• Food Science and Biotechnology
• /
• 제15권4호
• /
• pp.485-493
• /
• 2006

Lactococcus species are noninvasive and nonpathogenic microorganisms that are widely used in industrial food fermentation and as well-known probiotics. They have been modified by traditional methods and genetic engineering to produce useful food-grade materials. The application of genetically modified lactococci in the food industry requires their genetic elements to be safe and stable from integration with endogenous food microorganisms. In addition, selection for antibiotic-resistance genes should be avoided. Several expression and secretion signals have been developed for the production and secretion of useful proteins in lactococci. Food-grade systems composed of genetic elements from lactic acid bacteria have been developed. Recent developments in this area have focused on food-grade selection markers, stabilization, and integration strategies, as well as approaches for controlled gene expression and secretion of foreign proteins. This paper reviews the expression and secretion signals available in lactococci and the development of food-grade markers, food-grade cloning vectors, and integrative food-grade systems.

• Journal of Microbiology and Biotechnology
• /
• 제18권7호
• /
• pp.1286-1289
• /
• 2008

The ability of Lactococcus strains to inhibit the growth of intestinal bacteria was examined. In in vitro cocultures, we observed that among eighteen Lactococcus strains tested, the ability to inhibit growth of Escherichia coli varied, with the L. lactis N7 showing the greatest growth inhibition. Strain N7 ($8.94\times10^{10}$ CFU/day for 7 days) was orally administered to mice, and the viable count of strain N7 in feces appeared at a level of $10^{4-5}$ CFU/g. After administration, the proportion of Bacteroidaceae to total intestinal bacteria decreased. Lactococci may act as probiotic bacteria by inhibiting the growth of harmful bacteria.

• The Korean Journal of Ecology
• /
• 제22권1호
• /
• pp.45-50
• /
• 1999

김치 발효 초기에 bacteriocin 생성 유산균을 분리하고자 하였다. 분리 유산균은 형태, 배양 및 생리학적인 특징과 16S rDNA의 부분적인 염기서열로부터 Lactococcus lactis로 동정되었다. 분리균주가 생성한 bacteriocin은 Listeria monocytogenes, Staphylococcus aureus와 같은 그람 양성 병원성 세균과 몇몇 유산균에 대해 항균활성이 있었고, 그람음성균인 Yesinia에는 활성이 없었다. bacteriocin의 활성은 protease, protease ⅩⅣ, a-chymotrypsin과 pepsin에 대해서 활성이 소실되었으나, lipase, trypsin, lysozyme에 대해서는 활성이 유지되었다. bacteriocin의 활성은 pH 2∼11에서 안정적이며 l00℃에서 10분간 열처리시에도 변하지 않았다. 따라서 L. monocytogenes는 발효초기에 L. lactis에 의하여 억제될 수 있다.

• Journal of Microbiology and Biotechnology
• /
• 제16권11호
• /
• pp.1799-1808
• /
• 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.

