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Chūō-ku, Japan

Enterococcus faecalis strain N1-33 produces a peptide that shows bacteriocin activity, enterocin MR10A (EntMR10A). We cloned and sequenced the structural gene of the EntMR10A locus (entMR10A), and the peripheral region of entMR10A was analyzed. Inverted repeat-like structure was found for at both ends of entMR10A and so that it suggested entMR10A of strain N1-33 could be formed from an insertion sequence. Attempting to eliminate the plasmid from strain N1-33 using the plasmid-curing agent novobiocin was obtained the mutant strain of N1-33 which seemed to overproduction of bacteriocin than the original strain, termed N41-51. The number of originated bacteriocin structural genes detected by real-time PCR showed that its level of the strain N41-51 was about twofold higher than that of N1-33, and correspondingly appeared to produce about twofold more bacteriocin compared with the original strain in these culture broth, respectively. This difference as a food preservative potential was examined whether it proves the same result in the actual food systems, by using steamed rice. The culture supernatant of strain N41-51 has confirmed to inhibit the growth of Bacillus cereus by significantly less amount added to steamed rice than that of N1-33. Practical Applications: Besides having it with their usual meals, Japanese often eat steamed rice such as rice balls and in their lunch. The most important point we care at these cases is the food poisoning caused by Bacillus cereus, and thus, growth inhibition of B. cereus in steamed rice has been an important task for food industry. In this study, efficacy of the bacteriocin produced by Enterococcus faecalis N1-33 against B. cereus in steamed rice had shown. The difference in bacteriocin activity of the wild-type strain N1-33 and its mutant strain N41-51, which showed about twice as much bacteriocin production, was also reshown well in the actual food system. Also that, the high bacteriocin producing strain of N1-33, termed N41-51, could be obtained with a simple plasmid curing process. © 2015 Wiley Periodicals, Inc. Source

Terato K.,Chondrex Inc. | Do C.T.,Chondrex Inc. | Cutler D.,Chondrex Inc. | Waritani T.,Chondrex Inc. | Shionoya H.,Asama Chemical Co.
Journal of Immunological Methods

To study the possible involvement of potential environmental pathogens in the pathogenesis of autoimmune diseases, it is essential to investigate antibody responses to a variety of environmental agents and autologous components. However, none of the conventional ELISA buffers can prevent the false positive and negative reactions attributed to its principal, which utilizes the high binding affinity of proteins to plastic surfaces. The aims of this study are to reveal all types of non-specific reactions associated with conventional buffer systems, and to re-investigate antibody responses to potential environmental pathogenic and autologous antigens in patients with autoimmune diseases using a newly developed buffer system "ChonBlock™" by ELISA.Compared to conventional buffers, the new buffer was highly effective in reducing the most intense false positive reaction caused by hydrophobic binding of immunoglobulin in sample specimens to plastic surfaces, "background (BG) noise reaction", and other non-specific reactions without interfering with antigen-antibody reactions. Applying this buffer, we found that IgG antibody responses to Escherichia coli O111:B4, E. coli lipopolysaccharide (LPS) and peptidoglycan polysaccharide (PG-PS) were significantly lower or tended to be lower in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), whereas IgA antibody responses to these antigens were equal or tended to be higher compared to normal controls. As a consequence, the IgA/IgG antibody ratios against these agents were significantly higher in patients with RA and SLE, except for Crohn's disease, which showed significantly higher IgG responses to these antigens.To assay antibodies in human sera, it is indispensable to eliminate false positive and negative reactions by using an appropriate buffer system, and to include antigen non-coated blank wells to determine BG noise reactions of invidual samples. Finally, based on our preliminary analysis in this study, we propose that low IgG antibody responses to potential pathogenic environmental factors may be the fundamental disorder in autoimmune diseases. © 2014 Chondrex, Inc. Source

Katayama K.,Katayama Orthopedic Rheumatology Clinic | Katayama K.,Asahikawa Medical College | Matsuno T.,Asahikawa Medical College | Waritani T.,Chondrex Inc. | And 2 more authors.
Nutrition Journal

