Optimization Of Mulberry Polysaccharide Preparation And Its Probiotic Effect Of Hydrolysates

Mulberry(Fructus Mori) is a mature fruit of mulberry mulberry(Moraceae)mulberry(Morus L.).It is rich in bioactive substances such as polysaccharides,anthocyanins and polyphenols.It has many health functions such as reducing blood sugar,lowering blood lipids and improving constipation.It has been listed in the first batch of “medicine and food homologous” list by the National Ministry of Health.In this study,mulberry was used as the raw material,and the enzyme-microwave assisted method was used to optimize the extraction technology of mulberry polysaccharides.Mulberry polysaccharides were hydrolyzed by physical hydrolysis,chemical hydrolysis and enzymatic hydrolysis in three ways.The effects of mulberry polysaccharides and their hydrolysates on the in vitro growth of four probiotics such as Lactobacillus plantarum,Leuconostoc mesenteroides,Lactobacillus casei and Lactobacillus rhamnosus were investigated.Finally,after the co-cultivation of polysaccharide hydrolysates and Lactobacillus rhamnosus was simulated-digestion,human intestinal fecal co-culture experiments were conducted to explore the changes of human intestinal bacterial community structure.The major results were listed below:(1)Optimization of enzyme-microwave preparation of polysaccharides processThe effect of each factor on the extraction of mulberry polysaccharides was investigated by single factor test and optimized by the Box-Behnken test.The results showed that the enzyme-microwave assisted optimum conditions were as follows: cellulase addition was 9mL(1000 U/mL),microwave time was 54 s and microwave power was 600 W.The extraction rate of mulberry polysaccharides was 3.01% under the above conditions.(2)Preparation of the hydrolysate of mulberry polysaccharideMulberry polysaccharides,physical hydrolysates and enzyme hydrolysate can stimulated the proliferation of the four probiotics.Among them,the enzymatic hydrolysis of mulberry polysaccharides had the best effect on the proliferation of Lactobacillus rhamnosus,and the proliferation rate was up to 252%.Afterwards,the proliferative effects of four commercially available enzymolysis products on Lactobacillus rhamnosus were explored.The results showed that β-mannanase was the best and superior to commercial prebiotics galacto-oligosaccharides and isomaltooligosaccharides.(3)Evaluation of the probiotic effect of co-cultures of mulberry polysaccharide hydrolysates with Lactobacillus rhamnosusThe co-culture of mulberry polysaccharide hydrolysate with Lactobacillus rhamnosus retained a certain number of viable cells after artificially simulated stomach and intestine digestion.After co-cultivation of the digest with human feces,Illumina high-throughput sequencing technology was used to determine the composition of microbial colonies in the experimental and control groups,respectively.The dilution curves drawn by the chao,ace,shannon and coverage indices indicate that the sequencing of all the samples basically covers all the species of the sample and can describe the sample microbial community more accurately.A total of 244 OTU were detected in the control group,and 234 OTU in the experimental group.The three algorithms based on cluster analysis pointed that the experimental group and the control group are basically clustered together at each time.The principal component analysis of PCA and PCoA showed that there is a relatively clear separation from 0h and after 24 h.After co-culture of digestibles and feces for 48 hours,the value of Bacteroidetes/Firmicutes in the experimental group was 0.848 higher than that of the control group 0.659,indicating a certain degree of regulation of the intestinal microflora.On Family level,the Lactobacillaceae of the experimental group gradually increased during the culture period.On Genus level,the proportion of Bacteroides in the experimental group was 15.7%,which was higher than that in the control group.The research results provide a theoretical basis of optimizing the composition of intestinal microbes for the product development of the prebiotics in the next step.