Analysis Of Microbial Communities And Its Effect On Flavor Substance Of Raw In Chinese Strong-flavor Liquor Pit Muds

The production of Chinese Strong-flavor Liquor is characterized by solid-state fermentation.The growth and metabolism of microorganisms in pit mud show an important influence on the formation of flavor substances.However,the effect of complex microbial community in the pit mud on the flavor compounds is still unclear.In this study,the pit mud samples from different aged cellar were collected from Songhe Company in Henan province.Aerobic and anaerobic bacteria were isolated from different cellar aged pit muds.The changes of the prokaryotic communities in different cellar ages were detected by high-throughput sequencing of 16 S rRNA gene.The volatile metabolic substances of these isolates and liquors from different cellar age are determined by gas chromatography-mass spectrometry(GC-MS).In addition,a connection between the microbial community in pit mud and liquor flavor were established,which may provide a theoretical basis for qualitative improvement of pit mud and liquor.The main results of this research are listed as follows:(1)Aerobic and anaerobic bacteria from different aged pit mud were isolated using traditional culture-dependent methods.The isolates were identified by 16 S rRNA gene sequence analysis.The results indicated that 45 strains of aerobic bacteria were isolated,belonging to 9 different species: Aneurinibacillus migulanus,Bacillus subtilis,Brevibacillus borstelensis,Ba.licheniformis,Br.laterosporus,Ba.pumilus,P.lautus,Br.frigoritolerans and P.cellulositrophicus.It is indicated that A.migulanus,Brborstelensis and P.lautus were seldom reported to distribute in pit mud of Chinese strong-flavored liquor.Moreover,the number of aerobic bacteria in pit mud reduced gradually along with the increase of cellar age.(2)In this study,media of RCM and ES were used to isolate the Clostridium in pit mud and identified mainly based on sequence analysis of 16 S rRNAgene.The results suggested that 100 strains of anaerobic bacteria were isolated,and 78 strains showed high sequence similarity(>91%)with 8 Clostridium bacteria in GenBank,including Clostridium celerecrescens,C.cochlearium,C.carboxidivorans,C.sporogenes,C.sartagoforme,C.thermopalmarium,C.aurantibutyricum,and C.butyricum.These Clostridium isolates showed difference among 10-year pit mud with those of 6-year and 1-year samples.The main reason may be that the physicochemical properties changed in pit mud with the increase of pit mudage,thus causing alertation of the microbial community structure accordingly.(3)The high-throughput sequencing of 16 S rRNA gene accurately revealed the changes of the prokaryotic community in the pit mud from different aged cellar(1-year,6-year and10-year).The results indicated that the prokaryotic community structure was significantly changed with cellar age,and most of the core genera in different aged pit muds were significantly changed.In Firmicutes phylum,relative abundance ofLactobacillus and Caloramator were significantly decreased with cellar age,which was 2.87 and 6.17 fold lower in 1-year sample than in 10-year sample,respectively.However,relative abundance of most genera in Firmicutes phylum we detected was lower in 1-year than in older samples,such asClostridium,Syntrophomonas,Sedimentibacter,Unclassified Clostridia.In Euryarchaeota phylum,relative abundance of Methanosarcina,Methanobacterium,and Methanobrevibacter,the detected methanogens in our pit mud samples,were significantly increased by 5.31-fold,3.26-fold and 3.99-fold in 10-year sample relative to 1-year sample.Relative abundance of Aminobacterium in phylum Synergistetes and Unclassfied_Coriobacteriaceae in phylum Actinobacteria had the same increasing trend with cellar age.Relative abundance of Unclassfied_Porphyromonadaceae,Blvii28 and Unclassfied_BA008 in Bacteroidetes phylum was significantly increased from 1-year sample to 6-year sample,though it was decreased in10-year pit mud.Relative abundance of T78 in Chloroflexi phylum was increased from 0.82% in 1-year pit mud samples to 3.43% in 6-year sample,however,it was decreased to 0.21% in 10-year pit mud.Furthermore,environmental variables of pH,NH4+,available phosphate(AP),available kalium(AK),and Ca2+had significant correlation with prokaryotic community as revealed by Redundancy analysis.(4)The flavor substances in liquor with different cellar age(1-year,6-year and 10-year)were analyzed using liquid-liquid extraction and GC-MS.The volatile metabolic substances of these isolates were extracted by headspace solid phase microextraction(HS-SPME)and determined by GC-MS,and the relationship between them was analyzed.The results indicated that these aerobic and anaerobic bacteria could produce a variety of flavor substances or precursors of flavor substances.The aerobic isolates of O2.4(Bacillus subtilis),O2.10(Bacillus licheniformis),O2.11(Brevibacillus laterosporus),O2.15(Bacillus pumilus),O2.17(Paenibacillus lautus),O2.18(Bre.frigoritolerans)and O2.19(Paenibacillus cellulositrophicus)could produce ethyl caproate,which is the main flavor of Luzhou flavorliquor.Meanwhile,the anaerobic isolates of R50D1(C.celerecrescens),R50D4(C.cochlearium),R50zhong7(C.sporogenes),R50zhong10(C.sartagoforme)and R70D1(C.aurantibutyricum)could produce butyric acid in RCM.As one of the major acid and precursor of esters in liquor,butyric acid is beneficial for the formation of characteristic liquor flavors in wine.