 and 4) and nisin (100 and 200IU/ml) on heat resistance of Listeria monocytogenes Scott A were determined using citrate-phosphate buffer system. Heat resistance of vegetative and starved cell was decreased as pH value was lower at 65 and 72C. Starved L. monocytogenes was more resistant than vegetative cell at both temperature. Heat resistance of vegetative and starved cell was decreased significantly with treatment of nisin. The effect of nisin was increased significantly at low pH(5, 4). Adherent microcolony was more resistant to heat and nisin than planktonic cell. Contamination of L. monocytogenes may be prevent by using nisin in food and food processing environments.

• 한국미생물·생명공학회지
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• 제26권6호
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• pp.562-565
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• 1998

Nisin-producing and nisin resistant L. lactis subsp. lactis ATCC7962 harbored six plasmids. To find a plasmid containing a nisin resistant gene, these plasmids were transformed into L lactis LM0230 of plasmid-free and nisin sensitive strain. After screening on nisin selection media containing nisin (150 $\mu\textrm{g}$/$m\ell$), several nisin resistant transformants were obtained and the level of nisin resistance was very similar to that of wild type L lactis subsp. lactis ATCC7962. A 26.5 kb plasmid, named as pCS100, which confers resistance to nisin, was identified in transformants. The pCS100 was digested with EcoRI and Southern blot hybridization was done with nisI probe to localize the nisin resistant gene. A 4 kb EcoRI fragment showed a strong positive signal, and it was cloned into pBluescript for the potential selection marker.

• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.217-223
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• 1993

To investigate the nature and location of the nisin-resistance determinant of Lactococcus lactis subsp. lactis 7962 (L. lactis 7962), a total plasmid DNA prepared from L. lactis 7962, a nisin producer, was used to transform L. lactis subsp. lactis LM0230, a plasmid-free and nisin-sensitive strain, by protoplast mediated transformation procedures. All of the nisin-resistant transformants acquired the ability to utilize sucrose at the same time, confirming the close linkage between these two determinants in L. lactis 7962. The plasmid DNA profiles of a few selected nisin-resistant transformants were examined by agarose gel electrophoresis. No common plasmid was found among the transformants and some small plasmids previously not present in L. lactis 7962 were detected. These transformants were named as L. lactis KL1, KL2, KL3, KL4, or KL5, respectively based on their plasmid profiles. Growth curves of all transformants were similar to that of L. lactis LM0230, but different from that of L. lactis 7962. L. lactis KL5 showed the highest level of resistance to nisin, growing up to 1, 200 IU nisin/ml after 40 hr incubation. Some nisin-sensitive derivatives of KL1 or KL2 were obtained by plasmid curing experiments. The plasmid DNA profiles of the nisin-sensitive KL1 derivatives were apparently the same as that of the KL1. All of the nisin-sensitive KL2 derivatives were plasmid-free, but a nisin-resistant strain with no apparent plasmid was also obtained. These results indicate that the nisin-resistance of the $Nis^r$ transformants is presumably mediated by the chromosomally located gene(s) rather than plasmid-encoded gene(s).

• Preventive Nutrition and Food Science
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• 제12권4호
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• pp.259-266
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• 2007

It has been proposed that the mode of action of nisin against vegetative cells and spores of Clostridium botulinum is different. However, clear explanation is not available. Therefore, nisin action against vegetative cells and spores of C. botulinum was investigated in this study. Nisin was added at various stages of spore-to-vegetative cell transition and changes to sensitivity to the bacteriocin were observed. Different nisin preparation (Nisaplin or pure nisin) was compared for their activity against different stages of spore transformation of C. botulinum ATCC 25763. Germination was measured by determining loss of heat resistance and observing phase darkening of spores under phase-contrast microscope. Nisin acted bactericidally against vegetative cells, but acted sporostatically against spores of C. botulinum under the same concentration. This bactericidal and sporostatic action of nisin was dependent on the concentration of nisin used. Presence of nisin during spore activation by heat increased subsequent phase darkening and germination rates. However, nisin inhibited the germination and the outgrowth, when it was added after heat activation stage. Findings from this study suggest that the time of addition of nisin is very important for the effective control of spores during the heating process of foods. In addition, it may be possible to apply nisin at the stage of processing that coincides with the most sensitive stage of spore transformation.

