Lactis Biovar Research Articles

Significance of low molecular weight nitrogen constituents in cheese milk and of proteinase-negative variants of a Lactococcus lactis subsp. cremoris strain on the production and maturation of Danbo cheese

Abstract Danbo cheeses were produced according to a factorial design in order to elucidate the role of two factors on the production and maturation of the cheese. Frozen, defined-strain LD-cultures were used as starters. The LD-cultures were composed of L-type strain(s): Leuconostoc mesenteroides subsp. cremoris or Leuconostoc lactis; D-type strain(s): Lactococcus lactis subsp. lactis biovar. diacetylactis; and O-type strain(s): Lactococcus lactis subsp. cremoris and/or lactis. Three proportions (0, 40 and 80%) of proteinase-negative (prt−) variants of the O-component were incorporated as the first factor and two levels of low molecular weight nitrogen constituents (LMWN) in cheese milk (−/+ addition of 0.15%, w v ) as the second factor. Physiological characteristics of the starter were studied. The specific proteinase activity was reduced about 50% by the addition of LMWN. Bitterness, which played a key role as discriminator for the experimental cheeses, was reduced significantly by increasing the proportion of prt− cells. A linear correlation was established between bitterness scores and early eluting peaks from Sephadex G25 of the water soluble nitrogen fraction (WSNF) of eight selected experimental cheeses. Finally, reverse phase FPLC analysis of these early-eluting peaks from gel filtration showed that the release of hydrophobic peptides was also reduced by addition of LMWN.

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 μg/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 μg/ml and 4.5 ± 1.5 μg/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 109 cfu/ml and decreased to 1.5 (± 0.2) × 109 cfu/ml and 0.5 (± 0.3) × 109 cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.

Proton-dependent kinetics of citrate uptake in growing cells of Lactococcus lactis subsp. lactis bv. diacetylactis

The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.

Proton-dependent kinetics of citrate uptake in growing cells ofLactococcus lactissubsp.lactisbv.diacetylactis

The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l−1 lactose and 2 g·l−1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.

Comparison of ?-acetolactate synthase and ?-acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp.

Cell-free extracts of Leuconostoc and Lactococcus species were tested for their α-acetolactate synthase and α-acetolactate decarboxylase activities. In Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. mesenteroides and Leuconostoc lactis, the Km of α-acetolactate synthase for pyruvate was close to 10 mM whereas it was 30 mM in Lactococcus lactis subsp. lactis biovar. diacetylactis. The Km of α-acetolactate decarboxylase for α-acetolactic acid was very low (0.3 mM) in Leuconostoc species in comparison to Lactococcus lactis subsp. lactis biovar. diacetylactis (60 mM). In the latter bacterium, α-acetolactate decarboxylase showed a sigmoidal dependance upon α-acetolactic acid and was activated by the three branchedchain amino acids: leucine, isoleucine and valine.

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, Xg (growth + acidification) and Xng (acidification), and one inactive, Xi. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fermentations, and the use of this type of model for determining and optimizing fermentation parameters is discussed.

The physiology of Lactococcus lactis subsp. lactis biovar. diacetylactis immobilized in hollow-fibre bioreactors: glucose, lactose and citrate metabolism at high cell densities

Lactococcus lactis subsp. lactis biovar. diacetylactis was selected to study the physiological influences of immobilization and growth to high cell densities. Cells were cultivated on glucose or lactose medium in the presence and absence of citrate. With excess glucose the cells produced mainly lactate as the fermentation product (homofermentative) providing that not all of the substrate was consumed. The population so cultivated was exposed to extreme gradients of pH and lactate concentrations. When the glucose concentration was reduced the population showed a mixed product profile with half of the glucose being fermented to lactate, the remainder to formate, acetate, ethanol and 2,3-butanediol. Inclusion of citrate in the medium shifted the population to homofermentation, with respect to the amount of glucose or lactose consumed. The citrate was metabolized via the pyruvate-formate lyase and α-acetolactate synthase routes. The pH of the medium was shown to strongly influence the product profile from citrate, presumably by affecting the activity of the key enzymes of pyruvate metabolism. The lactococci immobilized at high cell densities show product profiles typical of carbohydrate limitation at low dilution rates.

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, φD1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a con-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.

