Genome-based assessment of safety characteristics of Lacticaseibacillus paracasei NY1301 and genomic differences in closely related strains marketed as probiotics.

The probiotic attributes of Lacticaseibacillus paracasei NY1301 were comprehensively characterized, and a comparison between the closely related LcA (Actimel) and LcY (Yakult) probiotic strains was conducted using genomic tools. All strains exhibited high genetic similarity and likely shared a common ancestor; differences were primarily expressed as minor chromosomal re-arrangements, substitutions, insertions, and deletions. Compared with LcY, NY1301 exhibited 125 single-nucleotide polymorphisms. NY1301 lacked virulence factors, antibiotic resistance genes, and mutations associated with antibiotic resistance and had a 46-kbp prophage. This prophage is spontaneously induced at low levels and remains in a non-lytic state under standard culture conditions. The observed causal adaptive mutations were likely related to niche adaptation within the respective laboratory or manufacturing processes that occurred during the maintenance of the strains. However, the phenotypic effects of these genomic differences remain unclear. To validate the safety of NY1301, we conducted an open-label trial with healthy participants who consumed excessive amounts of NY1301 (3.0 × 1011 cfu) daily for 28 days. The results of this trial and those of other in vivo studies, coupled with the long history of human consumption without established risks to humans, provide strong evidence confirming the safety of NY1301.

Analysis of free radical production capacity in mouse faeces and its possible application in evaluating the intestinal environment: a pilot study.

Complex interplay between the intestinal environment and the host has attracted considerable attention and has been well studied with respect to the gut microbiome and metabolome. Oxygen free radicals such as superoxide and the hydroxyl radical (•OH) are generated during normal cellular metabolism. They are toxic to both eukaryotic and prokaryotic cells and might thus affect intestinal homeostasis. However, the effect of oxygen free radicals on the intestinal environment has not been widely studied. Herein, we applied electron spin resonance spectroscopy with spin trapping reagents to evaluate oxygen free radical production capacity in the intestinal lumen and the faeces of mice. •OH was generated in faeces and lumens of the small and large intestines. There were no remarkable differences in •OH levels between faeces and the large intestine, suggesting that faeces can be used as alternative samples to estimate the •OH production capacity in the colonic contents. We then compared free radical levels in faecal samples among five different mouse strains (ddY, ICR, C57BL/6, C3H/HeJ, and BALB/c) and found that strain ddY had considerably higher levels than the other four strains. In addition, strain ddY was more susceptible to dextran sulphate sodium-induced colitis. These differences were possibly related to the relative abundance of the gut bacterial group Candidatus Arthromitus, which is known to modulate the host immune response. From these results, we suggest that the production capacity of oxygen free radicals in mouse faeces is associated with intestinal homeostasis.

Effects of lifelong intake of lemon polyphenols on aging and intestinal microbiome in the senescence-accelerated mouse prone 1 (SAMP1).

Polyphenols have been examined for their beneficial effects on health, particularly in rodents, but their lifelong effects are unclear. Lemons (Citrus limon), containing lemon polyphenols (LPP), are widely consumed but the effects of LPP on aging are unknown. Therefore, we examined the effects of LPP on aging such as aging-related scores, locomotor activity, cognitive functions, and intestinal microbiome using senescence-accelerated mouse prone 1 (SAMP1) and senescence-accelerated resistant mouse 1 (SAMR1). All mice had ad libitum access to water (P1_water group, SAMR1) or 0.1% LPP (P1_LPP group). In the P1_LPP group, LPP intake prolonged the lifespan by approximately 3 weeks and delayed increases in aging-related scores (e.g., periophthalmic lesions) and locomotor atrophy. The P1_water group showed large changes in the intestinal microbiome structure, while the R1 and P1_LPP groups did not. The phylum Bacteroidetes/Firmicutes, which is associated with obesity, in the P1_water group was significantly lower and higher than that in the P1_LPP and R1 groups, respectively. Although the relative abundance of Lactobacillus significantly increased in both P1 groups with aging, the P1_LPP group showed a significantly lower increase than the P1_water group. Thus, lifelong intake of LPP may have anti-aging effects on both phenotypes and the intestinal environment.

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function.

