Analysis Of Microbial Community In The Brewing Of Light Aroma Style Fenjiu Using PCR-DGGE

Liquor is a Chinese traditional fermented food. There are no practical qualitative andquantitative detection methods and insufficient understanding of liquor brewing biology,which are the main limiting factors to cause brewing theory and production technology ofliquor to progress at a slow pace. Light aroma style is one of the basic types of Chinese liquorand represented by Fenjiu, which is deemed as origin of liquor. Low-temperature Daqu isused as starter culture to produce light aroma style. Lower manufacturing temperature ofDaqu results in more complex microbial diversity in the brewing system of light aroma stylethan that in other types of liquor using medium-temperature Daqu or high-temperature Daquas starter. Moreover, special brewing techniques of light aroma style leads to simpler andstabler fermentation system. Therefore, light aroma style is a more suitable object to analyzeChinese microbiota.Based on international advanced techniques of microbial population, molecular ecologymethods to be suitable for Chinese liquor brewing system were established. Microbialcommunity structure in preparation of Daqu and fermentation process of Fenjiu was analyzedin order to reveal ecological characteristics of liquor brewing and identify key functionalmicroorganisms. These are vital and valuable for enriching brewing scientific theory onChinese liquor and modernizing liquor production. The main results were shown as follows:1) In order to solve problems of complex microbiota and solid-state matrix interference inthe brewing system of liquor, the methods of pretreatment of Daqu and fermented grains andextraction of genomic DNA were established. After amplifying16S rRNA gene V3regionusing a primer pair of P2/P3and overcoming defects in general DGGE, PCR-DGGEconditions were optimized. This method could detect bacterial species including those whichwere previously found through traditional isolation methods, low in the fermented system anddiffcult to culture such as lactic acid bacteria (LAB). Compared with16S rRNA clone library,PCR-DGGE could quickly analyze bacterial community succession and abundant speciesduring the fermentation process, and reveal the effect of production techniques andmicroenvironment in the fermentation vessel on bacterial composition. The results providedresearchers with a sound technical basis for later study.2) Qualitative and quantitative means of fungal community structure in the brewing ofChinese basic styles of liquor were established. After genomic DNA of Daqu and fermentedgrains was extracted using improved beatbeader method,26S rRNA gene D1/D2region and18S rRNA gene were amplified using two primer pairs of NL1-GC/LS2and GC-Fung/NS1toanalyze yeast and fungal composition, respectively. Moreover, four primer pairs to quantifyfour main yeast which were Zygosaccharomyces bailii, Pichia anomala, Saccharomycescerevisiae, Issatchenkia orientalis were designed. Primer speciality was validated throughcommon PCR and qPCR. After optimizing qPCR reaction conditions, standard curves of fouryeast species were drawn. The detection limit of S.cerevisiae was10CFU mL-1and that ofother three species was100CFU mL-1. There was no significant difference betweenquantitative results of solid and liquid mixed cultures under the lab conditions obtained through qPCR and plate-counting method. With regard to solid-state fermented grains,although qPCR results were slightly higher than those obtained by dilution plating method,qPCR could real-time monitor yeast content in the production prosess.3) Based on special manufacturing characteristics of Fenjiu Daqu, physico-chemicalproperties, microbiota and flavor compounds in Daqu were detected to reveal the effect oftechniques on Daqu characteristics. Production operations of Qingchaqu, Houhuoqu andHongxinqu were basically the same. The difference of pile temperature lied in the stagesbetween Shangmei and Houhuo and that of Daqu center temperature lied in the stagesbetween Liangmei and Yangqu. At the stage of Dahuo, pile temperatures reached the highestlevels which were44°C,46°C,48°C, and center temperatures also reached the peak value at49°C,54°C and55°C. The different temperature dynamics caused distinction of watercontent and acidity appearing in the corresponding periods. Because heat tolerance of yeastand moulds was lower than that of bacteria, fungi content was similar and bacteriaconcentration was obviously different between three types of Daqu. Using PCR-DGGE, maingenera in Daqu were detected including Lactobacillus, Staphylococcus, Pseudomonas,Thermoactinomyces, Bacillus, Saccharomycopsis, Saccharomyces, Candida, Hanseniaspora,Debaryomyces, Pichia, Issatchenkia, Rhizopus, Aspergillus, Absidia, Mucor. The researchwork found more species than results obtained through traditional microbial methods. Whenthe preparation of Daqu was completed, special heat-tolerant and heat-sensitive fungalcompositions were formed in three types of Daqu. After storing three monthes, differentpredominent moicrorganisms and flavor components were re-formed in three types of Daqu.So mixed usage of Daqu can result in complementation of important functionalmicroorganisms and metabolite substrates.4) The physico-chemical characteristics, microbial composition and metabolite profile inthe fermentation process of Fenjiu were analyzed using culture-dependent method,PCR-DGGE, qPCR and HS-SPME-GC-MS. Population dynamic succession was directlyrelated with fermentation course. Bacillus, Lactobacillus, Pichia, Saccharomycopsis,Saccharomyces, Issatchenkia, Hanseniaspora, Torulaspora, Candida, Rhizopus, Amylomyces,Aspergillus, Absidia were the main genera in the fermented grains and Lactobacillus was themost abundant microbial gtroup in the later process of fermentation. LAB might have anantagonism against Bacillus and yeast. LAB inhibited the growth of Bacillus and yeast in theinitial stage and led to a decrease in content of Bacillus and yeast. There were high amount ofnon-Saccharomyces at the beginning of fermentation. S. cerevisiae propagated quickly andinhibited non-Saccharomyces. PCA analysis revealed that in the former period (2-15d)microorganisms propagated and Bacillus, yeast were contributing factors, and in the laterperiod (15-30d) flavor compounds were synthesized and LAB was influencing factor. LABmight play important roles in the fermentation of liqur. LAB not only regulate micro-environment in the fermentation vessel and composition of other functional microorganisms,but also contribute to the transformation of flavor components such as acids, alcohols andesters.The relationship of microbial composition (bacteria, yeast, moulds), environmentconditions (physico-chemical characteristics, production techniques) and system function (flavor components) was investigated through combination of microbial ecology methods andother fields of technology. Key microorganisms and regulation factors important tomicrobiota were preliminarily determined. The results of work provided with microbialknowledge of Chinese liquor and valuable guidance of theory and practice for modernizationdevelopment of liquor industry.