Studies On Foam Stability Of Unpasteurized Beer Based On Saccharomyces Cerevisiae

Yeast proteinase A (PrA) [EC3.4.23.25], a proteolytic enzyme found in the vacuole of Saccharomyces cerevisiae, is involved in non-specific proteolysis during starvation and proteolytic maturation of other vacuolar enzymes. Proteinase A can. be excreted into beer under stress or by yeast autolysis and is detrimental to beer foam stability. Three detecting methods in yeast proteinase A (PrA) assay including UV absorbance, Lowry method and modified Bradford method were compared when using casein or hemoglobin as substrates. The result indicated that PrA was hardly detected by UV absorbance and Lowry method was not ideal. Modified Bradford method was found to be appropriate for determining PrA when substrates with low affinity to its enzyme. By comparing the effect of erizyme'and substrate concentration on PrA activity, the method for measuring PrA was established, using casein as substrate and detecting products by modified Bradford method Saturation of ammonium sulfate for salting out PrA was also compared, the result showed that 70 to 80% and 50% saturation were suitable for extracting PrA from yeast and unpasteurised beer, respectively.The condition of elctrophoresis was optimized with different Tris and acrylamide concentrations and different staining methods. The patterns of SDS-PAGE of these samples indicated that 12.5% acrylamide with 0.75 M Tris or 15% acrylamide with 0.6 M Tris concentration and silver-staining method was found to be appropriate for directly separating beer and its foam proteins without any concentration and purification. There were at least seven protein bands shown on the silver-stained gel after separating beer and foam with electrophoresis, and the main foaming proteins of beer-were protein Z (40 KDa) and lipid transfer protein (LTP1, 10 KDa). The SDS-PAGE pattern of beer samples hydrolyzed by crude yeast proteinase A indicated that 10 KDa of protein was degraded.Head retention, enzyme activity of yeast proteinase A, protein content and pH in unpasteurised beer stored at different temperatures were tested. The result indicated that all of the four indexes decreased during the storage period. The composition of beer foam proteins also changed during the storage time identified by SDS-PAGE. The pattern of electrophoresisindicated that 10 KDa of protein was degraded, which indicated that this protein was closely relative to yeast proteinase A and foam stability.Yeast proteinase A from fresh beer was firstly purified with Sephadex G-100 column chromatography and the active fractions reached to 5.3-fold purification with 7% of yield. After purification with DEAE Sephadex A50, proteinase A activity increased to be 10.1 times of the initial with 1% of yield. When identifying the sample from chromatography by SDS-PAGE, only one protein band with 42 kDa was observed, this indicated that the enzyme was purified. The pattern of electrophoresis of hydrolyzed beer by crude proteinase A did not show lipid transfer protein on the gel. The result of SDS-PAGE of interaction mixture of purified proteinase A and beer also indicated that lipid transfer protein was decomposed: PCR-mediated gene knock-out was used in this study. PUG6 was used as template for polymerase chain reaction, region 5' of the upprimer and downprimer was respective homologous to the region upstream and downstream of the ORF of proteinase A and region 3' was respective homologous to 3' of the KanMX gene in PUG6. The PGR product could be directly used for gene disruption. The method of transformation of intact yeast cells with lithium acetate was used and the transformants could be obtained from YEPD plus geneticin(G418) plates. By PCR verification of transformants genome DNA, the transformants were confirmed to PEP4 gene deletion strains, which were Saccharomyces cerevisiae WZ65/a, WZ65/b, WZ65/c 和 WZ65/d.The physiological characteristics of the transformants were tested including genetic stability, growing performance, fermentablity, flocculation ability, heat-resistance temperature, etc. Proteinase A activity was also measured and the proteins from the transformants were identified by electrophoresis. The results indicated that proteinase A was not expressed in these transformants, however the KanMX gene was lost and the transformants were not resistant to geneticin. The genetic performance was stable and the growing performance, fermentablity, flocculation ability and heat-resistance temperature of these transformants were similar to each other and also similar to the control strain.Saccharomyces cerevisiae WZ65/a was used to brew beer in 250L fermentation tank.