| PYF is a phenomenon whereby flocculation competent yeast settles out of the fermentation medium abnormally early during primary fermentation, resulting in a poorly attenuated wort and a final product with undesirable flavor characteristics.At present the beer groups implement centralized management and unified purchase in malt procurement, in order to guarantee taste consistency of the production. If once purchasing the malt with PYF problems, the quality of malt will directly affect yeast physiological performance and finally affect negative impacts on the beer flavor consistency of dozens of brewery, which will cause inestimable economic losses and reputation losses.Aware of the seriousness of this problem, beer manufacturing enterprise gradually tried to incorporate PYF into malt procurement evaluation, and set purchasing standards for PYF index according to the yeast flocculation sensitive properties and quality demands. However, current PYF evaluation method must rely on yeast flocculation performance and is a comprehensive index which reflects the malt PYF activity, yeast flocculation property and yeast vitality on yeast flocculation performance. Malt manufacturers refuse agreeing with this provision because the assay method lacks of objectivity. At the same time each beer group uses yeast strains with different flocculation properties, so PYF assay methods are different, which also brings some obstacles to incorporate the malt PYF index into purchasing standards.In this study, a rapid and sensitive PYF assay method using competitive ELISA detection technology was established for the first time. Compared to the current PYF assay methods, the new one has the advantages of stable, high reliable, and no need to use yeast, which can be applied to the third party assessment to evaluate malt quality. The causative and regulation mechanism of PYF was also researched according to assaying PYF activity of different batches of malt.The main research results are as follows:(1)A rapid and sensitive PYF activity assay method to determine purification efficiency was established at first on the basis of previous study by adjusting several indexes, such as the concentration of yeast suspension, the pH and the concentration of Ca2+of buffer solution.(2)A barley malt factor inducing premature yeast flocculation (PYF) was successfully concentrated using lager yeast as an affinity particle, followed by tangential flow filtration technology, anion-exchange chromatography and gel filtration chromatography. The purified PYF polysaccharide is proved as a single component by HPLC which could significantly promote yeast flocculation, and consists of protein component by UV spectrum assay and SDS-PAGE, with a relative molecular mass of40kDa. It was also found that protein structure is not the necessary trigger component of PYF phenomenon through the protein dispel test.(3)PYF-carrier protein conjugates (PYF-BSA/PYF-KLH) were artificially synthesized by the method using NaI04, and the conjugates of PYF-KLH was determined by UV scanning technology.(4)Polyclonal antibodies were raised through immunizing Balb/c mice and New Zealand white rabbits with PYF-KLH, and the titers of antiserum had reached1:25600and1:204800separately. The optimal working concentration was optimized by ELISA as follows:the dilution ratio of coated antigen was50μg/mL, the dilution ratio of rabbit antiserum and IgG was both10000.(5)A series of PYF samples from brewery have been determined by the competitive ELISA, and the results showed more sensibility, reliability (C.V.<5%) and reproducibility (C.V.<10%) than the former method.(6)According to the influence of the malt PYF activity on the yeast flocculation performance, the malt purchase standard based on this competitive ELISA method was established as follows:OD450of the malt sample<0.513:refuse;OD450of the malt sample>0.579:accept;OD450of the malt sample between0.513and0.579:discount purchase.(7)Methods to decrease the content of PYF activity including malt collocation^malt wash and peel off were studied and proved to be effective. It was also found that the malt polysaccharide was water-soluble and continued to have the PYF patent in the malt-wash water.Because the PYF activity of malt didn’t have additive property, so after mixture it must be assayed again. It was also showed that the PYF activity of mixture malts display a marked difference when the PYF(-)-malt was mixed with different PYF(+)-malts. If mixed with severe PYF(+) malt, the PYF activity will be very high even if accounts for only of10%.So it was strongly recommended that severe PYF(+)malt would not be suitable used for malt collocation. |
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