Lactobacillus Helveticus Strains Research Articles

Synthetic peptides as substrate for assaying the proteolytic activity of Lactobacillus helveticus.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.

Modulation of the cell-surface proteinase activity of thermophilic lactobacilli by the Peptide supply.

The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation of flavor and texture in fermented products. We investigated the influence of peptide content on the cell surface proteinase and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains. The proteinase activities were remarkably reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared with those found in a synthetic medium. The degree of inhibition observed was strain dependent. When proteinase activities were analyzed by their hydrolytic patterns of alpha- and beta-casein degradation, four types of P(III)-caseinolytic cleavage specificity were distinguished. Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L. delbrueckii subsp. lactis strains. However, the aminopeptidase activities were not influenced by the peptide content of the medium.

Recombinant Lactococcus starters as a potential source of additional peptidolytic activity in cheese ripening.

The aim of this study was to modulate the lactococcal proteolytic system for enhancement of the cheese ripening process. The genes encoding PepN, PepC, PepX and PepI peptidases of a highly proteolytic Lactobacillus helveticus strain were transferred into Lactococcus lactis in a food-grade cloning system. A comparison of the relative peptidase activities from the transformants with those from the untransformed host, determined in the conditions of maturing cheese, showed that an increase in peptidase activity could be achieved by introducing a selected peptidase gene from Lact. helveticus into L. lactis. Recombinant L. lactis starter strains, carrying a peptidase gene from Lact. helveticus, may have an important contribution to the proteolysis of maturing cheese by producing an additional peptidolytic enzyme activity. The results will be of importance in shortening the ripening period and production of special cheeses (e.g. reduced-fat cheeses) with improved characteristics.

Interaction between propionibacteria and starter / non-starter lactic acid bacteria in Swiss-type cheeses

Thermophilic lactic acid bacteria, propionibacteria (PAB) and facultatively heterofermentative lactobacilli (FHL) form the main flora of Swiss-type cheeses. The aim of this work was to investigate their interactions and impact on product quality, and in particular on the de- fect of late fermentation. For this purpose Emmental model cheeses were produced according to a twice-replicated 2 4 full factorial experimental design. The four factors were: (1) The type of cultures of PAB, one with a weak (Prop 96) and the other one with a strong aspartase activity (Prop 90). (2) The addition or not of a culture of FHL composed of 3 Lactobacillus caseistrains. (3) The addition or not of a culture of 4 Lactobacillus helveticusstrains. (4) The season: winter (hay feeding) or summer (grass feeding). FHL and PAB counts, organic acids, proteolysis, eye formation were followed during ripening. Prop 90 showed a higher growth and fermentation role resulting in a higher number of eyes and an increased risk of late fermentation compared to Prop 96. PAB growth was also favoured in cheeses manufactured with winter milk which had a slightly higher water content. The addition of L. helveticustended to increase the risk of late fermentation. On the other hand the addition of FHL in- hibited lactate fermentation, with a more marked effect on Prop 96 culture. In conclusion the defect of late fermentation can be prevented by using PAB with weak aspartase activity, by adding FHL and by omitting L. helveticus. Emmental cheese / propionibacteria / aspartase activity / Lactobacillus casei / Lactobacillus helveticus

Aspect qualitatif de l'activité protéolytique des lactobacilles thermophiles utilisés en fabrication de fromages à pâte pressée cuite

Qualitative aspect of proteolytic activity of thermophilic lactobacilli using in Swiss cheeses. The average peptidic pattern was established from 253 French Emmental cheeses sampled in 50 factories. It contained 53 peptidic peaks, 4 of which were the most important in quantity. Two Lactobacillus helveticus strains and five Lactobacillus delbrueckii ssp. lactis strains were tested in Emmental experimental cheesemaking in association with three different propionic strains (21 cheeses). The Propionibacterium freudenreichii strains did not affect the peptidic pattern of ripened Emmental cheese. On the other hand, 13 peptidic peaks were significantly influenced by the thermophilic lactobacilli strain. The two species of thermophilic lactobacilli were discriminated by three peptidic peaks. Seven peptidic peaks were correlated with the ammonia amount in ripened Emmental cheese. No significant correlation was found between peptidic peaks and SN/TN and NPN/TN. At the same time, the peptidic pattern produced by the seven lactobacilli strains were stud- ied in laboratory. The most specific peptidic peak of the French Emmental cheese were not found by using the Hammarsten casein as substrate. Another substrate, similar to hard cooked cheeses, lyophilized and sterilized by ionisation was tested. This substrate was obtained with microfiltered milk and Streptococcus thermophilusstrains only. This substrate digested by thermophilic lactobacilli allowed to find the specific peptidic peaks of the French Emmental cheese. For example, the 14th peak obtained in laboratory was significantly different according to lactobacilli species, as it was observed in the Emmental cheese experiments. This peak distinguished the strains inside the Lb. delbrueckii ssp. lactis too. peptidic pattern / proteolysis / thermophilic lactobacilli / Emmental

