Study On Structure And Properties Of Water-soluble And Water-insoluble Polysaccharides From Termitomyces Albuminosus(Berk.) Heim From Guizhou Province

Termitomyces albuminosus(Berk.)Heim is a wild and rare edible fungus,which mainly grows in the southwest and southeast area in China.It is abundant in essential nutrients and bio-active compounds,so a series of studies have been reported in recent years.It is acknowledged that polysaccharide is one of the active components in T.albuminosus,and the studies are concentrated in the physiological activities of water-soluble polysaccharides.However,structure of water-soluble polysaccharides,as well as the structure and activities of water-insoluble polysaccharides,is rarely reported.Chitin is an important component of fungal water-insoluble polysaccharides.Traditionally,chitin is obtained from crustaceans,but there are many limitations in the extraction and application.However,edible fungi is abundant in chitin as well,with many advantages involved the easy-obtained materials,mild extraction conditions and stable product properties,which make it an alternative of crustacean-derived chitin in the industry.Therefore,in this study,the fruiting body of T.albuminosus from Guizhou Province was used as the research object,from which the water-soluble polysaccharide WSP and water-insoluble chitin-glucan complex CGC were obtained.The neutral fraction NWSP was isolated from WSP,and the water-insoluble glucan ISP-3 was isolated from CGC.What's more,the properties,structure and physiological activities were investigated as well.The main contents and corresponding results of this study are presented as follows.1.Based on single-factor test and response surface methodology,the water-soluble polysaccharide(WSP)was extracted from the fruiting body of T.albuminosus,and its structure was analyzed by gas chromatography(GC),gel permeation chromatography(GPC),FT-IR,Gongo-red test and scanning electron microscope(SEM).Meanwhile,its antioxidant activity was investigated in vitro as well.Extraction temperature,extraction time and ratio of water to material were proved to have significant effects on the yield of crude water-soluble polysaccharide,with extraction temperature being the most significant.The optimum extraction condition was as follows:extraction temperature 93?,extraction time 3 h,ratio of water to material 31:1 m L/g,with the theoretical yield of 15.21%.WSP was extracted under the optimal extraction condition after depigmentation and deproteinization,with a yield of 1.32%.The contents of total sugar,water,ash and protein in WSP were 76.04%,6.69%,2.15%and 4.92%,respectively.WSP was mainly composed of mannose,glucose and galactose in a molar ratio of 8.72:24.41:1.In addition,WSP was a heterogeneous polysaccharide composed of at least three components.The number-average molecular weight of the components were 303823,1767 and 844 Da,respectively,of which the high-weight fraction was in the dominant percentage.Moreover,the pyranose ring,glycosidic bond of?-type and helical structure possibly existed in WSP.WSP had good DPPH and hydroxyl radical scavenging ability and ferrous ion chelating ability in the experimental concentration,with the highest values of 73.38%,53.50%and 86.07%,respectively.2.The separative fractions of WSP obtained from ion-exchange column were applied to gel column chromatography and GPC to estimate the purity.The structure of purified fraction was analyzed by the methods of GC,FT-IR,methylation analysis,nuclear magnetic resonance(NMR)spectroscopy and atomic force microscope(AFM),and its antioxidant,immunoregulatory and antitumor activities in vitro were investigated.Four fractions,named NWSP,ACWSP-1,ACWSP-2 and ALWSP respectively,were obtained,but only the neutral part NWSP was homogeneous which was used for subsequent analysis.NWSP was mainly composed of fucose and galactose in a molar ratio of 1:3.09,and its number-average molecular weight was 9636 Da.The repeated unit of NWSP was?2-?-L-Fucp-1?(6-?-D-Galp-1)₃?.In addition,the molecules of NWSP were not in a single-stranded structure but were in the multi-stranded or ring form.NWSP had strong DPPH radical scavenging ability in the experimental concentration,since its highest scavenging rate was 79.01%.However,its reducing power,hydroxyl radical scavenging ability and ferrous ion chelation ability were relatively weak,with the highest values of 0.55,35.42%and 37.64%,respectively.Moreover,among the experimental concentration,the phenomenon of promoting the proliferation of mice spleen cells could not be seen,indicating the absence of immunoregulatory activity in NWSP.NWSP had no inhibitory effect on the proliferation of human hepatoma cell Hep3B,colon cancer cell SW480 and mammary cancer cell MCF-7 in the experimental concentration,since the highest inhibition rates were 10.57%,15.24%and 7.40%,respectively.However,a weak effect on leukemia cell K562 could be found,with the highest inhibition rate of 24.13%.3.The residue of T.albuminosus was used to isolated chitin-glucan complex CGC after deproteination,demineralization and depigmentation.The structure and properties of CGC were analyzed by elemental analysis,GC,FT-IR,X-ray diffraction(XRD)spectroscopy,¹³C solid NMR,SEM and simultaneous thermal analyzer,and its prebiotic activity was investigated as well.CGC was extracted with a yield of 13.46%.The contents of water,protein and ash in CGC were 3.99%,0.11%and 1.31%,respectively,and the contents of residual metals were in a decreasing order of sodium,iron,calcium,aluminum,magnesium and so on.Glucan and chitin were in a molar ratio of 1:1.17,indicating a high content of chitin in CGC.The content of nitrogen was 3.34%,by which chitin content in CGC was calculated to be 48.43%.CGC and chitin from shrimp shells had certain similarities in the chemical structure and properties,but the similarity to laminarin was unobvious.All the characteristic groups of chitin could be found in the spectra of CGC,and the chitin was possible to be in?-configuration.The crystallinity index of CGC was 64.81%,which was lower than that of chitin from shrimp shells,while its degree of deacetylation(34.60%)was higher.In the thermodynamic property analysis,the peak temperature of degradation and peak enthalpy were 314.88?and-55.31 J/g,respectively,providing clues to the result that the thermal stability of CGC was weaker than that of chitin from shrimp shells.Both WSP and CGC had resistance to gastric juice and?-amylase,while CGC was more resistant with the highest degree of hydrolysis being 1.34%and 1.01%,respectively.Both of them showed certain promoting effect on the growth and acid production of Bifidobacterium bifidum,Lactobacillus plantarum,and strong promoting effect on Enterococcus faecalis.That is,both of WSP and CGC had obvious prebiotic activity.4.Separation of various water-insoluble glucans from CGC was performed under the treantment of different concentrations of sodium hydroxide and acetic acid solutions,and the structure and properties of ISP-3 which was of the highest content were analyzed by GC,FT-IR,methylation analysis,SEM and simultaneous thermal analyzer.ISP-3 was mainly composed of glucose,with traces of mannose,galactose and chitin.The glycosidic bond of?-type possibly existed in ISP-3,and the main chain of ISP-3was likely to be consisted of the glycosyl of?6-D-Glcp-1?,combined with a small amount of?4-D-Glcp-1?and?4,6-D-Galp-1?,and with a branch at the position of the galactosyl(?4,6-D-Galp-1?)linked by the glycosyl of?3-D-Manp-1?.In the thermodynamic property analysis,the initial temperature,peak temperature and end temperature in the thermal degradation process of ISP-3 were 284.18?,325.93? and355.75?,respectively.