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Plant-derived lactic acid bacteria are major fermentation organisms that can grow in medicinal herb extracts enriched with phytochemicals like glycosides, phenolic acids, flavonoids, and tannins. Fermentation with strain-specific Lactobacilli harboring metabolic enzymes can increase the bioactivity and bioavailability of medicinal herbs. Lactobacillus (Lact.) plantarum SN13T and Pediococcus (Ped.) pentosaceus LP28 are plant-derived probiotic strains with clinically reported health effects. In the present study, the bioactivity of the extract of the herb, Mentha, against lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells were increased when fermented with Lact. plantarum SN13T as compared to the unfermented extract or extract fermented with Ped. pentosaceus LP28. This higher bioactivity was associated with the metabolism of rosmarinic acid (RA) and caffeic acid (CA), the major bioactive phenolic acids reported in Mentha, along with the production of the metabolite dihydrocaffeic acid (DHCA). DHCA was found to be a more potent LPS-induced nitric oxide (NO) inhibitor than its precursor phenolic acids. The metabolism of RA to DHCA via CA could be mediated by the enzymes cinnamoyl ester hydrolase and hydroxycinnamate reductases, encoded by the ceh gene and the hcrRABC gene operon, respectively, which were identified in the complete genome sequence of Lact. plantarum strain SN13T but were absent in Ped. pentosaceus LP28. The genes hcrA, hcrB, and hcrC were significantly and time-dependently overexpressed in Lact. plantarum SN13T when grown in the Mentha extract, suggesting the role of phenolic acid metabolism in enhancing its bioactivity.

A lactic acid bacterial strain, Lactobacillus plantarum SN35N, which has been isolated from the pear, secretes negatively charged acidic exopolysaccharide (EPS) to outside cells. We have previously found that the SN35N-derived acidic EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35) promoting inflammation. The aim of this study is to find other health benefits of EPS. EPS has been found to exhibit an inhibitory effect against the influenza virus (Alphainfluenzavirus Influenza A virus) and feline calicivirus (Vesivirus Feline calicivirus), which is recognized as a model of norovirus. Although more studies on the structure-function relationship of EPSs are needed, SN35N-derived EPS is a promising lead for developing not only anti-inflammatory agents, but also antiviral substances.

Our previous clinical study has shown that the exopolysaccharide (EPS) produced by a plant-derived lactic acid bacterium

A lactic acid bacterial strain, Lactobacillus plantarum SN35N, which has been isolated from the pear, secretes negatively charged acidic exopolysaccharide (EPS) to outside cells. We have previously found that the SN35N-derived acidic EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35) promoting inflammation. The aim of this study is to find other health benefits of EPS. EPS has been found to exhibit an inhibitory effect against the influenza virus (Alphainfluenzavirus Influenza A virus) and feline calicivirus (Vesivirus Feline calicivirus), which is recognized as a model of norovirus. Although more studies on the structure-function relationship of EPSs are needed, SN35N-derived EPS is a promising lead for developing not only anti-inflammatory agents, but also antiviral substances.

We have recently demonstrated that the exopolysaccharides (EPSs) produced by a plant-derived lactic acid bacterium, Lactobacillus paracasei IJH-SONE68, prevent and ameliorate allergic reaction on contact in dermatitis model mice. In the present study, we conducted a clinical trial using a capsule containing spray-dried powder from pineapple juice broth fermented with the LAB strain as an experimental diet. The clinical trial was conducted as a double-blind and placebo-controlled randomized comparative study from May 2019 to July 2021. Males and females between the ages of 21 and 70 who experience chronic allergies participated in the study. Sixty subjects were instructed to orally take a capsule containing the IJH-SONE68 powder or placebo, every day for 12 weeks. After the clinical trial was over, the scores based on subjects’ self-assessment of allergic status were significantly improved in the intervention group, as compared with the placebo group. Some serum biochemicals associated with inflammation response were also significantly improved by intake of the experimental diet. In conclusion, the IJH-SONE68-derived EPS improves chronic allergy status in humans and is expected to decrease their inconvenience.