Expression of the phospho-beta-glycosidase ArbZ from Lactobacillus delbrueckii subsp. lactis in Lactobacillus helveticus: substrate induction and catabolite repression.

ArbZ from Lactobacillus delbrueckii subsp. lactis was previously shown to enable utilization of the beta-glucoside arbutin by Escherichia coli. The arbZ gene was cloned and expressed in the industrially used beta-glucoside-negative strain Lactobacillus helveticus 3036(62). The transformants were able to ferment not only arbutin, but also cellobiose, salicin and methyl-beta-glucoside (MbetaGlc). Cleavage of beta-glucosides by the transformants depended on the integrity of the cytoplasmic membrane, whereas in cell-free extracts only C(6)-phosphorylated substrates were hydrolysed. This suggested that ArbZ is a phospho-beta-glycosidase. ArbZ activity in transformants of Lb. helveticus was subject to substrate induction mediated by the beta-glucosides arbutin, salicin and MbetaGlc, whereas cellobiose or the beta-galactoside lactose had no inducing effect. Northern blot analysis proved that induction by MbetaGlc was due to enhanced transcription of arbZ. Catabolite repression of arbZ induction was observed with glucose, mannose, fructose and galactose. The induction kinetics observed in the presence of these sugars indicated that at least two different mechanisms are operative in catabolite repression of arbZ in Lb. helveticus.

Performance of Lactobacillus helveticus Spontaneous Phage-resistant Mutants in Hard Cheese Production

Abstract From the strain Lactobacillus helveticus ATCC 15807, a total of 66 clones were isolated using the lytic phages hv and ATCC 15807-B1. Phenotypic parameters related to their phage resistance capacity (efficiency of plaquing, phage resistance stability, lysogeny and adsorption rates) were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying power, proteolytic activity and slime production) characteristics of the isolates were also studied. Phage resistance stability was a variable property among the isolates but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of acquired lysogeny was demonstrated. The phage-resistant variants were completely or partially unable to bind phages and did not show differences with Lb. helveticus ATCC 15807 in their biochemical characteristics. However, the technological properties (acidifying powers and proteolytic activities) were heterogeneously distributed among the mutants. The variant RB1-28 (isolated under selective pressure of the phage ATCC 15807-B1) was evaluated for the technological performance in Sardo cheese production in comparison with Lb. helveticus ATCC 15807. There were no significant differences between the two types of cheeses, taking in consideration physicochemical parameters (proteolysis level, dry matter, total and soluble proteins, moisture), microbiological counts or sensory analysis. These phage-resistant variants could be employed in starter strain rotation programs for cheesemaking.

Genetic (RAPD‐PCR) and technological diversities among wild Lactobacillus helveticus strains

Diversity in 25 Lactobacillus helveticus strains isolated from natural whey cultures for Argentinian hard cheese production was studied by means of RAPD-PCR patterns and technological parameters (acidifying and proteolytic activities, salt tolerance, diacetyl, H2O2 and slime production, phage sensitivity). In the RAPD diversity study, 10 Lact. helveticus strains from the CNRZ collection were also included. The clustering of RAPD patterns from the Argentinian strains revealed the existence of two Lact. helveticus biotypes. Cluster 1 contained 22 strains (15 wild and seven CNRZ collection strains), Cluster 2 grouped 10 wild strains and Cluster 3 contained only three CNRZ collection strains. RAPD groups could be related to specific cheese-making characteristics (cheese plants). Analysis of technological characteristics in the Argentinian strains showed the occurrence of different natural variants. According to their capacity for growing in milk, they were classified as ‘fast’, ‘intermediate’ and ‘slow’ variants. Among the strains, low salt tolerance and widespread phage resistance were demonstrated. The genetic diversity (RAPD-PCR clustering) did not show any clear relationship with phenotypical diversity. Study of genetic and technological diversity allows a better characterization of wild strains belonging to Lact. helveticus.

Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics.

The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities. In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains. Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance. The possibility of choosing Lact. heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.

Incidence of Inducible Bacteriophage in Lactobacillus helveticus Strains Isolated from Natural Whey Starter Cultures

We studied the incidence of inducible Lactobacillus helveticus strains among 74 isolates from natural whey starter cultures that are used for the production of Italian Grana and Provolone cheeses. Mitomycin C was used for induction of cultures. Of the 77 strains tested, 35 strains (47%) released supernatant particles in the culture that were able to inhibit up to a maximum of 47 (61%) sensitive indicator strains. The presence of bacteriophages in culture supernatants of L. helveticus strains LH38 and LH60 was verified by electron microscopy. None of the positive culture supernatants formed plaques, and inhibition zones were observed only on agar plates that had been inoculated with the indicator strains. Fourteen of the 17 positive culture supernatants were able to propagate on some indicator strains, indicating the presence of completely infective virions in liquid medium. The particles were insensitive to proteolytic enzymes, were unable to pass through a microfiltration membrane, were sensitive to heat, and were sedimented by ultracentrifugation. Some of these particles showed killer activity against nonproliferating cells of indicator strains. The lysogenic state of the L. helveticus strains was related to a stronger resistance of these strains to the products of the induction; higher prevalence of indicator strains were found among the noninducible strains.

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

Mapping of two plasmids from Lactobacillus helveticus ILC 54, plasmid curing and preliminary studies on their involvement in lactose metabolism and peptidase activity

Two plasmids isolated from Lactobacillus helveticus strain ILC 54 were analysed by restriction digestion. A restriction map of the 14.3 and 5.6 kb plasmids is presented. Plasmid-curing studies suggest that these plasmids are involved in lactose metabolism and peptidase activity.

Molecular characterization, over-expression and purification of a novel dipeptidase from Lactobacillus helveticus.

A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.

Reduction of membrane-bound ATPase activity in a Lactobacillus helveticus strain with slower growth at low pH

One acid-sensitive variant of Lactobacillus helveticus, designated L. helveticus CPN4, was isolated from L. helveticus CP790 using neomycin resistance as a selective marker. In a milk medium, the variant strain showed similar growth to the parent strain until the pH reached 4.5, afterwards growth was slower than the parent strain. The variant strain had lower activity of membrane bound adenosine triphosphatase (ATPase, EC 3.6.1.3) over several batch growth ranges from pH 3.5 to 7.5. Membrane vesicles of the variant strain showed lower uptake of quinacrine than vesicles of the parent strain. The variant strain had lower uptake ability for tyrosine, leucine and alanine.

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

The X-prolyl dipeptidyl aminopeptidase gene (pepX) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 degrees C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

Structure of an exocellular polysaccharide of Lactobacillus helveticus TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2

Lactobacillus helveticus strain TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2, produced an exocellular polysaccharide from reconstituted skim milk. On the basis of the results of methylation analysis, enzymatic digestion, mild Smith degradation, mild acid hydrolysis, acetolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a d-galactofuranose containing hexasaccharide repeating unit with the following structure: ▪

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus

An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus.

An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.

Isolation of Galactose-Fermenting Thermophilic Cultures and Their Use in the Manufacture of Low Browning Mozzarella Cheese

The objectives of this study were to isolate galactose-fermenting, galactose-nonreleasing strains of Streptococcus and Lactobacillus and to use these strains as starter cultures in the manufacture of low browning Mozzarella cheese. Four Streptococcus isolates having the desirable phenotype, combined with a Lactobacillus helveticus strain, were acceptable as starter cultures based on activity tests. Fifteen vats of Mozzarella cheese were produced, in triplicate, representing four experimental treatments (made with galactose-nonreleasing strains) and one control (made with a galactose-fermenting, galactose-releasing Streptococcus). Analyses were performed on cheese after 5 and 28 d of refrigerated storage. The lactose and galactose content of cheese remained constant or decreased over the 28-d testing period for all experimental treatments. The galactose content in the control cheese increased over the testing period and was significantly higher by d 28 than in the test cheeses. Browning was greatest on d 28 and at the more severe baking conditions. Control cheese browned more than experimental cheeses did. Melt behavior and free oil formation were generally not affected by treatment. The results indicated that the galactose-fermenting, galactose-nonreleasing strains could be used to make low browning Mozzarella cheese.

Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase.

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -49.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepC- and ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol-aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.