Study On Metabolic Response Mechanism Of Saccharomyces Cerevisiae To Patulin Stress

Patulin?PAT?is a highly toxic fungal secondary metabolite,which is mainly found in moldy fruits and fruit products.It is one of the main factors that affect the quality of fruits and fruit drinks.Research has shown that Saccharomyces cerevisiae can effectively degrade PAT during fermentation and reduce the pollution of related products by PAT.However,the mechanism of response metabolism of yeast under PAT stress is not clear at present.In order to further clarify the mechanism of the influence of PAT on the fermentation process of Saccharomyces cerevisiae,it is particularly important to carry out research on the metabolic response mechanism of Saccharomyces cerevisiae under PAT stress.In this project,Saccharomyces cerevisiae CICC 31084 was used as the fermentation strain.Based on the concentration-effect-time relationship model,the effect of PAT stress on the growth characteristics of Saccharomyces cerevisiae was studied.Then,an apple juice simulation system was constructed to analyze the influence under PAT stress on the fermentation characteristics of Saccharomyces cerevisiae;Finally,the differential protein and related genes of Saccharomyces cerevisiae metabolized were analyzed under PAT stress that based on proteomics and transcriptomics technology,The main findings are as follows:1. Saccharomyces cerevisiae CICC 31084 had strong toxin tolerance to PAT. Flow cytometry?FCM?was used to determine the physiological and biochemical indicators of yeast cells under PAT stress.The results showed that the effect of PAT on the membrane permeability of yeast cells was positively correlated with the action time and the concentration of PAT?p<0.05?,the greater the concentration of PAT,the more obvious of increasing in cell membrane permeability.The effect of PAT on intracellular ROS content and MMP gradually increased with the growth of action time and PAT concentration.With the increase of PAT concentration,The internal MDA content would increase significantly,and the MDA content in the bacteria was positively correlated with reaction time and PAT concentration?p<0.05?.At the same time,the presence of PAT woulf affect the activity of Na⁺K⁺-ATPase?Ca²⁺Mg²⁺-ATPase and total ATPase activity in Saccharomyces cerevisiae,of which Na⁺K⁺-ATPase and total ATPase activity would show a continuous downward trend,With the extension of the processing time,the trend of Ca²⁺Mg²⁺-ATPase activity was to increase first and then decrease.2. In the early stage of fermentation,with the yeast cells'growth,glucose content and SSC content decreased rapidly,pyruvate content accumulated in large amounts and the content of main volatile aroma substances in fermentation gradually increased.PAT treatment would accelerate the rate of glucose consumption and pyruvate production during fermentation.In the absence of PAT,PDC,ADH,and acetyl-Co A were positively correlated with the formation of each characteristic aroma;after low-dose 1?g/m L PAT treatment,the production of PDC and ethyl acetate,phenethyl alcohol,caprylic acid were negative Related.The positive correlation between the content of acetyl-Co A and the production of ester aroma and phenylethanol was enhanced;after high-dose 10?g/m L PAT treatment,PDC was significantly positively correlated with the formation of ethyl acetate,isoamyl acetate and phenylethanol?p<0.05?,which had a very significant positive correlation with the formation of phenethyl acetate and ethyl octoate?p<0.01?.The content of acetyl-Co A was significantly positively correlated with phenethyl alcohol and caprylic acid?p<0.05?,and negatively correlated with the formation of ethyl butyrate?r=-0.047?.The greater the concentration of PAT,thehigher positive correlation between ADH and each aroma.The high concentration of PAT treatment would activate the expression of PDC in yeast cells and inhibit the expression of intracellular ethanol dehydrogenase and acetyl-Co A.3. Studying was performed on analysising the response of Saccharomyces cerevisiae to PAT in proteins expression levels based on proteomics technology.The results showed that the expression of 52 proteins were significantly down-regulated,and the expression of 135 proteins were significantly up-regulated.Down-regulated proteins were mainly involved in basic metabolic processes and biosynthetic processes in yeast cells.Twenty of the up-regulated proteins were annotated to oxidoreductase.Among them,glutathione peroxidase?GPX2?,glutathione transferase?GTT2?,alcohol dehydrogenase?ADH6?,glucose-6-phosphate dehydrogenase?ZWF1?were activated.Glutathione synthase?GSH2?,glutaredoxin?GRX2?,thioredoxin?TRX2?and other proteins involved in antioxidant stress were also significantly up-regulated.In addition,ABC transporter,iron transporter?MRS4?,and protein kinase?CAK1?were also activated.It was shown that Saccharomyces cerevisiae responded to oxidative stress by activating the expression of the above-mentioned proteins that caused by PAT for maintaining the redox balance inside the cell.4. Studying was performed on analysising the response of Saccharomyces cerevisiae to PAT in proteins expression levels based on transcriptomics technology.The results showed that the stimulation of PAT caused the expression level of 969genes in Saccharomyces cerevisiae to be significantly reduced,and the expression level of 1133 genes to be significantly increased.The down-regulated genes were mainly clustered into biological metabolism,protein metabolism,and biosynthesis of organic nitrogen compounds,which indicated that the metabolism of Saccharomyces cerevisiae was inhibited to a certain extent under the action of PAT.Significantly up-regulated genes OYE3?GPX2?TRX2?SOD1 were mainly involved in redox processes,antioxidant processes,and cells'response to drug transmembrane transport and biological stress.In addition,the expression levels of four genes related to the transcription factor Yap1p?copper metal chaperone protein ATX1,flavin hemoglobin gene YHB1,and type B cyclin gene CLB6 were significantly up-regulated and involved in the regulation of Saccharomyces cerevisiae response to PAT.