| Citric acid is the main organic acid in fruit wine and has an important influence on the quality offruit wine.However,the acidic environment caused by high organic acid content is often encountered during the fermentation of fruit wine,which inhibits the growth of microorganisms and limits the development of citric acid-based fruits.In this study,acid-tolerant yeasts(F8,F11 and F14),which were obtained by adaptive evolution of high citric acid concentration in the early stage,and were used to investigate their tolerance to different concentrations of citric acid and different organic acids.For the strains with strong acid tolerance,the tolerance mechanism of Saccharomyces cerevisiae to high concentration citric acid stress was preliminarily explored by analyzing the activity of related enzymes and transcriptome sequencing results under oxidative stress.The obtained acid-tolerant strain was used for Cabernet Sauvignon wine fermentation,and the fermentation performance,wine quality,and biogenic amine safety of Cabernet Sauvignon wine were evaluated to provide a theoretical basis for better wine and fruit wine production.The main research results are as follows:(1)The acid tolerance of the evolved strain was verified by observing the growth of the strain under(0,0.05,0.1,0.2,0.3 mol·L-1)citric acid and 0.25 mol·L-1organic acid(citric,malic,lactic,tartaric and acetic acid).Compared with the departure strain EC1118,strains F8 and F14 were not significantly affected by the inhibition of citric acid at low concentrations,while F11 growth was promoted and growth was inhibited at high concentrations of citric acid,but F11 growth was the best and the most tolerant to citric acid;among the different organic acids at 0.25 mol·L-1,lactic acid and malic acid did not inhibit yeast growth,and tartaric acid would inhibit yeast growth,where the departure strain EC1118 almost stopped growing,F8 and F14 grew slowly,F11 had the strongest growth ability,and all strains could not grow in acetic acid.In summary,the evolved strain F11 has the strongest tolerance to organic acids.(2)Scanning electron microscopy and lens electron microscopy analysis of the differences between the cell surface and internal structure of strain EC1118 and F11 under 0.25 mol·L-1citric acid stress,the results showed that:the cell surface of the unstressed strain was smooth and full,oval or round in shape,neat and robust cell morphology,yeast cells were vigorous,but there was adhesion between F11cells and cells;under stress the cell The surface of the cells became rough and crinkled under stress,among which EC1118 was the most obvious crinkled and adhesive phenomenon under stress,and F11 was the most obvious crinkled and adhesive phenomenon.It was observed by transmission electron microscopy that the intracellular microsomes increased and the cell wall thickened under citric acid stress of the starting strain EC1118 and the evolved strain F11 under stress.The oxidative stress-related enzyme activities of the strains were detected,and it was found that the evolved strain F11 had lower ROS level,higher CAT activity and no significant difference in SOD activity compared with the starting strain EC1118 under citric acid stress for 12 h.In summary,citric acid stress causes surface wrinkling and adhesion of yeast cells,thickening of cell walls,increase of intracellular microsomes and increase of ROS level,and the strain obtained by adaptive evolution can reduce ROS level by regulating the specific activity of intracellular CAT.(3)By transcriptome analysis of EC1118 and F11,a total of 596 and 1396 differentially expressed genes were obtained for EC1118 and F11 under stress compared with their non-stress,respectively,of which 756 and 386 were significantly up-regulated genes and 210 and 640 were significantly down-regulated genes,respectively.By analyzing transcriptomic data and referring to related literature,it was concluded that the cell membrane components(number of up-regulated genes 114,256,and number of down-regulated genes 66,176,respectively),amino acid metabolism(number of up-regulated genes 6,45,and number of down-regulated genes 16,56),oxidative stress stress-related(number of up-regulated genes1,7,and number of down-regulated genes 0,1)in the departure strain EC1118 and the evolved strain F11The key genes related to oxidative stress(Pdr12,Erg4,Grx2,Ahp1,Ctt1 and Tsa2)are important for acid tolerance in Saccharomyces cerevisiae.(4)Through fermentation experiments on Cabernet Sauvignon wines,the results of natural fermentation and fermentation with yeast EC1118 and F11 on Cabernet Sauvignon grapes showed that the residual sugars were 6.25,2.47,and 2.51 g/L,the alcohol content was 11.81,13.05,and 11.99(v/v),the detected biogenic amines were 0.34,0.52,and 0.54mg/L,putrescine content was 2.43,2.88,3.13mg/L cadaveric amine content was 0.93,0.9,0.94mg/L.There are 14,14 and 16 kinds of alcohols,4,5 and 7kinds of esters,6,4 and 5 kinds of acids.The detection types of amino acids are the same as those of biogenic amines,18 amino acids and 6 biogenic amines respectively.F11 compared to EC1118 alcoholic substances in hexanol,phenylethanol,heptanol with rose,peach,pineapple,banana and other fruit fragrance and floral fragrance content has increased,with bitter almond flavor isobutanol content decreased;ester substances increased by two,with pineapple,banana,honey and other fruit fragrance of ethyl caproate,ethyl caprylate,phenethyl acetate content increased.The content of glutamic acid,histidine and proline was low,and the content of other amino acids was high. |
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