Effects Of Exogenous Trehalose On The Physiological States States Of Brewer's Yeasts

During industrial beer production,the brewing process and the reusing of yeast slurry cause stress to yeast cells.Yeast storage is an indispensable period in industrial beer fermentation.With prolonged storage period and increased temperature,yeast cells experience compromised physiological states and even the autolysis process,causing negative effects on the quality of beer products.Trehalose serves as a source of energy and carbon in organisms and plays a protective role under a variety of stress conditions.It is of great importance to study physiological responses of yeast and the protective effects of trehalose on yeast in the storage period of beer production,which has a practical significance to beer production and contributes to the promising application of trehalose in the beer-brewing process.In this research,the physiological evaluation indicators of yeast are studied;then the effects of trehalose on yeast in storage and its protective mechanism are evaluated by determination of cell viability,cell vitality,stress tolerance,regulation levels of mRNA and etc.Two lager yeasts with different fermentation abilities,G-03?Saccharomyces pastorianus?and Pilsner?Saccharomyces pastorianus?,as well a typical ale yeast,M79?Saccharomyces cerevisiae?,were adopted in this research.The main results of the research are as follows:?1?The study of parameters for evaluating the physiological states of brewer's yeasts:during 4-day ageing at 25?,the physiological state of G-03 deteriorated.The ATP content correlated with the viability?r=0.9943?;and the intracellular pH value correlated with the viability as well?r=0.9160?.At 4?,a correlation was also observed between the ATP content and the viability?r=0.7640?,while no correlation was found between the intracellular pH value and the viability.The intracellular pH value failed to reflect the physiological differenced that could be shown by ATP contents and viability.When the relationship between intracellular ATP contents and viability in other strains was investigated,it was found that no matter viability changed significantly or not,intracellular ATP contents changed significantly.Therefore,intracellular ATP content is a sensitive indicator that could reflect subtle changes in yeast physiology.?2?The effects of trehalose on the physiological states of brewer's yeast in storage:the three yeast strains were stored at 25?.Compared with normal storage condition in water,the addition of trehalose increased the intracellular trehalose contents of the three strains by over 20 fold.The addition of trehalose increased the intracellular ATP contents of the two lager yeasts and maintained their viability.After the storage in trehalose solutions,the osmotic tolerance and the oxidative tolerance of the three strains,as well as the ethanol tolerance of Pilsner were promoted.Therefore,the addition of trehalose in yeast storage period can improve physiological states of brewer's yeast,especially the lager yeasts.?3?The analysis of the protective effects of trehalose on brewer's yeast under heat stress and its protective mechanism:the addition of trehalose increase the vitality and the cell viability of the 3strains.Compared with G-03 stored in water,the addition of 50 mmol·L^-11 trehalose 37?changed the regulation mode of 2505 genes.The two most enriched KEGG pathways are ribosome biogenesis pathway and ribosome biogenesis in eukaryotes pathway.And most differentially expressed genes in these two pathways were up-regulated.75%of the differentially expressed genes in the HSP family were also up-regulated.It was therefore hypothesized that the addition of trehalose promoted self-defense mechanism including the ribosome functions and the Hsp protein functions.Genes related to ATP production in the glycolysis pathway as well as genes related to glucose transport were up-regulated.It was therefore hypothesized that a part of the trehalose was degraded into glucose outside the cell and then the extracellular glucose was transported into the cell,which participated in the glycolysis pathway and promoted ATP production,increasing cell vitality.Compared with water,the addition of trehalose improved the intracellular trehalose level of G-03;genes involved in extracellular trehalose degradation were down-regulated.There was still trehalose outside the cell.Genes involved in unsaturated fatty acid degradation were down-regulated.It was therefore hypothesized that a part of exogenous trehalose was directly transported into cells and the degradation of extracellular trehalose was inhibited,making sure there was trehalose both inside and outside the cells,which helped to prevent the oxidation of unsaturated fatty acids on the cell membrane,resulting in the protection of cell membranes.?4?The preliminary study of the effects of the addition of trehalose on lager yeasts'storage period of high gravity brewing:as two lager yeasts were used over 3 generations,their cell viability,and intracellular ATP contents declined.Compared with water,the addition of trehalose to the storage environment of the 3^rd generation of the two strains improved the viability and the intracellular ATP contents of the cells.