• Microbiology and Biotechnology Letters
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• v.46 no.2
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• pp.91-101
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• 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.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.8-13
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• 1991

Bacteriocin-producing microorganisms were screened from raw milk and tested their antimicrobial activities against Lactobacillus plantarum ATCC 8014 as target organism, Antimicrobial substances isolated showed broad antimicrobial spectra against Gram positives and negatives. Strain 1112-1 was selected as a test organism due to its highest antimicrobial activity among the isolates. Antimicrobial substance produced by 1112-1 completely suppressed the growth of Lactobacillus plantarum at 230 IUIml and showed 11% growth inhibition of E. coli at 500 IUIrnl level. The antimicrobial substance was found to be proteinaceous material which was inactivated by carboxypeptidase, elastase, alpha amylase, amyloglucosidase, pronase, protease IV, alpha chymotrypsin, ficin, cellulase, phosphatase and lipase. The molecular weight was estimated by SDS-PAGE as 5,900. The isolate 1112-1 was identified as one of the related strains of Lactococcus sp. The strain was different from Lactococcus lactis in the following characteristics: late positive in maltose and sucrose fermentation; positive in mannitol and salicin fermentation; negative in lactose fermentation.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.623-625
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• 2000

Bacteriocin-producing lactic acid bacteria was isolated from Kimchi using MRS as selective media and Lactobacillus delbruekii subsp. delbruekii as an indicator strain. Strain JC-3 was tentatively identified as Lactococcus latis subsp. lactis through the API test and the bacteriocin produced by JC-3 showed the inhibitory activity against Grampositive pathogens and other lactic acid bacteria. The antimicrobial substance was inactivated by Protamax, Aroase AP-10, Neutrase, R-AMANO and was confirmed to be heating at $100^{\circ}C$. However, it was lost at high pH values showed the highest bacteriocin activity at a culture temperature of $30^{\circ}C$. The bacteriocin was partially purified by ammonium sulfate precipitation, Sep-pak $C_{18}$ cartridge. The apparent molecular mass of the bacteriocin was about 8 Kda, which was determined through the direct detection of bactericidal activity using SDS -PAGE.

• Journal of Microbiology
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• v.41 no.2
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• pp.63-72
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• 2003

Chronic inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis, affects around 2 in every 1000 individuals in western countries and its incidence, particularly amongst children, is increasing. IBD shows extreme morbidity with impact on all aspects of quality of life. If left untreated, IBD can lead to death. Conventional treatment of IBD involves powerful immunosuppressive chemotherapies and surgical intervention. Long-term anti-inflammatory medication is required and so patients are often subject to a spectrum of unpleasant side effects. Interleukin-10 (IL-10) is a cytokine that acts to suppress inflammation. When however administered by injection, the high levels of IL-10 that are distributed throughout the body also lead to side effects. Lactococcus lactis can be genetically engineered to secrete biologically active cytokines. When applied to the mucosa, these L. lactis can actively deliver such cytokines. By use of this principle we developed a new therapeutic approach for IBD. Administration of L. lactis that secretes murine IL-10 cures and prevents IBD in mice. The use of the engineered L. lactis gets around the problem of delivering IL-10, allowing dramatic reduction of the effective dose. A sincere concern exists about the possible dangers of uncontrolled, deliberate release of genetically modified microorganisms, such as could occur following application in healthcare. We engaged in the establishment of adequate means for biological growth control of engineered L. lactis by targeted gene exchange between thyA and hIL-10.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.386-390
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• 1996

The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.50-53
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• 1996

A cultured milk product was made by fennenting 10$\%$ reconstituted skim milk with Lactococcus lactis subsp. lactis TA29 and Saccharomyces exiguus SK2. L. lactis TA29 and S. exiguus SK2 grew up to 1.0 $\times 10^9\;and\;2.0 \times 10^6$ cfu/ml, respectively. After the fermentation 21$\%$ of lactose was hydrolyzed, pH was lowered to 4.2, and titratable acidity and alcohol concentration were increased to 0.96 and 0.023$\%$, respectively. When the fermented milk was stored at $4{\circ}C$ for 9 days, the viable cell counts for L. lactis TA29 and S. exiguus SK2 were $6.5 \times 10^5\;and\;1.6 \times 10^6$ cfu/rnl, respectively. The alcoholic fermented milk prepared in this experiment was more inhibitory against some pathogenic bacteria including C. perfringens than commercial yoghurt products tested.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.393-397
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• 1999

The gene (galE) encoding UDP-galactose-4-epimerase, operative in the galactose metabolic pathway, was cloned together with the $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis ATCC7962 (L. lactis 7962). galE was found to have a length of 981 bps and encoded a protein with a molecular mass of 36,209 Da. The deduced amino acid sequence showed a homology with GalE proteins from several other microorganisms. A Northern analysis demonstrated that galE was constitutively expressed by its own promoter. When galactose or lactose was added into medium, the galE transcription was induced by several upstream promoters. The structure of the gal/lac operon of L. lactis 7962 was partially characterized and the gene order around galE was galT-lacA-lacZ-galE-orfX.

• Food Science and Biotechnology
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• v.15 no.4
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• pp.485-493
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• 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.

• Food Science and Biotechnology
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• v.14 no.3
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• pp.405-409
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• 2005

Cellular components of Lactococcus lactis ssp. lactis (heat-killed whole cells, cytoplasm, and cell walls) were tested for their in vivo immunopotentiating activities. Peritoneal macrophages from mice injected intraperitoneally with cell-wall fractions exhibited significantly greater phagocytic activity than groups injected with whole cells or cytoplasm fraction. Cytotoxicity of natural-killer cells was highest in cytoplasm fractions. Production of cytokines (IFN-${\gamma}$, IL-2, IL-6, and IL-12) in spleen cells was significantly higher when cellular components were injected intraperitoneally, and tended to be higher in whole-cell and cytoplasm groups than in cell-wall group. These results demonstrate lactic acid bacteria whole cells and their cytoplasm and cell-wall tractions have immunopotentiating activities.

• Food Science of Animal Resources
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• v.38 no.6
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• pp.1315-1321
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• 2018

Bacteriocin is ribosomally synthesized by bacteria and inhibits closely related species. In this study we aimed at isolating lactic acid bacteria producing bacteriocin presenting anti-staphylococcal activity. A Lactococcus lactis strain was isolated from kimchi for the purpose and identified by 16S rRNA gene sequencing. As preliminary tests, optimal culture conditions, stabilities against heat, solvents, and enzymes treatments, and type of action (bacteriostatic or bactericidal) of the bacteriocin were investigated. The optimal culture conditions for production of the bacteriocin were MRS broth medium and $25^{\circ}C$ and $30^{\circ}C$ culture temperatures. The bacteriocin was acidic and the activity was abolished by a protease treatment. Its stability was maintained at $100^{\circ}C$ for 15 min and under treatments of various organic solvents such as methanol, ethanol, acetone, acetonitrile, and chloroform. Finally, the bacteriocin showed bactericidal action against Staphylococcus aureus where 200 AU/mL of the bacteriocin decreased the viable cell count (CFU/mL) of S. aureus by 2.5 log scale, compared with a control (no bacteriocin added) after 4-h incubation.