The Effect of Nutrients on Subjective Accomplishment at Work: Results from a Health Survey and a Single-Arm Dietary Intervention Study.

In Japan, many workers are exposed to chronic stress, sleep deprivation, and nutritional imbalance. They tend still to go to work when ill, leading to decreased work performance and productivity, which has become a major social problem. We conducted a human entry study with the aim of finding a link between these two factors and proposing an optimized diet, believing that a review of diet may lead to an improvement in labor productivity. In this study, we used subjective accomplishment (SA) as a measure of productivity. First, we compared nutrient intake between groups with high and low SA using data from a health survey of 1564 healthy male and female adults. Significant differences were found in the intake of 13 nutrients in males and 15 nutrients in females, including potassium, vitamin A, insoluble fiber, and biotin. Recommended daily intake of these nutrients was determined from survey data. Next, we designed test meals containing sufficient amounts of 17 nutrients and conducted a single-arm intervention study (registration code UMIN000047054) in Kameyama City, Mie Prefecture, Japan. Healthy working adults (males and females aged 20-79 years) were recruited and supplied with test meals, which were eaten once a day 5 days a week for 8 weeks. SA was significantly higher and daytime sleepiness (DS) was significantly lower after lunch on workdays in younger participants (under 60 years) when they ate the test meals as breakfast or lunch. Our results suggest that SA and DS, which change daily, are strongly influenced by the meal eaten before work, and that taking the 17 nutrients may help prevent presenteeism and improve labor productivity.

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose.

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-336, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and d-mannose. Among four mannobioses, α-1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 µM). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or α-1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

Complete Genome Sequence of Lactococcus cremoris Strain 7-1, a Lactic Acid Bacterium Isolated from a Traditional Mongolian Milk Product Possessing Mucin-Adhesive Ability.

We report the complete genome sequence of Lactococcus cremoris strain 7-1, which was isolated from urum, a traditional Mongolian milk product. Strain 7-1 adhered to porcine gastric mucin in a carbon source-dependent manner. The genome consists of a circular chromosome (2,557,589 bp; GC content, 35.7%) and two circular plasmids.

Complete Genome Sequence of Lactobacillus plantarum Strain LQ80, Selected for Preparation of Fermented Liquid Feed for Pigs.

Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids.

New lactic acid bacteria for skin health via oral intake of heat-killed or live cells.

Lactic acid bacteria play an essential role in the food industry in the manufacture of many fermented products (cheese, yogurt, fermented vegetables, etc.). Application of these organisms is now being extended to the area of health improvement, as their probiotic activities become known. Probiotics are defined as viable microorganisms that exert a beneficial effect on the health of the host when they are ingested in sufficient quantity. Lactic acid bacteria and bifidobacteria isolated from the human intestine are the most common probiotics used for human consumption. The development of new probiotics with new beneficial effects is eagerly awaited in the food industry. This review introduces Lactococcus, which are one of the genera of lactic acid bacteria and are mainly isolated from dairy products and fermented vegetables, as new probiotics, focusing especially on Lactococcus lactis H61, which improves skin status in Japanese women with oral intake of heat-killed or live cells. The deduced mechanisms associated with the beneficial effects of strain H61 are also discussed.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G50 with Immunostimulating Activity, Isolated from Napier Grass.

Lactococcus lactis subsp. lactis G50 is a strain with immunostimulating activity, isolated from Napier grass (Pennisetum purpureum). We determined the complete genome sequence of this strain using the PacBio RS II platform. The single circular chromosome consists of 2,346,663 bp, with 35.03% G+C content and no plasmids.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

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

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.

Dietary intake of heat-killed Lactococcus lactis H61 delays age-related hearing loss in C57BL/6J mice.

Age-related hearing loss (AHL) is a common disorder associated with aging. In this study, we investigated the effect of the intake of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) on AHL in C57BL/6J mice. Measurement of the auditory brainstem response (ABR) demonstrated that female mice at 9 months of age fed a diet containing 0.05% strain H61 for 6 months maintained a significantly lower ABR threshold than control mice. The age-related loss of neurons and hair cells in the cochlea was suppressed by the intake of strain H61. Faecal analysis of bacterial flora revealed that the intake of strain H61 increased the prevalence of Lactobacillales, which is positively correlated with hearing ability in mice. Furthermore, plasma fatty acid levels were negatively correlated with hearing ability. Overall, the results supported that the intake of heat-killed strain H61 for 6 months altered the intestinal flora, affected plasma metabolite levels, including fatty acid levels, and retarded AHL in mice.

Effect of sodium acetate on the adhesion to porcine gastric mucin in a Lactococcus lactis strain grown on fructose.

The association of lactic acid bacteria with mucosal surfaces plays important roles in the beneficial effects of these bacteria on human health, such as colonization of the gastrointestinal tract for pathogen antagonism. Previously, we found that the adhesion of Lactococcus lactis 7-1 to porcine gastric mucin was higher with fructose than with lactose, galactose or xylose as the carbon source. In this study, we examined the effect of growth conditions on the adhesion of strain 7-1 grown on fructose. Medium components affect the adhesion: the adhesion of strain 7-1 grown with sodium acetate was higher than that without it. The enhancement of adhesion by sodium acetate was not observed under aerobic conditions. Cellular properties grown with or without sodium acetate were characterized: strain 7-1 grown with sodium acetate had similar sugar contents, and different fatty acid composition to those grown without it. Strain 7-1 grown with sodium acetate showed significantly lower cell yield and significantly higher hydrophobicity than those grown without it, which is associated with higher adhesion. Fructose and sodium acetate are frequently used in the food industry; this study may reveal a simple way to enhance the adhesion of lactic acid bacteria by growing them with these substances.

