The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis. Received: 8 February 1996 / Accepted: 11 March 1996     

UV-inducible proteins and UV-induced cross-protection against acid,ethanol, H2O2 or heat treatments in Lactococcus lactis subsp. lactis

The relationship between UV-irradiation-induced tolerance to different environmental stresses and change in protein synthesis was examined in Lactococcus lactis subsp lactis IL1403. The results showed that preirradiation of cultures of L. lactis subsp. lactis with UV_254nm light led to increased tolerance of usually lethal challenges to acid (pH 4.0), ethanol (20%, v/v), H₂O₂ (15 mM), or heat (52° C). This suggests that there is an overlapping regulation between the UV-induced pathway and the other stress responses. Whole-cell protein extracts from UV-treated (100 J/m²) and untreated cultures were compared using two-dimensional polyacrylamide gel electrophoresis. At least 14 polypeptides were induced in response to damage after UV irradiation, which indicated an SOS-like response in this species. The RecA protein, however, seemed not to be significantly induced in Lactococcus lactis subsp. lactis IL1403. Some of the UV-induced polypeptides overlaped with stress proteins induced by the other treatments.     

Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis

Abstract

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25?°C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80?°C for 1?h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50?mM NaCl in 50?mM sodium acetate buffer (pH 4.0) as a wash buffer and 400?mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.     

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pH_e) on the intracellular pH (pH_i) of wild-type and citrate negative variants (cit⁻) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pH_i of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pH_e was decreased from 7 to 4, the pH_i decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pH_i of wild-type and cit(−) variant of L. lactis LD, nor on the pH_i of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pH_i, which was maintained at about 6.5–7.0 when the pH_e was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides. Received: 7 March 1997 / Accepted: 14 April 1997     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L

The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C₈ column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0–10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at ?20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0–30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.     

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at ?20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5–6.5 and 5.0–5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.     

Heat shock-induced protein synthesis inLactococcus lactis subsp.lactis

Heat shock inLactococcus lactis subsp.lactis may induce as many as 16 proteins after a temperature shift from 30° to 40°C. Five induced proteins were found to be immunologically related to theEscherichia coli GroEL, DnaK, DnaJ, and GrpE proteins, and to theBacillus subtilis ⁴³ factor. From these initial studies we conclude that, inL. lactis subsp.lactis, a heat shock response similar to that known to occur in other prokaryotes might exist.     

Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate

β‐(2,1)‐linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non‐digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related β‐(2,6)‐linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the preferred substrate for growth and biomass formation, FS exhibited a comparable cell growth (8.4 × 10⁷ counts/mL and 1.0 × 10⁷ counts/mL, respectively) and acidification power (84 °T and 74 °T, respectively) during 48 h of fermentation, as well as an increase in lactic acid and decrease in acetic acid formation. Bifidobacterium lactis Bb 12 did not utilize inulin as a sole carbon source as judged from the 60 % decrease in cell count and the insignificant (0.1 pH unit) acidification of the growth medium, whereas levan provided a noticeable increase in cell count and acidification (0.4 pH units) during 48 h of fermentation. FOS preparation appeared to be a satisfactory carbon source for this strain, but lower acidification power (56 °T) and cell counts were observed as compared to glucose‐ or FOS‐containing media (2.6 % and 22 %, respectively). The products obtained under conditions of mild lactic acid hydrolysis of levan (37 °C, pH 3.3, 24 h) enhanced the cell count (7–10 %) and acidification power (by a factor of 2.7) of Bifidobacterium lactis Bb 12.     

Purification,crystallization, and preliminary X-ray analysis of PepX,an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 Å, b = 102.6 Å, and c = 101.6 Å, space group P2₁2₁2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 Å and are suitable for high-resolution structural analysis. © 1995 Wiley-Liss, Inc.     

Isolation,Purification, and Characterization of Acetolactate Synthase from a Lactococcus lactis Culture

Acetolactate synthase catalyzing the synthesis of -acetolactate was isolated from lactic acid bacteria Lactococcus lactissubsp. lactisbiovar. diacetylactis4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the K _Mfor pyruvate was 70 mM. The curve relating the dependence of enzyme activity to pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had pH optimums of 5.8 and 6.5–7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38–40°C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.     

Nucleotide sequence and thermostability of pND324, a 3.6-kb plasmid from Lactococcus lactis

A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal θ-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 °C and 37 °C, whereas derivatives that lack cisE were highly unstable at 37 °C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal θ-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324, an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 °C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 °C. Received: 30 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999