Preparation,Characterization And Bioactivities Of Athelia Rolfsii Polysaccharide-zinc Complex (AEPS-Zn)

Athelia rolfsii exopolysaccharides（AEPS）are isolated from culture fermentation broth.It mainly consists of scleroglucan,a water-soluble glucan,featuring stability at high temperature,broad range of pH 0-13 and three-dimensional network structures even at low concentrations.AEPS also exhibit high biological activities and great absorption effect on heavy metals.In order to develop a bio-zinc preparation with high bioactivity,and characterize its structure,the hereinafter studies were done:1 Deproteinization rate and polysaccharide loss rate were selected as indexes;Sevage method,papain method and papain-Sevage method were used to remove protein.Based on single factor experiments,the response surface methodology was employed to optimize the deproteinization conditions of culture fermentation broth.The optimized conditions were as follows:reaction temperature 62℃,enzyme dosage 2.1%,reaction time 60 min and pH 6.2.Under those conditions,deproteinization rate was 76.05%.The three methods were compared:take the deproteinization rate into consideration:papain-Sevage method>papain method>Sevage method;take the polysaccharide loss rate into consideration:Sevage method>papain-Sevage method>papain method.Therefore,papain method was selected to remove protein.2 AEPS were purified by Sephacryl S-300,S-200 and HPLC,two fractions AEPSI and AEPSII were obtained and the physicochemical characteristics were assayed by HPGPC and GC methods.The results showed that both fractions were dissolved in water,dilute acid and dilute alkali,and insoluble in ethanol,methanol,acetone and other organic solvents.All of them contained glucuronic acid,not containing reducing sugars and starch.Both of them had three helical structure and no phenolic substances was exist except for trace protein.AEPSI consisted of glucose,galacturonic acid,talose,galactose,mannose and xylose,the relative contents of them were 24.74,19.60,33.65,8.77,7.97 and 5.28%,respectively.AEPSII consisted of glucose,inositol,galacturonic acid,ribitol,gluconic acid,talose and xylose,whose relative contents were36.06,21.21,12.78,11.07,6.58,5.45 and 6.82%,respectively.The Mw and Mn of AEPSI were6.1324×10⁴and 1.4218×10⁴ Da,those of AEPSII were 517 and 248 Da.3 Based on single factor experiments,the response surface methodology was employed to optimize the chelation conditions of Athelia rolfsii polysaccharide-zinc and its antioxidant activities were evaluated.The results of optimum chelation conditions were as follows:polysaccharide concentration 1.6 g/L,reaction temperature 39℃,reaction time 62 min,pH 8.3.Under these conditions,the chelation rate was 75.71%and the effect of AEPSII was better than AEPSI.4 The structures of AEPS and AEPS-Zn were characterized by SEM,FTIR and NMR.SEM observations showed that the AEPS was in the shape of irregular and loosely thinnest strip or stick that with many rough bulges,while the AEPS-Zn had a smooth surface and compact thick flake-like morphology.FTIR analysis showed that O–H,C-H and C-O in the spectrum of AEPS transferred to some extent in the spectra of AEPS-Zn,the C=O was disappeared,and a new absorption bands appeared in the carbohydrate fingerprint region.NMR analysis indicated thatα-pyranose residue,β-D-glucopyranose,uronic acid,acetamino-and C-6 deoxidized sugar methyl groups existed in AEPS.Compared with the spectra of AEPS,a“blinded”region appeared in the ¹H NMR spectrum of AEPS-Zn and the intensity of proton signals in ¹³C NMR spectrum of AEPS-Zn were weakened.The above results might illustrate that AEPS was bonded to zinc.5 The antioxidant activities of AEPS were studied by the scavenging rates of DPPH,hydroxyl,superoxide anion free radical and the reducing power.The scavenging rates of DPPH free radical of AEPS and AEPS-Zn were 61.24%and 79.54%,the scavenging rates of hydroxyl free radical were 65.32%and 71.32%,the scavenging rates of superoxide anion free radical were 55.32%and 62.02%,and the reducing power of AEPS and AEPS-Zn increased as the concentration enhanced.The results showed that compared with AEPS,AEPS-Zn had a better antioxidant activity.6 The bio-stability of AEPS-Zn was measured by simulating the gastrointestinal environments.After digestion in gastrointestinal environments,there still had 45.37%of zinc which would come into duodenum.The result showed that AEPS-Zn was stable.7 The effect of AEPS-Zn on zinc supplementation and antioxidant activities were studied in vivo.The results showed that AEPS-Zn could promote the increase of body weight,viscera indexes,and the absorption of Zn,Fe,Ca,Cu and Mg.It also could improve the differentiation of osteoblast,the activities of LZM,T-SOD and GSH-Px.