Effect Of Ca²⁺ On Growth,metabolism And Antioxidant Properties Of Yeast Under High Glucose Environment

Saccharomyces cerevisiae is a fungus widely used in food production.In the brewing and baking industries,yeast is often exposed to high sugar stress,which leads to slow growth,increased cell mortality,and changes in metabolites,resulting in reduced organoleptic properties of alcoholic products and poor fermentation of the dough.Ca²⁺plays an important role in the growth and metabolism of yeast cells,participating in transcriptase synthesis to stabilize yeast expression.At the same time,Ca²⁺is easily accessible and it is more convenient to improve the stress resistance of yeast by exogenous substances.Therefore,in this study,a control group（30 g/L glucose）,a high glucose low Ca²⁺group（300 g/L glucose+8.5 mmol/L EGTA）,and a high glucose with Ca²⁺group（300 g/L glucose+0.09 mmol/L Ca Cl₂）by exogenous addition were set up to study the effect of Ca²⁺on yeast under high glucose stress.The results of the study were as follows:（1）Effect of Ca²⁺signaling on growth characteristics,metabolites,and antioxidant capacity of yeast under high glucose stress.The results showed that the yeast in the high glucose with Ca²⁺group had a higher dry weight,which reduced the mortality of yeast at 6 and 12 h,increased the ethanol metabolic output of yeast at 12 h,increased the glycerol metabolic output of yeast at 24 and 36 h,and improved the growth and metabolic capacity of yeast.At 24 and 36 h,the intracellular glycerol synthesis capacity of yeast in the high glucose with Ca²⁺was reduced,and throughout the growth process,the yeast alginate synthesis capacity was reduced and the intracellular ROS content increased,but at the same time,the superoxide dismutase and catalase activities of yeast were increased,and the glutathione content was increased,which enhanced the antioxidant capacity of yeast.The intracellular malondialdehyde content of yeast in the high glucose with Ca²⁺was significantly reduced compared with that in the high glucose low Ca²⁺group except for 6 h.The carbonyl protein content of yeast in the high glucose with Ca²⁺was reduced compared with that in the high glucose low Ca²⁺group except for 12 h.The addition of Ca²⁺was able to reduce oxidative damage in yeast.Therefore,the moderate addition of Ca²⁺could effectively enhance the growth and metabolism of yeast,reduce the dependence on glycerol and alginate protectants,significantly enhance the antioxidant capacity and reduce the oxidative damage of yeast under high glucose environment,while yeast may increase the respiratory metabolism of mitochondria.（2）Effect of Ca²⁺on carbon metabolism in yeast under high glucose.The results showed that yeast in the high glucose with Ca²⁺was able to increase the intracellular Ca²⁺content of yeast under a high glucose environment under the regulation of Ca²⁺-ATPase;hexokinase（HK）activity in yeast in the high glucose with Ca²⁺weakened the elevation of enzyme activity by high glucose environment while ensuring a certain degree of elevation compared with the control group,and also weakened the elevation of yeast glucose-6-phosphate dehydrogenase（G6PDH）activity,reduced the intracellular pyruvate content,delayed the elevation of ethanol dehydrogenase（ADH）and elevated acetaldehyde dehydrogenase（ALDH）and acetyl coenzyme A synthase（ACS）activity in the high glucose environment.The elevated membrane potential was more significant,and the low leakage of intracellular cytochrome C elevated the activities of TCA-related pyruvate dehydrogenase（PDH）,isocitrate dehydrogenase（ICDH）,andα-ketoglutarate dehydrogenase（α-KGDH）,and the activity of cytochrome C oxidase was restricted.Therefore,the moderate addition of Ca²⁺can weaken the intensity of pentose phosphate metabolism in yeast under a high glucose environment,and make the carbon source flow to mitochondria-related metabolism through the action of ALDH and ACS under the premise of ensuring ethanol output,which can be sufficient to provide energy for yeast growth and stress resistance despite the reduced activity of respiratory chain metabolizing enzymes,so that yeast has more balanced carbon utilization under high glucose conditions,thus enhancing yeast cell viability and enhance yeast response to high glucose stress.（3）Effect of Ca²⁺on the expression of antioxidant genes in yeast under a high glucose environment.The expression of yeast Cu/Zn-superoxide dismutase（SOD1）,Mn-superoxide dismutase（SOD2）,catalase（CTT1）,γ-glutamylcysteine synthase（GSH1）,glutathione peroxidase（GPX2）and glutathione transferase（GTT1）genes were investigated in different treatment groups.The results showed that the high glucose environment was all able to significantly increase the expression of yeast antioxidant genes within a short period of 6 h and maintain the expression of SOD1 gene at the same level as the control yeast expression during the subsequent growth,while the expression of GSH1,GPX2,and GTT1 genes were significantly decreased.In addition to the SOD1 gene,the yeast in the high glucose with Ca²⁺was able to significantly enhance the antioxidant enzyme activity in the high glucose environment,possessing significantly higher CTT1 expression than that in the control yeast,and the yeast had a significant enhancement of SOD2 gene expression in the late growth period,and the yeast had a weaker inhibition of GSH1,GPX2,and GTT1 gene expression in the high glucose environment.Based on the comparison of gene expression with antioxidant enzymes and glutathione content in different groups of yeast,it was found that there was a possibility of destabilization of gene expression in a high glucose environment,while a sufficient Ca²⁺environment could promote the stability of gene expression.Therefore,the addition of exogenous Ca²⁺could effectively enhance the expression of antioxidant genes in yeast under a high glucose environment and promote stable gene expression.