• 한국식품영양과학회지
• /
• 제31권1호
• /
• pp.75-80
• /
• 2002

된장 제조과정 중 불쾌취와 점질물을 생성하는 부패균인 B. subtilis의 생육억제를 위해 nisin을 생성하는 L. lactis subsp. lactis ATCC 7962, ATCC 11454, IFO 12007을 이용하여 유산발효를 수행하여 pH변화, B. subtilis의 생육저해도등을 검토하였다. 증자콩에서 nisin 생성유산균들의 생육특성을 살펴본 결과, 세 가지의 균주 모두가 증자대두 1g 당 $10^{6}$ CFU 접종하여 24시간 이내에 $10^{9}$ CFU로 급격하게 자라므로 영양요구성이 복잡한 유산균이 증자대두에서 다른 영양소의 첨가없이도 잘 생육함을 확인하였다. 증자콩에서 7962와 11454는 생육에 따라 pH가 급격히 저하되어 된장발효용 종균으로 사용할 수 없었다. 증자한 콩 중에 잘 증식하고 유산발효 후 pH 변화가 완만한 12007을 된장발효의 종균으로 전혀 문제점 없어 사용시 부패균인 B. subtilis의 생육이 효과적으로 저해됨이 확인되었다. 생성된 nisin은 황국균이 생성하는 protease에 의해 분해되며 콩 속에 다량으로 존재하던 유산균은 사입시 첨가되는 식염으로 불화성화되거나 사멸하여 된장의 산패를 막아준다. 그리고 8% 식염을 첨가하여 된장 제조시, 즉 저염 된장 제조시 B. subtilis의 생육이 효과적으로 억제되고 다수로 존재하던 유산균은 담금 후 점차 줄어들어 시간경과 후에는 관찰되지 않았다. 8%함유 식염된장의 숙성 중 pH 변화 역시 12% 함유 식염된장과 유사한 값을 나타내었다.

• Journal of Microbiology and Biotechnology
• /
• 제10권4호
• /
• pp.507-513
• /
• 2000

A bacteriocin-producing organism, Enterococcus sp. T7, was isolated from human fecal samples. Bacteriocin T7, named tentatively as the bacteriocin, was produced by Enterococcus sp. T7 and it inhibited some strains of Lactobacillus. Staphylococcus, Enterococcus, and Streptococcus, but not all the lactococci and gram-negative bacteria tested. Bacteriocin T7 inhibited the growth of Listeria monocytogenes Scott A, but the degree of inhibition was less than those for other sensitive gram-positive vacteria. Bacteriocin T7 in MRS broth started to produce at the middle of the exponential growth phase and the inhibitory activity reached its maximum level during the stationary growth phase. Bacteriocin T7 was stable against heat treatments, pH variations (pH 2-10), and exposure to organic solvents. The molecular weight of bacteriocin T7 was estimated to be 6.500 Da by SDS-PAGe. All these facts, including physico-chemical stabilities, small molecular size, and inhibition of Kisteria monocytogenes, indicate that bacteriocin T7 is likely to be a member of the class IIa bacteriocins.

• 한국미생물·생명공학회지
• /
• 제46권2호
• /
• pp.91-101
• /
• 2018

The aim of this study was to transfer the 18.5 kb gene clusters coding for 17 genes from Lactobacillus rhamnosus to Lactococcus lactis subsp. cremoris MG1363 in order to determine the effect of host on exopolysaccharide (EPS) production and to provide a model for studying the phosphorylation of proteins which are proposed to be involved in EPS polymerization. Lactobacillus rhamnosus RW-9595M and ATCC 9595 have 99% identical operons coding for EPS biosynthesis, produced different amounts of EPS (543 vs 108 mg/l). L. lactis subsp. cremoris MG1363 transformed with the operons from RW-9595M and ATCC 9595 respectively, produced 326 and 302 mg/l EPS in M17 containing 0.5% glucose. The tyrosine protein kinase transmembrane modulator (Wzd) was proposed to participate in regulating chain elongation of EPS polymers by interacting with the tyrosine protein kinase Wze. While Wzd was found in phosphorylated form in the presence of the phosphorylated kinase (Wze), no phosphorylated proteins were detected when all nine tyrosines of Wzd were mutated to phenylalanine. Lactococcus lactis subsp. cremoris could produce higher amounts of EPS than other EPS-producing lactococci when expressing genes from L. rhamnosus. Phosphorylated Wzd was essential for the phosphorylation of Wze when expressed in vivo.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권1호
• /
• pp.60-66
• /
• 2005

