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PubMed2001

[Continuous production of nisin by calcium alginate-immobilized Lactococcus lactis subsp. lacits SM526].

The attempts were made to produce nisin by immobilized cells of Lactococcus lactis subsp. lactis SM526 entrapped in calcium alginate beads. The results showed that the immobilized cell beads were intact for 90 hr under 2% of calcium alginate in 10 mmol/L CaCl2 solution. The physical stability of beads was improved by removing phosphate, citrate and acetate salts supplied in SYS3 medium. Repeated-batch fermentation utilizing immobilized cells were performed in mSYS3 medium without agitation at 32 degrees C, the concentration of nisin produced during the third cycles increased to 850 IU/mL, and the beads were still intact with little cell leakage. The immobilized beads were loaded into a sterilized glass column to continuous fermentation, nisin concentration increased to 1150 IU/mL, as high as that obtained in free-cell fermentation, and the high productivity was stable for up to 70 h under a constant supplied of the fresh mSYS3 medium.

Alginates↗

PubMed1999

[Expression in Lactococcus lactis of catalytically active phenylalanine ammonia-lyase from parsley].

The phenylalanine ammonia-lyase (PAL) cDNA of parsley (Petroselinum crispum) was subcloned into constitutive expression vector pMG36e downstream of the P32 promoter. The resulting plasmid pMG36ePAL was introduced into Lactococcus lactis subsp. lactis MG1363 by electroporation. The recombinant strain showed its PAL activity conversing the L-phenylalanine in the culture medium into trans-cinnamic acid. A new secretory vector pXHS was constructed by recombination of pMG36e with a Lactococcal usp45 secretion leader coding sequence and a translational coupling sequence. Then the pXHSPAL was constructed and used for expression of PAL in L. lactis, the PAL activity was also detectable. The L. lactis dnaJ promoter sequence was cloned and used to construct a heat inducible vector pXHJ. PAL cDNA was cloned into pXHJ and the L. lactis IL1403 was transformed with the recombinant plasmid pXHJPAL. After a heat shock from 30 degrees C to 37 degrees C, the PAL activity of the pXHJPAL strain could increase approximately onefold. The prospect of using these engineering L. lactis strains for PKU therapy was also discussed.

Amino Acid Sequence↗

PubMed2003

Heterologous protein production and delivery systems for Lactococcus lactis.

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.

Animals↗

PubMed2003

Challenges when transferring technology from Lactococcus laboratory strains to industrial strains.

Many genetically modified Lactococcus strains have been constructed in research laboratories around the world. Most of these have originated from laboratory strains and therefore there are several barriers to using them in an industrial setting. Laboratory strains are often plasmid-free and consequently Lac- and Prt-, rendering them unable to grow in milk. Many of the commonly used techniques have been optimised for laboratory strains and their application to industrial strains may require a great deal of effort. Often genetically modified organisms produced in the laboratory do not fit the published definition of 'food-grade' (Johansen, 1999, Encyclopedia of Food Microbiology, Academic Press, London, pp. 917-921) and a great deal of effort is required to eliminate undesirable DNA sequences. As a consequence, it is often necessary to recreate the strains in industrial backgrounds before the innovations described in the scientific literature can be applied to the real-world dairy industry.

Biotechnology↗

PubMed1992

Colonization of the digestive tract of germ-free mice by genetically engineered strains of Lactococcus lactis: study of recombinant DNA stability.

The ability of genetically engineered Lactococcus lactis strains to become established in the digestive tract (DT) of germ-free mice was examined together with the stability of their genetic markers. Seven L. lactis strains were genetically modified by insertion of genetic markers on different replicons: chloramphenicol resistance gene cat was carried by self-transmissible plasmid pIL205, a derivative of plasmid pIP501; erythromycin resistance gene erm, originating from pAM beta 1, was inserted into non-transmissible plasmids pIL252 and pIL253 of low and high copy number respectively; erm gene from plasmid pMS1.5B was inserted into the chromosome. All strains carried a common wild-type plasmid pIL9 involved in lactose fermentation. It was observed that the DT of mice was rapidly and efficiently colonized with either the inoculated parental strain or with its derivatives or with both of them, but plasmid-free derivatives were always at dominant levels. Both plasmids pIL9 and pIL205 were lost, but the parental strains and the plasmid-lacking derivatives were at codominant levels, indicating that there is an equilibrium between plasmid loss and plasmid transfer in the DT. Strains that carried non-transmissible and low copy number plasmid pIL252 were rapidly eliminated from the DT, which in turn was colonized with the respective pIL252-less derivatives; this is probably due to the high segregational instability of pIL252.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Oxidative stress in Lactococcus lactis.

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.

Cell Survival↗

PubMed2004

Experimental evaluation of pathogenicity of Lactococcus garvieae in black rockfish (Sebastes schlegeli).

