Effects Of Metschnikowia Pulcherrima On PDH Bypass Of Saccharomyces Cerevisiae

In order to explore the influence of Metschnikowia pulcherrima on the PDH bypass of Saccharomyces cerevisiae,four strains of M.pulcherrima(mp3001,mp3002,mp3004 and mp3007)were selected in this study mixed fermentation(simultaneous inoculation and sequential inoculation)with three commercial S.cerevisiae(D254,QA23 and CY3079),under the conditions of different factors affecting the production of acetic acid(initial sugar concentration and inoculum amount).Firstly,different mixed fermentation methods of simultaneous inoculation and sequential inoculation were adopted,and high-performance liquid chromatography(HPLC)was used to detect the fermentation products glycerol,acetic acid and ethanol,and seq CY3079+mp3007 was selected as the combination with the lowest accumulation of acetic acid.Secondly,this combination was further explored under different initial sugar concentrations and different inoculation amounts of non-S.cerevisiae,and the combination with the lowest accumulation of acetic acid was selected.Then the activity of three key enzymes and the expression level of coding genes in the PDH bypass in S.cerevisiae of these combination was tested,pyruvate decarboxylase(PDC),acetaldehyde dehydrogenase(ALD)and acetyl-Co A synthase(ACS)and the coding genes(PDC1,PDC5,ALD6 and ACS2).The determination of the gene expression level of S.cerevisiae was used to explore the influence of the acetic acid synthesis metabolic pathway due to the competition between S.cerevisiae and non-S.cerevisiae in mixed fermentation.Finally,making ice red wine with above combination,and headspace solid-phase microextraction and mass spectrometry(GC-MS)were used to detect the volatile substances to further proves that M.pulcherrimai potential in ice red wine making.The result showed that(i)The inoculation method has a significant effect on the acetic acid content.When exploring the influence of inoculation method and strains on the content of acetic acid,it was found that mp3007 produced the highest glycerol content during sequential inoculation fermentated with D254,reaching 5.8g/L;in the early stage of fermentation simultaneous inoculation produced more glycerol than sequential inoculation.Compared with the control group,sequential inoculation can reduce the acetic acid content,while simultaneous inoculation can increase the acetic acid content.The combination seq CY3079+mp3007 had the lowest acetic acid content 0.31g/L.In terms of the ability of different strains and inoculation methods to affect the acetic acid content,the inoculation method has more obvious effects on the acetic acid content(ii)After determining the fermentation combination and fermentation method,further investigations will be made on the other factors which affect the acetic acid content substrate concentration and the inoculum amount.The results showed that same inoculation ratio but different substrate sugar concentration,the acetic acid content will increase as the substrate concentration increases;when same substrate sugar but different inoculation ratio,different substrate sugar concentrations have rules.At 200g/L,the acetic acid content will increase as the inoculation amount of M.pulcherrima.At 290g/L,as the inoculation amount of M.pulcherrima increases,the acetic acid content will first increase and then decrease.At 380g/L,with the increase of M.pulcherrima inoculation amount,the acetic acid content showed negative correlation with the inoculation amount of M.pulcherrima as a decreasing trend,.(iii)After determining the substrate sugar concentration and the M.pulcherrima inoculation amount,the enzyme activity in the samples taken at three key points during the fermentation process was measured.Results showed that at the early stage of the fermentation compared with control group pyruvate decarboxylase(PDC),aldehyde dehydrogenase(ALD)and acetyl-coa synthetase(ACS)activity were suppressed,with the passage of the fermentation process,the inhibition effect is abate,suggests that at the early stage of the fermentation M.pulcherrima had an adverse effect on S.cerevisiae PDH bypass,and with the increase of the substrate concentration of sugar,the trend shows attenuate after increase.(?)Sequential inoculation of M.pulcherrima on the acetic acid production of S.cerevisiae is reflected in the key genes expression of pyruvate decarboxylase(PDC),acetaldehyde dehydrogenase(ALD)and acetyl-Co A synthase(ACS)in the PDH bypass.The results showed that the interaction between M.pulcherrima and S.cerevisiae in sequential inoculation would affect the physiological metabolism of each other.The influence is reflected in the process of fermentation,pyruvate decarboxylase(PDC),aldehyde dehydrogenase(ALD)and acetyl-coa synthetase(ACS)amount of key genes expression have been affected,Compared with the control group,PDC1,PDC5,ALD6,and ACS2 all showed higher expression levels,indicating that M.pulcherrima has a promoting effect on the gene expression of the PDH bypass of S.cerevisiae,but it can inhibit the PDH bypass.Inhibition of the activity of PDC and ALD will reduce the content of acetate,and the increase of ACS activity will promote the consumption of acetate,which leads to a decrease in the final acetate content and a decrease in the content of acetic acid(V)After the fermentation of mp3007 and CY3079 inoculated in sequence,determineded the physical and chemical indicators and volatile substances of ice red wine,it was found that mp3007 has great potential and development prospects in the field of ice red wine making.The results showed that the mixed fermentation can reduce the alcohols in ice red wine by 3.2% and increase the esters by 21.2%.After the sensory evaluation of ice red wine,it was found that compared with the control ice red wine,mp3007 can reduce the greenness e and give more floral,fruity and caramel flavor of the ice red wine.Generally speaking,the sequential inoculation fermentation of mp3007 and CY3079 has a good development prospect for improving the flavor of wine and improving the quality of ice red wine.