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by chronic inflammation of the gastrointestinal tract. IBD includes Crohn’s disease (CD) and ulcerative colitis (UC). CD can occur in any part of the gastrointestinal tract, whereas UC mainly occurs in the colon and rectum. We previously demonstrated that a novel exopolysaccharide (EPS) produced by a plant-derived bacterium, Lactobacillus paracasei IJH-SONE68, prevents and improves the inflammation in contact dermatitis model mice via oral administration. To evaluate the preventive effect of the EPS against other inflammatory diseases, in the present study, we employed dextran sulfate sodium (DSS)-induced UC model mice. The stool consistency, hematochezia, and colonic atrophy of the mice were improved by the orally administered EPS. We also evaluated the cytokine transcription. Overexpression of the mouse macrophage inflammatory protein 2 mRNA in the colon as a functional homolog of human interleukin-8 was decreased by the orally administered EPS. However, the expression of interleukin-10, which is known as an anti-inflammatory cytokine, was stimulated in the EPS-administrated group. Based on these results, we conclude that the IJH-SONE68-derived EPS is a promising lead material for the development of drugs useful in treating inflammatory diseases such as UC.

Fermentation of medicinal herbs can be a significant technique to obtain bioactive compounds. Paeoniae Radix (PR) used in the present study is a well-known herbal medicine that exhibits anti-inflammatory and immunomodulatory activity. The aim of this study is to explore the possibility that a bioactive compound is newly generated in PR extract by fermentation with a plant-derived lactic acid bacteria Lactobacillus brevis 174A. We determined the anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The PR extract fermented with Lactobacillus brevis 174A markedly increased the total phenolic content, decreased intracellular ROS levels, inhibited the release of nitric oxide (NO). It also suppressed inflammatory cytokines IL-6, TNF-ɑ, while simultaneously downregulating the gene expressions of iNOS, IL-6, TNF-ɑ, and IL-1β compared to the unfermented PR extract. Furthermore, the bioactive compound newly generated from the fermentation was identified as pyrogallol. It inhibits the inflammatory responses in a dose-dependent manner suggesting that fermentation of the herbal extract used as a medium together with the plant-derived lactic acid bacterial strain may be a practical strategy to produce medicines and supplements for healthcare.

Cariogenic bacteria, such as Streptococcus (S.) mutans and S. sobrinus, produce insoluble and sticky glucans as a biofilm material. The present study demonstrates that a lactic acid bacterium (LAB) named BM53-1 produces a substance that inhibits the sticky glucan synthesis. The BM53-1 strain was isolated from a flower of Actinidia polygama and identified as Lactobacillus reuteri. The substance that inhibits sticky glucan synthesis does not exhibit antibacterial activity against S. mutans. The cariogenic S. mutans produces glucans under the control of three glucosyltransferase (GTF) enzymes, named GtfB, GtfC, and GtfD. Although GtfB and GtfC produce insoluble glucans, GtfD forms soluble glucans. Through quantitative reverse-transcriptional (qRT)-PCR analysis, it was revealed that the BM53-1-derived glucan-production inhibitor (GI) enhances the transcriptions of gtfB and gtfC genes 2- to 7-fold at the early stage of cultivation. However, that of gtfD was not enhanced in the presence of the GI, indicating that the glucan stickiness produced by S. mutans was significantly weaker in the presence of the GI. Our result demonstrates that Lb. reuteri BM53-1 is useful to prevent dental caries.

As a bacterium used in industry for production of several amino acids, an endotoxin-free Corynebacterium (C.) glutamicum is well known. However, it is also true that the endotoxin-producing other Corynebacterium species is present. An aim of this study is to obtain a lactic acid bacterium (LAB) that produces ornithine and citrulline at high levels. We successfully isolated a strain, designated K-28, and identified it as Weissella (W.) confusa. The production of ornithine and citrulline by K-28 was 18 ± 1 and 10 ± 2 g/L, respectively, with a 100 ± 9% conversion rate when arginine was continuously fed into a jar fermenter. Although the ornithine high production using C. glutamicum is industrially present, the strains have been genetically modified. In that connection, the wild-type of C. glutamicum produces only 0.5 g/L ornithine, indicating that W. confusa K-28 is superior to C. glutamicum to use a probiotic microorganism. We confirmed that W. confusa K-28 harbors an arginine deiminase (ADI) gene cluster, wkaABDCR. The production of ornithine and the expression of these genes significantly decreased under the aerobic condition rather than anaerobic one. The expression level of the five genes did not differ with or without arginine, suggesting that the production of amino acids in the K-28 strain was not induced by exogenous arginine.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.

