The Mechanism Of The Effect Of Pitching Rate On The Metabolism Of Higher Alcohols By Top-fermenting Yeast

Wheat beer is a type of alcoholic beverage which is made from wheat malt and fermented by top-fermenting yeast at high temperature.Compared with barley beer which is common in the market,top-fermented wheat beers,with their huge yield in our country,full-bodied taste and protein rich raw materials,has a good developing prospect.Whereas,it contains a high concentration(300 mg/L)of higher alcohols,which generate incompatible flavors and even damage to human health,thus,the development of top-fermented wheat beers is inhibited.There are many factors affecting the content of higher alcohols in wheat beer.The pitching rate is one of the important factors,which can affects fermentation time,lag phase,the maximum biomass and concentration of flavor-active substances.Despite many studies evaluating the impact of inoculum density on fermentation performance,the results remain inconclusive and has never been assessed systematically.Besides,so far,there are few studies focusing on effect of pitching rate on the production of higher alcohols by top-fermenting yeast in wheat beer fermentation.In this study,the effect of pitching rate on wheat beer fermentation and production of higher alcohols was studied using a top-fermenting industrial brewer's yeast to investigate the impact of pitching rates on the level of higher alcohols and wheat beer quality.We monitored the fermentation process of wheat beer by top-fermenting yeast S-17 and analyzed the relevant indicators,which including yeast growth,fermentation performance and concentration of higher alcohols.The results suggest that the inoculum density is likely not associated with yeast cell proliferation rate during the exponential growth phase when sufficient nutrients are not limiting.Besides,alcohol concentration and the real degree of fermentation exert no significant effects at different inoculum sizes.We also observed that the final free amino nitrogen(FAN)consumption was decreased by approximately 14.58%at the lowest inoculum size.And the final concentrations of n-propanol,isobutanol,isoamylol,active amyl alcohol and 2-phenylethanol produced by top-fermenting yeast were decreased by 25.01%,25.27%,19.51%,25.87%and 22.24%respectively at the lowest inoculum size.For further study,higher alcohols production and FAN consumption during the exponential growth phase and the stationary phase were investigated.We observed that FAN consumption and concentrations of n-propanol,isobutanol,isoamylol,active amyl alcohol and 2-phenylethanol were decreased by 81.3%,70.74%,71.76%,83.60%,80.60%and 77.78%during the stationary phase at the lowest inoculum size.The dynamics analysis result shows that the correlation between higher alcohol synthesis and yeast cell growth is negatively regulated by the inoculum size during the fermentation process.Besides,the result of acidification power(AP)test suggest that a decrease in yeast cell viability at lower inoculum sizes during the stationary phase,which indicating that the ability of yeast to synthesize higher alcohols during the stationary phase is reduced with decreasing pitching rates.We utilized sequencing to investigate the transcript changes under different inoculum sizes.Compared with normal inoculum size,36 significant differentially transcribed genes were obtained,including 3 upregulated and 33 downregulated genes when using the lower pitching rate.The result of Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis shows that the most significant enriched pathways were related to amino acid metabolism.The differentially transcribed genes ALD4,ALD6,AR09,ARO10,PUT1 and DAL80 involved in these pathways,which may affects the formation of higher alcohols in top-fermenting yeast.The recombinant strains were constructed by delete the ALD4,ALD6,AR09,AROIO,PUT1 and DAL80 respectively in the top-fermenting yeast S-17.The result of fermentation experiments shows that the concentration of higher alcohols decreased significantly after the gene ALD4,ALD6,AR09 and AROIO were knocked out,which indicating that these genes are the key functional gene that regulate higher alcohol metabolism at different inoculum sizes.