Effects of Lactococcus lactis subsp. cremoris YRC3780 daily intake on the HPA axis response to acute psychological stress in healthy Japanese men.

BACKGROUND: Lactococcus lactis subsp. cremoris (YRC3780), which is isolated from kefir, has been associated with anti-allergic effects in humans. However, it remains unknown whether daily intake of YRC3780 attenuates the response to psychological stress in humans in parallel with changes to the gut microbiome. We examined the fundamental role of YRC3780 in the gut microbiome, stress response, sleep, and mental health in humans. METHODS: Effects of daily intake of YRC3780 on the hypothalamic-pituitary-adrenal (HPA) axis response to acute psychological stress were investigated in a double-blind, placebo-controlled clinical trial involving 27 healthy young men (mean age and body mass index: 23.5 years and 21.5 kg/m2) who were randomly assigned to placebo (n = 13) or YRC3780 (n = 14) groups. The HPA axis response to acute psychological stress, the diurnal rhythm of HPA axis activity, and gut microbiome were assessed and compared between the two groups. RESULTS: The results showed that daily intake of YRC3780 significantly lowered morning salivary cortisol levels compared with placebo. In addition, salivary cortisol levels following a social stress test significantly decreased +40 min after beginning the TSST in the YRC3780-treated group compared to placebo. There were no significant differences between the two groups in terms of actigraphy-based sleep quality, but the subjective sleep quality and mental health were significantly improved in the YRC3780-treated group compared to placebo. CONCLUSIONS: Our study suggests that daily intake of YRC3780 improves the HPA axis response to acute psychological stress, which might be associated with a decrease in morning cortisol levels.

Hypothalamic-pituitary-adrenal axis differentially responses to morning and evening psychological stress in healthy subjects.

AIM: The hypothalamic-pituitary-adrenal (HPA) axis responds to changing environmental demands including psychological stressors. The aim of the present study was to assess whether the time of day effects on the acute response of HPA axis activity to acute psychological stress. METHOD: We studied 27 healthy young subjects. The subjects were participated two experiments as follows. In the first experiment, subjects were instructed to keep their regular sleep schedule for 2 weeks which were measured by using a wrist-worn activity monitor. Afterward, to evaluate a diurnal rhythm of salivary cortisol, eight saliva samples were collected during waking period every 2 hours from when the subjects woke up. In the second experiment, the subjects were randomly assigned to two groups. The Trier Social Stress Test (TSST) was performed either in the morning (n = 14) or in the evening (n = 13). We measured diurnal rhythm of salivary cortisol and stress response of salivary cortisol and heart rate by the TSST. Morning and evening tests were started at 2 hours and 10 hours after woke up, respectively. RESULTS: All subjects showed a normal diurnal rhythm of salivary cortisol concentration, with a peak in the morning immediately after awaking and a minimum in the evening. The salivary cortisol response after the TSST was significantly increased from the prestress level in the morning but not in the evening. CONCLUSION: The HPA response to acute psychological stress was more pronounced in the morning than in the evening, correlating with the circadian regulation of cortisol synthesis.

One year investigation of the prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido.

We investigated the seasonal prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido. Samples of raw milk were collected quarterly from May 2013 through February 2014. The mean clostridial spore count for the raw milk from 336 milk tankers was 27.6 CFU/100 ml. The clostridial species isolated most frequently from raw milk samples was Clostridium tyrobutyricum. The dominant species was C. tyrobutyricum regardless of the season. The percentage of samples with low spore counts (<10 CFU/100 ml) was highest (60.9%) during winter (February) and lowest (34.5%) in autumn (November). In comparison, the percentage of samples with high spore counts (>100 CFU/100 ml) was highest (5.7%) in autumn (November) and lowest (1.1%) during spring (May).

Characterization and expression analysis of the exopolysaccharide gene cluster in Lactobacillus fermentum TDS030603.

Part of the exopolysaccharide gene cluster of Lactobacillus fermentum TDS030603 was characterized. It consists of 11,890 base pairs and is located in the chromosomal DNA, 13 open reading frames of which were encoded. Out of the 13 open reading frames, six were found to be involved in exopolysaccharide synthesis; however, five were similar to transposase genes of other lactobacilli, and two were functionally unrelated. Expression analysis revealed that the exopolysaccharide synthesis-related genes were expressed during cultivation. Southern analysis using specific primers for the exopolysaccharide genes indicated that duplication of the gene cluster did not occur. The plasmid-cured strain maintained its capacity for exopolysaccharide production, confirming that the exopolysaccharide gene cluster of this strain is located in the chromosomal DNA, similarly to thermophilic lactic acid bacteria. Our results indicate that this exopolysaccharide gene cluster is likely to be functional, although extensive gene rearrangement occurs.

