Structural Identification,Rheological And Gel Characteristics,Antioxidant And Immunity Activities Of Sparassis Crispa Polysaccharides

[Objective]To gain insights into valuable evidence for applications of Sparassis crispa polysaccharides as functional food materials in the industrial process,the structure,processing characteristics,founction activities of Sparassis crispa polysaccharides were investigated.[Methods](1)SCPs were extractd by water extraction and alcohol precipitation with ultrasonic assisted.The SCPs were decolored by HZ-830 macroporous resin and purified by DEAE-52 with distilled water,0.1 mol/L,and 0.3 mol/L NaCl solution eluted,respectively.Different fraction of polysaccharides were further purified by Sephadex-G100 and then collected;The polysaccharide Structure was investigated using High performance gel permeation chromatography,infrared spectroscopy,ion chromatography,nuclear magnetic resonance,atomic force microscopy,scanning electron microscopy and Congo red.(2)The influence of different processing conditions on the rheological behavior of SCPs was investigated.The rheometer was used to determine the steady rheology at different conditions sucn as the polysaccharides concentrations were 0.5%,1%,2%,3%,and 5%;the processing temperature was 40?,60?,80?,100?,120?,NaCl concentrations were 0%,1%,2%,4%,8%and pH 3,5,7,9,11;Thixotropy was determined when the concentration of polysaccharide was 5%;and the oscillatory of SCPs solutions was detennainted when the concentration was 0.5%,1%,2%,3%,and 5%.The effect of different processing factors on the gel properties of SCPs was investigated.The polysaccharide gel texture and water holding capacity of SCPs throughtexture analyzer were investigated at different processing conditions when the olysaccharide concentrations were 2%,5%,10%,20%,NaCl concentrations were 0%,2%,4%,8%,sucrose concentrations were 0%,5%,10%,20%,and pH were 3,5,7,9.(3)The antioxidant activity of polysaccharides in vitro were studied through the determination of the total reducing power,the ability to remove free radicals such as DPPH-�,H2O2�,OH-�,and O2-� when the polysaccharides concentrations were 0.25 mg/mL,0.5 mg/mL,1 mg/mL,2 mg/mL and 4 mg/mL,respectivelyThe MTT method was used to measure the proliferation activity of macrophages RAW264.7 in mice with the polysaccharides concentrations ranged from 0 to 4000 ?g/mL.The components with the strongest macrophage proliferation were selected and its immune function was studied.Neutrophic red staining was used to determine the phagocytosis ability of this fraction;cell supernatants were collected and the productions of NO in the supernat were detect;Cells were collected and total RNA was extracted,then the expression levels of TNF-a,IL-1?,IL-6,IL-3,IL-10 and IFN-? mRNA in macrophages were determined using quantitative RT-PCR.[Results](1)After purified and isolated by HZ-830 macroporous resin,DEAE-52 and Sephadex-G100,four components of SCPs were obtained named SCP-1,SCP-2,SCP-3,SCP-4,respectively;The relative molecular mass of SCPs was 215 D-393 KD,and the relative molecular mass of SCP-3 and SCP-4 was 6456 Da and 3039 Da.SCPs was consisted of galactose,glucose,xylose,mannose,and fructose in a molar ratio of 1:13:2.4:3;SCP-1 was of xylose,mannose,and fructose in a molar ratio of 1:19:64;SCP-2 was consisted of galactose,glucose,xylose,and fructose in a molar ratio of 6:12:1:3;SCP-3 was consisted of galactose,glucose,xylose,fructose,in a molar ratio of 1:4:1;SCP-4 was consisted of galactose,glucose,xylose in a molar ratio of 2:7:1:2;The main chain of SCP-3 was ?-(1?3)-D-glucan and its branched chain is ?-(1?6)-D-glucan with a branching frequency of approximately thre;The triple-helix structure was existed in SCP-3 and SCP-4,while not existed in SCP-1 and SCP-2(2)SCPs solutions emerged as shear thinning pseudoplastic fluids;viscosity curves were evaluated by power law model fitting.Apparent viscosity increased with concentration but decreased with extreme conditions and was highest for 1%SCPs at 80 ? under neutral conditions;5%SCPs solutions formed a hysteresis loop exhibiting thixotropic properties.By oscillatory measurements,2%and 5%SCPs solutions were viscoelastic fluids,the latter showed elastic behavior and weak gel properties.SCPs gel textural properties and water holding capacity increased with concentration(to 20%)and decreased with salinity,extreme sucrose and pH.10%SCPs gels were optimized at 10%sucrose in neutral conditions(3)SCPs,SCP-1,SCP-2,SCP-3,and SCP-4 all have a certain ability to reduce and scavenge free radicals.The IC50 values of DPPH radical scavenging rates were 2.85 mg/mL,4.00 mg/mL,3.87 mg/mL,2 mg/mL,and 1.78 mg/mL,respectively;The IC50 values of H2O2 radical scavenging rates were 1.12 mg/mL,1.87 mg/mL,2.88 mg/mL,0.75 mg/mL,and 0.63 mg/mL,respectively;The IC50 values of OH' radical scavenging rates were 0.78 mg/mL,1.65 mg/mL,1 mg/mL,and 0.63 mg,respectively./mL,0.73 mg/mL;When the polysaccharides concentrations were 4 mg/mL,the O2-free radical scavenging rates were 30.66%,25.63%,27.59%,37.58%,40.86%,respectively;The total reducing force OD values were 0.18,0.19,0.16,0.35,0.38.Thus,SCP-3 and SCP-4 have stronger antioxidant capacity than SCPs,SCP-1,and SCP-2.SCPs,SCP-1,SCP-2,SCP-3,and SCP-4 can promote the proliferation of RAW264.7 macrophages and their valus were increased and then decreased with concentration of polysaccharides increased.The cell proliferation was the stronges twhen the concentration of each polysaccharide was 500 ?g/mL,31.25?g/mL,15.625 ug/mL,7.8125 ?g/mL and 31.25 ?g/mL,respectively.The order of promoting cell proliferation ability was SCP-3>SCP-2>SCP-4 =SCP-1>SCPs.The concentration of SCP-3 was in the range of 1.95 ?g/mL�15.62 ?g/mL,the phagocytic capacity of macrophages,NO secretion,and intracellular TNF-a,IL-1?,IL-6,IL-3,IL-10,IFN-? immune factor mRNA expression were significantly increased and showed a dose-effect relationship,which indicated that Sparassis crispa polysaccharides had a certain role in promoting the immune regulation of macrophages.[Conclution]The polysaccharides in a relative molecular mass of 6456 Da was isolated and acquired,its main chain was ?-(1?3)-D-glucan with the branched chain of ?-(1?6)-D-glucan,which exhibited antioxidant and immunity activity.SCPs were viscoelastic materials,their solutions behaved as shear thinning pseudoplastic fluids as well as exhibited thixotropic and weak gel properties.SCPs thus had the potential utilization as a new hydrocolloid source in food industries.The results can provid reference for the design of food processing parameters and functional food additives in food processing.