Premature Yeast Flocculation Induced By Barley Malt

The premature yeast flocculation(PYF) phenomenon has always been a problem for a long time in the brewing industry. Because of the mechanism of PYF, especially the PYF inducing substrates in barley or malt(PYF factors), have not been clearly elucidated, it effects on the beer brewing and restricts the development of beer industry. Also it causes great economic loss in brewing industries including the maltsters and brewers. In recent years, due to the environment pollution and climate change, PYF happened during brewing with increasingly frequency. In this study, the location of PYF factors in barley malt was identified firstly. And then, the characteristics and the formation of PYF factors were analyzed. Finally, feasible strategy of PYF improvement and control was put forward. The study will provide theoretical guidance and practical production methods for PYF elimination in the barley malt processing. The main process and results were as follows:(1) Nineteen malt samples collected from commercial maltsters were identified to exhibit variable PYF potential, even if that with same barley variety but produced in different maltsters or harvested in different years. The main factors that would affect the malt PYF potential include the malting conditions, the harvest year and growth area of barley.(2) Four PYF positive(PYF+) malts(PYF+1, PYF+3, PYF+5, PYF+6) were selected by detecting the suspended yeast cells during brewing process and the contents of residual sugar and alcohol in final fermentation liquids. By husk exchanging between PYF+ and the PYF negative(PYF-) malts, PYF potential was eliminated in PYF+1 malt, while remained in PYF+5, PYF+3 and PYF+6 malt. In addition, the PYF- malt got PYF potential when changed the husk of PYF+5, PYF+3 or PYF+6 malt. It could be deduced that PYF factors were present in both malt husk and non-husk part. By comparating the properties of two parts PYF factors, it was found that different molecular weight of polysaccharides could induce PYF. However, PYF factors from both part were with high branching extent of the arabinoxylan.(3) By comparing constitution of worts from PYF+ and PYF- malts that produced from the same variety by the same maltster, PYF+ wort was found to contain more bound ferulic acid(FA) rather than arabinxylan(AX). With fractional ethanol precipitation, the polysaccharides precipited by 40% ethanol in PYF+ wort could induce severe PYF, the more polysaccharides the severer PYF. Furthermore, these part of precipitants were found to with molecular weight of 20~27 k Da containing more FA and branches than that of PYF- wort.(4) Treated with either bacterial(Bacillus subtilis) or fungal(Aspergillus niger) xylanases, the PYF- malt husk could induce severe PYF. In addition, with both treated husks, the FA content and FA/AX value in 40% ethanol precipitants of wort were elevated. The effect of more bacterial xylanase on PYF factors formation seemed greater than that of more fungal xylanase. At the same time, addition of B. subtilis xylanase at the beginning of mashing process could also induce PYF, the FA content was significantly increased when 500 U·50 g-1 malt of xylanase was added. However, there was not the same effect with addition A. niger xylanase.(5) By the Illunima Miseq sequencing analysis, the diversity of bacteria and fungi microbial community in the PYF+ malt was greater than the PYF- malt, even at every microbial classification order, family, genus or species level. Based on the gene database, 14 bacteria species and 6 fungi species in the PYF+ malt, and 11 bacteria species and 6 fungi species in the PYF- malt belong to xylanase producing genus. The species probably including xylanase gene in the PYF+ malt were mainly Pseudomonas geniculata, Pseudomonas sp. S1, Pseudomonas anguilliseptica, Lactobacillus sakei, Lactobacillus mali, Chryseobacterium hispanicum, Chryseobacterium hominis, Flavobacterium sp., Pyrenophora Chaetomium, Pyrenophora teres, Aspergillus niger. For the PYF+ malt, the main microorgnisms associated with PYF were Lactobacillus sakei, Lactobacillus mali, Flavobacterium sp., and Aspergillus niger.(6) The proteome profiles(p H 6-11) of PYF+ and PYF- malt were compared by two-dimensional electrophoresis(2DE). Twenty-one spots with qualitative and quantitative differences were found and 8 spots were identified by MALDI-TOF/TOF. Among the different proteins, 2 spots identified as 26 k Da endochitinase 1 were found to be with more contents in PYF- malt than that in PYF+ malts. In addition, the chitinase activity in PYF- malt were significantly higher than that in PYF+ malt.(7) The recombinant Hordeum vulgare L. xylanase inhibitor(HVXI) was cloned and expressed in Pichia pastoris X33. With purified r HVXI adding to the mashing process, the PYF was slightly inhibited. In addition, the purified r HVXI was capable to inhibit the hydrolysis of AX by arabinoxylanase and thus deminish the PYF.