Black rockfish (Sebastes schlegeli) is an important mariculture species in Korea. The production of this fish is drastically declined due to bacterial diseases, particularly streptococcosis caused by Lactococcus garvieae. The bacterial surface characteristics of SJ7 and TY6 were found to have capsule but not NB13 and YS18. The experiential evaluation of L. garvieae pathogenicity, the capsular isolates showed high cumulative mortality i.e. SJ7 (100%) and TY6 (60%) compared to non-capsular isolates. Based on this result the capsular isolates L. garvieae were highly suspected as the causative agent of streptococcosis in rockfish.

Agglutination Tests↗

PubMed2005

[A case of femoral osteomyelitis caused by Lactococcus].

The authors report on a case of osteomyelitis of the femur, where the causative agent was Lactococcus. These bacteria are mainly pathogenic in endocardial implants and extremely rare in osteomyelitis. The osteomyelitis was complicated with endocarditis, cerebral and pulmonary abscess. In the reported case the occurrence of osteomyelitis was similar to bone tumor formation, so there was a challenging diagnostic approach. Based upon this case, the diagnostic algorithm, the treatment strategy and the outcome of the treatment are reported. Authors also give a short overview of the literature published on this rare microorganism in bone and joint diseases.

Adult↗

PubMed2005

[Influence of expression of transglutaminase on the growth of Lactococcus lactis].

To improve the aerobic growth performance of Lactococcus lactis subsp. cremoris NZ9000, the gene mtg encoding the mature microbial transglutaminase was amplified from the chromosomal DNA of Streptoverticillium mobaraense and then cloned into the nisin-inducible expression vector pNZ8148. The resulting plasmid pFL001 was transformed into strain NZ9000 by electroporation. Compared with strain NZ9000 harboring pNZ8148 (the control strain), strain NZ9000 harboring pFL001 (the recombinant strain) had a remarkably improved aerobic growth performance. When grown aerobically under non-pH-controlled conditions, the maximal biomass of the recombinant strain reached 4.13 g/L, which was 11-fold higher than the growth of the control strain (0.34 g/L). When grown aerobically with the pH controlled at 6.5 +/- 0.1, the maximal biomass of the recombinant strain reached 4.73 g/L, which was an 80% increase compared with the growth of the control strain. In addition, the efficiency of biomass synthesis relative to glucose consumption (Y(x/s)) of the recombinant strain, 71.7 g of biomass per mol of glucose, was 1.6-fold higher than that of the control strain. The significantly improved growth performance of the recombinant strain might be attributed to the expression of mtg in the recombinant strain, which might increase intracellular pH and save part of the energy(ATP) that was originally used for pumping the cytoplasmic H+, and as a consequence, the energy used for growth increased accordingly.

Hydrogen-Ion Concentration↗

PubMed1991

Transposon-encoded sucrose metabolism in Lactococcus lactis. Purification of sucrose-6-phosphate hydrolase and genetic linkage to N5-(L-1-carboxyethyl)-L-ornithine synthase in strain K1.

Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

[Study of intraspecific variations of the bacterium Lactococcus lactis in adaptation to high acidity of the medium].

This paper reports on the study of acid tolerance of lactic acid bacteria as a property of cells, determining their ability to divide efficiently and retain viability under conditions of increased nutrient medium acidity during bacterial growth. The bacteria of the strain TV2, isolated from a self-soured curd, similar to the bacteria of the strain STE05 (Russian National Collection of Industrial Microorganisms), were assigned to the species Lactococcus lactis according to their G+C composition (36.7-36.5 mol %) and the high level of DNA-DNA hybridization (93%). However, these strains were essentially different in the number and size of the plasmids and the chromosomal DNA restriction fragments, as well as in the sensitivity to phages of lactic acid bacteria. It was found that bacteria of the strain TV2 were stable (i.e., they divided efficiently at a pH as low as 5.3) and tolerant to the lactic acid that they produced while growing (i.e., they retained viability at pH 4.4). Bacteria of the strain STE05 lacked acid tolerance (at pH below 6.5, growth was retarded, and pH 5.0 was the lowest value at which the cells remained viable). The acid tolerance and phage resistance of TV2 bacteria are likely to characterize their higher adaptive capacity in comparison with STE05 bacteria. Acid tolerance is inherited in a stable manner and retained by the segregants of TV2 strain obtained in the course of long-term storage of the bacteria. Specifically, the strains TV29, TV13, and TV 229, which displayed this property, had altered physiological and biochemical characteristics (accumulation of biomass and fermentation of lactose) in spite of their genetic identity to the original strain (pulsed-field gel electrophoresis of chromosomal DNA restriction fragments).

Adaptation, Physiological↗

PubMed1991

Purification and properties of fructokinase I from Lactococcus lactis. Localization of scrK on the sucrose-nisin transposon Tn5306.

