Rapid Single-Cell Detection of Beer-Contaminating Lactic Acid Bacteria Using Bioluminescence/Rapid Microbe Detection.

The rapid microbe detection (RMD) method can detect a trace amount of adenosine triphosphate (ATP) equivalent to that generated by single cell of lactic acid bacteria (LAB). For the improved detection of LAB contamination in beer without cultivation, it is necessary to eliminate the influence of beer-derived ATP and to improve the signal-to-noise ratio. In this protocol, the beer sample is filtered using a membrane filter, thereby avoiding the formation of beer foam to the fullest extent. By washing the beer components remaining on the filter with an ethanol solution and a weakly alkaline solution and then culturing the filter in an agar medium, the beer-derived ATP remaining on the filter can be removed and the ATP of LAB cells is increased. As a result, the signal-to-noise ratio of the RMD method can be dramatically improved.

Dietary Heat-Killed Lactobacillus brevis SBC8803 Attenuates Chronic Sleep Disorders Induced by Psychophysiological Stress in Mice.

We previously reported that dietary heat-killed Lactobacillus brevis SBC8803 affects sleep in mice and humans. The present study examined whether SBC8803 improves psychophysiological stress-induced chronic sleep disorders (CSD) using a mouse model characterized by disrupted circadian rhythms of wheel-running activity and sleep-wake cycles. Mice were fed with a diet supplemented with 0.5% heat-killed SBC8803 for 6 wk and imposed stress-induced CSD for last 2 wk. Dietary SBC8803 suppressed the reduction in wheel-running activity induced by CSD. Electroencephalography (EEG) revealed that SBC8803 significantly restored wakefulness and increased non-rapid eye movement (NREM) sleep during the second half of the active phase during CSD. The CSD-induced reduction in EEG slow wave activity, a marker of NREM sleep intensity, during the beginning of the inactive phase was significantly improved by SBC8803 supplementation. These findings suggest that dietary heat-killed SBC8803 confers beneficial effects on insomnia and circadian sleep disorders induced by psychophysiological stress.

Prevotella cerevisiae sp. nov., beer-spoilage obligate anaerobic bacteria isolated from brewery wastewater.

Two obligate anaerobic, Gram-stain-negative, non-spore-forming bacilli (strains SBC 8034T and SBC 8065) were isolated from brewery wastewater. Cells of the two strains were rod-shaped and 0.8×2-5 µm in size. Strains SBC 8034T and SBC 8065 did not grow on Columbia agar or tryptic soy agar II with 5 % sheep blood, brain-heart infusion agar or chocolate agar, but did grow on peptone-yeast-glucose agar and de Man, Rogosa and Sharpe agar using beer instead of water. The organisms produced acetic acid and succinic acid as the major metabolic end-products. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains are clearly distinct from all recognized species within the genus Prevotella, but belong to the same species (DDH=85 %). Based on 16S rRNA and hsp60 gene sequencing, along with phenotypic, chemical and biochemical properties, strains SBC 8034T and SBC 8065 were considered to represent a novel species within the genus Prevotella, for which the name Prevotellacerevisiae sp. nov. is proposed. Strain SBC 8034T (=DSM 100619T=JCM 30867T) is the type strain of the proposed novel species.

Dietary heat-killed Lactobacillus brevis SBC8803 (SBL88™) improves hippocampus-dependent memory performance and adult hippocampal neurogenesis.

