Comparative Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) of Streptococcus thermophilus St-I and its Bacteriophage-Insensitive Mutants (BIM) Derivatives.

The CRISPR-Cas (CRISPR together with CRISPR-associated proteins) modules are the adaptive immune system, acting as an adaptive and heritable immune system in bacteria and archaea. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize, and clear infections. In this study, the homology of CRISPRs sequence in BIMs (bacteriophage-insensitive mutants) of Streptococcus thermophilus St-I were analyzed. Secondary structures of the repeats and the PAMs (protospacer-associated motif) of each CRISPR locus were also predicted. Results showed that CRISPR1 has 27 repeat-spacer units, 5 of them had duplicates; CRISPR2 has one repeat-spacer unit; CRISPR3 has 28 repeat-spacer units. Only BIM1 had a new spacer acquisition in CRISPR3, while BIM2 and BIM3 had no new spacers' insertion, thus indicating that while most CRISPR1 were more active than CRISPR3, new spacer acquisition occurred just in CRSPR3 in some situations. These findings will help establish the foundation for the study of CRSPR-Cas systems in lactic acid bacteria.

Effect of Lactobacillus Strains on Intestinal Microflora and Mucosa Immunity in Escherichia coli O157:H7-Induced Diarrhea in Mice.

This study investigated the effects of KLDS 1.8701 and AD1 administrations by gavage on intestinal microflora and mucosal immunity in diarrhea mice infected by Escherichia coli O157:H7 compared to normal mice. The levels of E. coli, Enterobacteria, and Enterococcus decreased significantly (P < 0.05), while viable counts of Lactobacilli and Bifidobacterium increased in diarrhea mice. Moreover, KLDS 1.8701 and AD1 improved secretion of secretory immunoglobulin A and enhanced the levels of interferon-γ and interleukin. Results indicate that KLDS 1.8701 and AD1 could effectively alleviate diarrhea in mice via modulation of intestinal microflora and improve the function of immune system. The study on the effect of KLDS1.8701 and AD1 supplementation in human flora-associated animal models was recommended.

Enterobacter xiangfangensis sp. nov., isolated from Chinese traditional sourdough, and reclassification of Enterobacter sacchari Zhu et al. 2013 as Kosakonia sacchari comb. nov.

A Gram-stain-negative bacterial strain, 10-17(T), was isolated from traditional sourdough in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, RNA polymerase ß subunit (rpoB) gene sequence analysis, DNA gyrase (gyrB) gene sequence analysis, initiation translation factor 2 (infB) gene sequence analysis, ATP synthase ß subunit (atpD) gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain 10-17(T) was phylogenetically related to Enterobacter hormaechei CIP 103441(T), Enterobacter cancerogenus LMG 2693(T), Enterobacter asburiae JCM 6051(T), Enterobacter mori LMG 25706(T), Enterobacter ludwigii EN-119(T) and Leclercia adecarboxylata LMG 2803(T), having 99.5%, 99.3%, 98.7%, 98.5%, 98.4% and 98.4% 16S rRNA gene sequence similarity, respectively. On the basis of polyphasic characterization data obtained in the present study, a novel species, Enterobacter xiangfangensis sp. nov., is proposed and the type strain is 10-17(T) ( = LMG 27195(T) = NCIMB 14836(T) = CCUG 62994(T)). Enterobacter sacchari Zhu et al. 2013 was reclassified as Kosakonia sacchari comb. nov. on the basis of 16S rRNA, rpoB, gyrB, infB and atpD gene sequence analysis and the type strain is strain SP1(T)( = CGMCC 1.12102(T) = LMG 26783(T)).

Proteomic analysis of the response to NaCl stress of Lactobacillus bulgaricus.

Lactobacillus bulgaricus is commonly used in dairy products as a starter culture. Its viability during freeze-drying is of commercial interest. Here a significant (p < 0.05) improvement in survival rate of L. bulgaricus ATCC 11842 was achieved during freeze-drying when it was prestressed with 2 % (w/v) NaCl for 2 h in the late growth phase. To understand the mechanism of this stress-related viability improvement in L. bulgaricus, protein synthesis was analyzed by 2D difference gel electrophoresis. Nine protein spots were significantly altered by NaCl and were subsequently identified by peptide mass fingerprinting. The functions of the proteins included stress-related protein synthesis, amino acid biosynthesis, nucleotide biosynthesis, sugar metabolism, transport systems, and vitamin biosynthesis. These findings provide a considerable background regarding the NaCl stress response of L. bulgaricus.

Lactobacillus heilongjiangensis sp. nov., isolated from Chinese pickle.

