Yasushi Kawai

Biosorption of heavy metals by lactic acid bacteria and identification of mercury binding protein

Heavy metals cause various health hazards. Using lactic acid bacteria (LAB), we tested the biosorption of heavy metals e.g. cadmium (Cd) (II), lead (Pb) (II), arsenic (As) (III), and mercury (Hg) (II). Cd (II) sorption was tested in 103 strains using atomic absorption spectrophotometery (AAS). Weissella viridescens MYU 205 (1 × 10(8) cells/ml) decreased Cd (II) levels in citrate buffer (pH 6.0) from one ppm to 0.459 ± 0.016 ppm, corresponding to 10.46 μg of Cd (II). After screening, 11 LAB strains were tested using various pH (pH 4.0, 5.0, 6.0, 7.0) showing the sorption was acid sensitive; and was cell concentration dependent, where the Cd (II) concentration decreased from one ppm to 0.042 (max)/0.255 (min) ppm at 1 × 10(10) cells/ml. Additionally, the biosorption of Pb (II), As (III), and Hg (II) were tested using an inductively coupled plasma mass spectrometer (ICP-MS). The Hg (II) concentration was reduced the most followed by Pb (II) and As (III). Many of the bacterial cell surface proteins of W. viridescens MYU 205 showed binding to Hg (II) using the Hg (II) column assay. Having a CXXC motif, a ∼14 kDa protein may be one of the Hg (II) binding proteins. LAB biosorption may aid the detoxification of people exposed to heavy metals.

Identification of a new adhesin-like protein from Lactobacillus mucosae ME-340 with specific affinity to the human blood group A and B antigens.

Aims:  To identify and characterize a new adhesin‐like protein of probiotics that show specific adhesion to human blood group A and B antigens.

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyers patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

Food preservative potential of gassericin A‐containing concentrate prepared from cheese whey culture supernatant of Lactobacillus gasseri LA39

Gassericin A (GA) is a circular bacteriocin produced by Lactobacillus gasseri LA39. In this study, GA-containing concentrate was prepared using a cross-flow membrane filtration device (30 kDa cut-off) from the culture supernatant of Lb. gasseri LA39 cultivated in a cheese whey-based food-grade medium. The bacteriocin activity titer in the concentrate was 16 times as high as that of the culture supernatant and was completely maintained through each incubation at 4°C for 3 months, 37°C for 2 months, 60°C for 5 h, and 100°C for 30 min. The GA-containing concentrate was used with glycine powder to make custard creams, and then four representative strains of custard cream spoilage bacteria (Bacillus cereus, Lactococcus lactis subsp. lactis, Achromobacter denitrificans and Pseudomonas fluorescens) were individually inoculated at c. 10(3) colony forming units/g in the custard creams. Throughout 30 days of incubation at 30°C, all of the inoculated bacteria were completely inhibited by the combination of 5% (w/w) of the GA-containing concentrate and 0.5% (w/w) glycine. This is the first highly practical application of GA to foods as a biopreservative, and the concentration method and the bacteriocin concentrate would contribute to biopreservation of several foods.

An Adhesin-Like Protein, Lam29, from Lactobacillus mucosae ME-340 Binds to Histone H3 and Blood Group Antigens in Human Colonic Mucus

A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation.

BackgroundPreviously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB).ResultsAll toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3).ConclusionsBIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.

New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin.

Nisin A extends the shelf life of high-fat chilled dairy dessert, a milk-based pudding.

The aims of this study were to evaluate the effectiveness of nisin A to control the growth of spore‐forming bacteria, Bacillus and Paenibacillus, in chilled high‐fat, milk pudding and to reduce heat treatment to improve aroma and flavour.

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time-consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle

Abstract We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.