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PubMed2003

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp cremoris P8-2-47.

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered (copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.

Amino Acid Sequence↗
PubMed2000

Growth of Lactococcus lactis strains at low water activity: correlation with the ability to accumulate glycine betaine.

Lactococcus lactis strains were divided into two groups based on their ability to grow in the presence of an upper limit of either 2% w/v NaCl (sensitive) or 4% w/v NaCl (tolerant). Growth inhibition of NaCl tolerant strains was substantially relieved by glycine betaine which was accumulated in significant amounts when growing at low water activities (a(w)). Very little accumulation of glycine betaine occurred during growth of the NaCl sensitive strains. The NaCl tolerant strains had substantial levels of glycine betaine transport activity in vitro, whereas the NaCl sensitive strains had little or no such activity. A low a(w) sensitive mutant of L. lactis subsp. cremoris MG1363 (NaCl tolerant) was isolated following ISS1 insertional mutagenesis. This mutant was inhibited at an a(w) of 0.988 produced by addition of 2% w/v NaCl or the equivalent glucose concentration (0.58 M). The mutant did not accumulate glycine betaine when growing at low a(w), and did not transport glycine betaine when assayed in vitro.

Betaine↗
PubMed2000

Changes in acid tolerance of Lactococcus lactis during growth at constant pH.

Cells of Lactococcus lactis MG1363 growing in batch culture in TYG (tryptone, yeast extract, glucose) medium at constant pH 7.0 became gradually more acid sensitive shortly after inoculation until a point of maximum sensitivity was reached in early log-phase. The acid tolerance then gradually increased in the mid- and late-log phase until maximum tolerance was reached at the onset of stationary phase. This pattern has been termed the growth-phase acid tolerance. The variation in acid tolerance seen in pH 7.0 grown cells of L. lactis MG1363 did not result from changes in internal pH or membrane H+ ATPase activity levels. Neither the amount of glucose present during mid-log phase nor the amount of lactate produced by the cells correlated with the pattern of the log-phase acid tolerance. Cells grown in partially spent TYG medium showed a reduced growth rate and increased acid tolerance compared to cells grown in fresh TYG medium. Supplementing the spent medium with tryptone or yeast extract or both restored the growth rate and cells became more sensitive to acid. Fractionation of tryptone yielded a fraction which stimulated the growth of MG1363 in partially spent medium and delayed the acquisition of acid tolerance. The active compound(s) has a putative molecular weight of about 1 kDa and was partially degraded by papain and trypsin.

Glucose↗
PubMed2000

Lactococcus lactis, a bacterial model for stress responses and survival.

The dairy organism, Lactococcus lactis, is continuously exposed to stress conditions generated during industrial processes. To identify the mechanisms that confer resistance to the lethal effects of oxygen and thermal stress, we isolated resistant strains by insertional mutagenesis. Mutated genes were identified and mutations were shown to confer resistance to multiple stresses (including non-selected stresses such as carbon starvation). Our results revealed that metabolic flux plays an important role in L. lactis stress response, and suggested that phosphate and guanine pools may be intracellular stress sensors. As previously shown, we also observed an increase of stress resistance during the stationary phase. We have evidence that stationary phase actually initiates very early during growth. Taken together, these data show that the stationary phase is a very complex system with multiple participants interacting altogether. These results reinforce the idea of the interdependence of stress response and the intimate relation between metabolic flux and stress responses in L. lactis.

Lactococcus lactis↗
PubMed2000

Metabolism of Lactococcus lactis subsp. cremoris MG 1363 in acid stress conditions.

The metabolism of glucose by Lactococcus lactis subsp. cremoris MG 1363 remains homolactic whatever the pH of the culture medium. The growth rate decreased with the acidification of the medium until a limit pH value of 4.0 for which no growth was observed. In contrast, the specific rate of glucose consumption decreased only for very low pH values, i.e., below 4.5. The efficiency of biomass synthesis relative to the energy supply decreased when the medium pH diminished, as illustrated by Y(ATP) values. This observation was related to the increase in both components of the proton-motive force when the pH decreased. The growth stopped when the internal pH reached a limit value of 5.4 due to organic acid accumulation.

Energy Metabolism↗
PubMed2000

Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress.

Lactococcus lactis subsp. cremoris NCDO763 accumulates glycine-betaine (betaine) when submitted to an osmotic stress with NaCl. Betaine transport activity increases with the extent of the osmotic upshock but also with growth temperature, and supplementation of the medium by Tween-80. Fatty acid analysis of the lipid fraction of L. lactis NCDO763 reveals significant modifications of the fatty acid composition of the membrane when cells are submitted to osmotic stress, high temperature or Tween-80 medium supplementation. The main modification in L. lactis membrane fatty acid composition in response to high osmolality is the increase of Cyclopropane Fatty Acid (CFA) deltaC19:0, whereas Unsaturated/Saturated ratio remains unchanged.

