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PubMed1998

Evidence for a mosaic structure of the Tn5481 in Lactococcus lactis N8.

The sequences of the left end of the nisin-sucrose transposon Tn5481 in Lactococcus lactis subsp. lactis N8, the adjacent 1.5 kb chromosomal region upstream of the junction site as well as a 5.0 kb region downstream of the nisZBTCIPRKFEG genes within the transposon have been determined. In the upstream chromosomal region, an incomplete open reading frame encoding a protein with strong N-terminal homology to the low-affinity branched chain amino acid carriers was identified. Within the transposon, downstream of the nisin gene cluster, a 186 bp almost identical copy of the left hand sequence was located. Further downstream, four new open reading frames were found. The codon usage in these reading frames as well as the G+C content of the region are clearly different from those of the nisin genes, suggesting that the functionally unrelated areas of Tn5481 are gathered from different origins during the evolution of the transposon.

ATP-Binding Cassette Transporters↗
PubMed1998

Characterization of the nisFEG operon of the nisin Z producing Lactococcus lactis subsp. lactis N8 strain.

Biosynthesis of the food additive nisin, a posttranslationally modified peptide antibiotic existing as two natural variants (A and Z), requires eleven genes (nisA/ZBTCIPRKFEG) involved in modification, secretion, regulation and self-immunity. The suggested self-immunity genes (nisFEG) of the nisin Z producer Lactococcus lactis subsp. lactis N8 were cloned and sequenced. Putative binding sites of the NisR transcription factor were recognized upstream of the nisF promoter. The hydrophilic NisF protein was expressed in Escherichia coli and shown to be associated with the membrane. Expression of the nisF gene from a plasmid in L. lactis MG1614, a strain lacking the nisin operons, did not increase the nisin resistance of the cells. This showed that NisF alone does not protect against nisin. Overexpression of the nisF gene in the N8 nisin producer did not affect the level of nisin immunity, indicating that the wild-type amount of NisF is not limiting the level of nisin immunity. Production of antisense-nisEG or antisense-nisG RNA in L. lactis N8 resulted in severe reduction in the level of nisFEG mRNA and a clearly reduced immunity showing that the nisFEG transcript is important for development of nisin self-immunity.

ATP-Binding Cassette Transporters↗
PubMed1999

Cloning, DNA sequence analysis, and deletion of a gene encoding diacetyl-acetoin reductase from Lactococcus lactis.

Diacetyl is produced by strains of lactic acid bacteria used in the dairy industry. Production of this important flavour compound could be increased by genetic manipulation of genes encoding enzymes involved in diacetyl metabolism. This paper reports the cloning and sequencing of the gene (dar) encoding diacetyl-acetoin reductase from Lactococcus lactis. Analysis of the DNA sequence of the dar gene and surrounding area revealed the presence of a putative operon with similarity to the family of ABC transporter systems. The dar gene has been deleted from the chromosome by double cross-over homologous recombination.

Alcohol Oxidoreductases↗
PubMed2000

Growth associated exopolysaccharide expression in Lactococcus lactis subspecies cremoris Ropy352.

A natural lactococcal isolate, Lactococcus lactis ssp. cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid). This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression. Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media. Ropy352 was examined for inducible lysogenic phages. No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction. The sugar compositions of the two phenotypically distinct exopolysaccharides were determined. The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively. The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively. Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.

Bacteriophages↗
PubMed2000

Short communication: salt extends the upper temperature limit for growth of Lactococcus lactis ssp. cremoris on solid M17 medium.

We have determined conditions for plating of the Lactococcus lactis ssp. cremoris laboratory strain MG1363 on solid M17 broth at 38 degrees C, which is required for the optimal use of the pGhost plasmids. The addition of 1% NaCl (or KCl, potassium acetate, or sucrose at 170 mM) to M17 agar plates results in extension of the upper temperature limit for growth from 37 to 40 degrees C; no decrease in plating efficiency was detected from 30 to 39 degrees C.

Culture Media↗
PubMed2001

Cell release from alginate immobilized Lactococcus lactis ssp. lactis in chitosan and alginate coated beads.

The effects of chitosan and alginate coatings of alginate beads with entrapped Lactococcus lactis ssp. lactis were studied in batch and continuous fermentations. Chitosan coating reduced the final concentrations of free cells, the initial release of free cells and the rate of lactate production in milk fermented batch-wise to a final pH of 4.7 in five consecutive batch fermentations. An alternative experimental system based on continuous fermentation with controlled pH and a high dilution rate was developed to better study the phenomenon of cell release. To estimate the effects of different bead coatings on cell release, alginate beads were coated with chitosan or alginate, or sequentially with chitosan/alginate or chitosan/alginate/chitosan. Chitosan coating alone seemed to reduce the rate of cell release only in the early stages of the fermentation, while sequential coatings with chitosan and alginate showed significant reduction throughout the whole test period. To examine whether the observed effects of bead coating could be explained only by a decrease in cell activity, the ratios between the rate of cell release and the rate of lactate production were examined during the fermentations for the different beads. This ratio showed qualitatively the same behavior as direct results of volumetric cell release.

