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PubMed1989

Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning.

In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides. We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon. Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1. These plasmids could be stably maintained in L. paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure. In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L. paramesenteroides. Expression of this heterologous gene in L. paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots). In addition, we transformed a lactose-deficient derivative of L. paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion. The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L. paramesenteroides.

Blotting, Western↗
PubMed1994

Cloning, sequencing and comparison of three lactococcal L-lactate dehydrogenase genes.

The conversion of pyruvate to lactate is a key feature of lactococcal strains. The enzyme which facilitates this conversion, L-lactate dehydrogenase (LDH), and the gene which encodes it (Idh), are therefore of great significance. This paper presents the cloning and DNA sequence analysis of three further lactococcal genes which are of key importance in the genetic manipulation of commercial starter strains. The Idh gene from Lactococcus lactis subsp. Lactis biovar diacetylactis BU2-60 has been isolated from a lambda library and sequenced. The Idh gene from L. lactis subsp. cremoris NCDO 762 and that from L. lactis subsp. Lactis IL1403 have been amplified by the polymerase chain reaction (PCR) and sequenced. These DNA sequences and deduced amino acid sequences have been compared with those from L. lactis subsp. Lactis MG1363. The LDHs from L. lactis subsp. Lactis MG1363 and L. lactis subsp. cremoris NCDO 762 are 99.4% homologous. The LDHs from L. lactis subsp. lactis MG1363 and L. lactis subsp. Lactis IL1403 are 96.4% homologous. The LDHs from L. lactis subsp. lactis IL1403 and L. lactis subsp. lactis biovar diacetylactis BU2-60 are 99.9% homologous. Our results provide further evidence that L. lactis subsp. Lactis MG1363 and other L. lactis subsp. Lactis NCDO 712 derived strains should be reclassified as Lactococcus lactis subsp. cremoris.

Amino Acid Sequence↗
PubMed1995

Expression of lactococcin A and pediocin PA-1 in heterologous hosts.

Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter. Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.

Anti-Bacterial Agents↗
PubMed1996

Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation.

Fast milk-coagulating (Fmc+) strains of lactococci are known to segregate slow milk-coagulating (Fmc-) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc- phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. In Lactococcus lactis subsp. lactis (L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. In Lactococcus lactis subsp. cremoris SK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid in L. lactis C2 encoded both the Prt and Opp systems. Fmc- derivatives of L. lactis C2 were missing the prt genes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc+ phenotype.

Animals↗
PubMed1994

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods.

Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22 degrees and 37 degrees C for 90 days. Spoilage was detected by the presence of gas and/or odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Cheese↗
PubMed1991

Effect of lactic cultures on Escherichia coli in ewes' milk stored at low temperatures.

The behaviour of Escherichia coli in pasteurized ewes' milk inoculated with different lactic starter cultures and incubated at temperatures in the range 4-16 degrees C for 96 h was investigated. Growth temperature of lactic starter cultures before inoculation had a significant effect on inhibition of E. coli. The growth temperature of lactic starter inoculum which resulted in the highest inhibitory activity was 24 degrees C. Size of lactic starter inoculum also significantly influenced growth of E. coli, with a higher inhibition for 1% inoculum than for 0.1% or 0.3% inocula. Single cultures of Lactococcus lactis showed a stronger inhibitory activity than single cultures of Leuconostoc cremoris or Leuconostoc dextranicum. A lactic starter culture comprising Lactococcus lactis, Leuconostoc cremoris and Leuconostoc dextranicum resulted in the strongest inhibition. Stimulation of E. coli by the lactic starter cultures was frequently recorded at 4 degrees C and 8 degrees C. However, none or a very limited growth of E. coli was seen at these temperatures.

Animals↗
PubMed1992

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.

Amino Acid Sequence↗
PubMed2001

Influence of adjunct cultures on ripening of reduced fat Edam cheeses.

The influence of four adjunct cultures [Brevibacterium linens (BL2), Lactococcus lactis ssp. diacetylactis, Lactobacillus helveticus (LH212), and Lactobacillus reuteri (ATCC 23272)] on chemical and sensory characteristics of reduced fat Edam cheese was studied. The aminopeptidase activity of Lactococcus lactis ssp. diacetylactis was higher than that of Lactobacillus helveticus, Lactobacillus reuteri, and Brevibacterium linens, respectively. Mean percent fat and moisture contents of reduced fat cheese were 20.85 +/- 0.69 and 42.95 +/- 0.43, respectively. Percentage of fat and moisture of full fat control cheese were 30.06 +/- 0.78 and 39.11 +/- 0.60. Titratable acidity increased in all cheese with aging while pH initially decreased but increased in cheese after 6 mo aging at 7 degrees C. Lactic acid bacteria counts were on average one log higher for reduced fat cheeses than for full fat control cheese and counts decreasing with aging. Free amino acids (FAA) in cheeses increased with aging, and were higher in reduced fat cheeses than in the full fat control cheese. Reduced fat cheeses containing L. helveticus exhibited the highest FAA content. Descriptive sensory panelists (n = 9) did not detect differences among cheeses after 3 and 6 mo ripening, but aged/developed flavors (fruity, nutty, brothy, sulfur, free fatty acid) and sweetness increased between 3 and 6 mo. Expert panelists (n = 6) detected differences in texture quality among the cheeses. Reduced fat control cheeses and reduced fat cheeses with L. helveticus and L. reuteri received the highest texture quality scores. Addition of L. helveticus and Lc. lactis ssp. diacetylactis, as adjunct cultures to reduced fat Edam cheeses increased proteolysis, while the addition of L. helveticus and L. reuteri increased texture quality of cheeses.