AIMS: Lactobacillus species are used widely as various food and supplements to improve health. Previous studies have shown that heat-killed Lactobacillus brevis SBC8803 induces serotonin release from intestinal cells and affects sleep rhythm and the autonomic nervous system. However, the effect of SBC8803 on cognitive function remains unknown. Here, we examined the effects of dietary heat-killed SBC8803 on hippocampus-dependent memory and adult hippocampal neurogenesis. METHODS: Hippocampus-dependent memory performance was assessed in mice fed heat-killed SBC8803 using social recognition and contextual fear conditioning tasks. Adult hippocampal neurogenesis was evaluated before, during, and after feeding heat-killed SBC8803 by measuring the number of 5-bromo-2-deoxyuridine (BrdU)-positive cells following systemic injections of BrdU using immunohistochemistry. RESULTS: Mice fed a heat-killed SBC8803 diet showed an improvement of hippocampus-dependent social recognition and contextual fear memories and enhanced adult hippocampal neurogenesis by increasing the survival, but not proliferation, of newborn neurons. CONCLUSION: Dietary heat-killed SBC8803 functions as memory and neurogenesis enhancers.

Rapid Single Cell Detection of Lactic Acid Bacteria in the Beer Using Bioluminescence Method.

　In this study, we developed a system, known as MicroStarTM Rapid Microbe Detection System (RMDS) , to detect Lactobacillus brevis, which usually requires 2-4 days for examination by the conventional plate count procedure, for beer quality control using a bioluminescence method within 24 hr and also aimed to develop a technology to detect bacterial growth without the need for cultivation. We used a highly sensitive luminous reagent that increased the activity of the luciferin- luciferase reaction to 2.5×10－18 mol ATP/0.2 µl and could detect even a single lactic acid bacterial cell. The limitation of the method was that ATP derived from the beer hindered bacterial measurement and the supply of energy source to secure ATP of lactic acid bacterial cell. The sample beer was filtered through a membrane filter, avoiding the formation of beer foam to the best extent, the filter was cleaned with 10% ethanol and 0.1% sodium hydrogen carbonate solution, and incubated on a GMY agar plate (1% glucose, 0.2% malic acid, 0.67% yeast nitrogen base, 1% agar; pH 5.2) at room temperature for 2 hr. Post incubation of the filter, bacterial cell count was measured with RMDS. This method could overcome the hindrance of ATP measurement and could stably detect lactic acid bacteria without the need for cultivation.

Effects of oral intake of heat-killed Lactobacillus brevis SBC8803 (SBL88™) on dry skin conditions: A randomized, double-blind, placebo-controlled study.

Lactobacilli are important in intestinal homeostasis, which involves the regulation of immune function, digestive health, cholesterol absorption and intestinal tumor growth amongst others. Our previous investigations have suggested that oral intake of heat-killed Lactobacillus brevis (L. brevis) SBC8803 (SBL88™) suppresses dermatitis by modulating the immune function in an atopic dermatitis mouse model. The aim of the present study was to investigate the effect of heat-killed L. brevis SBC8803 intake on skin hydration conditions in humans. A randomized, double-blind, placebo-controlled study was conducted with volunteers with slightly higher levels of transepidermal water loss (TEWL) on the forearm. The subjects (126 people aged between 21 and 59 years) were randomly allocated to three groups so that the level of TEWL and the age were distributed equally among the groups. The subjects took placebo or heat-killed L. brevis SBC8803 at a daily dose of 25 or 50 mg for 12 weeks. Following the exclusion of eight subjects for plausible reasons (two withdrawals from the study, two for study violations, one for not meeting exclusion criteria and three due to their physical condition), 118 subjects were subjected to the analysis. The results of the present study revealed that following the analysis of the whole populations, marginal differences were observed in TEWL (for example, suppression of skin water loss) at the neck in the 25 mg/day group at week 8 and at the lower eye region in the 50 mg/day group at week 4 (P=0.05 and 0.09, respectively, compared with the placebo group analyzed by Dunnett's test). A significant increase in corneal hydration was also observed at the neck in the 25 mg/day group at week 12 (P=0.06, as compared with the placebo group as analyzed by Dunnett's test). In the analysis of the subpopulations whose habitual frequency of taking lactic fermentation products was less than once per week, the levels of corneal hydration at the neck (in the 50 mg/day group) and lower eye region (in the 25 mg/day group) were significantly increased at week 12 (P<0.05). In conclusion, the results of the present investigation suggest that oral intake of heat-killed L. brevis SBC8803 is effective at improving skin hydration conditions in populations with low habitual frequency of taking lactic fermentation products.