Betaine↗
PubMed2001

Physiological function of exopolysaccharides produced by Lactococcus lactis.

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.

Bacteriophages↗
PubMed2001

Pre-inoculation enrichment procedure enhances the performance of bacteriocinogenic Lactococcus lactis meat starter culture.

Sodium nitrite and sodium chloride may inhibit growth and bacteriocinogenesis of protective starter cultures. To reduce sensitivity of a lacticin 3147-producing starter culture to nitrite, prior to production of salami, Lactococcus lactis DPC 4275 was placed in a number of pre-inoculation treatments, containing (a) 1% glucose, (b) 2.5 ppm manganese (Mn), (c) 250 ppm magnesium (Mg), (d) 2.5 ppm manganese + 250 ppm magnesium (Mn + Mg), and held at ambient temperature for 30 min and 4 degrees C for 2 h. The growth, pH reduction, and bacteriocin production was monitored in beaker sausage over a period of 10 days at 28 degrees C, corresponding to typical salami production time, and compared to untreated starter culture. The effect of 1% tryptone and inoculum level on growth and bacteriocin production was also determined. Challenge tests were performed using Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 as target strains. All treatments gave a significantly higher (P < 0.05) initial starter level than the untreated starter. Beaker sausage inoculated with either low (10(7)) or high (10(9)) levels of starter culture, treated with Mn + Mg reached significantly (P < 0.05) higher levels by day 10 than other treatments. Trends indicate that Mn + Mg also gave best pH reduction in sausage containing the low-level starter culture, sausage and significantly lower (P < 0.05) values for sausage produced with higher inoculum. Bacteriocin production was also higher in starter culture treated with Mn, or glucose. Pre-treatment with Mg gave a 2-fold increase in bacteriocin, the addition of Mn augmenting this increase further. The incorporation of tryptone gave no additional effect. In beaker sausage, both L. innocua and S. aureus populations showed significant reductions (P < 0.05) in the presence of the bacteriocinogenic strain compared to a non-bacteriocinogenic control strain.

Antibiosis↗
PubMed2001

Growth characteristics of Candida kefyr and two strains of Lactococcus lactis subsp. lactis isolated from Zimbabwean naturally fermented milk.

Lactic acid bacteria (LAB) and yeasts constitute part of the microflora in Zimbabwean traditional fermented cows' milk, amasi. The present study was carried out to investigate the growth characteristics of Candida kefyr 23, Lactococcus lactis subsp. lactis biovar. diacetylactis C1 and L. lactis subsp. lactis Lc261, previously isolated from amasi, in ultrahigh temperature (UHT)-treated cows' milk. The strains were inoculated into the UHT milk as both single and yeast

Animals↗
PubMed2002

Utilizing luminometry for monitoring growth of Listeria monocytogenes in its liquid or gelified monocultures and cocultures with "acid-only" Lactococcus lactis.

The light output of a bioluminescent recombinant strain of Listeria monocytogenes increased parallel with its viable cell counts during the exponential phase of the growth both in aerobic monocultures and mixed cultures, but dropped significantly at the commencement of the stationary phase. Suppression of L. monocytogenes by a nisin-less strain of Lactococcus lactis occurred only as an early induction of the stationary state of the target organism. In low-salt cocultures, an inverse linear correlation was found between the logarithmic initial counts of lactic acid bacteria and the extent of growth of Listeria. Decrease of luminometric activity of the bioluminescent test organism indicate sensitively the transformation of the cells into metabolically less active stationary state as a stress-adaptive response to nutrient depletion, "metabolic crowding", or, inimical processes.

Antibiosis↗
PubMed2003

Fermented soymilk with a monoculture of Lactococcus lactis.

Lactococcus lactis strain (LL3) isolated from mothers' milk was used to produce fermented soymilk. The strain survived at levels of over 7 log cfu/ml for 3 weeks in the fermented soymilk. A consumer survey was carried out to compare the acceptability of the fermented product with a similar product made with L. lactis ATCC11545 originally isolated from cow's milk. Blind samples produced by fermentation with the two strains were rated equally attractive, whereas information on the origin of the strains significantly enhanced the pleasantness of the fermented soymilk.

Animals↗
PubMed2003

Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage.

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.

Amino Acid Sequence↗
PubMed2003

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer.

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.

Cheese↗
PubMed2003

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection.

