Investigation Of Microbial Community Of Chinese Liquor Daqu By Polymerase Chain Reaction-denaturing Gradient Gel Electrophoresis

Solid state fermentation based on starters (Daqu in Chinese) is a major characteristic of Chinese liquor which is well known Chinese traditional fermented food. Daqu is prepared by solid state fermentation with uncooked grain such as wheat and barley. As the main provider of microorganism, the quality of Daqu directly affects the yield and quality of liquor. For a long period, due to the disperse research condition and backward technologies, researchers know very limited about the microbial communities in Daqu.To overcome the limitation of culture-dependent method and get the Daqu microbial community from a culture-independent method, the microbial diversity of Chinese liquor Daqu was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The investigation of Daqu microbial community based on culture independent method would have theory and practice sense in getting better understanding of Daqu microbial community structure and the mechanism of traditional brewing microbe.10 typical Daqu samples collected from different regions were analyzed for bacteria, yeast and fungi by PCR-DGGE as a culture independent method. Extracted DNA was used as a template for PCR to amplify 16S rRNA gene V3 region, 26S rRNA gene D1 region and 18S rRNA gene partial region using universal primers. The Daqu microbial community information was confirmed by sequencing of dominant bands cutting from DGGE gel.The result indicated that1)For bacteria, compared with culture-dependent method, the bacterial DGGE profile displayed high diversity. Lactic acid bacterias were one of the dominant bacterial groups in different Daqu samples. The LAB included Weissella cibaria, L. helveticus, L. fermentum, L. panis and L. pontis. Thermoactinomyces sanguinis, Pseudomonas spp., Staphylococcus xylosus, and Stenotrophomonas maltophilia were also detected in bacterial DGGE while they were not detected in.2)As for fungi, researches based on traditional culture dependent method and culture independent method published in recent years mainly focused on bacteria, so 26S rRNA gene D1 region and 18S rRNA gene partial region DGGE was proceeded in Daqu samples. The main yeast detected by traditional culture-dependent method were Saccharomyces cerevisiae, Hansenula spp., Pichia spp. and Endomycopsis spp.. Besides these yeast, the DGGE method also detected yeast such as Hanseniaspora guilliermondii, Debaryomyces hansenii, Trichosporon asahii and Issatchenkia orientalis. And the non-Saccharomyces yeasts revealed its high diversity in this study. The predominance of Saccharomycopsis fibuligera and Pichia anomala were proved in all the samples. The fungal 18S rRNA DGGE proved the dominance of Absidia spp. and Aspergillus spp. in Daqu. Saccharomycopsis fibuligera, Saccharomyces bulderi, Candida allociferrii and Candida tropicalis were identified as yeast in the fungal analysis. DGGE profiles of fungal 18S rRNA gene showed fewer bands than bacterial and yeast DGGE profiles. Daqu manufactured with only wheat showed lower Shannon-Wiener diversity than those manufactured with more than one variety of grains. 3)Bacillus licheniformis and Bacillus subtilis were proved to be dominant in Daqu Bacillus species. Due to the limitation of PCR amplification by bacterial universal primer, 16S rRNA V3 DGGE failed to discriminate Bacillus species in the Daqu samples. A nested-PCR based DGGE method was established. The gene which was special for Bacillus species was obtained by first round PCR and the Bacillus 16S rRNA V9 gene was obtained by the second round PCR. The 16S rRNA V9 gene DGGE was proved to be effective in profiling Bacillus community in Daqu.4)To get a better understanding of correlation between craftwork and bacterial community structure in Daqu. The bacterial community structure of 5 high and medium temperature Chinese liquor Daqu from the same region were investigated using PCR-DGGE. DNA sequencing was proceeded to obtain the dominant bacterial population information. The result of DGGE profiles showed that Weissella cibaria, L. helveticus, L. fermentum and L. panis were commonly detected in all the five Daqu. Thermoactinomyces sanguinis was detected only in the Jiangqu sample. The correlation between the craftwork of Daqu and the bacterial community structure was obvious. The Daqu bacterial Shannon-Wiener index decreased as the craftwork temperature of Daqu increasing. PCR-DGGE was proved to be a powerful tool for gaining detailed insight into the bacterial diversity of the Chinese liquor Daqu.Microbial community of different Chinese liquor Daqu was investigated using culture-dependent method in this study and information of dominant species in Daqu was obtained and the affection of craftwork on microbial community structure was studied. The combined analysis of microorganism community structure in Daqu obtained by DNA fingerprint methods with interrelated results using other experiment means, such as traditional microbial identification based on culture and separation, detection of liquor solid-form fermentation and analysis of aroma components in liquor, will help to isolate and identify important microbes corresponding to the formation of characteristic key flavor components, and direct improvement of production techniques.