Dietary heat-killed Lactobacillus brevis SBC8803 promotes voluntary wheel-running and affects sleep rhythms in mice.

AIMS: We previously reported that heat-killed Lactobacillus brevis SBC8803 enhances appetite via changes in autonomic neurotransmission. Here we assessed whether a diet supplemented with heat-killed SBC8803 affects circadian locomotor rhythmicity and sleep architecture. MAIN METHODS AND KEY FINDINGS: Daily total activity gradually increased in mice over 4 weeks and supplementation with heat-killed SBC8803 significantly intensified the increase, which reached saturation at 25 days. Electroencephalography revealed that SBC8803 supplementation significantly reduced the total amount of time spent in non-rapid eye movement (NREM) sleep and increased the amount of time spent being awake during the latter half of the nighttime, but tended to increase the total amount of time spent in NREM sleep during the daytime. Dietary supplementation with SBC8803 can extend the duration of activity during the nighttime and of sleep during the daytime. Daily voluntary wheel-running and sleep rhythmicity become intensified when heat-killed SBC8803 is added to the diet. SIGNIFICANCE: Dietary heat-killed SBC8803 can modulate circadian locomotion and sleep rhythms, which might benefit individuals with circadian rhythms that have been disrupted by stress or ageing.

Disruption of behavioral circadian rhythms induced by psychophysiological stress affects plasma free amino acid profiles without affecting peripheral clock gene expression in mice.

Disordered circadian rhythms are associated with various psychiatric conditions and metabolic diseases. We recently established a mouse model of a psychophysiological stress-induced chronic sleep disorder (CSD) characterized by reduced amplitude of circadian wheel-running activity and sleep-wake cycles, sleep fragmentation and hyperphagia. Here, we evaluate day-night fluctuations in plasma concentrations of free amino acids (FAA), appetite hormones and prolactin as well as the hepatic expression of circadian clock-related genes in mice with CSD (CSD mice). Nocturnal increases in wheel-running activity and circadian rhythms of plasma prolactin concentrations were significantly disrupted in CSD mice. Hyperphagia with a decreased leptin/ghrelin ratio was found in CSD mice. Day-night fluctuations in plasma FAA contents were severely disrupted without affecting total FAA levels in CSD mice. Nocturnal increases in branched-chain amino acids such as Ile, Leu, and Val were further augmented in CSD mice, while daytime increases in Gly, Ala, Ser, Thr, Lys, Arg, His, Tyr, Met, Cys, Glu, and Asn were significantly attenuated. Importantly, the circadian expression of hepatic clock genes was completely unaffected in CSD mice. These findings suggest that circadian clock gene expression does not always reflect disordered behavior and sleep rhythms and that plasma FFA profiles could serve as a potential biomarker of circadian rhythm disorders.

Effects of intraduodenal injection of Lactobacillus brevis SBC8803 on autonomic neurotransmission and appetite in rodents.

Lactobacilli provide several health benefits to mammals, including humans. We previously observed that in rats, intraduodenal injection of Lactobacillus johnsonii La1 elevated efferent gastric vagal nerve activity (efferent-GVNA), while Lactobacillus paracasei ST11 suppressed efferent-GVNA, and thereby increased or decreased food intake. To determine the function of Lactobacillus brevis (SBC8803), its effect on food intake was examined by providing food containing heat-killed SBC8803 to mice. We observed that administration of SBC8803 elevated food intake. Because the afferent intestinal vagal nerve (IVN) is hypothesized to be involved in efferent-GVNA changes, we examined the effect of intraduodenal administration of heat-killed SBC8803 on efferent-GVNA and afferent-IVN activity (IVNA) in rats. In this study, we found that intraduodenal administration of heat-killed SBC8803 increased both efferent-GVNA and afferent-IVNA in rats. Moreover, IV administration of the serotonin 3 receptor antagonist granisetron eliminated the effects of SBC8803 on efferent-GVNA and afferent-IVNA. These findings suggest that heat-killed SBC8803 enhances appetite by elevating digestion and absorption abilities via changes in autonomic neurotransmission that might be mediated by the serotonin 3 receptor.

