| Since ancient times,mushrooms have often been added to health products as nutritional supplements or dietary fiber due to their high nutritional value.Modern medical research has clearly stated the immunomodulatory activity of wild edible fungi.Among the several components of mushrooms,polysaccharides are thought to be the most potent in regulating the innate and adaptive immune systems.Mushroom polysaccharides are a class of macromolecules with different physical and chemical properties,and changes in these properties are considered to be the determinants of stimulating the immune system.Hericium erinaceus,as an edible and medicinal fungus,has the effects of invigorating the spleen,nourishing the stomach,calming the mind,and fighting cancer.In the past decades,it has been demonstrated that Hericium erinaceus polysaccharides have a variety of biological activities,including anti-tumor,anti-oxidation,anti-hyperlipidemia,anti-hyperglycemia,anti-fatigue,anti-aging,immunomodulatory,treatment of gastric ulcers,liver protection,neuroprotection and nerve regeneration.In this paper,Hericium erinaceus polysaccharides were isolated from the fruiting body of Hericium erinaceus,and the structural changes and metabolites of Hericium erinaceus polysaccharides in the digestive process of stomach and intestine were investigated.Furthermore,the polysaccharides of Hericium erinaceus were further isolated and purified to obtain relatively uniform polysaccharides components,and the structure,immunomodulatory activity and signaling pathway of each component were analyzed.The effects of Hericium erinaceus polysaccharides on the structure and diversity of intestinal microflora in mice of different ages were determined by high-throughput sequencing technology.To explore the immune regulatory activity of Hericium erinaceus polysaccharides by affecting intestinal flora.The results are as follows:(1)Study on the probiotics activity of crude polysaccharides from Hericium erinaceus.The crude polysaccharides were extracted from the fruiting body of Hericium erinaceus by hot water extraction.The polysaccharides of Hericium erinaceus were digested in gastric and intestinal digestible liquid model established in vitro.Under different digestion time(0,0.5,1,2,4,6 h),the molecular weight of Hericium erinaceus polysaccharides decreased from 1.68×106 Da and 2.32×104 Da to 529.3±7.2 Da,and the reducing sugar content increased from 0.610±0.007mg/mL to 22.698±0.752 mg/mL,accompanied by the release of free monosaccharides mannose and galactose.The prebiotic activity of Hericium erinaceus polysaccharides for 6 kinds of probiotics(Lactobacillus rhanosus IMC501,Lactobacillus plantarum BG112,Lactobacillus acidophilus LA3,Lactobacillus paracasei ET-22,Bifidobacterium BLC1,Streptococcus thermophilus ST064)before and after digestion respectively were compared.It found that the Hericium erinaceus polysaccharides after digesting can promote the growth of probiotics and production of acids,especially for Lactobacillus plantarum BG112.Furthermore,the prebiotic activity of Hericium erinaceus polysaccharides on Lactobacillus plantarum BG112under different digestion time of stomach and intestine were compared.The results showed that Hericium erinaceus polysaccharides had the strongest effect on promoting the proliferation of Lactobacillus plantarum BG112 after digestion for 2 h and digestion for 4 h in intestinal fluid.In addition,Lactobacillus plantarum BG112produced a variety of short-chain fatty acids,including acetic acid,iso-valerate,lactic acid and butyric acid.