In Vitro Study About Regulation Effect Of Polysaccharide From Dendrobium Officinale Leaves On Human Intestinal Microbiota

Dendrobium is rich in polysaccharides,flavonoids,alkaloids and polyphenols,among which polysaccharides are the main active components.Dendrobium officinale leaves are usually the by-products of Dendrobium Maple processing which have not been used effectively.The content of polysaccharide in the leaves of Dendrobium is about one third of that in stems,The output is large,but it has not been used effectively.In order to promote the full utilization of Dendrobium officinale resources,in our study,we took Dendrobium 1,Dendrobium 3 and Saint LAN 8 as raw materials to extract and purify the polysaccharide from their leaves(named DOP1,DOP2 and DOP3 in turn),and determinetheir primary structure.Firstly,the influence of three polysaccharides on the proliferation of the common beneficial bacteria and pathogens was studied in pure culture systems,respectively.Then,onein vitrocomplex culture system with human intestinal microbiota was established,in which the impacts of the three kinds of polysaccharides on intestinal microbiota of male/female were respectively studied.DOP3,which had better regulation effects,was selected.Moreover,the regulation effects of DOP3 on the composition of intestinal microbiota and metabolite of short chain fatty acids(SCFAs)in different parts of colon(ascending colon,transverse colon and descending colon),were studied by in vitro colon simulation system to establish the foundation for its further functional researches.The main research contents and results are as follows:(1)The polysaccharides from Dendrobium 1,3 and 8 leaves were extracted and purified.The results showed that DOP1 had two absorption peaks that their relative molecular weight were 2.56×10~5Da and 2.68×10~3Da,respectively.In addition,the relative molecular weight of DOP2 and DOP3 were 1.35×10~5Da and 3.26×10~5Da,respectively.These polysaccharides were all composed of four monosaccharides,including arabinose(Ara),galactose(Gal),glucose(Glc)and mannose(Man),but had different molar ratios.The results of scanning electron microscope showed that the structures of these polysaccharides were all reticular,the spherical and ellipsoidal aggregates were irregularly distributed on the smooth membrane structure of DOP1,and the other two polysaccharides had a smooth and uniform surface.All three polysaccharides could be the only carbon source to promote the growth of Lactobacillus plantarum,Lactobacillus pentosus and Lactobacillusrhamnosus,of which DOP3 was the best.The promotion effect became better when combined with glucose.On the contrary,allthree polysaccharides did not promote the proliferation of Escherichia,Staphylococcus aureus and Listeria monocytogenes,indicating that the three strains could not decompose and utilize the polysaccharides.(2)The feces of different genders were collected to perform in vitrocomplex culture system with human intestinal microbiota using three Dendrobium polysaccharides as the only carbon source.The fermentation broth was sequenced by16S rRNA sequencing,and detected SCFAs contents by gas chromatography(GC).The results showed that DOP3 increased the diversity of male gut microbes and the number of unique species.Three polysaccharides increased the relative abundance of Firmicutes and Bacteroidota,but reducedthat of Proteobacteria.At the genus level,all three polysaccharides promoted the growth of Bifidobacterium,Bacteroides,Collinsella and Prevotella,but inhibitedthat of Escherichia-Shigella.Furthermore,the regulation effects of three polysaccharides on female werebetter than that of male.In addition,polysaccharides significantly promoted the production of SCFAs,and the effects of DOP1 and DOP2 were similar while DOP3 group had highertotal SCFAs in male systems.(3)In vitro colon simulation system,we studied regulation effects of DOP3 on the composition of intestinal microbiota and metabolite of SCFAs.It was found that DOP3 improved the diversity of bacteria in ascending colon,transverse colon and descending colon.At the phylum level,DOP3 reduced the relative abundance of Proteobacteria and Actinobacteria,but increased that of Firmicutes and Bacteroides.At the genus level,it significantly reducedthe relative abundance of Escherichia-Shigella and Desulfurious,but increased that of Bacteroides,Bifidobacterium and Parabacteroides(P<0.05).Propionic acid,butyric acid and valeric acid increased in colon simulation system.