Growth characteristics of Lactobacillus brevis KB290 in the presence of bile.

Live Lactobacillus brevis KB290 have several probiotic activities, including immune stimulation and modulation of intestinal microbial balance. We investigated the adaptation of L. brevis KB290 to bile as a mechanism of intestinal survival. Strain KB290 was grown for 5 days at 37 °C in tryptone-yeast extract-glucose (TYG) broth supplemented with 0.5% sodium acetate (TYGA) containing 0.15%, 0.3%, or 0.5% bile. Growth was determined by absorbance at 620 nm or by dry weight. Growth was enhanced as the broth's bile concentration increased. Bile-enhanced growth was not observed in TYG broth or with xylose or fructose as the carbon source, although strain KB290 could assimilate these sugars. Compared with cells grown without bile, cells grown with bile had twice the cell yield (dry weight) and higher hydrophobicity, which may improve epithelial adhesion. Metabolite analysis revealed that bile induced more lactate production by glycolysis, thus enhancing growth efficiency. Scanning electron microscopy revealed that cells cultured without bile for 5 days in TYGA broth had a shortened rod shape and showed lysis and aggregation, unlike cells cultured for 1 day; cells grown with bile for 5 days had an intact rod shape and rarely appeared damaged. Cellular material leakage through autolysis was lower in the presence of bile than in its absence. Thus lysis of strain KB290 cells cultured for extended periods was suppressed in the presence of bile. This study provides new role of bile and sodium acetate for retaining an intact cell shape and enhancing cell yield, which are beneficial for intestinal survival.

New lactic acid bacterial strains from traditional Mongolian fermented milk products have altered adhesion to porcine gastric mucin depending on the carbon source.

Attachment of lactic acid bacteria to the mucosal surface of the gastrointestinal tract is a major property of probiotics. Here, we examined the ability of 21 lactic acid bacterial strains isolated from traditional fermented milk products in Mongolia to adhere to porcine gastric mucin in vitro. Higher attachment was observed with Lactobacillus delbrueckii subsp. bulgaricus strains 6-8 and 8-1 than with Lactobacillus rhamnosus GG (positive control). Lactococcus lactis subsp. cremoris strain 7-1 adhered to mucin as effectively as did strain GG. Heat inactivation decreased the adhesive ability of strains 6-8 and 8-1 but did not affect strain 7-1. The adhesion of strains 6-8, 7-1 and 8-1 was significantly inhibited when the cells were pretreated with periodate and trypsin, indicating that proteinaceous and carbohydrate-like cell surface compounds are involved in the adhesion of these strains. The adhesion of strain 7-1 was affected by the type of carbohydrate present in the growth medium, being higher with fructose than with lactose, galactose or xylose as the carbon source. The sugar content of 7-1 cells grown on various carbohydrates was negatively correlated with its adhesive ability. We provide new probiotic candidate strains and new information regarding carbohydrate preference that influences lactic acid bacterial adhesion to mucin.

Effects of strains of Lactococcus lactis on the production of nitric oxide and cytokines in murine macrophages.

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.

Cellular fatty acid composition and exopolysaccharide contribute to bile tolerance in Lactobacillus brevis strains isolated from fermented Japanese pickles.

Bile tolerance is a fundamental ability of probiotic bacteria. We examined this property in 56 Lactobacillus brevis strains isolated from Japanese pickles and also evaluated cellular fatty acid composition and cell-bound exopolysaccharide (EPS-b) production. The bile tolerance of these strains was significantly lower in modified de Man - Rogosa - Sharpe (MRS) medium (without Tween 80 or sodium acetate) than in standard MRS medium. Aggregating strains showed significantly higher bile tolerance than nonaggregating strains in MRS medium, but there was no significant difference in the modified MRS media. The relative octadecenoic acid (C18:1) content of the 3 most tolerant aggregating and nonaggregating strains was significantly higher when bile was added to MRS. In MRS without Tween 80, the relative C18:1 content was only marginally affected by addition of bile. In MRS without sodium acetate, only the 3 most tolerant nonaggregating strains increased their relative C18:1 content in the presence of bile. Meanwhile, culture in MRS without sodium acetate reduced EPS-b production in aggregating strains. In conclusion, both EPS-b and cellular fatty acid composition play important roles in bile tolerance of pickle-derived L. brevis.

Novel exopolysaccharides produced by Lactococcus lactis subsp. lactis, and the diversity of epsE genes in the exopolysaccharide biosynthesis gene clusters.

To characterize novel variations of exopolysaccharides (EPSs) produced by dairy strains of Lactococcus lactis subsp. lactis and subsp. cremoris, the EPSs of five dairy strains of L. lactis were purified. Sugar composition analysis showed two novel EPSs produced by strains of L. lactis subsp. lactis. One strain produced EPS lacking galactose, and the other produced EPS containing fucose. Among the eps gene clusters of these strains, the highly conserved epsD and its neighboring epsE were sequenced. Sequence and PCR analysis revealed that epsE genes were strain-specific. By Southern blot analysis using epsD, the eps gene cluster in each strain was found to locate to the chromosome or a very large plasmid. This is the first report on the identification of two novel EPSs in L. lactis subsp. lactis. The strains can be detected among other strains by using epsE genes specific to them.

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.

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.

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.

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.

Intraspecies discrimination of Lactobacillus paraplantarum by PCR.

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.