• Preventive Nutrition and Food Science
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• 제12권4호
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• pp.251-258
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• 2007

Thermal resistance and heat activation characteristics of Clostridium botulinum ATCC 25763 spores were evaluated. The effects of nisin and pH on the activation and subsequent germination were also investigated. Spores of C. botulinum were not inactivated by heat treatments up to $92^{\circ}C$ for 2 hr. Heat treatment at $85^{\circ}C$ for 90 min was selected as the optimal activation condition based on monitoring subsequent germination. L-alanine alone or in combination with L-cysteine was not sufficient to germinate the spores of this strain. Tryptone-Peptone- Glucose-Yeast extract (TPGY) broth supplemented with L-alanine was used as a suitable germination medium. Decreasing pH of activation suspension increased the degree of phase darkening, i.e., germination. In addition, the presence of nisin during activation increased the degree of phase darkening. The majority of spore populations were dormant at a pH of less than 2.8, and these populations required heat activation to increase the culturability on TPGY agar medium. However, extended heating in the presence of nisin at pH 2.8 decreased the spore count; however, heat activation was less necessary at pH 3.4, compared at pH 2.8.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제39회 학술심포지움
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• pp.82-82
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• 2003

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.180-184
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• 2003

선별된 7종의 균주는 넓은 범위의 산과 담즙산에 대해 기존에 보고된 결과에 비하여 높은 내성을 나타냈고, 장내세균인 E. coli에 대해서도 우수한 항균력을 나타냈고, 항생제 tetracycline, nisin, kanamycin, ampicillin. streptomycin에 대해서도 높은 내성을 보였다. 또한 7종의 균주는 어느 정도의 산을 생성하고 있었고, 네 가지 효소 amylase, protease, lipase, cellulase에 대해서도 높은 활성을 나타냈다. 균주 특성실험 결과, 7종의 균주는 그람음성균으로 모두 구형, 비운동성이고 균체의 색은 노란색이거나 흰색을 띠고 있고, 다양한 유기물에 대하여 분해력을 지니고 있었다.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• Food Science and Biotechnology
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• 제15권5호
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• pp.677-682
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• 2006

Strain HY701 was isolated from human feces for probiotic use by selecting highly resistant isolates to artificial gastric acid and bile acid. Strain HY701 was identified as Lactobacillus reuteri using 16S rDNA sequencing, and tentatively named L. reuteri HY701. The resistance of L. reuteri HY701 to artificial gastric acid (PH 2.5) was high with a survival rate of over 90%. L. reuteri HY701 also showed high tolerance to artificial bile acid after incubation in artificial gastric acid. Using the API ZYM test kit, the carcinogenic enzymes (${\beta}$-glucuronidase and (${\beta}$-glucosidase were not detected with L. reuteri HY70l, while the beneficial enzyme (${\beta}$-galactosidase was weakly detected. L. reuteri HY701 was sensitive to $100\;{\mu}g/mL$ nisin, $20\;{\mu}g/mL$ roxithromycin, $15\;{\mu}g/mL$ erythromycin, but resistant to $20\;{\mu}g/mL$ streptomycin, $10\;{\mu}g/mL$ tetracycline, $20\;{\mu}g/mL$ ciprofloxacin, $20\;{\mu}g/mL$ nystatin, $20\;{\mu}g/mL$ gentamycin, $10\;{\mu}g/mL$ doxycycline, $10\;{\mu}g/mL$ chloramphenicol, and $20\;{\mu}g/mL$ ampicillin. L. reuteri HY701 was shown to possess bactericidal activity as it inhibited the growth of Listeria monocytogenes ATCC 19111 and Escherichia coli JM109 completely within 24 hr of incubation. These results indicate that L. reuteri HY701 could be used as a probiotic strain.