Screening Of Polysaccharide-degrading Bacteria In Stinky Tofu Brine And Its Application In Microcapsule Biological Preparations

Stinky tofu brine contains complex microbial flora,it also has cellulose,xylan,chitosan,soybean oligosaccharides and other polysaccharides degradated by vegetable materials,soy materials and some animal raw materials.However,the type of these bacterias and the the interaction between them is unclear.The aim of this study is to separate the bacteria which can degrade polysaccharides from stinky tofu brine by using conventional bacterial isolation method and molecular biology method,and we hope to provide the material basis for the development and utilization of new microbial resources.The main results are as follows:(1)In this study,we prepared the modified MRS medium by using three kinds of oligosaccharides(SBOS,XOS and FOS)as carbon sources,then we used those modified medium to enrich the microorganisms in the stinky tofu brine samples without oxygen.Then we extracted the microbial total DNA and used high-throughput sequencing to analysis flora composition of these samples.The result showed that the diversity of microorganisms became lower after the enrichment.The result of venn diagram refleted that different group has common and unique OTUs.The number of OTU in the original sample(chou)was 1041,which was significantly higher than in the enrichment groups,this indicated that these oligosaccharide mediums had some selectivity to enrich the microbes.At the Phylum level,the Proteobacteria was significantly reduced while the Firmicutes was significantly improved and became the dominant Phylum category.In glucose(mrs)enrichment group,the percentage of Firmicutes was the highest which was up to 82.2%.At the genus level,the Pseudomonas and Arcobacter in oligosaccharide enrichment groups were significantly reduced and the proportion was less than 1%.However,the proportion of Streptococcus and Lactobacillus in the enrichment groups were both increased,they increased to 29.37%and 3.71%in SBOS enrichment group(dou);XOS enrichment group(mu)increased to 17.98%and 14.95%;FOS enrichment group(guo)was increased to 24.12%and 6.79%.Those indicated that compared with oligosaccharide enrichment groups,the ability of enriching Streptococcus and Lactobacillus was the strongest in mrs group.Compared with mrs group,streptococcu,Pseudomonas and clostridium in oligosaccharide enrichment groups were significantly increased.(2)We usd traditional bacterial isolation method to isolate and purify the colonies on the oligosaccharide solid plate.The results showed that we isolated the Lactobacillus fermentum YZUF1 in each solid plate.we isolated the Staphylococcus epidermidis YZUS2 in the XOS medium.From the results of the growth curve in all kinds of medium,it showed that the delay period of the Lactobacillus fermentum YZUF1 in SBOS and XOS medium was 4 hours,the highest maximum growth rate were 0.049 and 0.118 and the maximum growth were 0.609 and 0.872.The delay period of the Staphylococcus epidermidisYZUS2 were both 4 hours in each medium.The growth curve of the Staphylococcus epidermidisYZUS2 in FOS and MRS medium was close,their maximum growth rate were both 0.127,maximum growth were 1.123 and 1.072.We usd traditional bacterial isolation method to isolate the strain in COS medium from stinky tofu brine.As a result,the strain YZUc2-3 was obtained which can degrade COS best.The strain YZUc2-3 was identified as Corynebacterium vitaeruminis and and the homology was 99.6%by 16S rDNA sequencing.(3)We used YZUc2-3 and COS to feed the ICR mice,then we used high-throughput sequencing to analysis the intestinal flora of the mice.The results showed that after 5 days of continuous intragastric administration,we found that after 5 days of 108cfu/mL YZUc2-3 and 3%COS intaking,the Lactobacillus in the feces were higher than those in the saline group(1.32%),which were 3.96%and 13.4%.However,the proportion of Lactobacillus in YZUc2-3 + COS group was significantly lower than in the saline group,which was 0.57%.There was no significant difference in streptococcus between the treatment group and the control group,the proportion was about 2.5%.The results showed that the YZUc2-3 and COS could increase the proportion of Lactobacillus in the mouse intestine.With intaking YZUc2-3+ COS,YZUc2-3 can form the dominant flora in the intestine by using COS.(4)CS can effectively protect the bioactive material from degradation in the body,but how to release these bioactive material from CS is not reported at home and abroad recently.This study investigated the possibility of the obtained chitosan-degrading strain YZUc2-3 as a biodegradable agent.We used odium alginate and CS as materials to embedded the phage and strain YZUc2-3.The aim is to study the behavior of phage releasing in vitro with the presence of strain YZUc2-3.The results showed that the microcapsules have uniform size and regular size,and the average particle size is 630±37μm.Then we released the phage microcapsules in vitro.The results showed that the number of the phage was increasing along the time.The microcapsule phages which contained strain YZUc2-3(A and B group)releaseed the higher phage,which was 1 × 107PFU/mL and 5.6 × 106PFU/mL.This suggested that the phage in the microcapsule phages can be efficiently released under the induction of strain YZUc2-3.