Separation,Structural Analysis Of Haematococcus Pluvialis Polysaccharide And Its Function On Immune And Anti-aging Activity

Haematococcus pluvialis is an unicellular microalgae which contains quantities of astaxanthin,polysaccharides,proteins,vitamins and micronutrients,approved to be a new food raw material,following microalgae Spirulina and Dunaliella.At present,H.pluvialis is used mainly in the efficient production of astaxanthin andthe residues are wasted after extraction of astaxanthin.To utilize the H.pluvialis comprehensively,the polysaccharide rich in residues after defatting has potential development space.In recent years,microalgae polysaccharides have become the research focus with a variety of species and biological activity after large seaweed polysaccharides.Therefore,in-depth study onH.pluvialispolysaccharide can improve the comprehensive utilization of H.pluvialis and increase the added value.In this paper,the H.pluvialis residue after extraction of astaxanthin was used to optimizeextraction,isolation and purification process of polysaccharides.The structure analysis,immunomodulatory and anti-aging activity evaluation of H.pluvialispolysaccharide were studied.The main results are as follows:(1)The primary nutritional components of H.pluvialis residue after extraction of astaxanthin were: moisture 14.21%,ash 43.73%,crude fat 0.19%,carbohydrate 30.67%,protein 20.83%.H.pluvialis residue is rich in carbohydrates.(2)Research on extracting process of Haematococcus pluvialis polysaccharide.Aqueous,alkaline,microwave-assisted,ultrasonic-assisted and enzymatic extraction methods were used to extract polysaccharide from H.pluvialis residues.The best extractio n method was optimized to get the optimum extraction condition.The best extraction method is ultrasonic-assisted method and the best results were obtained after ultrasonic treatment at 400 W for 30 min at a solid-to-solvent ratio of 1:25(g/mL),water bath temperature of 90 ? for 3 h.After verification experiment,the extraction rate of H.pluvialis polysaccharide was approximately 3.48%.(3)Remove the proteins in H.pluvialis polysaccharide by Sevag treatment and get the optimization condition by single factor and orthogonal screening analysis.The condition is as follows: chloroform:n-butanol = 5:1,raw sugar solution:removing liquids=4:1,remove protein 5 times,shaking time 20 min.After verification experiment,the protein removal rate was approximately 88.14%,and the polysaccharide content was approximately 34.85%.(4)Study on the separation and purification of H.pluvialispolysaccharide.DEAE-52 cellulose anion exchange column chromatography and Sephacryl S400 gel permeation chromatography were used to separate and purificate H.pluvialispolysaccharide based on tracking immune activity using cell as a model in vitro.Five different polysaccharide HPP-c1,HPP-c2,HPP-c3,HPP-c4 and HPP-c5 were get from HPP after theDEAE-52 column chromatography.HPP-c3 had higher polysaccharide content and immune activity.Then HPP-c3 was separated by Sephacryl S400 gel permeation chromatography and get HPP-c3-s1,HPP-c3-s2 and HPP-c3-s3.HPP-c3-s1 had thebest immune activity and a dose-effect relationship.(5)HPGPC and polyacrylamide gel electrophoresis were used for purity and molecular weight analysis.Periodate oxidation,Smith degradation,IR,GC-MS,NMR and atomic force microscopy were used for the preliminary analysis.The results showed that HPP-c3-s1 was a homogeneous polysaccharide with 23413 k Da of the molecular weight;monosaccharide composition and proportionwere:D-ribose: D-arabinose:D-mannose:D-glucose= 2.15:1:1.15:1.85;polysaccharide had type 1?2,1?4 and 1?3 glycosidic linkages,which may contain extremely few 1?6 glycosidic bond type;HPP-c3-s1 was proved to be a pyranose containing an amino group and an O-acetyl group with polysaccharide characteristic absorption peaks,which sugar chain containing ?,? two kinds of glycosidic configuration,mainly made up of ?configuration;5.00 ?g/mL HPP-c3-s1 showed the existence of two forms,one was a branched chain forms,one was a straight-chain form,where in the linear polysaccharide molecule width of 23~36 nm,a height of 0.6~1.5 nm.50.00 ?g/mL HPP-c3-s1 appeared collective form of different size.(6)To determine the mechanism of immune activity of polysaccharide,the effect of proliferation of T lymphocytes and B lymphocytes stimulated with collaborative ConA and collaborative LPS respectively by different concentrations of the HPP-c3-s1 was studied.The results showed that HPP-c3-s1 had significant effect in LPS-stimulation only.Therefore,HPP-c3-s1 can stimulate B lymphocyte proliferation,but no activity for T lymphocytes.(7)Anti-aging C.elegans model was used to evaluate the anti-aging effect.The results showed that 200 ?g/mLHPP-c3-s1 can significantly slowed down the aging of C.elegans,andthe average life span extended 14.92% higher than the control group.Fecundity test showed no significant difference between HPP-c3-s1 group and control group,so HPP-c3-s1 had no side effect on reproductive capacity.In addition,a dose of 200 ?g/mL of the HPP-c3-s1 group improved aging related indicators in motility test,head swing frequency test,body bending frequency test and intestinal lipofuscinosis quality test.