Regulation Of The Growth And Metabolism Of Brewer's Yeast By Wheat Gluten Derived Bioactive Peptides

Osmotic stress and ethanol are the main stress factors for yeast during very high-gravity(VHG)fermentation,which would result to cell inactivation,the decrease of fermentation efficiency and ethanol production.Thus,improving multiple-stress tolerance as well as the growth and metabolism of brewer's yeast is one of the key strategies for improving fermentation efficiency during VHG fermentation.In this thesis,the effects of wheat gluten hydrolysates(WGH)and their fractions separated by several methods on the growth,viability and fermentation performance of yeast cells were investigated based on the tolerance to sorbitol/ethanol stress and the fermentation performance of yeast during the VHG fermentation.The potential mechanism of improving stress tolerance and fermentation performance of yeast was revealed.Then,the WGH fractions,which could significantly promote the proliferation and metabolism of yeast,were purified and identified,and the fine structures of active peptides were obtained.Finally,the potential molecular mechanisms of the multiple regulation of active peptides affecting the proliferation and fermentation performance of yeast during VHG fermentation were investigated using metabolomics.The new findings of this study would be beneficial to the improvement of high gravity fermentation efficiency and the development of high-quality nitrogen sources.The main research results are listed as follows:(1)The effects of sorbitol and ethanol on yeast growth and vitality were studied.The results showed that the inhibition degree of yeast growth and cell viability were enhanced with the increase of stress factors concentrations.WGH supplementation could significantly enhance the multiple-stress tolerance of yeast and further improve the proliferation and metabolism of yeast under 1.5 M sorbitol or 10%(v/v)ethanol stress.(2)WGH were used to examine their adsorption–desorption kinetics and thermodynamics characteristics on D101,DA-201 E,AB-8,XAD-16,DM130 and HPD-826 macroporous resins.The adsorption kinetics test results showed that WGH could be quickly adsorbed by six macroporous resins.The pseudo-second-order kinetic model was the most suitable model used for describing the whole adsorption process on these resins.The adsorption process of WGH was affected by both intra-particle diffusion and boundary layer diffusion on all resins.Thermodynamic experimental results showed that Langmuir and Freundlich model could illuminate the adsorption mechanism,and the adsorption process was physical,spontaneous and exothermic.Among them,XAD-16 resin showed the most outstanding adsorption and desorption capacity.And there were significant differences in the amino acid composition and molecular weight distribution of the WGH ethanol elution fractions,which was the main reasons for their different effects on the enhancement of proliferation and metabolic,and the improvement of multiple-stress tolerance of yeast.(3)The effects of WGH and WGH-ultrafiltration fractions on sorbitol stress and ethanol stress tolerance in yeast were evaluated.The component of Mw <1 kDa in permeate fraction(WGH2)could be markedly increased to 86.50% by ultrafiltration.It was found that WGH and WGH2 could strongly improve the tolerance of yeast to osmotic stress and ethanol stress,especially WGH2.In addition,the tolerance of yeast was closely related to the cell membrane integrity,mitochondrial membrane potential(??m)and intracellular reactive oxygen species(ROS)level.Meanwhile,multiple stress tolerance of yeast was enhanced due to the addition of WGH supplementation which could improve membrane integrity,reduce ROS,and maintain the stability of ??m in yeast cells.(4)The effects of WGH and their fractions(ultrafiltration fraction and macroporous resin fraction)on the physiological activities and fermentation performance of yeast in VHG fermentation of two fermentation broths were revealed.WGH and their fractions supplementations could improve the cell growth,cell viability and fermentation performance of yeast during VHG fermentation.Moreover,the XAD-16 macroporous resin 40% ethanol fraction(WGH5)showed the best fermentation-promoting activity in both fermentation broths.The physiological activity and fermentation performance of yeast were improved during VHG fermentation,which was mainly due to that WGH fractions supplementation enhanced the content of intracellular protective substances(such as glycerol and trehalose),and reduced the cell membrane permeability,and maintained the stability of ??m,and reduced the level of intracellular ROS.(5)The effects of WGH active fractions on the multiple-stress tolerance of yeast were investigated.The small molecule peptides of LL,LW,EP,LLL,LLW,LML,LPL and EFPL from the WGH5 were isolated and identified by C18 column chromatography combined with UPLC-ESI-MS/MS.And then the effects of 8 small molecule peptides on the fermentation performance of yeast under high gravity conditions were evaluated.The results showed that LL,LL and LML supplementations could improve proliferation and fermentation performance of yeast.Related mechanism studies found that the LL,LLL and LML supplementations increased alcohol dehydrogenase(ADH)activity of yeast,and improved the integrity of cell membrane,and decreased the levels of intracellular ROS.The decrease of ROS levels was mainly due to the increased enzyme activities of catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in antioxidant enzyme system,and increased the content of reduced glutathione(GSH)in non-enzymatic antioxidant systems.(6)The effects of LL supplementation on the expression of intracellular metabolites in yeast during VHG fermentation were investigated by metabolomics.12 major differential metabolites were screened by principal components analysis and partial least squares projection to latent structure-discriminant analysis.The addition of LL resulted in increases of 8 metabolites but decreases of 4 metabolites in the yeast cell compared to the control group,in which glycerol,DL-glutamine,lysine,phenylalanine,norleucine and malic acid have significant expression.These major differential metabolites were mainly participated in sesquiterpenoid and triterpenoid biosynthesis,sphingolipid metabolism,pyruvate metabolism,purine metabolism,glycerolipid metabolism,and so on.