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein-approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.

Xanthohumol prevents atherosclerosis by reducing arterial cholesterol content via CETP and apolipoprotein E in CETP-transgenic mice.

BACKGROUND: Xanthohumol is expected to be a potent anti-atherosclerotic agent due to its inhibition of cholesteryl ester transfer protein (CETP). In this study, we hypothesized that xanthohumol prevents atherosclerosis in vivo and used CETP-transgenic mice (CETP-Tg mice) to evaluate xanthohumol as a functional agent. METHODOLOGY/PRINCIPAL FINDINGS: Two strains of mice, CETP-Tg and C57BL/6N (wild-type), were fed a high cholesterol diet with or without 0.05% (w/w) xanthohumol ad libitum for 18 weeks. In CETP-Tg mice, xanthohumol significantly decreased accumulated cholesterol in the aortic arch and increased HDL cholesterol (HDL-C) when compared to the control group (without xanthohumol). Xanthohumol had no significant effect in wild-type mice. CETP activity was significantly decreased after xanthohumol addition in CETP-Tg mice compared with the control group and it inversely correlated with HDL-C (%) (P<0.05). Furthermore, apolipoprotein E (apoE) was enriched in serum and the HDL-fraction in CETP-Tg mice after xanthohumol addition, suggesting that xanthohumol ameliorates reverse cholesterol transport via apoE-rich HDL resulting from CETP inhibition. CONCLUSIONS: Our results suggest xanthohumol prevents cholesterol accumulation in atherogenic regions by HDL-C metabolism via CETP inhibition leading to apoE enhancement.

Xanthohumol, a prenylated chalcone from Humulus lupulus L., inhibits cholesteryl ester transfer protein.

High density lipoprotein (HDL)-cholesterol levels are correlated with a low risk of atherosclerosis. The inhibition of cholesteryl ester transfer protein (CETP), which catalyses cholesterol transfer between lipoproteins, leads to an increase in HDL-cholesterol and is expected to be the next anti-atherogenic target. This study revealed that xanthohumol, a prenylated chalcone, showed the highest inhibition against CETP from screening of natural products in various plants. We investigated the inhibitory activity of some chalcones and flavanones. Naringenin chalcone showed weak CETP inhibition compared with xanthohumol. In addition, isoxanthohumol and naringenin drastically decreased the inhibitory activity. These results suggest that the prenyl group and chalcone structure of xanthohumol were responsible for the CETP inhibitory activity.

Heat-killed body of lactobacillus brevis SBC8803 ameliorates intestinal injury in a murine model of colitis by enhancing the intestinal barrier function.

BACKGROUND: Probiotics have been clinically administered to improve intestinal damage in some intestinal inflammations. However, probiotic treatments are not always effective for these intestinal disorders because live bacteria must colonize and maintain their activity under unfavorable conditions in the intestinal lumen when displaying their functions. This study investigated the physiological functions of a heat-killed body of a novel probiotic, Lactobacillus brevis SBC8803, on the protection of intestinal tissues, the regulation of cytokine production, the improvement of intestinal injury, and the survival rate of mice with dextran sodium sulfate (DSS)-induced colitis. METHODS: Heat shock protein (Hsp) induction and mitogen-activated protein kinase (MAPK) phosphorylation in intestinal epithelia by heat-killed L. brevis SBC8803 were examined by Western blotting. The barrier function of intestinal epithelia was measured with [(3) H]-mannitol flux in the small intestine under oxidant stress. The effects of the bacteria on improving epithelial injury and cumulative survival rate were investigated with a DSS colitis model. RESULTS: Heat-killed L. brevis SBC8803 induced Hsps, phosphorylated p38 MAPK, regulated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß and IL-12, and improved the barrier function of intestinal epithelia under oxidant stress. The induction of Hsp and the protective effect were negated by p38 MAPK inhibitor. These functions relieve intestinal impairments and improve the survival rate in mice with lethal colitis. CONCLUSIONS: The administration of heat-killed L. brevis SBC8803 helps to successfully maintain intestinal homeostasis, while also curing intestinal inflammation. A therapeutic strategy using heat-killed bacteria is expected to be beneficial for human health even in conditions unsuitable for live probiotics because the heat-killed body is able to exhibit its effects without the requirement of colonization.

Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway.

Probiotics exhibit beneficial effects on human health, particularly in the maintenance of intestinal homeostasis in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we aimed to isolate the cytoprotective compound from a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine. L. brevis was incubated in MRS broth and the supernatant was passed through with a 0.2-µm filter. Caco2/bbe cells were treated with the culture supernatant, and HSP27 expression was evaluated by Western blotting. HSP27-inducible components were separated by ammonium sulfate precipitation, DEAE anion exchange chromatography, gel filtration, and HPLC. Finally, we identified that the HSP27-inducible fraction was polyphosphate (poly P), a simple repeated structure of phosphates, which is a common product of lactobacilli and other bacteria associated with intestinal microflora without any definitive physiological functions. Then, poly P was synthesized by poly P-synthesizing enzyme polyphosphate kinase. The synthesized poly P significantly induced HSP27 from Caco2/BBE cells. In addition, Poly P suppressed the oxidant-induced intestinal permeability in the mouse small intestine and pharmacological inhibitors of p38 MAPK and integrins counteract its protective effect. Daily intrarectal administration of poly P (10 µg) improved the inflammation grade and survival rate in 4% sodium dextran sulfate-administered mice. This study, for the first time, demonstrated that poly P is the molecule responsible for maintaining intestinal barrier actions which are mediated through the intestinal integrin ß1-p38 MAPK.

Oral administration of heat-killed Lactobacillus brevis SBC8803 ameliorates alcoholic liver disease in ethanol-containing diet-fed C57BL/6N mice.

We examined the effect of heat-killed Lactobacillus brevis (L. brevis) SBC8803 on the development of alcoholic liver disease using ethanol-containing diet-fed mice. Heat-killed L. brevis was orally administered at a dose of 100 or 500 mg/kg once a day for 35 days. Alcoholic liver injury was examined by measuring the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in a serum, and the alcoholic fatty liver was assessed from the content of triglyceride (TG) and total cholesterol in the liver. Quantitative RT-PCR was used to examine mRNA expression of tumor necrosis factor (TNF)-alpha, sterol regulatory element-binding protein (SREBP)-1, SREBP-2, and peroxisome proliferator-activated receptor alpha (PPARalpha) in the liver, as well as E-cadherin, Zonula occludens 1 (ZO-1), and heat shock protein (Hsp) 25 in the small intestine. Oral administration of L. brevis significantly inhibited an increase in the level of serum ALT and AST, as well as the content of TG and total cholesterol in the liver caused by ethanol intake. L. brevis supplementation suppressed the overexpression of TNF-alpha, SREBP-1, and SREBP-2 mRNA in the liver induced by ethanol intake and up-regulated the expression of Hsp25 mRNA in the small intestine. These results suggest that L. brevis ameliorated the ethanol-induced liver injury and the fatty liver by suppressing the up-regulation of TNF-alpha and SREBPs in the liver. We speculate that the inhibition of TNF-alpha and SREBPs up-regulation by L. brevis is due to the inhibition of gut-derived endotoxin migration into the liver through the enhancement of intestinal barrier function by the induction of cytoprotective Hsps.

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.

Oral administration of a hop water extract ameliorates the development of dermatitis induced by the periodical topical application of a mite antigen in atopic dermatitis model NC/Nga mice.

We investigated the inhibitory effect of an oral administration of a hop water extract (HWE) on the development of dermatitis by using NC/Nga atopic dermatitis model mice. The induction of allergic dermatitis was conducted by tape-stripping and topical application of a mite antigen (Dermatophagoides farinae) on to the ear once a week for 10 weeks. HWE was orally administered at a dose of 100 or 500 mg/kg. The total immunoglobulin E (IgE) concentration in serum and the ear thickness were periodically examined. Finally, the antigen-specific IgE level in the serum and the production of interleukin (IL)-4, IL-12 and interferon (IFN)-gamma from splenocytes and cervical lymph node cells were measured. The oral administration of HWE significantly inhibited the increase of total IgE production and ear swelling throughout the experimental period. The production of IL-12 was significantly lower in the HWE administered group than in the control group. The results suggest that the intake of HWE may be effective in preventing and alleviating the development of atopic dermatitis-like skin disease.

