Extraction,Structural Elucidation And Antioxidant Activity Of Water-soluble Polysaccharides Alkali Extracted From Agaricus Blazei Murill

Agaricus Blazei Murill?ABM?,also known as Brazilian mushroom,was native to Brazil.It was a valuable edible and medicinal mushroom used in both medicine and food.It was widely cultivated in China and Japan because of its medicinal value.Agaricus blazei Murill was rich in polysaccharides,proteins,nucleic acids,fats,saponins,tannins,sterols,minerals,trace elements and essential amino acids.It contained active ingredients such as sterols and polysaccharides,and had remarkable effects in anti-tumor,hypoglycemic,anti-fatigue and anti-oxidation.It can be used to treat diseases such as cancer,diabetes and heart disease.In this paper,an alkali-extractable and water-soluble polysaccharide?ABMP?was isolated from the residue of Agaricus blazei by sodium hydroxide solution,the extraction process was optimized and the antioxidant activity of ABMP was studied.ABMP was purified by ion exchange column and gel column chromatography to obtain the homogeneous polysaccharide ABMP-2-a,and its primary structure was identified.We have also analysed the conformation and morphological properties of the homogeneous polysaccharide ABMP-2-a.The dried fruiting bodies of A.blazei were extracted at a ratio of 1:20?w/v?for 2h by boiling water to obtain residue.The residue was extracted with NaOH solution.The supernatant was combined after centrifugation,then it was neutralized with acetic acid?3 mol/L?.After being filtered,the supernatant obtained was dialyzed and ethanol precipitation.The precipitate was dissolved in distilled water and then freeze-dried to obtain the crude polysaccharide ABMP.The extraction process of ABMP was optimized by Box-Benhnken response surface design.The optimum conditions were predicted as follows:5.33%of alkali concentration,60?of temperature,2.23 h of extraction time.Under this conditions yield of ABMP was 12.69±0.08%,close to the predicted value12.74%.However,excessive temperature may destroy the structure of polysaccharide.And considering the actual situation,we reset the extraction conditions as 5%of alkali concentration,50°C of temperature and 2 h of time to extract the polysaccharide for further study.Under this conditions the yield of polysaccharide was 12.44±0.12%,and the content of polysaccharide and protein were 37.80±0.20%,32.12±0.18%,respectively.The results of antioxidant activity showed that the IC₅₀ values of ABMP for DPPH,ABTS free radical scavenging abilities and reducing power were 0.104,0.237,0.0136 mg/mL,respectively.ABMP exhibited better antioxidant activity.ABMP was applied to a DEAE-Sepharose Fast Flow column?XK 26 mm×100cm?,eluted with 0.2 mol/L NaCl solution,dialyzed and lyophilized to obtain ABMP-2.After desalting by dialysis bag?MWCO:20000 D?,it was separated and purified by Sephacryl S-300 HR gel column chromatography to obtain homogeneous polysaccharide ABMP-2-a.The molecular weight of ABMP-2-a was 1.87×10⁵ Da measured by laser light scattering instrument.The results of Fourier transform infrared spectroscopy?FT-IR?and Ultraviolet-visible spectrometer?UV?showed that ABMP-2-a contained no nucleic acid and protein,and it was in conformity with polysaccharide characteristics.It was a?-configuration neutral polysaccharide.The acetylation and methylation of ABMP-2-a tested by gas chromatography-mass spectrometry?GC-MS?showed that it was mainly composed of L-Ara,D-Xyl,D-Man,D-Glc and D-Gal,and the molar ratio was 0.53:0.37:1.00:13.65:6.77,respectively.The main chain of ABMP-2-a was mainly composed of?-6-linked glcp.The conformational and morphological properties of ABMP-2-a were also studied.The results of Congo red and Circular dichroism spectroscopy?CD?experiments showed that ABMP-2-a had no triple helical conformation.The results of scanning electron microscopy?SEM?showed that ABMP-2-a was mainly existed in long strips.Long strips were linked and overlapped,and formed complex network structure.The surface of long strips was smooth with no hole structures.It might decrease the repulsive interactions,leading to a more stable structure.The thermal characteristic analysis of ABMP-2-a showed that it had a favorable thermal stability because no decomposition occurred in the range of 30-300°C.X-ray diffraction?XRD?experiments showed that ABMP-2-a was semi-crystalline and amorphous.AFM results showed that ABMP-2-a was a compliant chain conformation structure in solution.It had no triple helix structure,which was consistent with the Congo red experiment.