The stability of the lactose and citrate plasmids in Lactococcus lactis subsp. lactis biovar. diacetylactis

The ability of Lactococcus lactis subsp. lactis biovar. diacetylactis strain C17 to ferment lactose and citrate are stable functions when cells are continuously cultivated under carbon limitation with lactose and citrate present in the medium. Switching the carbohydrate source to glucose rapidly resulted in a population which could no longer ferment lactose. The presence of citrate had no effect on the rapidity of the loss of ability to utilize lactose. The ability of strain C17 to utilize citrate was unaffected by cultivation for more than two weeks on citrate-free medium, containing either lactose or glucose as fermentable carbohydrate. These results confirm the lack of stability of the lactose plasmid and demonstrate for the first time that the citrate plasmid in L. lactis subsp. lactis biovar. diacetylactis is remarkably stable.

The stability of the lactose and citrate plasmids inLactococcus lactissubsp.lactisbiovar.diacetylactis

The ability of Lactococcus lactis subsp. lactis biovar. diacetylactis strain C17 to ferment lactose and citrate are stable functions when cells are continuously cultivated under carbon limitation with lactose and citrate present in the medium. Switching the carbohydrate source to glucose rapidly resulted in a population which could no longer ferment lactose. The presence of citrate had no effect on the rapidity of the loss of ability to utilize lactose. The ability of strain C17 to utilize citrate was unaffected by cultivation for more than two weeks on citrate-free medium, containing either lactose or glucose as fermentable carbohydrate. These results confirm the lack of stability of the lactose plasmid and demonstrate for the first time that the citrate plasmid in L. lactis subsp. lactis biovar. diacetylactis is remarkably stable.

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant ofLactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.

Lactococcus Genus: A selective and Differential Agar Medium

ABSTRACTAn agar medium (Alsan), was selective for Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar. diacetylactis. The former developed white or yellow colonies; the latter gave blue‐green or blue colonies. Eleven strains of L. lactis subsp. cremoris did not grow in this medium. The medium contained phenylethanol agar, lactose, glycine anhydride, lithium chloride and trimethoprim. In comparison with M17 medium, Alsan was not significantly different (P > 0.05) in recovering either bacterium. Furthermore, the medium inhibited the common food genera Pseudomonas, Escherichia, Leuconostoc, Enterococcus and Lactobacillus. Bacteriophage plaque sizes of 0.4–0.5 cm were demonstrated on the medium using a lactococcal bacteriophage.

Isolation of chromosomal mutations ofLactococcus lactissubsp.lactisbiovar.diacetylactis18-16 after introduction of Tn919

The conjugative transposon Tn 919 was introduced at high frequency to L. lactis subsp. lactis biovar. diacetylactis 18-16 and transconjugants were screened for mutations in two chromosomally located genotypes; citrate metabolism and maltose utilization. A citrate negative mutant, lacking citritase activity, was isolated at a frequency of 1.18 × 10−4. The mutant, 18-16C5, contained a single copy of Tn 919 in a chromosomal location. A junction fragment of Tn 919 ::18-16C5 chromosomal DNA was cloned in Escherichia coli . Mutations in maltose metabolism were detected at a frequency of 4.0 × 10−4. No mutants were detected when Tn 919 was not introduced. Reversion to a Mal+ phenotype occurred at high frequency, but was not due to Tn 919 transposition.

Effect of antibiotic resistance on the flavour profile of lactic acid bacteria

The major problem encountered in the dairy industry is due to the failure of starter in the manufacture of fermented dairy products such as cheese, Yogurt buttermilk and also the Indian fermented milk products srikhand and dahi, in terms of low acid production and absence of flavour and aroma. Usually, in such cases the presence of antibiotics is detected. Little work has been done on the effect of antibiotic resistance in the starter on flavour production, though many workers have investigated acid production and proteolytic activity (Kondratenko et al. 1978 Ottogalli et al. 1975; Shahani & Harper 1958; Wilkowske & Krienke, 1951). The present investigation has been undertaken to develop antibiotic-resistant strains of lactic streptococci, i.e. Streptococcus lactis biovar. diacetylactis and Lactobacillus delbrueckii subsp. bulgaricus which could be used for the preparation of fermanted milk products.

Influence of lactic cultures in denaturation of whey proteins during fermentation of milk

SummaryUndenatured whey protein (UWP) content of skim milk acidified with lactic acid or cultured with lactic cultures was estimated by a dye-binding method. The UWP content decreased with increase in acidity and the denaturation was only partly reversible on neutralization to the original acidity. The decrease in UWP was higher in cultured milk than in the milk acidified to the same extent with lactic acid, indicating the effect of lactic cultures in denaturation of whey proteins during fermentation of milk. Among the lactic cultures the denaturation effect of Lactobacillus delbrueckii subsp. bulgaricus was highest, followed by Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. lactis and Lact. lactis biovar diacetylactis. Denaturation of whey proteins by lactic cultures was found to be partly irreversible.