Study On The Submerged Fermentation Of Hypsizigus Marmoreus And The Extraction, Purification, Structural Analysis Of Mycelia Polysaccharide

Hypsizigus marmoreus(Peck)Bigelow belongs to Basidiomycotina,Hymenomycetes,Agarcales,Tricholomataceae,Schizophyllum commune family,Tricholoma genera.Itcontains a variety of active substances with high nutritional and medicinal value.This paper take Hypsizigus marmoreus ZJ029 strain,which is preserved in the laboratoryof Food Science and Engineering College of Northwest A.&F.University because ofhigh-yield polysaccharide,as the object of studies.The suitable fermentation medium wasoptimized by investigations on the effects of 34 nutritional factors on the production ofpolysaccharide from ZJ029 strain by submerged culture.On this basis,the optimizationfermentation process conditions including initial pH value of medium,liquid seed age,inoculation amount,fermentation temperature,dissolved oxygen content in fermentor andfermentation time were researched by taking shake flasks and 5L fermentors as carriers.Under the optimization fermentation medium and fermentation process conditions,abundancemycelia of Hypsizigus marmoreus were prepared as the following tests materials.Thedifferent technical methods of the extraction,separation,purification of myceliapolysaccharide were compared and analyzed.The results show that using hot alkaline solutionas extraction solution,enzymolysis isolation removal protein,ion-exchange column andsephadex column chromatography purification polysaccharide are the suitable methods.Moreover,in the last chapters,the purity and structure of polysaccharides were analyzed bymeans of paper chromatography,UV spectrum,IR spectrum,high performance liquidchromatography,gas chromatography and other technologies.In this paper,the main resultsare as follows:(1)In the course of submerged culture of Hypsizigus marmoreus ZJ029 strain,thesignificant factors on extra-cell polysaccharide production(Y₁^) in sequence are maltose(X₂),MgSO₄(X₄),and glucose(X₁).The relationship between these three factors and extra-cellpolysaccharide production could be description with a formula i:Y₁^=1.21+0.093×X₁+0.14×X₂+1.11×X₄.The significant factors on inter-cell polysaccharideproduction(Y₂^) in sequence are cornstarch(X₃),MgSO₄(X₄),and glucose(X₁).Their relation model is a formula ii:Y₂^=-0.021+0.017×X₁+0.041×X₃+0.23×X₄.(2)The four factors of the higher ratio of polysaccharide yield to cost price arecornstarch(X₁),glucose(X₂),MgSO₄(X₃),soybean meal(X₄) respectively.The relationshipbetween these four factors and extra-cell polysaccharide yield(Y₃^) could be description witha formula iii:Y₃^=-1.27+0.86X₁+0.29X₂+1.26X₃+0.43X₄-0.025X₁²-0.003926X₂²-0.056X₃²-0.031X₄²-0.007072X₁X₂-0.093X₁X₃-0.004797X₁X₄-0.003744X₂X₃+0.00016566X₂X₄+0.002656X₃X₄.The adjusted determination coefficient of model iii is 0.8997.The relationship between thefour factors and inter-cell polysaccharide yield(Y₄^)could be description with a formula iv:Y₄^=-0.17+0.13X₁+0.044X₂+0.20X₃+0.047X₄-0.003852X₁²-0.0005903X₂²-0.009031X₃²-0.003582X₄²-0.001116X₁X₂-0.015X₁X₃-0.003954X₁X₄-0.0008063X₂X₃+0.00007187X₂X₄+0.0016556X₃X₄.The correlation between the true value and the predictive value for the formula iv is ashigh as 97%.(3)The optimization fermentation nutrition conditions for Hypsizigus marmoreus ZJ029are as follows:cornstarch 5.63g/L,glucose 25.11g/L,soybean meal 6.84 g/L,MgSO4 3.84g/L,KH₂PO₄ 3g/L,KNO₃ 1g/L,V_B1 10mg/L.The optimum fermentation process conditions are:natural culture medium pH value(at about 6.5),liquid seed age 6 days,inoculation amount10%,fermentation temperature 25℃,dissolved oxygen content in fermentor 80%,andfermentation time 8 days.Under the optimization nutrition conditions and fermentationprocess conditions,we can harvest inter-cell polysaccharide for 1.98g/L and extra-cellpolysaccharide for 9.45g/L.