This study was performed to know whether there is any change of physiological activity in DLMJ which is inoculated by lactic acid bacteria. Lactic acid bacteria were isolated from Dolsan leaf mustard Kimchi (DLMK) at $20^{\circ}C$. In the optimum ripening period, the population of Leuconostoc and Lactobacilli in the DLMK were found to be high. The Leuconostoc, Lactobacilli and Lactococci strains were identified as Leuconostoc mesenteroides, Leuconostoc gelidum, Weissella confusa, Lactobacillus plantarum, Lactobacillus raffinolactis, Lactococcus lactis and Weissella confusa using the Biolog system. The most predominant strain which was isolated from DLMK was Weissella confusa. As the results of the phylogenetic analysis using 16s rDNA sequence, the Weissella confusa turned out to be Weissella kimchii, with 99.0% similarity. To investigated the change of physiological activity in DLMJ by lactic acid bacteria, 7 predominant strains inoculated to DLMJ (Dolsan Leaf Mustard Juice). The cytotoxicity was found to be under $19.55\%$ all cases. Also, the antioxidative activity of the DLMJ treated with lactic acid bacteria was very low, which might have been due to the reduced antioxidative phytochemicals during the preparation of the sterile sample. The ACE inhibiting activity of DLMJ by inoculation with Weissella kimchii was shown to be the highest ($94.0\%$). This could be that the degradation of sulfur containing materials in DLMJ by Weissella kimchii gave rise to ACE inhibiting activity.

• Journal of Microbiology and Biotechnology
• /
• 제2권2호
• /
• pp.73-77
• /
• 1992

The effect of a nisin-producing Lactococcus lactis spp. lactis (L. lactis) on the growth and attachment of Listeria monocytogenes Scott A and Brie 1 on stainless steel and their growth in Brain Heart Infusion broth was determined. Viable cells of Listeria decreased rapidly after 9~12 hr of incubation at $21^{\circ}C$ and after 6~9 hr of incubation at $32^{\circ}C$ in the presence of L. lactis. The number of L. monocytogenes Scott A attached to stainless steel in pure culture was $2.5{\times}10^3/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}2.3{\times}10^3/\textrm{cm}^2{\;}at{\;}32^{\circ}C$ after 48 hr of incubation, but was only $10/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}1.1{\times}10/\textrm{cm}^2{\;}at{\;}32^{\circ}C$ in the presence of L. lactis. Results from L. monocytogenes strain Brie 1 were similar to those from strain Scott A. The population of L. monocytogenes Scott A which attached to stainless steel with previously adherent L. lactis was $1.8{\times}10^2/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}8.2{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$, whereas the population attached to sterile stainless steel was $1.2{\times}10^3/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}2.1{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$. For L. monocytogenes Brie 1, the attached population of the control was $1.6{\times}10^4/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}3.2{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$, and on stainless steel with adherent L. lactis, it was $1.1{\times}10/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}6.9{\times}10/\textrm{cm}^2{\;}at{\;}32^{\circ}C$. Surface adherent L. lactis was less inhibitory to attachment of L. monocytogenes on stainless steel than a liquid culture inoculum. Listeria attached to stainless steel survived dry storage for 20 days both in the presence and absence of adherent lactococci.

• 한국유가공학회:학술대회논문집
• /
• 한국유가공기술과학회 1997년도 춘계 제44회 유가공 심포지움
• /
• pp.41-61
• /
• 1997