Two electrophoretically distinct proteins with fructokinase (ATP:fructose-6-phosphotransferase) activity were detected in Lactococcus lactis subsp. lactis K1. Whereas fructokinase I was induced specifically by growth of the organism on sucrose, fructokinase II was derepressed during growth on ribose, galactose, maltose, and lactulose. Fructokinase I was purified about 1000-fold to electrophoretic homogeneity (specific activity 112 units/mg). The amino acid composition, N-terminal sequence, nucleoside triphosphate, and metal requirement(s) of the enzyme are reported. Ultracentrifugal analysis showed that the enzyme was primarily dimeric with subunits of 33.5 kDa (+/- 5%). When completely reduced, fructokinase I migrated as a single protein (Mr = 32,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in the absence of reducing agent two polypeptides (apparent Mr = 29,000 and 31,000) were detected. Isoelectric focusing also revealed two polypeptides (pI 5.6 and 5.8), and both species catalyzed the phosphorylation of fructose and mannose. Hybridization studies showed that: (i) a sucrose-negative mutant lacking the fructokinase I gene (scrK) retained fructokinase II activity and (ii) scrK is closely linked to scrA and scrB which encode Enzyme IIScr and sucrose-6-phosphate hydrolase, respectively. In L. lactis K1, these genes and the N5-(1-carboxyethyl)-L-ornithine synthase gene (ceo) are encoded on the sucrose-nisin transposon Tn5306 in the order ceo-scrKAB.

Carbohydrate Metabolism↗

PubMed2006

Portal vein thrombosis and liver abscess due to Lactococcus lactis.

A 26-year-old man was admitted with fever and abdominal pain. Abdominal ultrasonography and Doppler ultrasound eventually revealed portal vein thrombosis and a pyogenic liver abscess (17x11x11 cm). Lactococcus lactis was isolated from a culture of the abscess material. This organism is not a common pathogen in humans. This is the first published description of portal vein thrombosis and pyogenic liver abscess due to L. lactis.

Adult↗

PubMedDate not supplied

[Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116].

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.

Culture Media↗

PubMed2006

Stimulation of nisin production from whey by a mixed culture of Lactococcus lactis and Saccharomyces cerevisiae.

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.

Animals↗

PubMed2006

[Glutathione plays an anti-oxidant role in Lactococcus lactis].

To assess the physiological function of GSH in resistance to oxidative stress in Lactococcus lactis ssp. cremoris NZ9000, the recombinant strain NZ9000 (pNZ3203) capable of producing GSH was used as the experiment materials. The anti-oxidant role of glutathione was observed under higher H2O2 dosage, i.e., 150 mmol/L H2O2 treatment for 15 min. The resistance of NZ9000 (pNZ3203) cells grown for 3 h, 5 h, and 7 h (nisin-induced for 1 h, 3 h and 5 h) were 1.8-fold, 2.6-fold, and 2.9-fold that of NZ9000 (pNZ8148) cells, respectively. In addition, the survival of NZ9000 (pNZ3203) cells grown for 5 h (nisin-induced for 3 h), upon treatment of 20 mmol/L menadione for 60 min, was 6.2-fold that of NZ9000 (pN8148) cells. Therefore, introduction a new biosynthetic pathway of glutathione could confer higher resistance to oxidative stress on L. lactis NZ9000.

Antioxidants↗

PubMed2006

[Effects of gavage with lactococcus lactis recombinant heme oxygenase-1 gene on inflammation of intestine and bacterial translocation in rats with hemorrhagic shock].

OBJECTIVE: To evaluate the effects of gavage with lactococcus lactis (L. Lactis) recombinant heme oxygenase-1 (HO-1) gene on alleviation of intestinal inflammation and protection of the intestinal mucosa in rats with hemorrhagic shock. METHODS: A model of rats with hemorrhagic shock was reproduced in 30 healthy SD male rats. The rats were randomly divided into the L. Lactis recombinant HO-1 gene group (HO group, n=10), L. Lactis group (LL group, n=10) and phosphate buffer group (PBS group, n=10). These agents were respectively gavaged 24 hours before the experiment. Rats were re-anesthetized 1 hour after fluid resuscitation. The mortality, myeloperoxidase (MPO) activity, bacterial translocation, the pathologic changes, the contents of HO-1, tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in the intestine were determined and compared. RESULTS: Compared with LL group and PBS group, the mortality, Chiu's grade and the bacterial translocation rate of HO group were significantly decreased (all P<0.05) but the content of HO-1 and the level of IL-10 in HO group were markedly increased (both P<0.05). Compared with HO group and LL group, the MPO activity of PBS group was obviously increased (P<0.05). CONCLUSION: The recombinant L. Lactis has the effect to deliver HO-1, which has protective effect on the intestinal mucosa in lessening the inflammation of the intestine and the incidence of bacterial translocation.

Animals↗

PubMedDate not supplied

[Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk].

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow's milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951-DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.

Animals↗