Breeding Of Beer Yeast Strain With Lower-level Proteinase A

Beer foam is an important indicator of beer quality. With the rise of draft beer in both international and domestic market, more attention is focused on the foam stability of draft beer. The proteinase A excretes by yeast is recognized as the most important negative factor which influence foam stability. In this study, ultraviolet mutagenesis, ion-beam mutation was adopted for breeding yeast with low proteinase A excretion to solve the foam stability of draft beer fundamentally.Five screening methods were compared in this study, including acid denatured bovine hemoglobin plate, CPY plate, 96-well plate, Bradford method and fluorescent substrate. 96-well plates and Bradford method were used as preliminary and second screening method, respectively, fluorescent substrate is introduced to determine the final content of proteinase A.Ultraviolet mutagenesis is adopted commonly in mutagenesis. The original brewing yeast Mâ… 4 was induced by ultraviolet and four brewing yeasts with lower-level proteinase A were bred. Compared with the original strain, the proteinase A activity of four mutation strains decreased in different rate, in which three mutants were reduced with lager extent.N+-ion-beam mutation is a new method in recent years. The entire process experience physical, chemical and biological four stages, which is a composite mutagenesis, with advantages as high efficiency, little injury and so on. This study is the first report using this method on beer yeas. Mutating by this mean, two mutation strains were got from the original yeast Mâ… 4. Compared with original strain, the proteinase A activity of the two mutation strains decreased different rate, but the reduction is not significantly as the UV mutagenesis.Fermentation experiment found that fermentation performance of mutation strains reduced with distinct range. The results of EBC tube showed that comparing with erlemeyer flask fermentation, the fermentation capability of mutation strains improved with different rate, but which is still lower than the original strain. The fermentation indicators of mutations M^I₄-A and M^I₄-D are close to original strain, which have an industrial application foreground.Gene pep4 encode proteinase A. Analysis and comparison the gene of both original strain and mutation strains found that there are seven amino acid substitutions and one synonymous mutation in the four ultraviolet mutation strains. The acid amino of M^I₄-A are L8S and K387I, M^I₄-B are S16G, K39R, D393E, N397K and A402T, M^I₄-D had a synonymous mutation occurred in 399, and no mutation was occurred for M^I₄-C, the two N+-ion-beam mutation strains had a synonymous mutation for M^I₄-F and no mutation for Mâ… 4-E.