Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52

F. MATHIEU, I.S. SUWANDHI, N. REKHIF, J.B. MILLIERE AND G. LEFEBVRE. 1993. One hundred and sixty-five isolates of Leuconostoc spp. were tested for bacteriocin production. Only one strain, Leuc. mesenteroides ssp. mesenteroides FR 52, isolated from a raw milk, produced a bacteriocin which was named Mesenterocin 52. This bacteriocin inhibited other Leuconostoc strains and several strains of Enterococcus and Listeria spp. No activity was found against lactococci and lactobacilli. The antibacterial spectrum differed from that of previously described Leuconostoc bacteriocins. Mesenterocin 52 was secreted into the medium during the growth phase. It was inactivated with protease treatments. At pH 7.0 it had a relative stability after heating at 100C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80C. The apparent molecular mass was estimated to be less than 10 kDa by ultrafiltration. Mesenterocin 52 showed a bactericidal effect on Leuconostoc paramesenteroides DSM 20288.     

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.     

In vitro and in situ growth characteristics and behaviour of spoilage organisms associated with anaerobically stored cooked meat products

AIMS: Understanding spoilage caused by different types of spoilage organisms, associated with vacuum-packaged sliced cooked meat products (CMP). METHODS AND RESULTS: First, strains were characterized in a broth at 7 degrees C under anaerobic conditions to compare their growth rate, acidifying character and metabolite production under conditions simulating refrigerated vacuum-packaged conditions. Brochotrix thermosphacta grew faster than the lactic acid bacteria (LAB). Within the group of the LAB, all strains grew fast except Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc carnosum. Secondly, the organisms were inoculated on a model cooked ham to better understand the relationship between spoilage, microbial growth, pH, metabolite production and accompanying sensory changes. Most rapidly growing strains were Leuc. mesenteroides subsp. mesenteroides followed by B. thermosphacta, while Leuc. mesenteroides subsp. dextranicum and Leuc. carnosum grew very slowly compared with the other LAB. Brochotrix thermosphacta caused sensory deviations at a lower cell number compared with the LAB. The related pH changes, metabolite production and sensory perception are presented. CONCLUSIONS: In this pure culture study, B. thermosphacta and Leuc. mesenteroides subsp. mesenteroides had the highest potential to cause rapid spoilage on CMP. SIGNIFICANCE AND IMPACT OF THE STUDY: A systematic study on the behaviour of spoilage organisms on a model cooked ham to establish the relationship between microbial growth, pH, metabolite formation and organoleptic deviations.     

Random amplified polymorphic DNA analysis for differentiation of Leuconostoc mesenteroides subspecies isolated from Tenerife cheese

AIMS: In the present study, a RAPD-PCR fingerprinting method was developed to assign Tenerife cheese Leuconostoc mesenteroides strains to its three subspecies (mesenteroides, cremoris and dextranicum). METHODS AND RESULTS: Arbitrarily primed-PCR gave different DNA banding patterns for each type strain of Leuc. mesenteroides subspecies consisting in three major and intense bands of: 1800, 1600 and 1150 bp for subspecies mesenteroides 1800, 1150 and approximately 350 bp for subspecies cremoris; and 1800, 1600 and 1500 bp for subspecies dextranicum. DNA fingerprints of Tenerife cheese Leuc. mesenteroides subspecies were coincident to that of their respective type strain. RAPD profiles were reproducible with DNA template obtained by two different extraction methods. CONCLUSIONS: Tenerife cheese Leuc. mesenteroides strains were rapidly and unequivocally assigned to one of the subspecies by comparing their RAPD-PCR fingerprints with those displayed by type strains used as standards. This technique can be applied to complement preliminary identification of Leuc. mesenteroides to the species level by other molecular methods such as protein fingerprinting. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD-PCR allows reliable, reproducible and rapid molecular differentiation of Tenerife cheese Leuc. mesenteroides subspecies with no need to use time-consuming and often ambiguous biochemical tests.     

Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PRÉVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIÈS. 1996. The conversion from citrate positive (Cit⁺) to citrate negative (Cit^-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit⁺ phenotype was observed. The Cit^- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit^- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit⁺ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit^- mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.     

Peptide utilization by Lactococcus lactis and Leuconostoc mesenteroides

To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin. Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases. Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc. mesenteroides assimilated only shorter peptides containing up to seven amino acids. Growth was limited by the transport of peptides and not by their hydrolysis. The nutritional value of peptides varied with the strains and the composition of the peptides, L. lactis being advantaged over Leuc. mesenteroides.     

Oxygen and pyruvate as external electron acceptors for Leuconostoc spp.