Amino Acids↗
PubMed2003

A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening.

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period. CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.

Bacterial Proteins↗
PubMedDate not supplied

[Effect of products of alphaS1-casein proteolysis on the activity of angiotensin-converting enzyme].

The influence of alpha s1-casein proteolysis products of cow milk on the activity of angiotensin-converting enzyme (ACE) has been investigated. The peptide fraction has been obtained after incubation of alpha s1-casein with different strains Lactococcus lactis ssp. lactis and pepsin. The peptides with low molecular weight has been obtained with the help of a gel filtration. It is shown, that such peptides received with the help of some strains of Lactococcus lactis ssp. lactis and pepsin are capable to inhibit the activity of ACE. A conclusion about possibility of appearing antihypertensive peptides as a result of proteolitic processes in milk products has been made.

Caseins↗
PubMed2002

Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture.

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10(5) to 10(6) CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10(4) CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.

Cheese↗
PubMed2006

Total bacterial and species-specific 16S rDNA micro-array quantification of complex samples.

AIMS: We describe a novel DNA-micro-array-based method that targets 16S rDNA to quantify changes in both the total bacterial DNA and the species-specific DNA composition. METHODS AND RESULTS: Quantifications were achieved by combining competitive PCR for quantifying total bacterial DNA with quantification of species-specific DNA composition based on signature 16S rDNA sequences. We constructed 11 different probes, which were evaluated on 21 different strains, in addition to complex samples. The signals obtained with sequence-specific labelling of the probes corresponded well with what should be expected based on 16S rDNA phylogenetic reconstruction. The quantification of species-specific DNA composition showed that the micro-array approach could be used to accurately determine differential growth of bacteria in mixed samples. We analysed samples containing mixtures of Lactococcus lactis and different species of propionibacteria during a 2-week incubation period. Lactococcus lactis grew fast, reaching a maximum after 12 h, Propionibacterium acidipropionici and Propionibacterium freudenreichii reached a maximum after 48 h, whereas Propionibacterium jensenii showed a slow increase during the whole growth period. The 16S rDNA total bacterial DNA quantification was compared with real-time PCR, absorbance measurements (ABS600) and colony forming units (CFU). CONCLUSION: The accuracy of the array approach was in the same range or better than the alternative techniques. The potential of the 16S rDNA micro-array method was further demonstrated using a liquid cheese model. SIGNIFICANCE AND IMPACT OF THE STUDY: This is to our knowledge the first time quantification of the total bacterial DNA and the species-specific DNA compositions of mixed populations have been achieved in the same assay.

Cheese↗
PubMed1992

Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii P126.

The genus Propionibacterium includes cutaneous species typically found on human skin and the dairy or classical species (Propionibacterium freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici) used industrially for the production of Swiss cheese and propionic acid. Grinstead (1989, M.S. thesis, Iowa State University, Ames) has previously observed that some dairy propionibacteria inhibit other species in the classical grouping. We further investigated the inhibitor(s) produced by P. jensenii P126 (ATCC 4872). An antagonist(s) from anaerobic agar cultures of P126 strongly inhibited two closely related strains of propionibacteria, P. acidipropionici P5 and P. jensenii P54, and Lactobacillus bulgaricus NCDO 1489, Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lactococcus cremoris NCDO 799, and Lactococcus lactis subsp. lactis C2. The inhibitor, designated jenseniin G, was active at pH 7.0; inactivated by treatment with pronase E, proteinase K, and type 14 protease; insensitive to catalase; and stable to freezing, cold storage (4 degrees C, 3 days), and heat (100 degrees C, 15 min). Classification of the inhibitor as a bacteriocin is supported by its proteinaceous nature and its bactericidal activity against L. delbrueckii subsp. lactis ATCC 4797. The lack of detectable plasmids suggests a chromosomal location for the determinant(s) of jenseniin G.

Bacteriocins↗
PubMed2005

A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles.

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein) that binds the intron RNA to promote RNA splicing and intron mobility. Here, we used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to cellular poles in Escherichia coli and Lactococcus lactis. This polar localization occurs with or without coexpression of Ll.LtrB intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three nonoverlapping LtrA subsegments, possibly reflecting dependence on common redundant signals and/or protein physical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. The polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in the origin and terminus regions of the E. coli chromosome, may facilitate access to exposed DNA in these regions, and could potentially link group II intron mobility to the host DNA replication and/or cell division machinery.

Bacterial Proteins↗
PubMed2003

Survival of lactococci during passage through mouse digestive tract.

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.

Animals↗
PubMed1995

Novel insertion sequence-like element IS982 in lactococci.

A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp. cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster. The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats. IS982 contained an open reading frame encoding a putative transposase of 296 amino acids. An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp. cremoris Wg2 with 17-bp perfect inverted repeats. Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20. IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Amino Acid Sequence↗
PubMed1999

Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme.

DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolactic enzymes were distinct at the phylogenetic level, except for malic enzymes of yeast and Escherichia coli which were nearer the malolactic enzymes than the other malic enzymes. The analysis of conserved sites showed several interesting amino acids specific to either malic enzyme or malolactic enzyme.

Amino Acid Sequence↗
PubMed2006

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva.

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.

Bacterial Proteins↗