Effects of a bacteria-based probiotic on ruminal pH, volatile fatty acids and bacterial flora of Holstein calves.

Twelve ruminally cannulated Holstein calves (age, 12 ± 3 weeks) were used to identify the effect of a probiotic comprised of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum on ruminal components. The calves were adapted to a diet containing a 50% high-concentrate (standard diet) for 1 week, and then, the probiotic was given once daily for 5 days (day 1-5) at 1.5 or 3.0 g/100 kg body weight to groups of four calves each. Four additional calves fed the standard diet without probiotic served as the corresponding control. Ruminal pH was measured continuously throughout the 15-day experimental period. Ruminal fluid was collected via a fistula at a defined time predose and on days 7 and 14 to assess volatile fatty acid (VFA), lactic acid and ammonia-nitrogen concentrations, as well as the bacterial community. The probiotic at either dose improved the reduced 24-hr mean ruminal pH in calves. The circadian patterns of the 1 hr mean ruminal pH were identical between the probiotic doses. In both probiotic groups, ruminal lactic acid concentrations remained significantly lower than that of the control. Probiotic did not affect ruminal VFA concentrations. L. plantarum and C. butyricum were not detected in the rumen of calves given the high-dose probiotic, whereas Enterococcus spp. remained unchanged. These results suggest that calves given a probiotic had stable ruminal pH levels (6.6-6.8), presumably due to the effects of the probiotic on stabilizing rumen-predominant bacteria, which consume greater lactate in the rumen.

Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli.

A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588)--a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.

Production of apoptosis-inducing substances from soybean protein by Clostridium butyricum: characterization of their toxic effects on human colon carcinoma cells.

Microbial metabolism of soybean constituents is known to produce novel active substances as a chemopreventive agent during the fermentation, and enterobacteria are expected to produce chemopreventive agents as a consequence of metabolizing soybean constituents in the intestinal tract. Then, the conditioned medium was prepared by culturing an enterobacterium Clostridium butyricum (C. butyricum) with soybean protein, and its direct effect on human colon carcinoma HCT116 cells was examined. The conditioned medium was shown to induce the cell death, and suggested to contain novel apoptosis-inducing substances. Thus, enterobacteria are predicted to produce anti-tumor substances from food-derived proteins within the intestinal tract.

Suppressive effect of Clostridium perfringens-produced heat-stable substance(s) on proliferation of human colon adenocarcinoma HT29 cells in culture.

Clostridium perfringens has been regarded as one of the intestinal bacteria increasing colon cancer risk. In previous studies, we have shown that the oral administration of C. perfringens culture medium can inhibit the mutagen-induced formation of pre-neoplastic lesions in rat colon, thus proposing the existence of factor(s) preventing colon tumorigenesis in this culture medium. However, the properties of effective factor(s) and the mechanism of this inhibitory action still remain to be investigated. Then, the effect of C. perfringens culture medium on human colon adenocarcinoma HT29 cells was examined. The exposure of HT29 cells to C. perfringens culture medium was found to suppress the proliferation of these cells probably through the reduction of immediate early gene egr-1 expression. These observations suggest that C. perfringens culture medium has a cytostatic action on colon tumor cells, which may be responsible for the prevention of pre-neoplastic formation in rat colon.

Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions.

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.