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.

Adsorption↗
PubMed2003

Identification of proteins induced at low pH in Lactococcus lactis.

The Gram-positive bacterium Lactococcus lactis is of major importance to the dairy industry due to its conversion of lactose to lactic acid leading to the acidification of milk. To investigate which proteins are induced when L. lactis is exposed to conditions of low pH, we used two-dimensional gel electrophoresis to follow how protein expression changes with the degree of acidification. We found that reducing the pH of the growth medium with hydrochloric acid induced the synthesis of a small subset of proteins. The majority of these proteins were induced both after a minor (pH 5.5) and a major (pH 4.5) reduction in pH. Among the most strongly induced proteins, we identified the oxidative stress proteins superoxide dismutase and alkylhydroperoxidase as well as the autoinducer synthesis protein, LuxS. We also observed a differential induction of heat shock proteins by low pH as members of the CtsR regulon, ClpE and ClpP were induced at both pH 5.5 and 4.5, while HrcA-regulated chaperones, GroEL, GroES, DnaK and GrpE were induced only at pH 4.5. In addition, we identified two proteins repressed by low pH that proved to be the L. lactis HPr protein of the phosphoenolpyruvate sugar phosphotransferase system and the trigger factor known to participate in the folding of newly synthesized polypeptides.

Animals↗
PubMed1997

Autolysis of Lactococcus lactis ssp. lactis and Lactobacillus casei ssp. casei. Cell lysis induced by a crude bacteriocin.

Autolytic properties of Lactococcus lactis subsp. lactis IFPL359, its Lac Prt derivative Lc. lactis Tl and Lactobacillus casei subsp. casei IFPL731, used as starter and adjunct starter in goat's milk cheese making, have been studied. The lytic effect of a bacteriocin produced by a lactic acid bacterium isolated from raw goat's milk has also been analyzed. Lactococcal cells resuspended in phosphate buffer showed a peak of autolysis when they were harvested in the early growth phase. A more stable autolytic pattern through the exponential growth was obtained for Lb. casei IFPL731. Optimal autolysis was found in 0.1 M sodium phosphate buffer during incubation at 40 degrees C for Lb. casei IFPL731 and at 35 degrees C for the lactococci. Thermoinduction of cell lysis was not obtained in any of the cases under the conditions studied. Lytic effect of the crude bacteriocin assayed was strongest against Lc. lactis Tl. Lysis response to the bacteriocin seemed to be strain-dependent and related to growth conditions.

Bacteriocins↗
PubMed1998

Production of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp. lactis biovar. diacetylactis, isolated from Zimbabwean naturally fermented milk.

Malty compound-producing Lactococcus lactis subsp. lactis biovar. diacetylactis strain INF-DM1, originally isolated from naturally fermented milk in Zimbabwe was used to prepare fermented milk from ordinary milk, milk enriched with 2.5% (w/v) skimmed milk powder and by 2.5% (w/v) increase in dry matter by ultrafiltration. Inoculated milk was incubated at 22, 30 and 37 degrees C. Analyses were made after 0, 9, 18 and 24 h incubation and also after 24 h incubation followed by storage for one week at 4 degrees C. Samples were analysed for volatile compounds including malty compounds and for organic acids, pH and log cfu/g. All samples were also judged for sensory attributes. Products made from enriched milks showed increased viscosity which was most marked in ultrafiltrated milk incubated at 30 and 37 degrees C. The levels of certain compounds (lactic acid, citrate and diacetyl) were significantly affected by milk type. Incubation temperature had a significant effect on starter growth rate and the rate of production and amount of the malty compounds, lactate, diacetyl, ethanol, acetoin and acetaldehyde. 3-Methyl butanal concentrations were above the taste threshold level of 0.06 ppm in almost all products, including stored products. Although initial growth rate was fastest at 37 degrees C, an uncoupling of acid production and growth was observed after 9 h incubation, suggesting that this is above the optimum temperature for this strain. In addition, products incubated at 37 degrees C showed a tendency to whey separation, indicating that this temperature is also too high to give optimum product quality. All products attained good scores in sensory analysis provided that fermentation was complete. Variation in the levels of malty compounds during the fermentation had no significant effect on the sensory score for total flavour.

Acetaldehyde↗
PubMed1998

Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture.

Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments. A transconjugant of this strain, L. lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus. Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis. These products were compared with a salami manufactured with a conventional starter (Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a(w)-value, weight loss, colour development and sensory characteristics. Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a(w)-values below 0.90 which is favourable for preservation and hygienic stability. In addition, these salamis had good sensory and colorimetric qualities. A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L. lactis DPC4275.

Animals↗