Study On Mechanism Of The Effect Of Cu²⁺ On Alcoholic Fermentation And Response To Copper Stress In Saccharomyces Cerevisiae

Two strains of Saccharomyces cerevisiae (one industrial strain Freddo and one newly selected strain BH8in our lab) and a model fermentation system containing different concentrations of Cu2+and proanthocyanidins were used in this study. The aim of this study was to understand the effect of Cu2+and proanthocyanidins on yeast cells growth, metabolism, alcoholic fermentation and expression of ADH1encoding the key enzyme alcohol dehydrogenase in wine yeast alcohol biosynthesis, expression of GPD1encoding glycerol3-phosphate dehydrogenase in glycerol biosynthesis under copper stress; to determine the effect of trehalose on the tolerance of wine yeasts to copper. We can discuss the mechanism of resistance to copper, the influence of copper on fermentation in Saccharomyces cerevisiae, the regulation of proanthocyanidins on wine yeast fermentation under copper stress.From the three aspects above. The main results are as follows:Cu2+inhibited the growth of yeast cells, sugar metabolism and alcoholic fermentation process. The effect of Cu2+inhibiting fermentation and delaying fermentation time is positively correlated to its concentration. At the concentration of0.2mM, the fermentation was inhibited and forced to terminate early.0.05,0.1mM Cu2+promoted strain B on the use of sugar at late fermentation, when fermentation finished, ethanol yield was significantly higher than the control and0.05mM Cu2+treatment. Glycerol yield of0.05mM Cu2+treatment for strain B was significantly higher than control and the group treated with0.1mM Cu2+. To strain F, Cu2+treated groups compared with the control, there is no significant difference in terms of final yield of ethanol and glycerol. During the fermentation under copper stress, Cu2+greatly upregulated ADH1expression throughout the fermentation; early in the fermentation Cu2+inhibited the expression of GPD1, and then upregulated the expression of GPD1.During fermentation adding proanthocyanidins under copper stress, in early stage of fermentation, compared with control,0.1,1.0g/L proanthocyanidins inhibited slightly the viability of yeast cells. Throughout the fermentation process, compared with0.1mM Cu2+treatment,0.1mM Cu2++0.1g/L PAs more inhibited the growth of yeast cells and alcoholic fermentation.In the late fermentation, wine yeast maintained a higer biomass, cell viability and finished fermentation ahead in the mediums containing1.0g/L PAs. Finally, the treatment0.1mM Cu2++1.0g/L PAs finished the fermentation7-8days earlier than0.1mM Cu2+treatment. Late in the fermentation, Cu2+concentration of copper treatments adding proanthocyanidins was significantly lower than copper treatment. Ethanol yield of the treatments containing higher proanthocyanidins of strain B was significantly higher than control and the treatments containing lower proanthocyanidins, glycerol yield is higher than the treatments containing no proanthocyanidins. Ethanol yield of the treatments containing higher proanthocyanidins of strain F was higher than other treatments, glycerol yield was significantly higher than control and the treatments containing lower proanthocyanidins. Proanthocyanidins downregulated ADH1expression to some extent during alcoholic fermentatin; early in the fermentation, proanthocyanidins upregulated GPD1 expression. Adding proanthocyanidins under copper stress, the effect of Cu2+on the upregulation of ADH1played a dominant role; at the early stage of fermentation, the effect of proanthocyanidins on the upregulation of GPDl played a dominant role. The effect of Cu2+on the upregulation of GPD1played a dominant role at late fermentation1.OmM Cu2+increased reactive oxygen species (ROS), membrane lipid oxidation index malondialdehyde (MDA) levels, while inducing the synthesis of trehalose in wine yeast. Pretreated either with a mild heat treatment at37℃or with a50mM trehalose solution, the content of trehalose increased in the yeast cells, as compared with the direct copper treatment, the level of ROS and MDA in yeast cells were reduced, thereby increasing the viability of yeast cells, the effect of50mM trehalose pretreatment is the best. Obviously, the protection of trehalose on yeast under copper stress was related to its antioxidant function.