A Gram-stain-positive bacterial strain, S4-3(T), was isolated from traditional pickle in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain S4-3(T) showed 97.9-98.7 % 16S rRNA gene sequence similarities, 84.4-94.1 % pheS gene sequence similarities and 94.4-96.9 % rpoA gene sequence similarities to the type strains of Lactobacillus nantensis, Lactobacillus mindensis, Lactobacillus crustorum, Lactobacillus futsaii, Lactobacillus farciminis and Lactobacillus kimchiensis. dnaK gene sequence similarities between S4-3(T) and Lactobacillus nantensis LMG 23510(T), Lactobacillus mindensis LMG 21932(T), Lactobacillus crustorum LMG 23699(T), Lactobacillus futsaii JCM 17355(T) and Lactobacillus farciminis LMG 9200(T) were 95.4, 91.5, 90.4, 91.7 and 93.1 %, respectively. Based upon the data obtained in the present study, a novel species, Lactobacillus heilongjiangensis sp. nov., is proposed and the type strain is S4-3(T) ( = LMG 26166(T) = NCIMB 14701(T)).

Lactobacillus mudanjiangensis sp. nov., Lactobacillus songhuajiangensis sp. nov. and Lactobacillus nenjiangensis sp. nov., isolated from Chinese traditional pickle and sourdough.

Three Gram-stain-positive bacterial strains, 11050(T), 7-19(T) and 11102(T), were isolated from traditional pickle and sourdough in Heilongjiang Province, China. These bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain 11050(T) belonged to the Lactobacillus plantarum species group and shared 98.0-98.4 % 16S rRNA gene sequence similarities and 84.7-88.9 % dnaK gene sequence similarities with type strains of Lactobacillus plantarum subsp. plantarum, Lactobacillus plantarum subsp. argentoratensis, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus fabifermentans and Lactobacillus xiangfangensis and had 75.9-80.7 % pheS gene sequence similarities and 90.7-92.5 % rpoA gene sequence similarities with Lactobacillus plantarum subsp. plantarum LMG 6907(T), Lactobacillus plantarum subsp. argentoratensis LMG 9205, Lactobacillus pentosus LMG 10755(T), Lactobacillus paraplantarum LMG 16673(T), Lactobacillus fabifermentans LMG 24284(T) and Lactobacillus xiangfangensis 3.1.1(T), respectively. Strain 7-19(T) was phylogenetically related to Lactobacillus thailandensis, Lactobacillus pantheris and Lactobacillus sharpeae, having 94.1-96.7 % 16S rRNA gene sequence similarities, 71.5-82.3 % pheS gene sequence similarities and 71.2-83.4 % rpoA gene sequence similarities with type strains of Lactobacillus thailandensis, Lactobacillus pantheris and Lactobacillus sharpeae, respectively. Strain 11102(T) was phylogenetically related to Lactobacillus oligofermentans, Lactobacillus suebicus, Lactobacillus vaccinostercus and Lactobacillus hokkaidonensis. Strain 11102(T) had 99.2 % 16S rRNA gene sequence similarity, 81.3 % pheS gene sequence similarity and 96.1 % rpoA gene sequence similarity with Lactobacillus oligofermentans LMG 22743(T), respectively. Strain 11102(T) shared 96.0-96.8 % 16S rRNA gene sequence similarities, 73.3-81.0 % pheS gene sequence similarities and 74.6-76.9 % rpoA gene sequence similarities with type strains of Lactobacillus suebicus, Lactobacillus vaccinostercus and Lactobacillus hokkaidonensis, respectively. Based upon the data from polyphasic characterization obtained in the present study, three novel species, Lactobacillus mudanjiangensis sp. nov., Lactobacillus songhuajiangensis sp. nov. and Lactobacillus nenjiangensis sp. nov., are proposed and the type strains are 11050(T) ( = LMG 27194(T) = CCUG 62991(T)), 7-19(T) ( = LMG 27191(T) = NCIMB 14832(T) = CCUG 62990(T)) and 11102(T) ( = LMG 27192(T) = NCIMB 14833(T)), respectively.

Suppressive effects of oral administration of heat-killed Lactobacillus acidophilus on T helper-17 immune responses in a bovine ß-lactoglobulin-sensitized mice model.

