A Novel C-Terminal Truncated Bacteriocin Found by Comparison between Leuconostoc mesenteroides 406 and 213M0 Isolated from Mongolian Traditional Fermented Milk, Airag.

Bacteriocins produced by lactic acid bacteria are known to be useful tools for food biopreservation and fermentation control. Leuconostoc mesenteroides subsp. mesenteroides 406 and 213M0 isolated from different samples of Mongolian traditional fermented milk, airag, had been reported to produce listericidal bacteriocin-like inhibitory substances with similar but slightly different properties. In this study, the antibacterial properties and the related gene sequences of both strains were compared, and then their bacteriocins were purified and identified. Strain 406 was superior to strain 213M0 in cell growth and antibacterial activity against many strains. However, the activity of 213M0 was stronger than that of 406 against a few strains. DNA sequencing revealed two and three plasmids in 406 and 213M0, respectively, and each one of them harbored an almost identical mesentericin Y105-B105 gene cluster. Removal of these plasmids resulted in a complete loss of activity, indicating that the antibacterial activity of both strains was generated by bacteriocins encoded on the plasmids. Mesentericins Y105 and B105 were purified from both cultures, and another novel bacteriocin, named mesentericin M, was identified from the 213M0 culture only. Its structural gene was coded on a 213M0 plasmid and, surprisingly, its C-terminal three amino acid residues were post-translationally cleaved. To our knowledge, this is the first report of a C-terminal truncated bacteriocin. In conclusion, the novel bacteriocin should be mainly responsible for the difference in antibacterial properties between the two strains.

Production of a bacteriocin-like inhibitory substance by Leuconostoc mesenteroides subsp. dextranicum 213M0 isolated from Mongolian fermented mare milk, airag.

Strain 213M0 was selected with productivity of a bacteriocin-like inhibitory substance (BLIS) among 235 strains of lactic acid bacteria (LAB) isolated from Mongolian fermented milk 'airag'. Strain 213M0 was species-identified as Leuconostoc mesenteroides subsp. dextranicum by morphological observation, carbohydrate fermentation profiling and sequencing the 16S rRNA gene. Incubation temperature proper to produce the BLIS was 25°C rather than 30 and 37°C, and the production actively proceeded during the exponential growth phase of the producer cells. Antibacterial effect of BLIS 213M0 was limited to all nine strains of Listeria sp. bacteria and seven strains of LAB cocci among 53 tested strains, which corresponds to a typical feature of the class IIa pediocin-like bacteriocins. BLIS 213M0 was not inactivated in every broad pH range solution (pH 2.0-11.0), and was stable against storage at 25°C for 1 week and heating at 121°C for 15 min under pH 4.5. Peptide frame of BLIS 213M0 was confirmed by inactivation with some peptidases, and then its molecular weight was estimated to be 2.6-3.0 kDa using an in situ activity assay following sodium dodecyl sulfate polyacrylamide gel electrophoresis. The estimated size was different from the other Leuconostoc bacteriocins already reported. These results suggest that BLIS 213M0 would be a novel listericidal bacteriocin.

Draft Genome Sequence of Leuconostoc mesenteroides 213M0, Isolated from Traditional Fermented Mare Milk Airag in Bulgan Aimag, Mongolia.

Leuconostoc mesenteroides213M0 was isolated from traditional fermented mare milk airag in Bulgan Aimag, Mongolia. This strain produces a listericidal bacteriocin-like inhibitory substance. Here, we report the draft genome sequence of this organism.

Draft Genome Sequence of Leuconostoc mesenteroides 406 Isolated from the Traditional Fermented Mare Milk Airag in Tuv Aimag, Mongolia.

Leuconostoc mesenteroides406 was isolated from the traditional fermented mare milk airag in Tuv Aimag, Mongolia. This strain produces an antilisterial bacteriocin. Here, we report the draft genome sequence of this organism.

Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.

Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of ß-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of ß-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag.

Interaction between lactic acid bacteria and yeasts in airag, an alcoholic fermented milk.

