Analysis Of Microbial Community Construction In Different Self-made Enzyme Samples By PCR-DGGE Technique

Objective:In recent years, the study on enzyme resources have become more and more popular. The efficacy was concerned by the researchers and they had made some progress but composition exploration. Based on that, this experiment put the naturally fermented enzyme as the research object and the technique of PCR-DGGE was chosen to detect the composition of the bacterial community. With this method, the condition of microbial metabolism and the rule of evolution on bacterial community will be reflected in some degree. At the same time, we can figure out the relations between the difference of raw materials fermentation and enzyme products. It could provide a reference and complementary for the population of domestic enzyme.Methods:1. Taking in enzyme liquid which accord to the brown sugar: fermentation materials:water = 1:3:10(1kg,3kg,10L) for 10 months, and fermentation materials include plants, fruits and so on. We got about 100 mg bacteria mud after filtering and centrifuging.2. The method of CTAB/proteinase K was used to extract the total DNA in microorganism mud and it was purified with DNA agarose gel purification kit.3. The bacteria 16 Sr DNA V3 region and yeast 26 Sr DNA D1 region were amplified by PCR.4. After optimizing the fingerprints of bacteria and yeast,the Quantity one software was used to analyze the DGGE profiles.5. After sequencing in Beijing Genomics Institute(BGI),we constructed phylogenetic trees and compared the sequencing results with Clustal X and Mega5.0 softwares.Results:1. The comparability of bacterial community in different samples source varies from59.1% to 97.3%. At the level of 63%,6 kinds of enzyme samples belong to the same cluster.The bacterial community in the enzyme samples is diverse, the internal microbial community composition maintain stabilized with the extension of fermentation time.2. The bacterial diversity index, richness and evenness in various samples are close.No.6(carrot) sample has the highest diversity index and richness which are 1.60 and 5.00. On the contrary, these two indicators of No.1(apple) are lowest with the index 0.69 and 2.00.3. The bacterial phylogenetic tree shows that all of the sequences can be divided into 4 main clusters. Cluster 1 contained 3 kinds of bacteria of Lactobacillus genus, and it gets together with band E. The similarity with Lactobacillus kefiri is99%,so band E can be determined as Lactobacillus kefiri.Band A of cluster 2belongs to Bacteroides thetaiotaomicron, similarity is 99%. The similarity of band C in clusters 3 and Acetobacter pasteurianus reaches 99%. Band B and D are parts of cluster 4. They are Lactobacillus harbinensis and Lactobacillus acetotolerans. At the clusters’ nodes, the credibility of homology is bigger than90%, it illustrates that the relations is credible.4. The dominant bacterial communities in 6 kinds of enzyme samples distribute on 3genera and 5 species. Lactobacillus harbinensis, Lactobacillus acetotolerans and Lactobacillus kefiri belong to Lactobacillus; Acetobacter includes Acetobacter pasteurianus; Bacteroides thetaiotaomicron is a part of Bacteroides.5. Yeast community structure comparability fall in between 5.9% and 74.9%.Clustering analysis shows that at the level of 17%, yeast flora are divided into 2large groups and at the 75% level, the population structure of No.1(apple) and No.2(chayote) are special. Group 2 holds another 4 kinds of samples together at43%.6. No.1(apple) and No.2(chayote) sample have the highest diversity index and richness in the yeast, they are 1.45, 5.00 and 1.49, 5.00. Compared with them No.3(Alternantheraphiloxeroides) is lowest with a index of 0.98 and 3.00.7. According to the yeast phylogenetic tree, the cluster 1 contains the Pichia sequences, band I, F, G are parts of it and homology credibility is 66%. Cluster 2is Saccharomyces, the homology credibility of band J with Kazachstania exigua is 86%. Because the species of Issatchenkia scatteres in Pichia yeast and it can not be separated from Pichia. Therefore, cluster 3 contains sequences of Pichia and Issatchenki, and Band H and K have a close relationship, which belong to the Issatchenkia occidentalis.8. The dominant communities of yeast are Pichia, including Uncultured Pichia,Pichia membranifaciens,Pichia galeiformis and Pichia occidentalis.Issatchenkia occidentalis which belongs to Issatchenkia,and Kazachstania exigua which belongs to saccharomyces.Conclusions:1. After 10 months fermentation, the bacteria comparability index are bigger than60% and the community maintain stability in different enzyme samples.2. In the 6 enzyme samples, the dominant bacterial commutities are distributed on 3genera and 5 species,and the dominant yeast commutities with 3 genera and 6species distribution.This study prvides supplement for bacteria and yeast resource of domestic enzyme.3. The discovery of Lactobacillus and Bacteroides could expand the edible and medicinal development of enzyme.