The present study was planned to explore the effect of Lactobacillus (L.) acidophilus on the T helper-17 (Th17) immune response in a mouse model of ß-lactoglobulin (ß-lg) allergy. Bovine ß-lg sensitised BALB/c mice were orally administered with different doses of heat-killed L. acidophilus (low, 5×10(7) colony forming unit (CFU); medium, 5×10(8) CFU; high, 5×10(9) CFU) in 200 µL of phosphate buffered saline (PBS) three times a week, starting from 1 week before ß-lg sensitisation for 4 weeks. After the allergen challenge, the numbers of blood eosinophils and neutrophils were examined by light microscope; the levels of cytokine (interleukin (IL)-12, IL-4, tumor growth factor (TGF)-ß, IL-10, IL-6 and IL-17A), total immunoglobulin E (IgE) and ß-lg-specific IgE contents in the serum were measured with enzyme-linked immunosorbent assay (ELISA); The mRNA expression levels of TGF-ß, IL-17A,CD25, Foxp3, retinoic acid-related orphan receptor γt (RORγt) and IL-10 were analyzed using real-time polymerase chain reaction (PCR). The results showed that oral administration of L. acidophilus suppressed hypersensitivity responses, attenuated the numbers of inflammatory cells and inhibited IgE production. We found up-regulation of TGF-ß and down-regulation of IL-17A in the serum of L. acidophilus-treated group, along with IL-6 levels was significantly decreased than that of the allergy group (p<0.05). Moreover, the mRNA expression levels of CD25, forkhead box P3 and TGF-ß were significantly higher in the spleen of L. acidophilus-treated group, while the mRNA expression levels of IL-17A, RORγt and IL-10 were significantly lower than that in the allergy group (p<0.05). In conclusion, the suppression of major allergic symptoms by oral administration of L.acidophilus was probably due to improve the regulatory T (Treg)/Th17 balance and inhibit the IL-6 production.

Lactobacillus xiangfangensis sp. nov., isolated from Chinese pickle.

A Gram-positive bacterial strain, 3.1.1T, was isolated from traditional pickle in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Based upon the data obtained in the present study, a novel species, Lactobacillus xiangfangensis sp. nov., is proposed with the type strain 3.1.1T (=LMG 26013T=NCIMB 14687T).

Leuconostoc mesenteroides subsp. suionicum subsp. nov.

Strains LMG 8159 and LMG 11499 were reclassified by a polyphasic approach, including 16S rRNA gene sequence analysis, 16S-23S rRNA intergenic spacer (IGS) sequence analysis, (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid methyl ester analysis and an analysis of phenotypic features using API 50 CH. The two strains were closely related to the type strains of the three defined subspecies of Leuconostoc mesenteroides, showing 99.7-99.9% 16S rRNA gene sequence similarity, 99.2% 16S-23S rRNA gene intergenic spacer sequence similarity, 97.1-97.4% pheS gene sequence similarity and 98.0-98.2% rpoA gene sequence similarity. Low atpA gene sequence similarity (91.4-91.7%), (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid compositions and phenotypic features allowed us to differentiate strains LMG 8159 and LMG 11499 from all established subspecies within L. mesenteroides. Based upon the data obtained in the present and previous studies, a novel subspecies is proposed within the species L. mesenteroides, Leuconostoc mesenteroides subsp. suionicum subsp. nov., with the type strain LMG 8159(T) (=ATCC 9135(T) =DSM 20241(T) =NCIMB 6992(T)).

[Gene regulation to lactic acid bacteria for increasing production of flavor metabolite].

Abstract: The major flavor substances Lactic acid bacteria produced include the buttermilk aroma butanedione, the yoghurt flavor acetaldehyde and the amino acid. Metabolic engineering in LAB had focused primarily on rerouting of pyruvate metabolism towards butanedione or acetaldehyde to improve the flavor of fermented milk, which has considerable economic value. The typical yogurt flavor is caused by acetaldehyde produced through many different pathways. The attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase, which encoded by the glyA gene. In addition, the efficient conversion of glucose into acetaldehyde was achieved by over-expression of pyruvate decarboxylase and NADH oxidase in LAB. The concentration of acetaldehyde derived from this regulation is higher than the other strategy. As for the regulation of butanedione, it was focused on combinating inactivation of alpha-acetolactate decarboxylase with low lactate dehydrogenase activity, or with overproduction of NADH-oxidase, or with overproduction of alpha-acetolactate synthase. Then the possibility of co-regulation with certain three kinds of enzyme above was recommended.

Some current applications, limitations and future perspectives of lactic acid bacteria as probiotics.