L. NURAIDA, I. GRIGOLAVA, J.D. OWENS AND G. CAMPBELL-PLATT. 1992. Leuconostoc mesenteroides NCDO 518, Leuc, mesenteroides NCIB 8710, Leur. mesenteroides NCIB 8023, Leuconostoc sp. Pz 45, Leuconostoc sp. Pz 34 and Leuconostoc sp. Pz 10 were grown in a chemically defined medium with glucose and different external electron acceptors. All strains, except Leuconostoc sp. Pz 10, formed acetate under aerobic conditions. Leuconostoc Pz 10 produced only small amounts of acetate under aerobic or anaerobic conditions. This strain was the only strain not possessing NADH oxidase. All strains produced acetate from a mixture of glucose plus pyruvate. None of the bacteria used glycerol. Oxygen and pyruvate as external electron acceptors increased the cell yield on glucose.     

Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE

The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.     

Media and process parameters affecting the growth, strain ratios and specific acidifying activities of a mixed lactic starter containing aroma-producing and probiotic strains

AIMS: The effects of medium-composition and fermentation parameters on the properties of mixed mesophilic starters were studied. The starter was composed of Lactococcus lactis ssp. lactis (L. lactis), Lactococcus lactis ssp. cremoris (L. cremoris), Lactobacillus rhamnosus (Lact. rhamnosus) and Leuconostoc mesenteroides ssp. cremoris (Leuc. cremoris). METHODS AND RESULTS: The media used were reconstituted skim milk (RSM), and whey-based media with either citrate or phosphate buffers. The fermentation parameters were incubation temperature (22 degrees C or 32 degrees C), no pH control, and pH control in pH zones of either pH 6.0-5.8 or pH 6.0-5.2. The starter properties were strain ratio, specific acidifying activity (SAA), total population, residual carbohydrates and organic acids produced. The growth of L. lactis was favoured under pH control in whey-based media. High concentrations of Lact. rhamnosus were favoured in whey-based media prepared at 32 degrees C. The highest contents of Leuc. cremoris were obtained in starters prepared in RSM at 22 degrees C without pH control. Starters prepared under pH control gave the highest populations and made it possible for significantly lower inoculation rates (IR) to be used to carry out subsequent milk fermentations. However, the SAA of starters prepared under pH control were lower than the SAA of starters grown without any pH control. CONCLUSIONS: None of the conditions enabled the strain ratio at inoculation to be maintained. The data show that it is possible to prepare a mesophilic starter that has a significant probiotic Lact. rhamnosus content; this starter could be used in the preparation of probiotic-containing cheeses or in Leuc. cremoris for aroma production in fermented milks. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on what should be expected with respect to strain ratios and IR if cheesemakers decide to shift their aroma-producing starter production method from the traditional 'milk-based without pH control' method to whey-based media used with pH-zone control strategies.     

Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

Identification and characterization of an oligopeptide transport system in Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1463

AIMS: To identify and characterize an oligopeptide transport system in Leuconostoc mesenteroides CNRZ 1473. METHODS AND RESULTS: The uptake of a model substrate was monitored by determining intracellular concentrations of the corresponding amino acids by means of reversed-phase HPLC analysis. The oligopeptide transport system is specific for peptides containing at least four amino acid residues and operative under physiological conditions of growth. It is expressed maximally in the presence of oligopeptides, enhanced in the presence of Mg2+ or Ca2+ ions, and driven by ATP or a related energy-rich phosphorylated intermediate. CONCLUSIONS: The study showed evidence for and characterized the oligopeptide transport system of Leuc. mesenteroides for the first time. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Leuc. mesenteroides in mixed-strain cultures for the dairy industry.     

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).     

Leuconostoc mesenteroides SJRP55: A Bacteriocinogenic Strain Isolated from Brazilian Water Buffalo Mozzarella Cheese

The production of bacteriocins by Leuconostoc mesenteroides represents an important opportunity for exploration of their potential use for industrial purpose. The antimicrobial compounds produced by L. mesenteroides subsp. mesenteroides SJRP55 strain were characterized and purified. Cell-free supernatant of Leuc. mesenteroides subsp. mesenteroides SJRP55 produced antibacterial compounds against Listeria spp. strains and not inhibiting against Lactobacillus spp. The antimicrobial substances were stable at high temperatures (100 °C for 2 h and 121 °C for 20 min) and low pH (pH 2–4) values, but sensitive to proteolytic enzymes and resistant to α-amylase, lipase and catalase enzymes. The optimal temperature for active peptides production was 25 °C. The antimicrobial compounds were purified by ammonium sulfate precipitation, affinity column and reverse-phase chromatography. Mass spectrometry and amino acids analyses showed that the bacteriocins were identical to mesentericin Y105 and B105. The producer strain’s DNA analysis revealed presence of open reading frames possibly coding for virulence factors, such as enterococcal surface protein (esp), collagen adhesion (ace) and intrinsic vancomycin resistance (vanA); however, biogenic amines encoding genes were not observed. Leuc. mesenteroides subsp. mesenteroides SJRP55 is a promising biopreservative culture in fermented milk, and the purified bacteriocins can also be applied in food preservation.     

Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity. Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified. Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor. The production and activity of protease in culture medium was strongly dependent on pH. When L. mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.     

Molecular and physiological characterization of dominant bacterial populations in traditional mozzarella cheese processing

The development of the dominant bacterial populations during traditional Mozzarella cheese production was investigated using physiological analyses and molecular techniques for strain typing and taxonomic identification. Analysis of RAPD fingerprints revealed that the dominant bacterial community was composed of 25 different biotypes, and the sequence analysis of 16S rDNA demonstrated that the isolated strains belonged to Leuconostoc mesenteroides subsp. mesenteroides , Leuc. lactis , Streptococcus thermophilus , Strep. bovis , Strep. uberis, Lactococcus lactis subsp. lactis , L. garviae, Carnobacterium divergens , C. piscicola, Aerococcus viridans , Staphylococcus carnosus, Staph. epidermidis , Enterococcus faecalis , Ent. sulphureus and Enterococcus spp. The bacterial populations were characterized for their physiological properties. Two strains, belonging to Strep. thermophilus and L. lactis subsp. lactis , were the most acidifying; the L. lactis subsp. lactis strain was also proteolytic and eight strains were positive to citrate fermentation. Moreover, the molecular techniques allowed the identification of potential pathogens in a non-ripened cheese produced from raw milk.     

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

AIMS: To identify and characterize amino acid transport in Leuconostoc mesenteroides. METHODS AND RESULTS: The transport of labelled amino acids was measured in whole cells of Leuc. mesenteroides CNRZ 1273. Systems were operative under physiological conditions of growth, energy dependent and differed from peptide transport. Some of the systems were shared by several amino acids. Kinetic analysis indicated the presence of three transport systems with very high (VH), high (H) and low affinity (H) for the 11 amino acids studied. The K(t) values (micromol l(-1)) ranged from 0.088 to 0.815 (VH), 6-390 (H) and 320-4500 (L) and the V(max) values [nmol s(-1) (g dry weight)(-1)] from 0.015 to 0.8 (VH), 15-95 (H) and 90-470 (L). CONCLUSIONS: The study showed the presence of three transport systems in Leuc. mesenteroides for all amino acids tested, some of them being shared by several amino acids. SIGNIFICANCE AND IMPACT OF THE STUDY. The findings are discussed with reference to the growth of Leuc. mesenteroides in milk as pure or in mixed-strain culture with Lactococcus lactis.     

Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham

In an attempt to determine the composition and origin of the spoilage flora of refrigerated vacuum-packed cooked ham, the changes in microbial numbers and types were followed along the processing line. Results revealed Lactobacillus sake and Leuconostoc mesenteroides ssp. mesenteroides as the major causative agents of spoilage of sliced ham stored at 4 °C and 12 °C, due to recontamination in the cutting room. On the contrary, the progressive deterioration of whole ham under the same storage conditions was associated with a non-identifiable group of leuconostoc-like bacteria. Except for lactic acid bacteria, no other organism grew in vacuum packs of either sliced or whole ham. Although atypical leuconostocs could not be detected among isolates recovered from freshly produced whole ham, they appeared to survive cooking and proliferate during storage. Neither these organisms however, nor Lact. sake and Leuc. mesenteroides were important in curing and tumbling as carnobacteria, mainly Carnobacterium divergens, and Brochothrix thermosphacta dominated at this stage. A progressive inversion of the ham microflora from mostly Gram-negative at the beginning of processing to highly Gram-positive prior to cooking was noted. Listeria monocytogenes cross-contaminated ham during tumbling. However, the pathogen was always absent from the vacuum-packed product provided that heating to a core temperature of 70 °C occurred and recontamination during slicing and packing was prevented. The percentage distribution of different species of lactic acid bacteria as well as the uncommon phenotypic characteristics of some strains were discussed.     

Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays

The ability of selected lactic acid bacteria to carry out malolactic fermentation depends on the level of numerous wine characteristics. A Hadamard's experimental matrix was used to determine the main effects of 11 physico-chemical factors on malolactic activity of three Leuconostoc œnos strains and one Lactobacillus plantarum strain. Ethanol had the greatest inhibitory effect on the achievement of malolactic fermentation for all Leuc. œnos strains. An inhibitory effect of the L-malic acid was also found in the operating conditions. These strains show different degrees of sensitivity to pH. One of these strains was inhibited by SO₂. Malolactic activity of the Lact. plantarum strain is mainly affected by a low pH, and this strain is often less efficient than Leuc. œnos strains. This methodology could be used for the selection of strains for malolactic starters. Further work is in progress using factorial design in order to determine the interactions between influential factors.     

The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas

AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated. METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents. Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV. The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7. CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc. mesenteroides physiology. The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc. mesenteroides in the fermentation process.