| Lactic acid is a universal metabolite and growth inhibitor in the fermentation of fermented foods and beverages.For example,in the fermentation process of Moutai-flavor Baijiu,lactic acid is the organic acid with the highest content,up to 36.20 g·kg-1 fermented grain,which inhibits the growth and metabolism of core yeasts.However,the current researches on the lactic acid tolerance of yeasts are mainly at the level of single yeast,which is not suitable for the mixed yeasts system of Baijiu fermentation.Studying the lactic acid tolerance mechanism of the core yeasts group helps to maintain the stability of the yeasts group's fermentation capacity during Baijiu fermentation,and contributes to the standardization,precision and controllability of Baijiu production.In this work,the core yeasts in Baijiu production were taken as the study object.I employed transcriptomics,co-culture and reverse transcription-quantitative polymerase chain reaction to explore the adaptive responses of the core yeasts group to high-concentration lactic acid.The main results are as follows:?1?First,I explored the influence of lactic acid on the core yeasts P.kudriavzevii C-16 and S.cerevisiae C-3 in Baijiu fermentation.S.cerevisiae C-3 rarely grew under 40 g·L-1 lactic acid stress.The final OD600 of S.cerevisiae C-3 was reduced by 98%,and the ethanol production was reduced by 66%.While under 40 g·L-1 lactic acid stress,the final OD600 of P.kudriavzevii C-16 was only reduced by 46%,ethanol production did not decrease.It indicated that the lactic acid tolerance of P.kudriavzevii C-16 was significantly better than S.cerevisiae C-3.It was also found that P.kudriavzevii C-16 could degrade 18%of lactic acid in the medium,1.62 times that of S.cerevisiae C-3.It indicated that the degradation of lactic acid may be an important mechanism for yeast to adapt to lactic acid stress.?2?Then,the adaptive mechanism of P.kudriavzevii C-16 to lactic acid stress was studied.The lactic acid tolerance of P.kudriavzevii C-16 was significantly stronger than its model strain ATCC 24210.The amount of lactic acid degradation of C-16 was 2.83 times that of model strain.This indicated that C-16 had undergone adaptive evolution during long-term lactic acid stress,and its ability to degrade lactic acid had increased.Comparative transcriptomics analysis showed that the key enzymes in pyruvate metabolism pathway,ABC transporter,and GAD system of P.kudriavzevii C-16 were up-regulated,compared to the model strain.These indicated that the efficiency of degrading lactate,effluxing lactate ions and consuming H+was higher.In addition,four enzymes were found to be up-regulated during the synthesis of leucine and valine,and amino acids.It showed that P.kudriavzevii C-16 was more suitable for the Baijiu fermentation system than the model strain.?3?Then,the role of P.kudriavzevii C-16 in the adaptation of yeasts group to lactic acid stress was explored.Under lactic acid stress,when S.cerevisiae C-3 and P.kudriavzevii C-16co-cultured,the delay period of S.cerevisiae C-3 was shorter than that of mono-culture.It showed that P.kudriavzevii C-16 degraded lactic acid in the medium,which could provide a suitable growth environment for S.cerevisiae C-3.Under high lactic acid stress,P.kudriavzevii C-16 reduced lactate from 40.00 to 35.46 g·L-1 within 24 h culture.Compared with mono-culture,the transcription of S.cerevisiae C-3 lldD was significantly up-regulated at 24 h,indicating that S.cerevisiae C-3 restored its ability to degrade lactate at this time.In the end,the two strains together reduced lactate to 26.29 g·L-1.Compared with the mono-culture of P.kudriavzevii C-16?increased by 90%?or S.cerevisiae C-3?increased by 209%?,the co-culture significantly increased the consumption of lactate.It was also found that the transcription of VMA8 and STL1 was up-regulated in the yeasts group.These indicated that the catabolism of lactate,the efflux of H+and the transport of glycerol were important pathways for the yeasts group to tolerate lactic acid. |
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