The interaction between nine lactic acid bacteria (LAB) and five yeast strains isolated from airag of Inner Mongolia Autonomic Region, China was investigated. Three representative LAB and two yeasts showed symbioses were selected and incubated in 10% (w/v) reconstituted skim milk as single and mixed cultures to measure viable count, titratable acidity, ethanol and sugar content every 24 h for 1 week. LAB and yeasts showed high viable counts in the mixed cultures compared to the single cultures. Titratable acidity of the mixed cultures was obviously enhanced compared with that of the single cultures, except for the combinations of Lactobacillus reuteri 940B3 with Saccharomyces cerevisiae 4C and Lactobacillus helveticus 130B4 with Candida kefyr 2Y305. C. kefyr 2Y305 produced large amounts of ethanol (maximum 1.35 g/L), whereas non-lactose-fermenting S. cerevisiae 4C produced large amounts of ethanol only in the mixed cultures. Total glucose and galactose content increased while lactose content decreased in the single cultures of Leuconostoc mesenteroides 6B2081 and Lb. helveticus 130B4. However, both glucose and galactose were completely consumed and lactose was markedly reduced in the mixed cultures with yeasts. The result suggests that yeasts utilize glucose and galactose produced by LAB lactase to promote cell growth.

Production of bacteriocin by Leuconostoc mesenteroides 406 isolated from Mongolian fermented mare's milk, airag.

The purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides strain 406 that was isolated from traditional Mongolian fermented mare's milk, airag, were carried out. Leuconostoc mesenteroides strain 406 was identified on the basis of its morphological and biochemical characteristics and carbohydrate fermentation profile and by API 50 CH kit and 16S ribosomal DNA analyses. The neutral-pH cell-free supernatant of this bacterium inhibited the growth of several lactic acid bacteria and food spoilage and pathogenic organisms, including Listeria monocytogenes and Clostridium botulinum. The bacteriocin was heat-stable and not sensitive to acid and alkaline conditions, but was sensitive to several proteolytic enzymes such as pepsin, pronase E, proteinase K, trypsin, and α-chymotrypsin, but not catalase. Optimum bacteriocin production (4000 activity units/mL) was achieved when the strain was cultured at 25°C for 24-36 h in Man Rogosa Sharpe medium. The bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 1000), and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the bacteriocin had a molecular weight of approximately 3.3 kDa. To our knowledge, this is the first report of the isolation of a bacteriocin-producing Leuconostoc strain from airag. An application to fermented milks would be desired.

Characterization of bacteriocin produced by Streptococcus bovis J2 40-2 isolated from traditional fermented milk 'Dahi'.

A bacteriocin-producing strain Streptococcus bovis J2 40-2 was isolated from traditional fermented milk 'Dahi' in Bangladesh. Despite its narrow antimicrobial spectrum, it showed strong antimicrobial activity against extremely challenging and problematic organisms in foods, such as Listeria monocytogenes. Bacteriocin was sensitive to several proteolytic enzymes and showed antimicrobial activity over a wide pH range of 2.0-10.0. It was stable when heated to 110 degrees C for 20 min, but lost 25% of its activity when heated to 121 degrees C for 15 or 20 min. Optimum bacteriocin production (5600 AU/mL) was achieved when the strain was cultured at 37 degrees C for 24 h in MRS medium rather than in TYLG, GM17, or skim milk medium. Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 3500) and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that bacteriocin had a molecular weight of approximately 4.5 kDa.

Oral administration of lactic acid bacteria isolated from traditional South Asian fermented milk 'dahi' inhibits the development of atopic dermatitis in NC/Nga mice.

We investigated the suppressive effect of lactic acid bacteria (LAB) isolated from traditional South Asian fermented milk 'dahi' on the development of atopic dermatitis (AD) using NC/Nga AD model mice. In the initial evaluation, we confirmed the effect of LAB on serum total IgE using ovalbumin (OVA)-induced type 1 allergy model mice. Forty-one bacterial strains isolated from dahi were evaluated for their ability to induce interleukin (IL)-12 production and suppress IL-4 production in splenocytes obtained from OVA-sensitized mice. Of the 41 strains tested, Lactobacillus delbrueckii subsp. lactis R-037 exhibited the greatest IL-12 induction, suggesting that it is a potent Th1 inducer. Oral administration of heat-treated R-037 significantly suppressed the elevation of serum total IgE in OVA-induced type 1 allergy model mice. In NC/Nga AD model mice, oral administration of heat-treated R-037 reduced inflammatory auricular thickness and alleviated the AD clinical score although the effect on serum total IgE level was unclear. Histopathological findings showed a tendency toward improvement of inflammation. Hyperkeratosis in particular showed improvement in dermatitis skin lesions. These results suggest that oral administration of R-037 may alleviate AD.

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4.

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.

Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: construction of an Escherichia coli-Lactobacillus shuttle vector.

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.