S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.
Sake (Japanese rice wine) has been recognized as being low-risk in terms of its microbiological safety. However, a confirmation of the food safety aspects of sake based on scientific evidence is important for establishing consumer confidence, in part because consumer concerns regarding food safety have increased. The presence of Bacillus cereus spores in refined rice wine has been reported, and in light of consumers’ growing concern over food safety, the establishment of food and beverage safety is important for consumers’ reassurance. Herein, to confirm the microbiological safety of sake, we investigated the content and growth of B. cereus. We conducted a spore addition test to determine whether B. cereus spores grow during sake production, and we observed no growth or germination of B. cereus spores during the manufacturing process. We also observed that processes such as solid-liquid separation and filtration help remove the risk posed by B. cereus. We then conducted a survey to assess the density of B. cereus in various commercial sake products. We analyzed 162 samples of commercial sake and observed that 11 of the products had ≥1 CFU of living cells in 1 mL of sake (detection rate: 6.8%). There was no product in which ≥100 CFU/mL-sake of living cells was detected. Our findings confirmed that the density of these bacteria in sake is lower than that in other foods, and the probability of infection is very low. The emetic toxin produced by B. cereus was not detected in any of the sake samples. This is the first study based on experimental data demonstrating that B. cereus is not able to grow in sake or during the sake manufacturing process. We thus conclude that the safety risk of B. cereus in sake is negligible. Our findings indicate that Bacillus cereus is not a significant hazard in the sake brewing process, and they will contribute to the food hygiene management based on scientific evidence in sake breweries.
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