Studying On Improving The Stability Of Beer Flavour By Genetic Modification Of MET10

The poor flavour instability of beer is a obvious problem to the beer quality.So many researchers are very interested in this problem.The contribution of sulfite to control flavour instability of beer would be emphasized in this lecture.First,sulfite inhibits beer oxidation during storage by acting as an antioxidant and avoids the free radical chain reactions.Second, sufite can react with the carbonyl staling compounds in beer,masking stale flavours.Douring the beer brewing ,the concentration of sulfite in beer is rather low because suifite can be deoxidized by sulfite reducase.So constructing a Saccharomyces cerevisiae of high production of sulfite may be one of the best way to solve this problem.In S.cerevisae, MET10 gene encodes a subunit of sulfite reducase.Partial or full deletion MET10 gene resultes in inactivity of sulfite reducase.So sulfite couldn't be deoxidized and would be accumulated in beer.Beer brewed using this mutant S.cerevisae strain may prevent beer oxidation and stabilize the beer flavour.LoxP-KanMX-LoxP gene disruption cassette was constructed from plasmid pUG6 and S.cerevisae by PCR.It consisted of a G418-resistance gene with long fanking homology regions of MET10 gene. With the application of homologous recombination techniques,a resistant gene was transformed to Saccharomyces cerevisiae genome so as to inactivate MET10 by LiAc method and a new mutant strain was abtained。Then,eliminated G418-resistance gene by Cre/Loxp recombinase system from plasmid pSH47,thus an industrial strain with MET10 gene mutation was obtained by test of resistance certification,PCR product ,lead acetate plate.Assaying of sulfite reductase activity by colormetric method ,we discovered that mutant of the enzyme activity than the original strain reduced 22%. This showing that inactivation of MET10 genes could lead to the sulfite reductase activity reduced , While reducted sulfite further downward metabolism in fermentation liqud.At the end of fermentation ,the determination of sulfur dioxide by colorimetric method was carried out, we discovered that the sulphite produced by the mutant strain was 1.5 fold as much as that of the original strain.here was a consistency of their changes.this showing that we obtained modified yeast by genetic engineering technology.The original strain and the MET10 gene-knockouted mutant differed in growth rate and fermentation property in small scale fermentation. The results indicated the mutants have an advantage of acetaldehyde by contrast with the parents, but inferiored fermentation and diacetyl,DMS,Isopentanol . But beer taste was a synthetical index, small scale test and industrial production exsited difference. It still needed more further research.