Effect Of Polysaccharide From Dendrobium Officinale On Human Intestinal Flora In Vitro Fermentation

Dendrobium officinale is a traditional prized Chinese herbal medicine,polysaccharide which is its main bioactive ingredient has beneficial effects on colon health.Most of the polysaccharides used as dietary fibers in the digestive tract of the human body are not affected by various digestive enzymes and transport proteins,but are used as substrates for the degradation and utilization of intestinal microflora that are symbiotic to the human body,and metabolites from intestinal microflora also affect the intestinal health.This article explores the effects of D.officinale polysaccharides on human intestinal flora and microbial metabolites in vitro,the polysaccharides used in this study were extracted from Dendrobium officinale in Yandang Mountain.Intestinal microbiota collected at Zhongshan Hospital of Xiamen University from a healthy FMT(Fecal microbiota transplantation)donor.This research mainly include the isolation and purification of polysaccharides from Dendrobium officinale,the identification of monosaccharide components in polysaccharides,the effects of Dendrobium officinale polysaccharides on the changes of intestinal microflora structure and metabolites,and the correlation analysis.1.The content of polysaccharide of Dendrobium officinale was used as the evaluation index,the orthogonal experiment was conducted with the ratio of material to liquid,extraction temperature and extraction time as the influencing factors,the extraction process of Dendrobium officinale was optimized by the orthogonal experiment and the content of polysaccharide was detected by phenol-sulfuric acid method.The results show that the effect of three factors on the extraction rate of polysaccharide from D.officinale is as follows:temperature>solid-liquid ratio>extraction time,the optimum extraction conditions for D.officinale polysaccharides are extraction temperature 80?,solid-liquid ratio 1:40,extraction time 2 h,and the stability of the extraction conditions was proved by verification test.The extraction rate of D.officinale polysaccharide under this extraction condition was 31.85%.The protein content was detected by Coomassie brilliant blue method.The polysaccharide contained no protein,and the monosaccharide composition of the Dendrobium officinale polysaccharides were derivatized with PMP(1-phenyl-3-methyl-5-pyrazolone).The results showed that the monosaccharide in Dendrobium officinale polysaccharide is mainly composed of mannose(49.03%),glucose(16.54%)and galactose(14.83%).2.The in vitro fermentation method was used to study the effect of D.officinale polysaccharides on intestinal flora.In the experimental group,200 mg of D.officinale polysaccharide was added,and in the control group,an equal dose of ultrapure water was added.Sampling analysis was performed at 0,4,8,12,24,36,48 hours after inoculation.The experimental analysis results are as follows:(1)During 24 hours of in vitro fermentation,the total carbohydrate content in the experimental group's fermentation broth rapidly decreased from 739.45±11.74 mg L-1 to 270.86±20.47 mg L-1.After 24 hours,the consumption rate of polysaccharides slowed down,indicating that the Dendrobium officinale polysaccharides are absorbed by the intestinal flora,which makes the intestinal flora grow rapidly in fermentation.(2)4 hours after inoculation,three types of monosaccharides were found in the fermentation broth.The three monosaccharides were the main components of Dendrobium officinale polysaccharides,namely mannose,glucose,and galactose.With the progress of fermentation,the contents of these three monosaccharides decreased to a certain extent,glucose was not detected in fermentation broth at 12 h after inoculation,and no mannose and glucose were detected in fermentation broth at 36 h after inoculation.This result shows that Dendrobium officinale polysaccharides can be degraded into monosaccharides by the intestinal flora,and the monosaccharides degraded can continue to be utilized by the intestinal flora.(3)After 48 h of fermentation,the concentration of total short-chain fatty acids in the fermentation broth of D.officinale polysaccharide group was 51.76 ± 0.45 mM,which was significantly higher(P<0.05)than that of blank control group(35.74 ±0.26 mM after 48 h of inoculation).The differences in the production of short-chain fatty acids between the two groups during fermentation were consistent with the differences in pH.After 48 hours of fermentation,the pH of the fermentation broth of the D.officinalis experimental group was significantly lower(P<0.05),the pH of the fermentation broth was 6.95 when inoculated,the pH of the fermentation broth was 4.70 at 48 h after inoculation,and the pH value of the blank control group did not change significantly,the pH of the fermentation broth was 6.98 when inoculated,and the pH of the fermentation broth was 6.71 at 48 h after inoculation.At 48 h after inoculation,the concentrations of acetic acid,propionic acid,and butyric acid in the experimental group of Dendrobium officinale(the acid concentrations in the fermentation broth after fermentation for 48 h were 30.75 ± 0.37 mM,12.43 ± 0.05 mM,and 8.58 ±0.10 mM,respectively)were significantly(P<0.05)higher than those in the blank control group(the acid concentrations in the fermentation broth for 48 h were 18.67 ± 0.15 mM,9.06±0.11 mM,and 7.01±0.07 mM,respectively).(4)Illumina HiSeq 2500 system was used to analyze the 16s rDNA sequence of intestinal flora during fermentation to study the changes of intestinal flora structure.The results showed that during 48 hours of fermentation,the structure of gut microbiota in the experimental group and the control group always had a significant difference;Bacteroidetes and Firmicutes were significantly increased in the experimental group,while Proteobacteria were significantly reduced;At the taxonomic level of Genus,the beneficial bacteria of Prevotella,Bacterioides,Lachnoospiracea and Faecalibacterium increased significantly in the experimental group,while the conditional pathogens Citrobacter and Enterobacteriaceae decreased significantly;the experimental group can retain more OTUs during fermentation;D.officinale polysaccharides can increase the growth of probiotics and inhibit the growth of harmful pathogens.3.The effects of Dendrobium officinale polysaccharides on intestinal microflora metabolites were further studied by GC-MS.The small molecular metabolites were qualitatively and quantitatively analyzed.The effects of Dendrobium officinale polysaccharides on intestinal microbial metabolites were analyzed by PCA(principal component analysis)and PLS(partial least squares).The results showed that the metabolic level of short chain fatty acids and branched chain amino acids in the experimental group of Dendrobium officinale polysaccharides was relatively high,which had an important effect on the pH value of fermentation process.