Background. Environmental factors, particularly commensal bacteria in the gastrointestinal tract, may be involved in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to evaluate whether natural milk antibodies against a wide spectrum of pathogenic enteromicobes and their toxins modify the disease activity in RA. Methods. Twenty patients with RA, whose disease activity was uncontrolled by authentic medications due to drug resistance, complications and/or risk factors were treated for 3 months with an oral administration of a whey protein concentrate (WPC) containing high levels of natural milk antibodies. Eighteen background-matched RA patients, not supplemented with milk antibody adjunct, were used as controls. Results. Statistically significant reduction of arthritis symptoms and improvement of intestinal disorders were observed only in the test group: effective in 8 (44%), possibly effective in 2 (12%) and not effective in 8 (44%) of 18 patients treated (2 patients withdrew) based on an ad hoc "evaluation point", the sum of variables that are improved more than 20% among the 8 core variables used for the American College of Rheumatology (ACR) response criteria. This disease modifying effect of the WPC disappeared upon cessation of treatment, but was reappeared upon reintroduction of it. Importantly, 7 of 8 non-responders carry DR15 haplotype (DRB1-1501 and 1502), whereas only 1 of 7 responders was DR15 positive (risk ratio: 6.1). Furthermore, the pre-clinical serum anti-LPS and anti-type II collagen antibody levels in the responders were higher or tended to be higher than those in the non-responders, suggesting that there are 2 sub-types of RA based on an interaction between gastrointestinal pathogens and MHC class II haplotypes. Conclusions. The natural milk antibody preparation containing high levels antibodies against pathogenic enteromicrobes and their toxins seems to be effective in a certain RA subset, and deserves more attention as a potential adjunct in the treatment of RA. © 2011 Katayama et al; licensee BioMed Central Ltd. Source

Nakayama M.,Kao Corporation | Hosoya K.,Kao Corporation | Shu T.,Kao Corporation | Sato T.,Asama Chemical Co. | And 2 more authors.
Nippon Shokuhin Kagaku Kogaku Kaishi

Lactobacillus fructivorans has been identified as a spoilage bacterium in dressing, since it is strongly resistant to acetic acid. In recent years, the spoilage of dressing has increased in frequency because of the reduced concentrations of both acetic acid and NaCl in dressings. To develop new control methods for L. fructivorans in lowacetic acid dressing, the properties of L. fructivorans were investigated. Doubling time of the bacterium was longer than that of other lactic acid bacteria. In addition, the incorporation rate of fluorescent dye or esterase activity of the bacterium was also lower than observed with Lactobacillus brevis. These results suggest the importance of increasing the incorporation of acetic acid into the bacterial cells. For this purpose, chitosan was employed. Cells treated with chitosan showed a nearly 10-fold increase in the incorporation of SYTO green compared to control cells. Chitosan adsorbed onto the surface of L. fructivorans and increased the surface hydrophobicity of the cells. The chitosan-treated cells localized at the water-oil interface of the dressing, resulting in decreased combined antibacterial action of chitosan and acetic acid. The addition of 0.04% thiamine dilaurylsulfate, an amphipathic antibacterial compound, completely inhibited the growth of L. fructivorans in the dressing with 0.04% chitosan and 3.5% NaCl at pH 4.1, for 60 days at 30°C. Source

Iwatsuki S.,Asama Chemical Co. | Kijima Y.,Asama Chemical Co. | Shionoya H.,Asama Chemical Co.
Nippon Shokuhin Kagaku Kogaku Kaishi

The intake of antibodies in the diet is expected to improve intestinal flora and to have an effect against human pathogens, but reports in this field are scarce. We administered whey protein concentrate (WPC) that was rich in natural milk antibodies against human pathogens to healthy elderly volunteers and analyzed fecal flora by T-RFLP and FISH. T-RFLP data for the intestinal flora showed the presence of 29 bacterial operational taxonomic units (OTUs). The administration of milk antibodies for 3 weeks resulted in the reduction of OTU 369 (Clostridium cluster IV), OTU 469 (Bacteroides), and OTU 853 (Bacteroides), and the increase in OTU 366 (Bacteroides), OTU 443 (unidentified bacteria) and OTU 995 (Clostridium subcluster XIVa). Bifidobacteria and Lactobacilli were not influenced by the administration of milk antibodies. For FISH, fecal samples obtained for 8weeks of WPC treatment were analyzed for bacterial counts by 16S rRNA hybridization. No effect was observed on number of total bacteria and Bifidobacteria were observed. However, numbers of E. coli, Clostridium difficile and C. perfringens decreased. In contrast, Lactobacilli, Bacteroides spp., Prevotella, and Bacteroides fragilis increased. Measurement of milk 1gG in feces following administration of 320 mg of bovine immunoglobulin showed that 800μg (0.24%) was recovered. The influence of administration of milk antibodies on intestinal bacterial flora was discussed from the aspects of endotoxin translocation and improvement of rheumatoid arthritis. Source

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