The Regulation And Metabolism Characteristics Of Intracellular High Trehalose Fermentation In Zygosaccharomyces Rouxii

High-tolerance Zygosaccharomyces rouxii is the main fermenting yeast in soy sauce brewing,which plays a crucial role in the production of soy sauce aroma.The technology of adding soy sauce active dry yeast in the middle and later stages of soy sauce brewing has matured.However,the soy sauce active dry yeast has low activity during preservation,strict preservation conditions,and long shelf life,and this still limits the application of the yeast.As a natural environmental protection agent,trehalose is beneficial to the preservation and fermentation force recovery of Z.rouxii.Therefore,it is of great theoretical and production significance to increase the intracellular trehalose to prepare a highly active Z.rouxii dry yeast.Based on this,this study focused on the following aspects:1.10 L mechanical stirring ventilation fermenter fed fermentation regulation of Z.rouxii.Taking the intracellular trehalose content and biomass of yeast as the main indicators,the effects of feeding methods and feeding rates on trehalose fermentation were studied.The results of the study showed that both constant-speed feeding and variable-speed feeding can increase the intracellular trehalose content and biomass of Z.rouxii.The optimal constant-rate feeding strategy was: 80% glucose was fed at 140 mL/h from 8 h to 16 h as a constant-rate feeding strategy.The maximum biomass was 31.26 g/L and the maximum intracellular trehalose concentration was 1.59%.The optimal speed-feeding strategy was: 80% glucose was fed at 40 mL/h(8~12 h)and 100 mL/h(12~16 h)from 8 h to 16 h as a shift feeding strategy,and the maximum biomass was 35.95.g/L,the maximum intracellular trehalose concentration was 1.71%.2.In order to increase the intracellular trehalose content of Z.rouxii,the effect of constant feed couple with constant temperature induction,variable feed constant couple with constant temperature induction,and variable speed gradient couple with temperature induction on intracellular trehalose was studied on the basis of supplementation.The intracellular trehalose content was used as a reference indicator.In a 20 L mechanically agitated and ventilated fermenter,the temperature was regulated at 35 for 4 h at constant speed feeding and variable speed feeding intervals(12~16 h).?It was found that the intracellular trehalose content of the yeast reached 2.08% at constant temperature and variable speed feeding,increased by 31%,22% and 93% respectively,compared to constant-speed feeding,variable-speed feeding and constant-temperature constant-speed feeding;the maximum biomass reached 45.77 g/L.Further study and optimization of gradient heating and variable-speed feeding coupling strategies established the following optimal fermentation regulation strategies: 8-12 hours at 80 mL/h with 80% glucose,320 mL,and 12-20 hours at 200 mL/h.80% glucose 1600 mL;temperature control 35(12~14 h),37(14~16 h),39 ? ? ?(16~18 h),41 ?(18~19 h),43 ?(19~20)h).Under this strategy,the biomass of Z.rouxii reached 41.12 g/L(22 h),and the intracellular trehalose concentration reached 15.18%(20 h),which was higher than that of variable-speed feeding 7.7 times,constant-temperature constant-speed feeding 14.1 times and constant-temperature variable-speed feeding 7.3 times.3.In order to reveal the metabolic mechanism of intracellular trehalose accumulation in Z.rouxii,gas chromatography was used to analyze metabolic metabolites of glucose metabolism in the fermentation process of variable-speed feeding and variable temperature-controlled coupling.The acetic acid,butyric acid,isobutyric acid,propionic acid,acetoin was identified.Under high temperature induction conditions,the growth of Z.rouxii was inhibited,but the use of reducing sugars was not affected and trehalose accumulated significantly.It can be seen that glucose 6-phosphate,an intermediate metabolite of glycolysis,strengthens the metabolic flux to the trehalose synthesis pathway under temperature stress and weakens the biomass synthesis pathway.At the same time,the organic acid overflow of glucose has existed,but in the process of gradient heating,the metabolism of organic acid,which was originally metabolized by acetic acid in the growth metabolism,turned to the overflow metabolism of organic acid mainly composed of isobutyric acid and propionic acid.