A clinical study carried out previously by our group has demonstrated that yogurt manufactured with a plant-derived lactic acid bacterium, Lactobacillus plantarum SN13T, significantly reduces the &gamma, glutamyl transpeptidase (&gamma, GTP) level as a liver-function parameter. In the present study, we show that with the oral administration of live SN13T cells, alcohol-poisoning symptoms in mice are improved, and the condition does not become fatal. However, prior to the simultaneous administration with ethanol, when the cells were heat-killed or sonicated, the improvement was not observed, and almost all of the mice died. In addition, the dysbiosis of the intestinal microbiota observed in the mice administered with ethanol was restored by simultaneous administration with live SN13T cells. Furthermore, by analyzing the metabolites detected in contents from the mouse cecum, it was demonstrated that the increase in nonvolatile putrefactive amines observed in the ethanol-administration group was reduced by simultaneous administration with live SN13T cells. Judging from these results, the lactic acid bacterial cells capable of reaching the living bowels prevent ethanol-induced poisoning and restore the intestinal microbiota.

The liver dysfunction is a worldwide health problem. It has been suggested that an imbalance of gut microbiome is associated with a variety of diseases. The goal of the present clinical trial is to evaluate the effectiveness of Lactobacillus plantarum SN13T on hepatic function and fecal microbiota. This study enrolled 22 subjects, aged between 20 and 70 years, and who had any of the following conditions: 40

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0 : 5.7 : 1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626 bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425 bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

In the present study, we have obtained two exopolysaccharide (EPS)-producing thermophilic lactic acid bacteria (LAB) that were isolated from tropical fruits of Thailand. The two strains, designated LY45 and PY45, were identified as Pediococcus pentosaceus and Lactobacillus amylovorus, respectively. Both plant-derived LAB strains, which produce neutral EPSs together with the acidic one, can grow vigorously at 45°C and even at 50°C. Hyaluronidase (EC 3.2.1.35), which catalyzes the degradation of hyaluronic acid, activates an inflammatory reaction. Interestingly, EPSs produced by the LY45 and PY45 strains were found to inhibit hyaluronidase activity at the same order of IC50 values as did sodium cromoglicate and dipotassium glycyrrhizinate, which are well-known as anti-inflammatory agents. The LY45-derived neutral EPS consists of glucose and mannose as monosaccharide components, whereas the acidic one contains mainly mannose, together with glucose and galactose. On the other hand, although Lactobacillus amylovorus PY45 also produces neutral and acidic EPSs, the main monosaccharide in both EPSs is mannose, and glucose is a minor component. Furthermore, the PY45 strain may be probiotically and industrially useful because the microorganism can utilize starch and glycogen as carbon sources.

As a bacterium used in industry for production of several amino acids, an endotoxin-free Corynebacterium (C.) glutamicum is well known. However, it is also true that the endotoxin-producing other Corynebacterium species is present. An aim of this study is to obtain a lactic acid bacterium (LAB) that produces ornithine and citrulline at high levels. We successfully isolated a strain, designated K-28, and identified it as Weissella (W.) confusa. The production of ornithine and citrulline by K-28 was 18 ± 1 and 10 ± 2 g/L, respectively, with a 100 ± 9% conversion rate when arginine was continuously fed into a jar fermenter. Although the ornithine high production using C. glutamicum is industrially present, the strains have been genetically modified. In that connection, the wild-type of C. glutamicum produces only 0.5 g/L ornithine, indicating that W. confusa K-28 is superior to C. glutamicum to use a probiotic microorganism. We confirmed that W. confusa K-28 harbors an arginine deiminase (ADI) gene cluster, wkaABDCR. The production of ornithine and the expression of these genes significantly decreased under the aerobic condition rather than anaerobic one. The expression level of the five genes did not differ with or without arginine, suggesting that the production of amino acids in the K-28 strain was not induced by exogenous arginine.

