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A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017.

The influence of Nisaplin, which contains 2.5% nisin, on the growth of Listeria monocytogenes in Karashi-mentaiko (red-pepper seasoned cod roe) was investigated. The MICs of Nisaplin for L. monocytogenes (10(8) CFU/mL) were measured; seven isolates showed a value of 1,600 microg/mL and one isolate showed a value of 800 microg/mL. All L. monocytogenes isolates had a MIC of 800 microg/mL at 10(6) CFU/mL. The number of L. monocytogenes in Karashi-mentaiko stored at 4 degrees C was decreased by Nisaplin added at 60 and 600 microg/g. These results indicated that Nisaplin effectively inhibits the growth of L. monocytogenes in Karashi-mentaiko.

The effects of tea polyphenols on fecal flora and fecal metabolic activities were studied in eight 30-day-old pigs fed a basal diet (control) or the diet supplemented with 0.2% of tea polyphenols for 2 weeks. During tea polyphenol administration compared to before administration, the levels of lactobacilli were increased significantly (P < 0.05 and P < 0.01 on days 7 and 14 of administration, respectively), whereas the levels of total bacteria (P < 0.01) and bacteroidaceae (P < 0.01 and P < 0.001 on days 7 and 14 of administration, respectively) were decreased significantly. The detection rate of lecithinase positive clostridia including Clostridium perfrigens showed a tendency to decrease during administration. No detectable change occurred in the levels of other organisms throughout the experimental period. Fecal concentrations of ammonia were decreased significantly (P < 0.05 and P < 0.01 on days 7 and 14 of administration, respectively). Fecal phenol (P < 0.001), p-cresol (P < 0.05), and skatol (P < 0.01) were reduced significantly on day 14 of administration compared to before administration. The amounts of fecal short chain fatty acids, acetic acid (P < 0.05) and lactic acid (P < 0.05) were increased significantly on day 14 of administration. The pH values were decreased slightly during administration. The odour of the feces was reduced remarkably during tea polyphenol administration.

The effects of lactosucrose (4G-β-D-galactosylsucrose) on fecal flora and fecal putrefactive products were studied in 3 Himalayan and 5 Persian cats fed 175 mg of lactosutrose/each/day for 2 weeks. During lactosucrose administration, the counts of lactobacilli increased significantly (p

The present study is the first to report the hypocholesterolemic effect of chitosan on humans. When 3–6 g/day of chitosan was given in the diet to 8 healthy males, total serum cholesterol significantly decreased, and when the ingestion was stopped, the value increased to the level before ingestion. Serum HDL-cholesterol was increased significantly by the ingestion of chitosan. The excreted amounts of primary bile acids, cholic acid and chenodeoxycholic acid, into the feces was significantly increased by the ingestion of chitosan, and the amount of cholic acid excretion decreased significantly after the ingestion was stopped. These facts suggest that chitosan combined bile acids in the digestive tract, and that the combined product was excreted into the feces. This, in turn, deceased the resorption of bile acids, so that the cholesterol poool in the body was decreased and the level of serum chrolesterol consequently decreased.

The anti-infectious effect of probiotics has recently been reported and one mechanism may be the non-specific stimulation of immunity. This study was performed to elucidate the influence of a probiotic formula on intestinal microflora and local immunity in healthy children. A follow-up formula containing viable bifidobacteria was given to seven healthy Japanese children (15 to 31 months old) for 21 days. During intake of the formula, the administered strain was detected in feces from five subjects (71%) and total fecal bifidobacteria slightly increased. Fecal levels of total IgA and anti-poliovirus IgA during intake of the formula were significantly higher than those before intake (P < 0.05). The increase in local IgA levels resulting from ingestion of the probiotic formula may contribute to enhancement of the mucosal resistance against gastrointestinal infections.