| Baijiu is one of the most famous distilled spirits around the world,which occupies the largest consumption proportion of ardent spirit in China.According to the different characteristics of flavor,baijiu can be divided into three main types: Nong-flavor,Jiang-flavor and Qing-flavor.The process of baijiu brewing is deeply dependent on Jiuqu that provides most of microorganisms and enzymes during the following process of baijiu fermentation.It is the complex and different enzymes system formed in different Jiuqu that forms the distinctive flavor substances in different baijiu.As the enzymes remained poorly understood,the detailed enzymatic composition of Daqu was analyzed by metaproteomics in this study.The main results as follows:(1)In this study,the activities of common enzymes in Nong-flavor Daqu were examined at first.8 enzymes(except phytase and tannase)including ?-amylase,acid protease,neutral protease and acid cellulase etc.were detected,and the enzymatic properties of these enzymes were also explored.The result suggests that the optimum temperature of most detected enzymes is 45-60oC and the enzyme activity of most detected enzymes is the highest in the acidic range.The enzyme activity between premium and normal Nong-flavor Daqu was compared,and the result suggests the enzymes activities of premium Nong-flavor Daqu were all higher than that of normal Nong-flavor Daqu.(2)The detailed enzymatic composition of Nong-flavor Daqu was analyzed by metaproteomics.There was a total of 922 enzymes in Nong-flavor Daqu,including 52 plant enzymes,2 animal enzymes and 868 microbial enzymes.Biological functions of enzymes revealed that these identified enzymes are mainly involved in these biological pathways including oxidation-reduction process(29.89%),carbon metabolism(9.02%),glycolysis(7.33%)and TCA cycle(4.51%).(3)The proteins and enzymes were compared in different grades Nong-flavor Daqu.The analysis results of species indicate that the proportion of total proteins from fungi in premium Nongflavor Daqu is higher than that in normal Nong-flavor Daqu,but the proportion of total proteins from bacteria in normal Nong-flavor Daqu is higher than that in premium Nong-flavor Daqu.A total of878 differential abundant proteins was identified between the premium and normal Nong-flavor Daqu.Among them,the abundance of 577 proteins including 383 enzymes in premium Nong-flavor Daqu is higher,and 301 proteins including 192 enzymes in normal Nong-flavor Daqu is higher.The analysis results of biological functions reveal that the number of proteins involved in carbon metabolism and the biosynthesis of amino acids in premium Daqu is more than that in normal Daqu.Additionally,the classification results of differential enzymes show that oxidoreductases and transferases are the two types of enzymes with the largest difference between the two kinds of Daqu,accounting for 31.82%and 19.65% of the total differential enzymes respectively.(4)The metaproteomic results of Nong-flavor Daqu(NFD)and Jiang-flavor Daqu(JFD)were compared.The results show that the number of microbial enzymes and animal enzymes in JFD is higher than that in NFD,while the number of plant enzymes is lower than that in NFD.The species analysis results of proteins show that the proportions of total protein produced by various yeasts in NFD and JFD are 8.18% and 1.79%,and produced by various molds in NFD and JFD are 9.42% and9.12%,indicating that there is a significant difference in the proportion of proteins produced by yeast in the two kinds of Daqu,while the difference of mold protein proportion is very small.Then the difference of enzymes related to the formation of 1-propanol,isobutanol,acetic acid,lactic acid,ethyl caproate and furfural in two kinds of Daqu were compared.The results show that the enzymes related to the formation of these flavor substances in two kinds of Daqu are significantly different.The metaproteomic results of NFD and Qing-flavor Daqu(QFD)were also compared.The results show that the plant proteins are the most diverse group of proteins between them,accounting for 44.04% of the total differential proteins.Oxidoreductases(30.47%)and transferases(22.36%)are the two types of enzymes with the largest difference between the two kinds of Daqu.The difference of enzymes related to the formation of acetic acid,alcohol,lactic acid,isobutanol in two kinds of Daqu were analysed.The results show that the contents of aldehyde dehydrogenase related to the synthesis of acetic acid and alcohol dehydrogenase related to the metabolism of ethanol in NFD are higher than those in QFD,but the difference of the enzymes related to the formation of lactic acid and isobutanol in them were not detected.In total,this study characterized and classified the enzymes in Nong-flavor Daqu,and the differential abundant enzymes in the premium/normal Nong-flavor Daqu,Nong/Qing-flavor and Nong/Jiang-flavor Daqu comparison.The results obtained in this study will provide clues for further understanding the irreplaceable role of Daqu and lay a foundation for analyzing the formation mechanism of characteristic flavor substances for NF baijiu. |
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