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Fujimoto J.,Yakult Central Institute for Microbiological Research | Tanigawa K.,Yakult Central Institute for Microbiological Research | Kudo Y.,Yakult Central Institute for Microbiological Research | Makino H.,Yakult Honsha European Research Center for Microbiology | Watanabe K.,Yakult Central Institute for Microbiological Research
Journal of Applied Microbiology | Year: 2011

Aims: To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces.Methods and Results: The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 105-109 cells per g of faeces was enumerated. After 11 healthy subjects ingested 10.7 log CFU of BbrY daily for 10 days, 6.9 (±1.5) log CFU g-1 [mean (±SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7.5 (±1.0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8.1 (±0.8) log cells per g.Conclusions: Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately.Significance and Impact of the Study: Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.


Kurakawa T.,Yakult Central Institute for Microbiological Research | Kubota H.,Yakult Central Institute for Microbiological Research | Kubota H.,Yakult Honsha European Research Center for Microbiology | Tsuji H.,Yakult Central Institute for Microbiological Research | And 6 more authors.
Journal of Microbiological Methods | Year: 2013

A primer set specific for Escherichia coli/Shigella 16S rRNA was developed and used for RT-qPCR analysis of fecal samples from 35 healthy adult volunteers in combination with the previously reported primer set specific for Enterobacteriaceae. Enterobacteriaceae and E. coli were present in the 29 out of 35 volunteers tested as intestinal commensals at the average population levels of 107.1±0.9 and 106.8±0.7cellsg-1 of stools (mean±standard deviation), respectively. Among the 7 volunteers, the significant deviation between the count of Enterobacteriaceae and that of E. coli was observed, suggesting non-E. coli/Shigella species were predominant in their Enterobacteriaceae populations. The clone library analysis revealed that the non-E. coli/Shigella populations included Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae/variicola and Morganella morganii. These data suggested that the RT-qPCR method with the primer sets specific to both Enterobacteriaceae and E. coli/Shigella enabled the accurate enumeration of intestinal E. coli populations and the other Enterobacteriaceae species populations. © 2012 Elsevier B.V.


Ito M.,Yakult Central Institute | Oishi K.,Yakult Honsha European Research Center for Microbiology | Yoshida Y.,Yakult Central Institute | Okumura T.,Yakult Central Institute | And 4 more authors.
Beneficial Microbes | Year: 2015

We investigated the effects of Streptococcus thermophilus YIT 2001, a strain of lactic acid bacteria, on the susceptibility of low-density lipoprotein (LDL) to oxidation and the formation of aortic fatty lesions in hyperlipidemic hamsters. S. thermophilus YIT 2001 had the highest in vitro antioxidative activity against LDL oxidation among the 79 strains of lactic acid bacteria and bifidobacteria tested, which was about twice that of S. thermophilus YIT 2084. The lag time of LDL oxidation in the YIT 2001 feeding group was significantly longer than in controls, but was unchanged in the YIT 2084 group. After the feeding of YIT 2001, lag times were prolonged and areas of aortic fatty lesions were dose-dependently attenuated, although there were no effects on plasma lipid levels. These results suggest that YIT 2001 has the potential to prevent the formation of aortic fatty lesions by inhibiting LDL oxidation. © 2014 Wageningen Academic Publishers.


Makino H.,Yakult Central Institute for Microbiological Research | Makino H.,Yakult Honsha European Research Center for Microbiology | Fujimoto J.,Yakult Central Institute for Microbiological Research | Watanabe K.,Yakult Central Institute for Microbiological Research
International Journal of Food Microbiology | Year: 2010

Yeast contamination is a problem in the food industry as a cause of spoilage. Moreover, various species of yeasts are known to be capable of causing opportunistic infections in humans. We have developed a real-time quantitative PCR (qPCR) assay to directly detect and quantify nine emerging opportunistic yeast species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Filobasidiella neoformans, Issatchenkia orientalis, Trichosporon asahii, and Trichosporon jirovecii) in dairy product samples. We designed six primer pairs, conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, to detect the yeasts and demonstrated their specificity. The qPCR assay could accurately quantify emerging opportunistic yeasts in an artificially contaminated dairy product. qPCR with the primer pairs we designed, was very sensitive and will allow producers to enumerate contaminating yeasts and identify whether they are opportunistic pathogens, in only 4 to 5. h. This assay can easily be extended to other food items and to a variety of food-monitoring initiatives. © 2010 Elsevier B.V.


Akiyama T.,Yakult Honsha European Research Center for Microbiology | Kimura K.,Yakult Central Institute | Hatano H.,Yakult Central Institute
Bioscience, Biotechnology and Biochemistry | Year: 2015

Galactooligosaccharides (GOS) possess prebiotic properties that speci fically increase the number of bifidobacteria in the human intestine, thus giving health benefits to the host. Although the bifidogenic effect of GOS has been demonstrated in numerous studies, the utilization of GOS by specifi c bifidobacteria remains unclear. The goal of our study was to elucidate GOS consumption by specific bifidobacteria and gain insights into the mechanism. First, we examined GOS consumption by 14 bifidobacterial strains belonging to seven different species by comparing growth rate, carbohydrate consumption, and acid production. We then performed a transcription analysis in the case of one strong GOS consumer, Bifidobacterium adolescentis YIT 4011T, to predict the operon contributing to GOS use. The study indicated the contribution of an operon consisted of LacS symporter and β-galactosidase to bifidobacterial GOS consumption. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry.

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