The Composition Of Microbial Community And Its Relationship With Liquor Quality In The Brewing Process Of Maotai-flavor Liquor

Chinese liquor,is one of the world's oldest distilled spirit in the worldalcoholic beverages,has a long history.Nowadays,the four main Chinese flavor types of liquors,namely,Luzhou-flavor,Maotai-flavor,Fen-flavor liquor and rice-flavor liquor have been developed into twelve kinds of flavor liquors,including Feng,Jian,Dong,Te,Chi,Zhima,Laobaigan and Fuyu.With distinctive production process,the flavor components of Maotai-flavor liquor is received great popularity with numerous consumers attributed to is unique brewing process,which contributing to its aromatic species and content,thus making Maotai-flavor liquor superior to other types of flavor liquors.is far ahead of the rest in flavor type and content.Furthermore,the production and sales of Maotai-flavor liquor is obviously higher than that of other types.Daqu,the starter culture of Maotai-flavor liquor,Maotai-flavor Daqu,which acts as saccharifying,leaven and aroma-producing agents in during the liquor production of Maotai-flavor liquor,In addition,many microorganisms with high temperature resistance and aroma production abilities were trained due to the rising temperature in the brewing process.can produce beneficial aroma-producing microorganism of high-temperature resistance during brewing process with rising temperature.The unique stacking process is of great importanceplays great role oin the flavor production formation of Maotai-flavor liquor..In this process,various microorganisms and its produced various enzymes contributing to the biotransformation,enzymatic browning and Maillard reaction fermented grains,which gradually forming its elegant and full sauce flavor.Fermenting grains can bring about a series of biochemical reaction such as enzymatic browning and Maillard reaction through the environmental microorganism and all kinds of enzymes,and then the elegant and full bodied flavor of Maotai liquor is gradually produced.The two important processes of high temperature brewing and stack fermentation mentioned above have been used by many famous liquor-making enterprises.Two important process mentioned above have been of reference for many liquor-making enterprise.Starter-making and liquor-brewing are the two key processes in the production of Maotai-flavor liquor.In this study,microbial diversity sequencing technology and gas chromatography-mass spectrometry methods were used to study the changes of microbial community structure characteristics,enzymes and flavor substances of fermented grains from starter-making to distillation process,in order to 1)reveal the microbial community characteristics and analyze the key functional microorganisms;2)investigate the relationship between key microorganisms and their effects on the quality of Maotai-flavor liquor;3)analyze the main factors which affecting the quality of Maotai-flavor liquor and the quality factors of special technology.The results obtained would provide theoretical basis for improving the quality and production efficiency of Maotai-flavor liquor.The main results are as follows:This study aimed to investigate the characteristic of microbial community structure,related enzyme system and the relationship between flavor substance and important microorganisms in two Maotai-flavor liquor-making processes,namely Daqu making and fermentation,by making use of sequencing technology and gas chromatogram analytical method.Combined with the understanding of ecological characteristics of microbial community in Maotai liquor brewing period and the analysis of functional microorganisms,quality of Maotai-flavor liquor in special making process can be conducted.This study could provide a theoretical basis for the improvement of liquor quality and production efficiency.The main research content in this study followed these steps:1.A comprehensive analyzed on the community structure of bacteria and fungus in high-temperature Daqu making and stacking,and in-pit fermentation process was determined by Illumina HiSeq 2500 sequencing system.Microbial flora in high temperature Daqu making and stacking fermentation process showed biological diversity in quantity and structure distribution consistent with microbial flora species.The dominant bacteria included Escherichia-Shigella,Lactobacillus,Clostridium sensu stricto 1,Streptococcus,Actinobacillus,Bifidobacterium,and Citrobacter.And the dominant fungus contained Alternaria,Cyphellophora,Pyrenochaetopsis,Issatchenkia,Pichia and Candida.The analysis result demonstrated that bacterial diversity was significantly higher than that of fungi,because bacterium suited difference existing between the ecological environment of high-temperature Daqu and stacking fermentation,thus the abundance of dominant also different.Moreover,the network analysis was also used to explore the interaction among microorganism.Clostridium sensu stricto 1 had a negative correlation with Escherichia-Shigella,Actinobacillus,Bifidobacterium,and Citrobacter,however,it showed positive correlations with Sarcina.Escherichia-Shigella is postively related to Actinobacillus,Citrobacter and Enterobacter,but negatively related to Clostridium sensu stricto 1 and Sarcina.Lactobacilluss showed positive correlation with Bifidobacterium.2.The enzymatic activity of eight enzymes in fermented grain samples was tested by using Enzyme-linked immunosorbent