Study On The Preparation Of Hericium Erinaceus Active Peptide And Its Immunoregulatory Effect

Hericium erinaceus is a kind of edible fungus with high-protein,low-fat,and a variety of nutrients.Nowadays,the research on the active substances of Hericium erinaceus was mostly focused on exploring the activity of polysaccharides or extracts.However,research on Hericium erinaceus protein peptides was relatively scarce.This study mainly screened,prepared and determined active function of Hericium erinaceus immunomodulatory peptides through separation and purification,activity screening,structure analysis,and activity demonstrations.The main results of this paper were as follows:1.Hericium erinaceus protein was extracted by alkaline extraction and acid precipitation,and Hericium erinaceus polypeptide was prepared by enzymatic hydrolysis.Through analysis of molecular weight,degree of hydrolysis and amino acid composition,it was preliminarily proved that the Hericium erinaceus protein peptides after alkaline protease hydrolysis had immunological activity potentially.The mouse macrophage screening model was used to further verify that it contained highly immunologically active protein peptides.Through ultrafiltration combined with multistage chromatographic separation and mass spectrometry technologies,8 dominant peptides with immunological activity were purified and screened out,and the corresponding amino acid sequences were VVAL,AGLY,TLLV,ALLGF,ASSVGSG,KSPLY,PLLVPAS,LPGKVIAS.2.Eight dominant peptides were synthesized by Solid-phase synthesis technology.The synthetic peptides were verified and identified by high performance liquid chromatography and mass spectrometry,and obtained by the RAW264.7 mouse macrophage screening model.Among them,the molecular weight was 608.38 Da,and the amino acid sequence was KSPLY,which had high immunological activity.Griess Reagent method and ELISA were used to determine the effect of different concentrations of KSPLY on the secretion of typical cytokines in M0 type mouse macrophages.The results showed that KSPLY could significantly induce macrophages to secrete NO,IL-1?,IL-6 and TNF-? with a concentration-dependent increase.Through TLRs inhibitor treatment,it was proved that TLR4 was the specific receptor of KSPLY on the surface of mouse macrophages.The research on M1 macrophages showed that KSPLY reduced the secretion of NO and IL-6,and inhibited the inflammatory response induced by LPS;the research on M2 macrophages showed that KSPLY could promote the secretion of NO and IL-6 and inhibit the secretion of IL-10,which were also characterized by q RT-PCR.3.A Caco-2 cell monolayer membrane model was established.To study the effect of KSPLY on the secretion of cytokines by Caco-2 cells,the results showed that KSPLY can promote the secretion of cytokines such as IL-8,IL-10,IL-12 and INF-? by Caco-2 cells.Through the transmembrane transport inhibitor blocking experiment,it was cleared that KSPLY pass through small intestinal epithelial cells in a bypass transport mode,with P glycoprotein used as a carrier for the efflux process and the ATP used as the energy supplier for transport process.The establishment of a Caco-2/RAW264.7cell co-culture system proved that KSPLY can act on the underlying RAW264.7 mouse macrophages through Caco-2 cells.After passing through the monolayer membrane,KSPLY activated RAW264.7 cells to secrete cytokines such as TNF-?,IL-6 and NO,thereby exhibiting the potential effects of regulating intestinal immunity.