Hiromi Kimoto-Nira

Anti-ageing effect of a lactococcal strain: analysis using senescence-accelerated mice

The effects of oral administration of a lactococcal strain on physiological changes associated with ageing were investigated using senescence-accelerated mice (SAM). SAM develop normally, but then show an early onset and irreversible advancement of senescence. SAMP6 is a SAM strain that develops osteoporosis with ageing. Oral administration of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) to aged SAMP6 mice was associated with reduced bone density loss, a suppression of incidence of skin ulcers and reduced hair loss, compared with controls. Spleen cells from mice fed strain H61 produced more interferon-gamma and IL-12 than those from control mice, suggesting that administration of strain H61 altered immune responses. The numbers of viable cells of Bifidobacterium sp., Bacteroides sp. and Enterococcus sp. in faeces were similar for mice fed the strain H61 and control diets, but counts for Staphylococcus sp. were significantly lower (P < 0.05) in mice fed strain H61. Mice fed strain H61 had similar serum concentrations of thiobarbituric acid-reactive substances as in controls, indicating a lack of effect on lipid peroxidation status. Administration of living cells of strain H61 or fermented milk containing strain H61 was also associated with a suppression of incidence of skin ulcers and reduced hair loss. These results indicate that oral administration of strain H61 has the potential to suppress some of the manifestations associated with ageing.

Immunomodulatory and cytotoxic effects of various Lactococcus strains on the murine macrophage cell line J774.1

In a series of in vitro culture experiments using the murine macrophage-like cell line, J774.1, we investigated the ability of 46 different Lactococcus lactis strains to induce production of the cytokines interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-alpha. The extent of induction of IL-6, IL-12 and TNF-alpha was strain-specific and was not related to subspecies, biovariety, or the source of the isolate. When incubated with a high concentration of viable cells of some lactococcal strains, J774.1 cells hardly produced cytokines in which case the percentage of J774.1 cells that were double-stained with the apoptosis probe FITC-labeled annexin V and propidium iodide was significantly increased. This finding suggests that perturbation of cytokine induction is due to the cytotoxic effects of these strains. On the other hand, when incubated with living cells of other strains, even at a high concentration, J774.1 cells produced IL-6, IL-12 and TNF-alpha. In these cases, FITC-labeled annexin V interacted with these cells, suggesting that incubation with these strains causes phosphatidylserine to be exposed at the cell surface. The ability of these strains to induce TNF-alpha, but not IL-6 and IL-12, was lost after heat treatment, suggesting that the stimulus required for TNF-alpha induction is heat sensitive and is different from those required for IL-6 and IL-12 induction. The specificity of cytokine induction by different lactococci is discussed in terms of interaction of non-pathogenic bacteria with macrophages, as well as the implications for the use of lactococci as probiotics.

Bile resistance in Lactococcus lactis strains varies with cellular fatty acid composition: analysis by using different growth media.

Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that altering their fatty acid composition (i.e. increased hexadecanoic acid and octadecenoic acid and decreased hexadecenoic acid and C-19 cyclopropane fatty acid) by changing growth conditions may be a useful way to enhance their bile resistance in lactococci.

Survival of a Lactococcus lactis strain varies with its carbohydrate preference under in vitro conditions simulated gastrointestinal tract.

Lactococcus lactis G50 is a candidate probiotic bacterium with immunomodulatory activity. We evaluated the suitability of strain G50 as orally administered probiotics on the basis of its resistance to simulated gastrointestinal (GI) stress, including the presence of lysozyme, low pH, and bile, carbohydrate preference, and bacterial cell surface composition in vitro. This strain survived GI stresses but its resistance to lysozyme was affected by the type of available carbohydrate in the growth medium; it was unaffected with lactose, xylose and galactose as the carbon source but was significantly lower for fructose, sucrose and glucose. The resistance of strain G50 to low pH was unaffected by carbon source. Resistance to bile was determined by two methods; growth and survival study and varied with carbon source. The growth of strain G50 with 0.3% bile was lowest in lactose-containing broth, higher in broth containing xylose or galactose, and highest in broth containing sucrose, glucose, or fructose. In contrast, the survival of cells after 3h incubation with 0.3% bile was highest for lactose. The hydrophobicity of bacterial cells, which can be related to epithelial adhesion in certain cases, was also highest for lactose. The fatty acid composition of cells grown on lactose differed from that of cells grown on other carbon sources. These results suggest that survival of strain G50 in the GI tract depends on the kinds of carbohydrates available. Carbohydrate preferences were observed for other strains of lactic acid bacteria under conditions of GI stress, and this preference varied with the strain and the type of GI stress. Careful consideration should be given to the selection of carbohydrates for in vitro testing of the survival of lactic acid bacteria in the GI tract.