assay(ELISA).People's mouthfeel and liquor quality were affected by protease,lipase,tannase and esterase,while a-amylase,saccharifying enzyme,cellulase and pectinase mainly influenced utilization ratio of raw material and liquor yield.The content of a-amylase presented regularly change trend,namely rising firstly and then falling.And the microbial flora Clostridium sensu stricto 1 detected by sequencing test also shown similar change of tendency,which demonstrated that Clostridium sensu stricto 1 probably had directly effect on a-amylase activity leading to the decomposition reaction of starch or intermediate products.Finally it affected the taste and style of liquor.The change tendency of Clostridium sensu stricto 1,Issatchenkia,Pichia and Alternaria were also similar change of tendency with protease activity.Namely the content increased when tested in pit and the abundance of Clostridium sensu stricto 1 reached the highest level during the third round in-pit fermentation.The activity of a-amylase,proteinase and pectinase was higher in stacking fermentation than in in-pit fermentation,which was probably related to the stacking process.All in all,it was oblivious that stacking fermentation became great importance for enzyme production after microbial recomposition.The abundance change of Alternaria at each fermentation time point was consistent with the change of Glucoamylase activity and rose to the highest level after the second round of in-pit fermentation.This study intended to explore the enzyme-producing function of microorganism.The Dominant bacteria Clostridium sensu stricto 1 and Alternaria had important effect on the content of amylase,protease and Glucoamylase respectively,while their activity increased first and decreased later.3.Gas chromatography(GC)technique was applied for the detection of flavor substances in seven liquor rounds and in fermenting grain samples during fermentation stage.On the whole,the result showed that four kinds of flavor substances in the samples were alcohol,ester,aldehyde and acid,the content of the four compounds was in the following order: alcohol > ester>aldehyde >acid.The overall content of flavor substances remain high in the third,fourth and fifth liquor round,which provided material precondition for base liquor.This was in accordance with the results of physical and chemical index detection.The content of alcohols,acids and esters was higher in in-pit fermentation than in high-temperature stacking fermentation,which demonstrated that high-temperature stacking had brought some precursors into pit for decomposition and conducted effective extraction in liquor.This process was of great significance in liquor rounds.As to the alcohol of Maotai-flavor liquor,the content of n-propyl alcohol,which can made base liquor more crisp and refreshing,was the highest.It was generally considered that organic acid was produced by Lactobacillus using lactic acid decomposed via glucose as carbon source in liquor making process.The glucoamylase played a certain role in the biochemical process,the range of acid change was small,and the content of acetic acid was the highest.For fermented grains,the content of acetic ether was highest,followed by ethyl lactate.Both of them were important compounds for the formation of unique liquor flavor.Taking the abundance change of Lactobacillus into account,it was speculated that there was a significant correlation between Lactobacillus and,acetic acid,ethyl acetate and ethyl lactate.4.According to the Ren Huai Maotai-flavor liquor technical Standard System,this study was the physicochemical properties of liquor used for quantitative analysis.The result showed that the quality and yield of Maotai flavor liquor in the third,fourth and fifth liquor rounds was the best.Because the enzyme activitysome of protease and alpha-amylase were higher in stacking fermentation than in in-pit fermentation.And according to the abundance change of dominant bacteria Escherichia-Shigella,Lactobacillus,Clostridium-sensustricto-1 and Alternaria,it looked forward that the unique manufacturing process of Maotai-flavor liquor brought about stable microbial flora and each dominant bacteria produced corresponding enzyme.Also stacking provided prerequisite substance for in-pit fermentation.And then with the full decomposition of aroma components,the liquor flavor of each round could be special and unique.5.The results showed that the ethyl carbamate was detected and its content ranged from 41.07?g/kg to 284.11?g/kg,which conformed to the content limited 400?g/kg suggested in Risk assessment of ethyl carbamate in main alcoholic beverages in China.Only a few samples in the content of ethyl carbamate was higher than the foreign limitation standards of related alcohol.As the test results,it was found that the content of ethyl carbamate showed an upward trend on the whole,whether it was the fermented grains sample in the fermentation stage.Through cluster analysis of the dominant microbes in the fermented grain,it was found that lactic acid bacteria as well as some other genus such as Peptoclostridium,Actinobacillus,Turicibacter and Aquabacterium involved in the formation of ethyl carbamate formation.Therefore,this study provided valuable information for the pathways of ethyl carbamate formation in liquor fermentation,and also provided theoretical information for the development of standards related to production of sauce-flavor liquor.