Alginates↗
PubMed2001

Expression of clpX, an ATPase subunit of the Clp protease, is heat and cold shock inducible in Lactococcus lactis.

In this study, the clpX gene and surrounding sequences were cloned and sequenced from Lactococcus lactis. The putative clpX gene encodes a 411 amino acid polypeptide with a predicted molecular weight of 45.8 kDa. Analysis of the relative levels of clpX transcript revealed that in addition to a role in proteolysis of heat damaged proteins, ClpX may also be involved in cryoprotection.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2002

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552.

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.

Bacterial Proteins↗
PubMed2003

Technical note: Use of RFLP to characterize Lactococcus lactis strains producing exopolysaccharides.

Restriction fragment length polymorphism (RFLP) is used to differentiate microorganisms by analysis of their DNA restriction patterns. A modified RFLP procedure is proposed for the rapid characterization of Lactococcus lactis strains producing exopolysaccharides (EPS). The availability of such effective cataloging system is likely to benefit research aimed at identifying lactococcal strains that produce novel EPS.

DNA, Bacterial↗
PubMed2004

The effect of Lactococcus lactis starter cultures on the oxidative stability of liquid whey.

The oxidative stability of liquid Cheddar cheese whey was evaluated using 2 Lactococcus lactis starter cultures in combination and alone along with a control, utilizing glucono-delta-lactone for acid development. Fresh and stored whey were evaluated for volatile composition, free fatty acids, and flavor by descriptive sensory analysis. A significant increase in volatile lipid oxidation products, most notably, hexanal, occurred during storage, and a corresponding decline in the free fatty acid linoleic acid was found. The flavor and aroma characteristic, cardboardy, was correlated to the increase in volatile lipid oxidation products and the decline in linoleic acid. Evidence strongly suggested that lipid oxidation was initiated during whey production and escalated during storage and that the starter cultures significantly influenced the level of volatile lipid oxidation products. Further understanding of the impact of starter cultures on whey may allow for the production of higher quality whey ingredients with wider food application.

Cheese↗
PubMed2005

Lactococcal 936-species phage attachment to surface of Lactococcus lactis.

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.

Adsorption↗
PubMed2005

Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats.

Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milks, yogurts, and cheeses is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study was conducted to assess in a rat bioassay the response of administration of milk fermented by modified Lactococcus lactis on the riboflavin status of deficient rats. Rats were fed a riboflavin-deficient diet during 21 d after which this same diet was supplemented with milk fermented by Lactoccus lactis pNZGBAH, a strain that overproduces riboflavin during fermentation. The novel fermented product, with increased levels of riboflavin, was able to eliminate most physiological manifestations of ariboflavinosis, such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegaly, that were observed using a riboflavin depletion-repletion model, whereas a product fermented with a nonriboflavin-producing strain did not show similar results. A safety assessment of this modified strain was performed by feeding rodents with the modified strain daily for 4 wk. This strain caused no detectable secondary effects. These results pave the way for analyzing the effect of similar riboflavin-overproducing lactic acid bacteria in human trials. The regular consumption of products with increased levels of riboflavin could help prevent deficiencies of this essential vitamin.

Animals↗
PubMed2006

Altering renneting pH changes microstructure, cell distribution, and lysis of Lactococcus lactis AM2 in cheese made from ultrafiltered milk.

The objective of this study was to investigate the lysis of a highly autolytic strain of Lactococcus lactis ssp. cremoris AM2 in a model cheese made from concentrated ultrafiltered milk. From the same initial ultrafiltered retentate inoculated with L. lactis AM2, 5 cheeses were made by the addition of rennet at different pH values (6.6, 6.2, 5.8, 5.4, and 5.2). Lysis was monitored by measurement of the release of lactate dehydrogenase, an intracellular marker enzyme, and by immunodetection of intracellular proteins with species-specific antibodies. Confocal scanning laser microscopy (CSLM) was used to investigate the cheese microstructure by staining for protein and fat. Dual staining with a bacterial viability kit with CSLM was performed to reveal the integrity and localization of the bacterial cells. Levels of soluble calcium significantly increased when the pH at which the rennet was added decreased. In cheese renneted at pH 6.6, CSLM revealed an open porous structure containing a dense protein network with fat globules of different sizes distributed in the aqueous phase. In cheese renneted at pH 5.2, the protein network was homogeneous, with a less dense protein network, and an even distribution of fat globules. On d 1, bacterial cells were organized into colonies in cheese renneted at pH 6.6, whereas in cheeses renneted at pH 5.2, bacteria were evenly dispersed as single cells throughout the protein network. Lysis was detected on d 1 in cheeses renneted at high pH values and continued to increase throughout ripening, whereas induction of lysis was delayed in cheeses renneted at lower pH values until the end of ripening. This study demonstrates that alterations in the microstructure of the cheese and the distribution of cells play a role in lysis induction of L. lactis AM2.

Amino Acids↗
PubMed2006

Proteolysis of Hispanico cheese manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639.