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.

Clinical effects of a hop water extract on Japanese cedar pollinosis during the pollen season: a double-blind, placebo-controlled trial.

The clinical effects of an oral administration of a hop water extract (HWE) on the improvement of Japanese cedar pollinosis (JCPsis) symptoms were investigated. In a double-blind, placebo-controlled trial, 39 subjects took a drink containing either 100 mg of HWE or a placebo for 12 weeks during the pollen season. Nasal symptoms (sneezing attacks, nasal discharge, and nasal obstruction) were assessed from the subjects' diaries. A clinical examination and blood sampling were carried out before and 4, 8 and 12 weeks after the initiation of treatment. As a result, a significant difference was observed in the symptom score and in the symptom-medication score 10 weeks after the intervention in comparison with the placebo group. Improvements were observed in nasal swelling, nasal color, amount of nasal discharge, and characteristics of nasal discharge in the intervention group 12 weeks after the treatment. No significant eosinophil infiltration into the nasal discharge was apparent in the intervention group throughout the study period, although it was observed in the placebo group. These findings indicate that an oral administration of HWE may be effective in alleviating the allergic symptoms related to JCPsis.

Effects of a hop water extract on the compound 48/80-stimulated vascular permeability in ICR mice and histamine release from OVA-sensitized BALB/c mice.

The antiallergic properties of a hop water extract (HWE) were studied by evaluating the Evans blue leakage from ICR mice caused by compound 48/80 stimulation, and the histamine release from ovalbumin (OVA)-sensitized BALB/c mice. An oral administration of HWE significantly inhibited the vascular permeability and histamine release. HWE itself did not have any influence on the total and antigen-specific immunoglobulin E (IgE) production in OVA-sensitized mice. These results indicate that HWE exerted an antiallergic effect by inhibiting the release of chemical mediators from mast cells and basophiles.

Flavonoid glycosides extracted from hop (Humulus lupulus L.) as inhibitors of chemical mediator release from human basophilic KU812 cells.

The antiallergic properties of hop water extract (HWE) were studied by evaluating histamine release from human basophilic KU812 cells induced by calcium ionophore A23187. HWE significantly inhibited histamine release, but boiling water extract and chloroform-methanol extract did not show any inhibitory effect on it. A 50% methanol-eluted fraction separated from HWE by XAD-4 column chromatography (MFH) had a strong inhibitory effect as compared with HWE. Quercetin glycosides and kaempherol glycosides were identified in MFH, of which quercetin glycosides contributed to the inhibition of histamine release. Most quercetin in HWE existed in glycoside form and its quercetin content, obtained by acid hydrolysis, was about 200 mug/g. HWE and MFH significantly inhibited protein kinase C, which plays a pivotal role in the degranulation of chemical mediators. These results indicate that HWE can inhibit type-I allergic reactions.

Effects of hop extracts on nasal rubbing and sneezing in BALB/c mice.

The effects of hop extracts (Humulus lupulus L.) on histamine release from rat peritoneal mast cells and human basophilic KU812 cells were studied. Hop water extract (HWE) and XAD-4 50% methanol fraction of HWE (MFH) inhibited histamine release from rat mast cells induced by compound 48/80 at concentrations of 100 and 10 mug/ml, respectively. Almost the same findings were observed with A23187-induced histamine release from KU812 cells. Next, we studied the effects of hop extracts on antigen-induced nasal rubbing and sneezing in sensitized BALB/c mice. HWE caused a significant inhibition of nasal rubbing and sneezing at a dose of 500 mg/kg. MFH also inhibited nasal rubbing and sneezing dose-dependently. A significant difference was observed from 100 mg/kg in nasal rubbing and 200 mg/kg in sneezing. The effects of both extracts became clear after repeated administration. HWE and MFH significantly inhibited both nasal rubbing and sneezing, respectively, after consecutive treatment for 15 d at smaller doses compared with single administration. This finding indicates that the active component of hop is included in MFH, which was absorbed to Amberlite XAD-4 and eluted with 50% methanol. These results clearly demonstrated that hop extracts may be effective in the relief of symptoms of allergic rhinitis.