| Chinese liquor fermentation has been carried out in a spontaneous way and multiple species are involved. Complex microbial interactions among the species bring desirable changes in the fermentation process, which determine the quality and safety of Chinese liquor. Therefore, it is significant to study microbial interactions during fermentation. That is not only benefit for increased control over and rational design of mixed-culture fermentation systems, but also provide extremely important practical and theoretical value for technical innovation of Chinese liquor.This dissertation chose Saccharomyces cerevisiae and Bacillus licheniformis, two functional strains during Maotai-flavor liquor fermentation, as a model system to study their interactions in mixed culture. The physiological metabolic characteristics of both strains were studied and the interaction mechanism was analyzed for the first time through transcriptome analysis, gene deletion and proteomic analysis. The main contents and results are as follows :(1) The physiological metabolic characteristics of both strains in single and mixed fermentation systems were measured. Results indicated that S. cerevisiae inhibited the growth of B. licheniformis in mixed culture with different inoculation ratios, while the growth of S. cerevisiae was not influenced by B. licheniformis. However, the ethanol production have increased by 11.8%. Pyruvic acid, citric acid, succinic acid, lactic acid, malic acid and tartaric acid produced by S. cerevisiae in mixed culture was 1.23, 2.11, 1.76, 2.02, 1.04 and 1.63 times higher compared with its single culture, respectively. In addition, different inoculation ratios of the two strains led to distinct volatile compound profiles during the fermentation process. More alcohols, acids, terpenes and aromatic compounds were produced with S. cerevisiae and B. licheniformis at a 1:1 ratio, while more ketones, acetaldehydes and esters at 1:100. Correlation analyses showed 12 volatile compounds could only be produced by S. cerevisiae were significantly correlated with the initial inoculation amount of B. licheniformis, independent of biomass, suggesting that the addition of B. lichenifomis improved metabolic activities of S. cerevisiae.(2) The S. cerevisiae genomic transcriptomics data in single and mixed culture was analyzed. It showed 119 and 86 genes were remarkably upregulated and downregulated(fold change>1.5 and p<0.05) in mixed culture system, respectively. The clusters of orthologous group of proteins(egg-NOG) analysis indicated that these remarkable changed genes mainly associated with translation, amino acid transport and metabolism, ion transport and metabolism, et al.. The analysis integrated with metabolic pathway suggested that S. cerevisiae induced ion transport and metabolism encoding genes expression, many enzymes encoding genes in thiamine metabolism pathway, as well as enzymes encoding genes in alcohol metabolism and organic acid metabolism pathway. These may change intracelluar ionic homeostasis and enzyme activity, which will influence the metabolic characteristics of S. cerevisiae. In single culture mode, the sulfur metabolism pathway, which related to cellular respiration and energy metabolism, of S. cerevisiae was down-regulated.(3) The gene function of FRE7 in S. cerevisiae and B. licheniformis was studied by gene deletion. FRE7 had no effect on the microbial growth, ethanol and organic acid production in single and mixed culture. However, the deletion of FRE7 decreased the acetic acid, octanoic acid, decanoic acid, ethyl octanoate and 2-phenylethyl hexanoate metabolism of S. cerevisiae in mixed culture. Meanwhile, gene involved in thiamine metabolism was downregulated by the deletion of FRE7 in mixed culture. That may change celluar enzyme activity of S. cerevisiae. These results suggested that FRE7 play a role in interactions between S. cerevisiae and B. licheniformis.(4) Two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) coupled with matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) was used to analyze and identify differentially expressed proteins of S. cerevisiae in single and mixed culture. The proteomic profile of S. cerevisiae showed 39 and 30 proteins were remarkably upregulated and downregulated(fold change>2 and p<0.05) in mixed culture system, respectively. Among them, 24 protein spots were identified, including protein related to glycolysis, ethanol fermentation, cell wall integrity, stress response, et al.. The protein related to glycolysis and ethanol fermentation were found to be upregulated in S. cerevisiae when in coculture with B. licheniformis. These proteins play important role in changing the metabolic behavior of S. cerevisiae. The other differentially expressed proteins, such as anti oxidant proteins and stress proteins, were related to adaptive mechanism of microbes in mixed culture systems. |
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