Oral administration of heat-killed Lactobacillus brevis SBC8803 ameliorates the development of dermatitis and inhibits immunoglobulin E production in atopic dermatitis model NC/Nga mice.

We have previously shown that the oral administration of heat-killed Lactobacillus brevis (L. brevis) SBC8803 strain inhibits IgE production in ovalbumin (OVA)-sensitized BALB/c mice through improvement of the type-1 helper T (Th1)/Th2 balance toward Th1 dominance. Atopic dermatitis is one of the most common skin diseases and is frequently associated with elevated immunoglobulin E (IgE) antibodies against many kinds of allergens. In this study, we investigated the inhibitory effect of oral administration of L. brevis SBC8803 on the development of dermatitis and IgE elevation using the NC/Nga atopic dermatitis model mice. Male 8-week-old NC/Nga mice were sensitized by the topical application of picryl chloride to foot pads and shaved abdomen. These mice were boosted with picryl chloride by topical application onto the ears once a week for 9 weeks. The mice (n=10 per group) were fed a diet containing 0%, 0.05% or 0.5% of heat-killed L. brevis SBC8803 from 2 weeks before the first sensitization to the end of the study. Total IgE concentration in serum, clinical score, and ear thickness were periodically examined throughout the study. Finally, cytokine (interleukin (IL)-4, IL-5, IL-6, IL-10, IL-12, IFN-gamma and transforming growth factor (TGF)-beta) productions from splenocytes and Peyer's patch (PP) cells of mice were measured. Oral administration of L. brevis SBC8803 significantly inhibited IgE production and ear swelling, and suppressed the development of dermatitis in a dose-dependent manner. Immunosuppressive cytokines such as IL-10 and TGF-beta production from PP cells significantly increased in the 0.5% group compared to the control group although Th1-type and Th2-type cytokines production was not affected.

Effect of oral administration of heat-killed Lactobacillus brevis SBC8803 on total and ovalbumin-specific immunoglobulin E production through the improvement of Th1/Th2 balance.

We examined the effect of 59 strains of heat-killed Lactobacillus brevis on interleukin (IL)-12 and interferon (IFN)-gamma production from mouse Peyer's patch (PP) cells. L. brevis has a great variety of strains that induce the production of these cytokines. Some L. brevis strains, which were selected for their ability to induce a strong Th1 immune response, inhibited both total immunoglobulin E (IgE) and antigen specific IgE production, and improved the Th1/Th2 balance by enhancing IL-12 and IFN-gamma and inhibiting IL-4 production from ovalbumin (OVA)-sensitized mouse splenocytes. Based on the results of this screening, we selected L. brevis SBC8803 as a potent inhibitor of IgE production, and investigated the effect of oral administration of heat-killed SBC8803 on IgE production in OVA-sensitized mice. OVA-sensitized mice were fed SBC8803 0% (control), 0.05%, or 0.5% added diet for 4 weeks during the period of the experiment. Total and OVA-specific IgE in the serum of mice, which were fed the 0.5% added diet, was significantly lower than that of the control diet fed mice. The IFN-gamma/IL-4 value, which represents the Th1/Th2 balance, from the 0.5% added diet fed mice splenocytes was also significantly higher than that of the control diet fed mouse splenocytes. Histamine release from OVA-sensitized mice into sera that were induced by the intraperitoneal antigen challenge decreased following the oral administration of SBC8803. The inhibition of IgE production and histamine secretion by the oral administration of heat-killed SBC8803 was probably due to the improvement of the Th1/Th2 balance toward Th1 dominance.