(4)The relation model between the key factors,extration temperature(X₁),extrationtime(X₂)and extration solution-dry mycelia ratio(X₃),and the extration rate of polysaccharide(Y^) is simulated by a quadratic polynomial v:Y^=339.43+6.68X₁+16.45X₂+2.56X₃-0.035X₁²+1.66X₂²-0.055X₃²-0.23X₁X₂+0.007X₁X₃-0.25X₂X₃.The correlation between the true valueand the predictive value for the formula v is as high as 95.26%.And this model couldexplain 84% of the total variation.When the extration temperature at 83～88℃,extrationsolution-dry mycelia ratio for 1:21～1:24(g/mL),extration time for 3.4～3.8h,the extrationrate of polysaccharide is not less than 5.8%.The suitable extration process conditions for thehighest extration rate of polysaccharide are as follows:extration temperature 83.95℃,extration time 3.80h,the extration solution-dry mycelia ratio 1:21(g/mL),extration solutionpH 13,and alcohol volume 3 times of extration solution.In this conditions,the extration rateof mycelia polysaccharide from fermentable Hypsizigus marmoreu ZJ029 is up to 7.97%.(5)The appropriate conditions of removing protein from the crude myceliapolysaccharide of Hypsizigus marmoreu ZJ029 by Serag method are:shocking time 30min,the ratio of sample to Sevag reagent 2:1(V/V),shocking times 1 times.Under this conditions,the removal ratio of protein is 33.8%,and the loss ratio of polysaccharide is25.1%.Compare with papain,acid protease,neutral protease,trypsin,and pepsin,alkalineprotease is the best protease for removing protein from the crude mycelia polysaccharide ofHypsizigus marmoreu Z J029.Under the suitable conditions which are reaction temperature50℃,enzyme dosage of alkaline protease 2.687 u/mg,reaction time 3.5h,the removal ratio ofprotein is 63.4%,and the loss ratio of polysaccharide is 8.3%.The results show that thescheme of enzyme is better than the scheme of Sevag.(6)There are 2 components(A-25-1 and A-25-3) are obtained through further isolatingploysaccharide of deproteinization with DEAE-Sephadex A-25 column chromatography.Purity identifications indicate that they are homogeneous compositions by means of paperchromatography,and no protein or nucleic acids by means of UV spectrum,but hetergeneouscompositions by means of Sephadex G-200 column chromatography and HPLCchromatography.So A-25-1 and A-25-3 are isolated by Sephadex G-200 columnchromatography again.Then 7 components,Tâ… -1,Tâ… -2,Tâ…¡-1,Tâ…¡-2,Tâ…¢-1,Tâ…¢-2,Nâ…£,are obatined.Three of them,Tâ…¡-2,Tâ…¢-2,Nâ…£,are identified as homogeneous compositionsby means of HPLC chromatography.(7)By means of different methods such as chemical methods,UV spectrum,IR spectrum,HPLC chromatography,GC chromatography,the structures of polysaccharides were analyzed.The results show that sample Tâ…¢-2 is composed of Fuc,Gal,Glc,Man,in molar ratio of1.00:3.39:0.63:1.28.The Mw of Tâ…¢-2 is estimated to be 19136Da.Its main chain is made upofβ-D-1,3-Gal,and the side chains are formed withβ-D-1,3-Glc and a few ofβ-D-1,3-Man.In addition,there are a few of-1,4 and -1,6 residues in the chains.Nâ…£is composed of Fuc,Rha,Ara,Gal,Glc,Xyl,and Man,in molar ratio of 1.00:0.59:1.11:2.80:2.25:1.25:0.62.TheMw of Nâ…£is estimated to be 522981Da.The main chain of sample Nâ…£is made up ofα-D-1,3-Glc linkedα-D-1,3-Gal,and the side chains are formed withα-D-1,4-Ala residues.Inaddition,there are some -1,6 residues in the chains.The new ideas and innovative points of this paper lie in the following:(1)It's the first time to study the extraction,isolation and purification of myceliapolysaccharide from fermentable Hypsizigus marmoreus.More valuably,two polysaccharides,Tâ…¢-2 and Nâ…£,with Mw for 19136Da and 522981Da,are obtained for the first time.Thestructures of Tâ…¢-2 and Nâ…£are analyzed,too.The structures of these two polysaccharidesare different from the polyscaaharides of Hypsizigus marmoreus fruit body.It is proved thatthey are not reported in former studies.(2)Moreover,in previous studies,just taking mycelia biomass;as a uniqe measure indexand shake flasks as the fermentation carrier,only more than 10 nutritional factors were investigated.But in this paper,taking the production of extra-cell polysaccharide andinter-cell ploysaccharide as major measure indexes,and using fermentor as the fermentationcarrier,34 nutritional factors were investigated.