The Diversity Of Four Main Enzymes Producers In Strong Flavor Daqu During Fermentation And Maturation Stages

With the functions of saccharification,fermentation,esterification and aroma-producer,Daqu provides both the microorganism,aromatic precursors and plenty of enzymes for the fermentation and flavour formation of Daqu liquor.Daqu,as an important guarantee for the quality and production of daqu liquor,has always been the focus of liquor industry research.Tannase(EC3.1.1.20),also known as tannic acid hydrolytic enzyme,tannase.Can tannin enzyme hydrolysis esters of gallic tannin and condensation of phenol carboxy keys,generate glucose and gallic acid,influence on liquor flavor and quality at the same time,the tannin enzyme has been widely used in the fields such as food,chemical and pharmaceutical applications.Esterifying enzyme is one of the most important enzymes in the production of luzhou-flavor liquor.The amount of enzymatic enzyme and enzyme activity are closely related to the rate of liquor quality.Protease can not only hydrolyze the protein of fermentation materials and bacteria,but also promote the production of wine and flavor and reduce the content of alcohol oil.Lipase is a kind of microbial growth and reproduction,and liquor flavor substances have certain effects.This study was focused on the isolation and screening producers of tannase,protease,esterase and lipase,coupled with the calculation of ratio of the diameters of the transparent circle and the colony,description of colonial morphologies,and identification by 5.8S PCR and 16 S PCR methods.The detailed information of every enzyme producers were exhibited below.The number of colonial morphologies of prokaryote was 9 out of total 16 types of tannase-producers,which is more than that of prokaryote by two types.The diversity of eukaryotic colonial morphologies of eukaryote was significantly richer during Daqu fermentation than maturation stage.Three eukaryotic genera,including Penicillium,Aspergillus and Byssochlamys were identified by molecular identification.Among these genera,six species,accounting for 60% of all tannase eukaryotic producers,belonged to Aspergillus.Followed by Aspergillus,Penicillium has three species,such as Penicillium commune strain,Penicillium chrysogenum strain and Penicillium robsamsonii.For prokaryote producing taanase,three genera all have one species identified in this study,namely Bacillus siamensis strain,Oxalophagus oxalicus strain and Pedobacter sp.Among them,Byssochlamys spectabilis strain and Bacillus siamensis strain isolated more frequently in the samples from fermentation stage,however,the species of Aspergillus and Pedobacter were more frequently obtained in maturation stage.In addition,tannase produced by Penicillium robsamsonii and Oxalophagus oxalicus strain has not been reported.The number of colony morphologies in the eukaryote secreting esterase was 11 types and most of them were white colonies.The prokaryotic colony morphologies were 22 types with characteristics of round,white or slightly yellow color.Among all the Daqu samples,five types of eukaryote and thirteen types of prokaryote were found in both two stages,which exhibited higher morphologies diversity.Fourteen species belonged to three prokaryotic genera(Bacillus,Paenibacillus and Cronobacter)were identified,of which 11 species of Bacillus were the most abundant microflora in both two stages.However,there are only 11 species in 6 eukaryotic genera.Three species in both Aspergillus and Lichtheimia were the abundant eukaryotic microflora during the two stages.Among all the species,Lichtheimia ornate,Bacillus wiedmannii strain and Paenibacillus relictisesami were rare reported producing esterase.As for the protease producer,the eukaryotic and prokaryotic colony morphologies were 10 and 23 types.Compared with the colony morphologies of protease producers,the number of eukaryotic colony morphologies of lipase producers was same that of eukaryotic protease producer,but the number of prokaryotic colony morphologies in lipase producers was lower than that of protease producers by 78%.Based on the molecular identification,five eukaryotic genera with the ability of secreting protease were identified,including Lichtheimia,Mycocladus,Penicillium,Fungal and Aspergillus.Among them,the genera Aspergillus and Penicillium also can secret lipase in this research.The prokaryotic producers of two enzymes have three genera(Bacillus,Hafnia and Bacillus),and only three species of Bacillus(Bacillus licheniformis,Bacillus sonorensis strain and Bacillus paralicheniformis)have the ability to produce lipase.Lichtheimia ornata and Aspergillus oerlinghausenensis were reported for the first time producing protease and lipase,respectively.There are few of study,focused on the same batch of Daqu samples,which involve in the diversity of microorganisms producing some important enzymes during the whole fermentation and maturation stages of Daqu.Therefore,with the deep desires to explore the mystery of diversity,dynamic,alternation of microorganism relating to mainly functional enzymes,the study by cultivable way were carried out on isolation,screening,and identification of producers of main enzymes using the Daqu samples derived from the whole fermentation and maturation stages,after descripting colonial morphologies and analyzing information of distribution and dynamic of eukaryotic and prokaryotic enzymes producers.This investigation will solid the foundation of researches on enzyme preparations,strengthen Daqu,enzymatic spectrums of microflora and their catalytic mechanism.