Cell-bound exopolysaccharides of Lactobacillus brevis KB290: protective role and monosaccharide composition.

We examined the survivability of Lactobacillus brevis KB290 and derivative strain KB392 in artificial digestive juices and bile salts. The strains have similar membrane fatty acids but different amounts of cell-bound exopolysaccharides (EPS). In artificial digestive juices, KB290 showed significantly higher survivability than KB392, and homogenization, which reduced the amount of EPS in KB290 but not in KB392, reduced the survivability only of KB290. In bile salts, KB290 showed significantly higher survivability than KB392, and cell-bound EPS extraction with EDTA reduced the survivability of only KB290. Transmission electron microscopy showed there to be a greater concentration of cell-bound EPS in KB290 than in either KB392 or EDTA-treated or homogenized KB290. We conclude that KB290's cell-bound EPS (which high performance liquid chromatography showed to be made up of glucose and N-acetylglucosamine) played an important role in bile salt tolerance.

Relationships between fatty acid composition and bile tolerance in lactobacillus isolates from plants and from non-plant materials.

Twenty plant-derived and 18 non-plant-derived strains of Lactobacillus casei were compared for their growth in tryptone - yeast extract - glucose broth containing 0.3% bile by measuring absorbance at a wavelength of 620 nm after 24 h of incubation at 37 °C. Bile tolerance - a fundamental probiotic property - was calculated by dividing the experimental data by control values (growth without bile). We found that bile tolerance was strain specific but that the average bile tolerance of the plant-derived strains was significantly (P < 0.05) lower than that of the non-plant-derived strains tested. All tested strains could not deconjugate sodium taurocholate, indicating that the difference in bile tolerance was not due to the ability to deconjugate bile. The fatty acid compositions of the test strains with and without exposure to 0.3% bile were investigated, and a statistical correlation analysis between these compositions and their bile tolerance was conducted. The fatty acids correlated with bile tolerance differed between plant and non-plant lactobacilli. This is the first report to show that the origin (i.e., growth environment) of lactobacilli affects their fatty acid composition, which in turn, appears to be related to their bile tolerance.

Isolation stress for 30 days alters hepatic gene expression profiles, especially with reference to lipid metabolism in mice.

To elucidate the physiological responses to a social stressor, we exposed mice to an isolation stress and analyzed their hepatic gene expression profiles using a DNA microarray. Male BALB/c mice were exposed to isolation stress for 30 days, and then hepatic RNA was sampled and subjected to DNA microarray analysis. The isolation stress altered the expression of 420 genes (after considering the false discovery rate). Gene Ontology analysis of these differentially expressed genes indicated that the stress remarkably downregulated the lipid metabolism-related pathway through peroxisome proliferator-activated receptor-alpha, while the lipid biosynthesis pathway controlled by sterol regulatory element binding factor 1, Golgi vesicle transport, and secretory pathway-related genes were significantly upregulated. These results suggest that isolation for 30 days with a mild and consecutive social stress regulates the systems for lipid metabolism and also causes endoplasmic reticulum stress in mouse liver.

Assessment of antibiotic resistance in probiotic strain Lactobacillus brevis KB290.

Our purpose was to investigate the safety of the probiotic strain Lactobacillus brevis KB290. The European Qualified Presumption of Safety (QPS) evaluation approach was applied to the strain. We determined the strain's antibiotic resistance, verified it at the genetic level, and determined whether it could be transferred to intestinal microflora. Of 14 antibiotics tested, 11 showed MICs within the limits of the QPS criteria. However, the L. brevis KB290 MICs of ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin were two, four, and eight times, respectively, the breakpoint MICs suggested by the European Scientific Committee on Animal Nutrition, and the MIC of tetracycline was eight times the breakpoint MIC suggested by the European Scientific Panel on Additives and Products or Substances Used in Animal Feed. Using analysis of gapped-genome sequences, we found no known transferable determinants for tetracycline or vancomycin resistance, and we found no mutations in the quinolone resistance-determining regions of the genes encoding GyrA or ParC for ciprofloxacin resistance associated with insertion sequences, integrons, or transposons. These data were confirmed by using PCR primers specific for the respective genes. We assessed the transferability of the resistance traits in conjugation experiments with enterococci and obtained no transconjugants, strongly suggesting that the resistance traits were not transferable. This study demonstrated that the antibiotic resistance observed in L. brevis KB290 was due not to dedicated mechanisms but to intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of L. brevis KB290 as a probiotic.

6-shogaol, a major compound in ginger, induces aryl hydrocarbon receptor-mediated transcriptional activity and gene expression.

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.

Lactobacillus brevis KB290 enhances IL-8 secretion by Vibrio parahaemolyticus-infected Caco-2 cells.

Vibrio parahaemolyticus in uncooked seafood causes acute gastroenteritis. The microorganism has two sets of type III secretion systems and two hemolysins. When it injects its effector proteins into a host cell via type III secretion system 1, one of the type III secretion systems induces secretion of interleukin (IL)-8, a proinflammatory chemokine, through the phosphorylation of ERK 1/2 and p38 MAPK. Although probiotics have beneficial effects on hosts and can help control some infectious diseases, there is little research on the efficacy of probiotics in V. parahaemolyticus infection. Here we pretreated V. parahaemolyticus-infected human intestinal epithelial cells with heat-killed Lactobacillus brevis KB290, a probiotic isolated from fermented vegetables (traditional Japanese pickles) and utilized as an ingredient of beverages and supplementary foods, and demonstrated its efficacy in enhancing IL-8 secretion from V. parahaemolyticus-infected cells. Among the three heat-killed lactic acid bacterial strains we tested, L. brevis KB290 induced the highest level of IL-8 secretions in the infected cells. Relative to control cells (Caco-2 cells pretreated with PBS), V. parahaemolyticus-infected Caco-2 cells pretreated with heat-killed L. brevis KB290 secreted IL-8 earlier, although concentrations were similar 450min after infection. Heat-killed L. brevis KB290 pretreatment also induced earlier ERK 1/2 phosphorylation, greater p38 MAPK phosphorylation, and enhanced IL-8 mRNA expression. Heat-killed L. brevis KB290 accelerated IL-8 secretion, a host cell immune response, in V. parahaemolyticus-infected cells. We consider this to be beneficial because IL-8 plays an important defensive role against infection, and would contribute to the repair of injured epithelial cells.

Genomic analysis by deep sequencing of the probiotic Lactobacillus brevis KB290 harboring nine plasmids reveals genomic stability.

We determined the complete genome sequence of Lactobacillus brevis KB290, a probiotic lactic acid bacterium isolated from a traditional Japanese fermented vegetable. The genome contained a 2,395,134-bp chromosome that housed 2,391 protein-coding genes and nine plasmids that together accounted for 191 protein-coding genes. KB290 contained no virulence factor genes, and several genes related to presumptive cell wall-associated polysaccharide biosynthesis and the stress response were present in L. brevis KB290 but not in the closely related L. brevis ATCC 367. Plasmid-curing experiments revealed that the presence of plasmid pKB290-1 was essential for the strain's gastrointestinal tract tolerance and tendency to aggregate. Using next-generation deep sequencing of current and 18-year-old stock strains to detect low frequency variants, we evaluated genome stability. Deep sequencing of four periodic KB290 culture stocks with more than 1,000-fold coverage revealed 3 mutation sites and 37 minority variation sites, indicating long-term stability and providing a useful method for assessing the stability of industrial bacteria at the nucleotide level.