Inhibition of Paenibacillus larvae by lactic acid bacteria isolated from fermented materials

We evaluated the potential application of lactic acid bacteria (LAB) isolated from fermented feeds and foods for use as probiotics against Paenibacillus larvae, the causal agent of American foulbrood (AFB) in vitro. We also assessed the ability of LAB to induce the expression of antimicrobial peptide genes in vivo. Screening of the 208 LAB isolated from fermented feeds and foods revealed that nine strains inhibited the in vitro growth of P. larvae. The LAB strains were identified by 16S rRNA gene sequencing as Enterococcus sp., Weissella sp. and Lactobacillus sp. These strains were screened for their abilities of immune activation in honeybees by real-time RT-PCR using antimicrobial peptide genes as markers. After oral administration of several of the screened LAB to larvae and adults, the transcription levels of antimicrobial peptide genes, such as abaecin, defensin and hymenoptaecin, were found to increase significantly. These findings suggested that selected LAB stimulate the innate immune response in honeybees, which may be useful for preventing bacterial diseases in honeybees. This is the first report to characterize the probiotic effects of LAB isolated from fermented feeds and foods in honeybees.

Oral administration of live Lactococcus lactis C59 suppresses IgE antibody production in ovalbumin-sensitized mice via the regulation of interleukin-4 production

To explore the potential probiotic effects of diary starter strains to suppress an IgE allergic response, 10 strains of live dairy lactic acid bacteria were screened for their ability to stimulate the T-helper (Th) type 1 response that counteracts the Th2 response. Four strains with distinct patterns of interleukin(IL)-12p70 and interferon-γ production by murine splenocytes were then orally administered to Balb/c mice, and serum IgE antibody production was examined after ovalbumin sensitization. Oral administration of live Lactococcus lactis strain C59 significantly reduced the total IgE antibody levels, whereas oral administration of the other three strains had no effect on the total IgE levels in ovalbumin-sensitized mice. This inhibitory effect on IgE antibody production was lost when heat-killed C59 was used for oral administration. Ex vivo experiments showed that IL-4 production upon stimulation with the anti-CD3 antibody was significantly reduced in splenocytes of mice with an oral administration of live strain C59 compared with the control. These results indicate that the inhibition of IgE antibody production in mice treated with live strain C59 was due to the suppression of IL-4 production.

Identification of Antioxidants Produced by Lactobacillus plantarum

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC50 values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.

Oral intake of heat-killed cells of Lactococcus lactis strain H61 promotes skin health in women.

We conducted a double-blind, placebo-controlled trial to evaluate the effect of heat-killed cells of Lactococcus lactis strain H61 on various skin properties of Japanese women. Volunteers (age 31–62 years) were randomly assigned to receive test food with or without 60 mg of heat-killed strain H61 (fifteen women in each group; H61 and control groups, respectively) daily for 8 weeks. Results were analysed for three age categories (30s, 40s and 50–60s). Compared with that at week 0, skin hydration at the inner forearm at weeks 4 and 8 decreased in all volunteers (except those in their 50–60s) because of the environmental change from autumn to winter. The oldest H61 group maintained skin hydration at the inner forearm throughout the study. Skin elasticity and melanin content in the cheek decreased and sebum content increased throughout the test period due to seasonal environmental change, regardless of age or H61 treatment. Self-evaluation scores for apparent hair follicles and dryness of the throat at week 8 were higher in the overall H61 group than in the combined placebo group. The 30s H61 group noted marked improvements in self-surveyed skin elasticity at week 8 compared with at week 0 and with the placebo group at week 8. The results of the present study indicate that oral intake of heat-killed cells of L. lactis strain H61 can improve some skin properties and body characteristics in women. This strain would probably be useful in increasing the quality of life in an ageing population.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology

ABSTRACT The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

Influence of long-term consumption of a Lactococcus lactis strain on the intestinal immunity and intestinal flora of the senescence-accelerated mouse.

The senescence-accelerated mouse develops normally until 5-6 months of age and then displays rapid and irreversible advancement of senescence manifesting as clinical signs and gross lesions. To clarify the effect of lactic acid bacteria on the physiological changes with increasing age, heat-killed Lactococcus lactis G50 was administered to 1-month-old senescence-accelerated-prone mouse (SAMP)6 mice for 11 months, a senescence-accelerated mouse strain that develops senile osteoporosis. Mice fed G50 gained more weight than the control mice (not fed G50) during the feeding experiment. Faecal IgA levels in the mice fed G50 at 3 months were higher than those of the control mice but decreased to control levels with increasing age. The numbers of viable cells of Bacteroides sp., Lactobacillus sp., Staphylococcus sp., Enterococcus/Streptococcus sp. and Enterobacteriaceae sp. in faeces were similar for mice fed the G50 and control diets at any age, but strain G50 suppressed the intestinal growth of H2S-producing bacteria. Bone density of the thigh bone did not differ between aged G50 and control mice. Strain G50 would be a beneficial bacterium for the enhancement of intestinal immunity during youth and to suppress the growth of harmful intestinal bacteria. The applicability of strain G50 for the food and animal industries has been proposed in the present study.