The population of the obese is increasing worldwide. Prevention and improvement of obesity are indispensable for decreasing the risk of metabolic disorders. We have recently shown that obesity and fatty liver are reduced by a plant-derived lactic acid bacterium, Pediococcus pentosaceus LP28 (LP28), in high-fat diet-induced obese mice. The aim of the present clinical study is to prove that LP28 is effective for reducing body fat and body weight, as shown in the experiment using mice.The clinical trial was carried out as a double-blind, randomized, placebo-controlled study comprising 62 subjects (20-70 years of age, BMI 25-30 kg/m(2)). These subjects were randomly assigned to three groups that received living LP28, heat-killed LP28 or a placebo powder, administered orally once a day for 12 weeks.Heat-killed LP28 reduced BMI (0.45 kg/m(2), 95% CI (0.04, 0.86), P=0.035), body fat percentage (1.11%, (0.39, 1.82), P=0.002), body fat mass (1.17 kg (0.43, 1.92), P=0.004) and waist circumference (2.84 cm (0.74, 4.93), P=0.009) when compared with a placebo group. Fasting plasma glucose, HbA1c, fasting insulin, HOMA-IR and serum lipids levels did not change by either living LP28 or heat-killed LP28 intake.Heat-killed LP28 displays an antiobesity effect that reduces BMI, body fat and waist circumference, suggesting that the plant-derived lactic acid bacterium LP28 would be a promising preventive of metabolic syndrome.

A lactic acid bacterium Lactobacillus paracasei IJH-SONE68, which was isolated from a fig leaf by our group, was found to produce both acidic and neutral exopolysaccharides (EPSs). The nuclear magnetic resonance analysis demonstrates that the former EPS is composed primarily of mannose, and the latter one consists of the α-1, 6-linked glycan chains made of N-acetylglucosamine (GlcNAc). The presence of α-1, 6-linked GlcNAc polysaccharide is first reported in prokaryotes. Furthermore, to reveal the EPS-biosynthetic gene organization in the IJH-SONE68 strain, in the present study, we determined the whole-genome sequence.

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo -derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes , Staphylococcus aureus , and Streptococcus mutans . In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins.

In the present study, we isolated a lactic acid bacterium (LAB) from a citrus iyo fruit and identified it as Lactobacillus brevis. This plant-derived LAB strain, designated 174A, produces bacteriocin consisting of two polypeptides designated brevicin 174A-β and 174A-γ. Although each polypeptide itself displays antibacterial activity, the ability is enhanced 100 fold by mixing both polypeptides at a 1 : 1 ratio. Significantly, brevicin 174A inhibits even the growth of several pathogenic bacteria that are more resistant to a lantibiotic bacteriocin, nisin A, which is commonly utilized as a preservative added to foodstuffs. Structural analysis of the 174A bacteriocin using a program that predicts secondary structure suggests that both component polypeptides have a positively charged N-terminal region, as well as two cysteine residues in both the N- and C-terminals. Judging from a mutational analysis of the antibacterial polypeptides, these unique amino acid sequences of 174A-β might be important for the expression of the synergistic activity that occurs in the presence of the two polypeptides combined.

Background: The comparison of traits in twins from opposite-sex (OS) and same-sex (SS) dizygotic twin pairs is considered a proxy measure of prenatal hormone exposure. To examine possible prenatal hormonal influences on anthropometric traits, we compared mean height, body mass index (BMI), and the prevalence of being overweight or obese between men and women from OS and SS dizygotic twin pairs.

A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.

Despite the need to provide evidence-based health policy, most developing countries suffer from a lack of resources for sound epidemiologic evidence. Most twin registers have been established in developed countries and there are relatively fewer twin registers in developing countries. Considering the immense potential of twin research, it will be worthwhile to attempt to establish a new twin register in Mongolia, where biomedical studies are still scarce. Our objectives were to initiate the process of establishing a nation-wide twin register in Mongolia, based on a nation-wide, population-based database. With the approval and support of the Ministry of Population Development and Social Welfare of Mongolia, we were able to access an initial list of 411 twin pairs who live in the district of Ulaanbaatar, the capital city of Mongolia. By developing a questionnaire to estimate zygosity, we conducted a pilot survey. Those who registered consisted of 822 individuals or 411 twin pairs (same sex: male — 178; female — 157; different sex — 76), two sets of triplets (same sex: female — 2). The age of twins ranged from 1 to 81 (mean age 7.3 ± 11.3), and 52.4% were males. The first twin survey in Mongolia not only resulted in interim data for the Mongolian Twin Register, but has the potential for establishing a larger register by using the national database. It has been proven possible to establish a twin register for research purposes in Mongolia.