Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Complete genome sequence of Leuconostoc mesenteroides subsp. mesenteroides strain J18, isolated from kimchi

Leuconostoc mesenteroides subsp. mesenteroides is one of the most predominant lactic acid bacterial groups during kimchi fermentation. Here, we report the complete genome sequence of L. mesenteroides subsp. mesenteroides J18, which was isolated from kimchi. The genome of the strain consists of a 1,896,561-bp chromosome and five plasmids.     

Specific Detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA Primers Identified by Randomly Amplified Polymorphic DNA Analysis

Randomly amplified polymorphic DNA analysis using primer 239 (5′ CTGAAGCGGA 3′) was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.     

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.     

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.     

A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

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.     

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Leuconostoc mesenteroides SJRP55: a potential probiotic strain isolated from Brazilian water buffalo mozzarella cheese

The probiotic potential of Leuconostoc mesenteroides subsp. mesenteroides SJRP55, isolated from water buffalo mozzarella cheese was evaluated. The microorganism presented resistance to stressful conditions that simulated the gastrointestinal tract, and to the best of our knowledge, Leuconostoc mesenteroides subsp. mesenteroides SJRP55 was the first of this species with the ability to deconjugate bile salts. Tolerance to NaCl was temperature dependent, as well the results obtained by aggregation capacity. The strain presented good adhesion properties, β–galactosidase activity, viability in fermented milk during storage, inactive against Streptococcus thermophilus and sensitive to most of the tested antibiotics. Some analgesic medications inhibited the growth of the strain. Leuconostoc mesenteroides subsp. mesenteroides SJRP55 exhibited in vitro probiotic potential, and it can be better characterized through future in vivo tests. This bacterium presents higher functional properties compared to other studied strains, and therefore, it is a potential candidate for the application as a probiotic strain, which could be used by industries in the manufacture of functional milk-based products.     

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10 degrees C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.     

Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.     

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins

A.M. REVOL-JUNELLES, R. MATHIS, F. KRIER, Y. FLEURY, A. DELFOUR AND G. LEFEBVRE. 1996. Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence o two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti- Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M_r of Mesenterocin 52A appeared identical to the previously described Mesentericing Y105. In contrast, Mesenerocin 52B possessed a M_r of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins:     

Characterization of pFR18, a small cryptic plasmid from Leuconostoc mesenteroides ssp. mesenteroides FR52, and its use as a food grade vector

A 1.8-kb cryptic plasmid pFR18 was isolated from Leuconostoc mesenteroides ssp. mesenteroides FR52 and characterized. The identification of single-stranded DNA intermediate (ssDNA) in Leuconostoc demonstrated that the replication of pFR18 is directed by a rolling-circle mechanism (RCR). Sequence analysis revealed a single open reading frame (rep18) encoding a putative 335-amino acid protein homologous to the pT181 replicase. Furthermore, a putative double strand origin similar to that of the pT181 plasmid family was identified. A cloning vector was developed on the basis of the pFR18 replicon by inserting an erythromycin resistance cassette within a non-essential region of the plasmid. The resulting construction was able to transform Lactobacillus sake and various species of Leuconostoc. It was stable in L. mesenteroides, however, the segregational stability of a pFR18 derivative containing large Escherichia coli DNA fragments was affected. Nevertheless, the new RCR plasmid pFR18 may be useful for the construction of food grade vectors.     

Molecular diversity of leuconostoc mesenteroides and leuconostoc citreum isolated from traditional french cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification

For a long time, the identification of the Leuconostoc species has been limited by a lack of accurate biochemical and physiological tests. Here, we use a combination of RAPD, 16S rDNA sequencing, and 16S rDNA fragment amplification with specific primers to classify different leuconostocs at the species and strain level. We analysed the molecular diversity of a collection of 221 strains mainly isolated from traditional French cheeses. The majority of the strains were classified as Leuconostoc mesenteroides (83.7%) or Leuconostoc citreum (14%) using molecular techniques. Despite their presence in French cheeses, the role of L. citreum in traditional technologies has not been determined, probably because of the lack of strain identification criteria. Only one strain of Leuconostoc lactis and Leuconostoc fallax were identified in this collection, and no Weissella paramesenteroides strain was found. However, dextran negative variants of L. mesenteroides, phenotypically misclassified as W. paramesenteroides, were present. The molecular techniques used did not allow us to separate strains of the three L. mesenteroides subspecies (mesenteroides, dextranicum and cremoris). In accordance with previously published results, our findings suggest that these subspecies may be classified as biovars. Correlation found between phenotypes dextranicum and mesenteroides of L. mesenteroides and cheese technology characteristics suggests that certain strains may be better adapted to particular technological environments.     

Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

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.     

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.