Several mechanism and non-mechanism-based studies supporting the claim that lactic acid bacteria (LAB) strains confer health benefits and play immune-modulatory roles were examined in this review. Probiotic applications of LAB on global burdens such as obesity and type-2 diabetes were discussed as well as the use of yoghurt and ice cream as important vehicles to convey several beneficial LAB strains. Probiotic and symbiotic dairy products may be used in the nearest future to treat a variety of health disorders. Current studies suggest that lactic acid bacteria possess anti-obesity and anti-diabetic propensities on their hosts and thus can play a crucial role in human health care. Research in the rheological and physicochemical properties of ice cream as well as its applications are also on the increase. These applications face certain hurdles including technological (for less developed countries), consumer acceptability of new functional foods may be influenced by culture, ethics or religion. There is need for more studies on the genetic basis for probiotic properties which will give further understanding regarding novel manipulation skills and applicability in nutrition and health sectors. More studies confirming the direct effects of probiotic LABs in lowering the spread of food-borne and other pathogens are also anticipated.

Complete genome sequence of Bifidobacterium animalis subsp. lactis KLDS 2.0603, a probiotic strain with digestive tract resistance and adhesion to the intestinal epithelial cells.

Bifidobacterium animalis subsp. lactis KLDS 2.0603 (abbreviated as KLDS 2.0603) is a probiotic strain isolated from the feces of an adult human. Previous studies showed that KLDS 2.0603 has a high resistance to simulated digestive tract conditions and a high ability to adhere to intestinal epithelial cells (Caco-2). These two characteristics are essential requirements for the selection of probiotic bacteria. To explore the stress resistance mechanism to the digestive tract environment and the adhesive proteins of this strain, in this paper, we reported the complete genome sequence of KLDS 2.0603, which contains 19,469bp and encodes 1614 coding sequences(CDSs), 15 rRNA genes, 52 tRNA genes with 1678 open reading frames.

In vitro assessment of the antimicrobial potentials of Lactobacillus helveticus strains isolated from traditional cheese in Sinkiang China against food-borne pathogens.

Lactobacillus helveticus, an obligatory hetero-fermentative LAB, is Generally Recognized as Safe (GRAS) and is gaining popularity for application in dairy products. Lactic acid bacteria (LAB) play a remarkable role in inhibiting the growth of pathogenic bacteria in food products, without disturbing the sensory attributes of the food. In this study, the screening of the antimicrobial potential of Lactobacillus helveticus KLDS 1.8701 against four food-borne pathogens including Listeria monocytogenes ATCC 19115, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, and Escherichia coli O157:H7 ATCC 43889 in vitro was inspected using the Oxford cup method and mixed culture inhibition assays. The organic acid production and antimicrobial potential of the cell-free supernatants (CFS) have been evaluated via different treatments and analysis using high performance liquid chromatography (HPLC). The analysis results revealed that KLDS 1.8701 exhibited the highest antimicrobial potential compared to other antimicrobial strains. The antimicrobial activity of KLDS 1.8701 resulted from the organic acids in the culture and CFS. From the study, it was found that carbon sources, as well as organic acid production, accelerate the antimicrobial activity of KLDS 1.8701 and the fructooligosaccharides (FOS) were considered the best for improving the proliferation of KLDS 1.8701 and supporting its antimicrobial action. Results of the mixed culture inhibition assays showed that part of the antimicrobial activity resulted from the inhibitory action of the bacteria itself in culture, and this action required cellular contact between the food-borne pathogens and KLDS 1.8701. Conversely, the results of the antimicrobial spectrum assay revealed that some Lactobacilli remained unaffected by KLDS 1.8701. KLDS 1.8701 might also be favorable for use as a supplementary starter in fermented dairy productions. Furthermore, KLDS 1.8701 could survive well under GI tract conditions. Further studies on in vivo inhibition assays and the probiotic effects are recommended.

Comparison of immunomodulating properties of Beta-lactoglobulin and its hydrolysates.

Cow's milk allergy is one of the most common food allergies in childhood. Beta-lactoglobulin (ß- lg) is a dominant allergen in cow's milk. Hydrolysis is known as an effective method to reduce the allergenicity of proteins. Thus, the objective of this study was to compare the allergenicity of ß-lg and its hydrolysates using an animal model. Twenty four BALB/c mice were divided into three groups and subcutaneously injected with native bovine ß-lg and its hydrolysates on days 0, 7 and 14. During the sensitization period, a number of systemic anaphylactic indicators were observed in mice sensitized by ß-lg compared to those sensitized by hydrolysates of ß-lg. Mice sensitized by hydrolysates of ß-lg showed a significantly lower spleen lymphocyte proliferation level than that sensitized by intact ß-lg. Antibody levels of ß-lg-specific IgE in serum induced by native ß-lg were significantly high. Plasma histamine levels were also evaluated and showed the same trend as IgE. Moreover, the hydrolysates of ß-lg significantly down-regulated IL-4 and IL-5 secretions in serum. These results suggested that enzymatic hydrolysis could reduce the allergenicity of ß-lg.