Generation of a uracil auxotroph strain of the probiotic yeast Saccharomyces boulardii as a host for the recombinant protein production

Saccharomyces boulardii [S. boulardii] is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA [5-Fluoroorotic acid], uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmaceutics into the intestinal lumen

[ antimicrobial effect of zatariamultifloraboiss essential oil against E.coli O157: H7 in minced beef during refrigerated storage as a replacement for chemical preservatives in order to maintain the consumers health]

Today, the consumers have more knowledge about the unwanted effects of synthetic antimicrobials in foods and they want food with more natural ingredients. There are many researches about antimicrobial effects of essential oils, but we did not found researches about Zatariamultiflora Bios on E.coli O157: H7 in minced beef. The aim of this study was to determinre the effect of Zatariamultiflora Bios essential oil on E.coli O157: H7 in minced. The antimicrobial effect of different concentrations of Zatariamultiflora Boiss essential oil at supplementation levels of [0, 0.005, 0.015, 0.03%] on E. coli O157: H7 was examined in minced beef. All of the above concentrations showed acceptable organoleptic properties in minced beef. Eo at 0.03% possessed a strong antibacterial activity against E.coli O157: H7 in minced beef. The correlation coefficient of different concentrations of Zatariamultiflora Boiss essential oil with logarithm of the numbers of E. coli O157: H7 was -0.701, -0.599 at 4 and 10°C respectively. It was found that effect of different concentrations of essential oil on growth rate of E. coli O157: H7 was statistically significant [p<0.01]. In this study, it was found that treatment of minced beef with different concentrations of essential oils showed an inhibitory activity against E. coli O157: H7 during storage at 4°C, but not at 10°C [p<0.01]. According to present study, storage at a proper refrigerated storage like 4°C is suggested. Zatariamultiflora Bioss essential oil can use as a natural preservative instead of chemical preservatives for meat products