Microbiota analysis of Caspian Sea yogurt, a ropy fermented milk circulated in Japan.

We analyzed the microbiota of domestic ropy fermented milk, Caspian Sea yogurt (or 'kasupikai yohguruto' in Japanese), circulated in Japan. We collected six varieties from five localities. Lactococcus (L) lactis ssp. cremoris was isolated from all samples as the dominant strain at levels of 10(8)-10(9) CFU/g. We show this strain produces an extracellular polysaccharide (EPS) that causes the unique characteristic viscosity of the product. From analysis of the RAPD pattern of 60 bacterial isolates from the six samples, we found that 59 strains from a total of 60 isolates were identical and produced this viscosity. Furthermore, PFGE analysis of representative strains from each sample indicated that the isolates could be classified into four subgroups. This suggests these L. lactis ssp. cremoris strains found in Caspian Sea yogurt may have been slightly mutated during subculture in Japan. In addition, Lactobacillus (L.) sakei ssp. sakei were isolated from three samples; L. plantarum, Gluconoacetobacter sacchari and Acetobacter aceti were isolated from two samples; and L. paracasei, L. kefiri, Leuconostoc (Leu.) mesenteroides were isolated from one sample.

Identification of polypeptides constituting lactophorin by monoclonal antibody to bovine milk lactophorin.

A monoclonal antibody to lactophorin (LP) was prepared by creating hybridoma from mouse myeloma cells and spleen cells from mice immunized with PAS-4 concentrated fraction from bovine milk fat globule membrane. The prepared antibody recognized a polypeptide moiety of LP27, the major component constituting LP, but not a carbohydrate moiety. Immunoblot analysis showed that all polypeptides (LP17, LP20, LP27, LP40, and LP50) constituting LP were recognized by the antibody. The identities of LP20, LP40, and LP50 were verified by N-terminal and internal amino acid sequencing. LP20 contains hydrolysate of LP27 besides LP27 without the O-glycosyl sugar chain. These results suggest that LP40 and LP50 are homo- or heterodimers of LP20 and LP27. This is the first report to the effect that LP was constructed from several forms of polypeptides, derived from LP27.

Oral administration of freeze-dried kefir reduces intestinal permeation of and oral sensitization to ovalbumin in mice.

An increase in plasma ovalbumin concentrations after intragastric administration of ovalbumin was suppressed by concomitant freeze-dried kefir in BALB/c mice. Serum levels of ovalbumin-specific immunoglobulin G and proliferation of splenic mononuclear cells in mice immunized orally with ovalbumin were suppressed by feeding freeze-dried kefir. We propose that kefir reduces intestinal permeation of food antigen, which contributes to suppression of oral sensitization.

Content and chemical compositions of cerebrosides in lactose-assimilating yeasts.

Cerebrosides were found in ten lactose-assimilating yeasts. Representative component ceramide moieties of cerebrosides from nine of these yeasts contained 9-methyl-4-trans, 8-trans-sphingadienine, and 2-hydroxy fatty acid with carbon chain lengths of 16 or 18. The major ceramide moieties in Brettanomyces anomalus, however, differed from those in other yeasts, and were predominately moieties containing 2-hydroxymyristic acid. Thus we found that various cerebroside molecular species are present in yeasts.

Purification, characterization, and gene cloning of lysyl aminoeptidase from Streptococcus thermophilus YRC001.

We purified and characterized an aminopeptidase from Streptococcus thermophilus YRC001 to obtain an enzyme for the application of reducing bitter-defect in cheese manufacturing. The purified enzyme was a monomer, and its molecular mass was estimated to be 90-100 kDa. It had a broad substrate specificity, and mostly hydrolyzed lysyl and leucyl peptides. The optimal temperature and pH for the enzyme were 35 degrees C and pH 6.5, respectively. EDTA, o-phenanthroline, and p-chloromercuribenzoate inhibited its activity, therefore it was considered to be a metallopeptidase. The purified enzyme efficiently reduced the bitterness of a trypsin digest of reconstituted skim milk. Therefore, we cloned a gene for the enzyme from YRC001. The nucleotide sequence of a 2,940-bp XbaI fragment containing the gene was analyzed. The gene encoded 849 amino acids, and the calculated molecular mass for the mature enzyme (initial methionine is removed) was 96,434. The deduced amino acid sequence showed high homology with the known bacterial lysyl aminopeptidase (aminopeptidase N).

Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose.

The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively. A4-beta-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.