Hispánico cheese was manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639, bacteriocin-nonproducing L. lactis ssp. lactis INIA 437, or a combination of both strains, as starter cultures. Lactobacillus helveticus LH 92, a culture of high amino-peptidase activity sensitive to lacticin 481, was added to all vats. Milk inoculation with the bacteriocin producer promoted early lysis of Lb. helveticus cells in cheese. Cell-free aminopeptidase activity in cheese made with the 3 lactic cultures was 1.8 times the level reached in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 15 d of ripening. Proteolysis (as estimated by the o-phthaldialdehyde method) in cheese made with the 3 lactic cultures was twice as high, and the level of total free amino acids 2.4 times the level found in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 25 d of ripening. Hydrophobic and hydrophilic peptides and their ratio were at the lowest levels in cheese made with the 3 lactic cultures, which received the lowest scores for bitterness and the highest scores for taste quality.

Amino Acids↗
PubMed1991

Functional alteration of macrophages by a slime-forming Lactococcus lactis ssp. cremoris.

The effect of a slime-forming, encapsulated Lactococcus lactis ssp. cremoris KVS20 on macrophage function has been examined in vivo and in vitro in short-term studies. Peritoneal macrophages in which 21 to 34% of macrophage was presenting Fc gamma-receptor positive macrophages were elicited by intraperitoneal injection of 10 to 50 mg/kg of L. lactis ssp. cremoris KVS20. The peritoneal macrophage exhibited cytotoxic activity against Sarcoma-180 cells in which the maximum activity was obtained in macrophage from mice injected with 10 mg/kg on d 5. However, L. lactis ssp. cremoris KVS20 rendered the elicited macrophage cytotoxic in vitro. The cytotoxicity was significantly augmented by 6- and 24-h treatment at the concentration of 50 to 500 micrograms/ml. These results obtained in the short-term studies demonstrated that the antitumor activity of L. lactis ssp. cremoris KVS20 may be mediated through the enhanced cytotoxic activity of macrophage.

Animals↗
PubMed1992

Growth and activities of Lactococcus lactis in milk enriched with low mineral retentate powders.

The growth and activities of three strains of Lactococcus lactis ssp. cremoris (Wg2, E8, and HP) and their proteinase-negative variants were studied in skim milk enriched with three types of retentate powder. The performance of these strains in enriched milks was compared with that determined in reconstituted skim milk. Proteinase-positive strains of L. lactis ssp. cremoris exhibited higher maximum specific growth rates than protease-negative variants. Moreover, maximum specific growth rates of lactococci were lower in skim milk than in enriched milk with a high buffering capacity. The performance of proteinase-positive strains was better than that of proteinase-negative variants. Growth of proteinase-positive lactococci in milk media increased alpha-amino groups as determined by the increase of equivalent glutamic acid concentration. Available alpha-amino groups decreased with proteinase-negative variants. Proteinase-positive strain Wg2 exhibited the most proteolytic activity but showed the least specific overall productivity of lactic acid despite high biomass concentration in milk. Among proteinase-positive lactococci, strain E8 produced more lactic acid than other strains, and, among proteinase-negative variants, strain HP had the best specific overall productivity of lactic acid.

Animals↗
PubMed1992

B-cell mitogenic activity of slime products produced from slime-forming, encapsulated Lactococcus lactis ssp. cremoris.

The mitogenic activities of whole cell lyophylized preparations, cell-wall components, and slime products obtained from Lactococcus lactis ssp. cremoris KVS20 were examined on murine spleen cells. Whole cell lyophylized preparations and slime products significantly (P < .05) stimulated mitogenic responses of the cells. The highest activity was induced by slime products in which the optimal concentration was 116 microg/ml. The significant (P < .05) increase of mitogenic activity induced by slime products occurred at 24 h, and the peak response was obtained 48 h after the stimulation. The activity was much higher in the fraction enriched with B cells than in the fraction enriched with T cells. In addition, slime products induced mitogenic activity to spleen cells of athymic nu/nu mice. The chemical analysis of lipopolysaccharide and the minimal concentration for mitogenic response eliminated the possibility that the activity of slime products may be due to the contamination of lipopolysaccharide. The data demonstrate that slime products are a potent B-cell-dependent mitogen.

Animals↗
PubMed1993

B-cell mitogen produced by slime-forming, encapsulated Lactococcus lactis ssp. cremoris isolated from ropy sour milk, viili.

A substance, active as a B-cell mitogen, was isolated from the slime products produced by Lactococcus lactis ssp. cremoris KVS20. The mitogenic substance was prepared by anion-exchange chromatography and gel filtration chromatography and then purified by proteinase digestion and HPLC. Chemical analysis determined that the mitogenic substance was a phosphopolysaccharide and consisted of rhamnose, glucose, galactose, and phosphorus. The activity of the mitogenic substance was higher than that of the slime products. The optimal concentration for the activity was approximately 120 micrograms/ml. The mitogenic substance also had substantial mitogenic activity to spleen cells from C3H/HeJ mice, which are resistant to lipopolysaccharide. The findings indicated that a B-cell mitogen different from lipopolysaccharide is produced from L. lactis ssp. cremoris KVS20.

Animals↗