젖산균의 유전자 연구를 촉진하기 위해 간단하고 신속한 plasmid DNA의 분리방법과 electro-poration을 이용하여 vector plasmid의 간단하고 신속한 전이방법을 얻기 위해 젖산균의 형질전환에 영향하는 요인에 대하여 연구하였으며 연구결과는 다음과 같다. 1. O'Sullivan과 Klaenhammer의 방법을 개선하여 젖산균 plasmid DNA의 분리에 좋은 결과를 얻을 수 있는 신속하고 쉬운 방법을 고안하였으며, genomic DNA 분리에 이용되는 guanidium thio-cyanate 처리방법을 plasmid의 분리에 적용할 수 있었다. 2. L. casei, L. acidophilus. L. delbruekii var. bulgaricus. L. brevis와 L. plantarum 균주에서 plasmid를 확인하였으며, 돼지 분에서 분리된 L. lactis ssp. lactis. L. fermentum과 L. plantarum에서도 plasmid를 분리 확인하였다. 3. Lactococci의 plasmid분리는 lactobacilli와는 달리 mutanolysin의 처리없이도 잘 되었으며, L. lactis ssp. lactis와 Ent. faecalis에서 plasmid를 확인하였다. 4. E. coli plasimd 분리에 이용되는 MPS membrane filter 방법으로 젖산균 plasmid pLZ12의 분리가 가능하였으나, 세포파편이 filter를 막아 사용에 어려움이 있는 것으로 확인되었다. 5. Plasmid 분리없이 electroporation을 이용한 세포 대 세포 전이법으로 간편하고 빠르게 E. coli DH5${\alpha}$에 E. coli Jm109의 plasmid pBX19, pBR322를 전이시켰다. 6. L. lactis ssp. lactis 균주에 lysozyme 처리시 30${\sim}$80%의 생존율을 보였으며, 대부분의 L. acidophilus 균주의 경우 약 70%의 생존율을 보였다. L. casei 102S의 경우는 45분간 처리 시에도 100%의 생존율을 보였다. 8. L. lactis ssp. lactis 균주에 pLZ12를 6.0kV에서 전이시킨 결과 12.5kV에서보다 형질전환 효율이 훨씬 높았으며 lysozyme 처리에 의해 형질전환 효율이 증가되었다. 9. L. acidophilus 균주에 pLZ12를 전이시 6.0kV에서는 전이가 모두 이루어졌으나, 12.5kV에서는 L. acidophilus WIESBY와 NCFM에서 전이가 이루어지지 않았으며, lysozyme 처리 후 pLZ12를 전이시켰을 때 12kV보다 6.0kV에서 형질전환 효율이 증가되었다. 10. Gene Pulser와 Progenitor II를 사용하여 pLZ12를 L. lactis ssp. lactis 균주에 전이하였을 때 Gene Pulser에 비해 Progenitor II의 형질전환 효율이 현저히 떨어졌다. L. acidophilus HY7008과 HY7001은 두 기기 모두 형질전환이 이루어졌으나, L. acidophilus WEISBY와 NCFM은 Progeni-tor II에서 전이가 일어나지 않았으며, Gene Pulser에서 전이균주를 얻어 두 electroporator간에 형질전환 효율의 차이를 보였다. 11. L. casei 102S에 pLZ12를 electroporation시 낮은 전압에서 형질전환 효율이 비교적 좋았으며, 배양 시기를 달리하여 전이시켰을 때 대수생장 말기의 세포가 형질전환 효율이 좋았다. 12. L. casei 102S세포를 각각 10% glycerol, EB, 2차 증류수 등에 녹여 electroporation을 실시하였을 때 각각 $3.8{\times}10^3$, $5.0{\times}10^2$,1.5${\times}10^2$cfu의 형질전환 효율을 보였으며, 1.0mM HEPES, TE buffer를 사용하였을 때에는 전이가 이루어지지 않았다. 13. Plasmid pLZ12의 농도를 달리하여 electroporation을 하였을 때 형질전환 효율이 농도에 비례하여 증가하였다. 14. L. casei 102S에 대수생장 말기의 세포를 채취하여 10% glycerol, 200 Ohms, 25 ${\mu}$FD, 10kV/cm로 plasmid pLZ12를 electroporation할 때 최대 형질전환 효율인 3.8${\times}$10$^{3}$cfu를 얻었으며, lysozyme 처리가 다른 젖산균과는 달리 형질전환 효율을 증가시키지 못하였다. 15. L. casei 102S 세포를 10% glycerol과 EB에 녹여 -20$^{\circ}C$에서 냉동시킨 다음 1일과 7일 후의 세포를 electroporation한 결과 냉동시 세포에 손상을 주는 것으로 인식되었다.