(2)The structure of the isolated and purified H.erinaceus polysaccharides was analyzed.The extraction rate of Hericium erinaceus polysaccharides was 2.735%,the total carbohydrate content was 53.36%,and the reducing sugar content was 4.43%.The content of uronic acid was 32.56%.The crude polysaccharides of Hericium erinaceus were isolated from the fruit body of Hericium erinaceus by hot water extraction.The crude polysaccharides were separated and purified by DEAE-cellulose52 ion exchange column and Sephadex G-100 gel column,and five relatively uniform components of Hericium erinaceus polysaccharides were obtained,namely,HEP-1,HEP-2,HEP-3,HEP-4 and HEP-5.HEP-1 is neutral polysaccharides,HEP-2,HEP-3,HEP-4 and HEP-5 are acidic polysaccharides.The polysaccharides were mainly composed of fucose,galactose and glucose,and the molar ratio was 0.331:2.884:1.The molecular weight of HEP-1,HEP-2,HEP-3,HEP-4 and HEP-5 were 2.12×106Da,9.27×105 Da,1.10×105 Da,1.99×104 Da and 1.09×104 Da,respectively.Infrared spectrum analysis showed that the polysaccharides of Hericium erinaceus contained O-H,C-H,C=O,-CH2,S=O,C-O-H or C-O-C functional groups.By methylation analysis and nuclear magnetic resonance(NMR)analysis,the main chain of HEP-1,HEP-2 and HEP-3 is(1?6)-linked?-D-galactose,and the side chain is(1?3)-linked?-Dglucose.The main chain of HEP-4 is(1?3)-linked?-D-glucose chains with and(1?6)-linked?-D-glucose.The main chain of HEP-5 is(1?4)-linked?-glucose,and the side chain is(1?6)-linked?-D-glucose.(3)Study on the effects of Hericium erinaceus polysaccharides on intestinal flora in vivo.In this chapter,adult mice and middle-aged and aged mice were given high and low doses of Hericium erinaceus polysaccharides for 28 days,respectively.Changes in the structure and distribution of microbiota in the feces of mice were analyzed by 16S DNA high-throughput sequencing technology,and their functions were predicted by Tax4Fun method.In addition,the effect of Hericium erinaceus polysaccharides on the immune system was studied.The polysaccharides of Hericium erinaceus were fermented in vitro with bacteria broth isolated from small intestine,colon,cecum and feces.The effects of polysaccharides fermentation broth obtained from Hericium erinaceus under different fermentation time on NO production and phagocytic activity of macrophages were analyzed and compared.The production of four cytokines(IL-6,IL-10,TNF-?and INF-?)and the expression of related immune proteins in three pathways(NF-?B,MAPK and PI3K/Akt signaling pathways)were detected in an intestinal absorption model in vitro.Hericium erinaceus polysaccharides can enhance the species richness and microbial diversity of intestinal microflora in adult mice and middle-aged and aged mice,and increase the number of OTU of intestinal microflora in mice.In order to improve the composition and structure of intestinal microbiota,we changed the relative abundance of intestinal microbiota in adult,middle-aged and aged mice at phylum,family and genus levels.In addition,the Hericium erinaceus polysaccharides fermented by colon and feces significantly promoted NO production and phagocytic activity of RAW 264.7,and promoted the secretion of cytokines IL-6,IL-10,TNF-?and INF-?by stimulating NF-?B,MAPK and PI3K/Akt signaling pathway,thus playing an immunomodulatory role.(4)Study on the immunomodulatory activity and mechanism of five kinds of polysaccharides components of Hericium erinaceus.First of all,the five polysaccharide components of Hericium erinaceus HEP-1,HEP-2,HEP-3,HEP-4and HEP-5 were determined to have no toxicity to macrophages in the concentration range of 25-800?g/mL.The effects of the five polysaccharides components of Hericium erinaceus on NO production and phagocytic activity of macrophages were compared,and HEP-1 was selected as the best one.In an intestinal absorption model established in vitro,HEP-1 significantly promoted the production of IL-6,IL-10,IFN-?and TNF-?in RAW 264.7 cells at a concentration of 200?g/mL.Western Blotting showed that HEP-1 induced NF-?B p65 nuclear transduction in macrophages and I?B-?degradation,and enhanced p38,JNK and ERK in MAPK pathway and Akt phosphorylation in PI3K/Akt pathway.To further confirm these results,five pathway inhibitors were used.The production of NO and TNF-?in macrophages was almost completely inhibited by NF-?B,JNK and ERK pathway inhibitors,but the inhibition of I?B-?,NF-?B p65 and Akt protein expression on NF-?B and PI3K/Akt signaling pathway was alleviated by HEP-1.The results indicated that NF-?B,MAPK and PI3K/Akt signaling pathway were involved in the immunomodulatory process of Hericium erinaceus polysaccharides. |
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