Novel Enzymatic Preparation Of Yeast β-glucan And Mannoprotein Using Myxobacteria β-1,6-glucanase And Investigation On Their Properties

β-glucan and mannoprotein are biopolymers with multiple biological activities and high commercial value,located in the cell wall of Saccharomyces cerevisiae.β-glucan is able to bind the immune cell receptor specifically to improve the immunity,which plays important roles of anti-tumor,anti-inflammation,reducing blood fat and cholesterol and regulation of intestinal microbial community structure.In terms of food preservation,mannoprotein has emulsification stability,and could maintain the quality of red wine and delay corruption of vegetables and fruits.At present,yeast polysaccharides have extracted by physical method,acid-alkali method,induced autolysis,hot water extraction.However,the above methods have some disadvantages such as inefficiency,environmental pollution,low yield and activity of product or complex process.As a mild and green method,enzymatic method has also been used to extract β-glucan and mannoprotein.However,the existing methods related to protease and lipase could only remove impurities in the extraction process.β-1,3-glucanase could greatly destroy the natural structure of β-glucan,resulting in the decrease of the activity and yield of the extracted products.Therefore,the establishment of a green and environmentally friendly extraction method is of great significance for the extraction of yeast biopolymers and further processing yeast.According to the structural model of yeast cell wall,β-1,6-glucan has a central role of crosslinking in the organization of the yeast cell wall.It is deduced that destruction ofβ-1,6-glucan could effectively collapse the yeast cell wall and release β-1,3-glucan and mannoprotein.GluM employed in this study was a novel outer membrane β-1,6-glucanase with high catalytic activity from myxobacterial strain Corallococcus sp.EGB.In this study,the fermentation conditions of GluM were established and optimized;The toxicity of strain EGB fermentation broth was evaluated;a novel green and efficient extraction method for preparation of β-glucan and mannoprotein based on β-1,6-glucanase GluM was first established;the structural analysis,physicochemical properties and biological functions ofβ-glucan and mannoprotein prepared by GluM enzymolysis were analyzed.1.The liquid fermentation process of GluM and preparation of β-glucan and mannoprotein by enzymolysis of GluMBy exploring the technical parameters of strain EGB liquid fermentation,YST culture medium composition for producing GluM was determined as follows:yeast cell 1.0%,soluble starch 0.07%,tryptone 0.01%,MgSO4 0.06%,pH 7.0.The maximum activity of GluM was 560 U/L in triangular flask fermentation,660 U/L in 50 L fermenter,and 670 U/L in 500 L fermenter.The toxicological safety of GluM was evaluated.The results of acute and subacute toxicity and genotoxicity tests showed that GluM was safe for the preparation of yeast β-glucan and mannoprotein,and did not cause harm to animal health.A new mild,green,and highly efficient extraction method for the preparation ofβ-glucan and mannoprotein from baker’s yeast based on GluM was established and optimized.The optimal reactions for GluM hydrolysis of yeast were determined at 10 U/g,33℃ and pH 7.3 for 17.5 h and subsequent papain hydrolysis were determined at 10 000 U/g,65℃ for 10 h.The sediment was β-glucan with 17.4%yield and 85.4%purity.The supernatant after GluM hydrolysis was precipitated by 75%alcohol,dialyzed with 7 kDa membrane,separated by chromatographic column to prepare mannoprotein.The final yield of mannoprotein was 11.1%with the purity of 90.7%.2.Structural analysis of β-glucan and mannoprotein prepared by enzymolysis of GluMThe structure of β-glucan and mannoprotein prepared by GluM enzymolysis were analyzed.The monosaccharide composition of β-glucan contained glucose and mannose,and the content of glucose accounts for 96.5%of the total carbohydrate.The results of NMR showed that β-glucan(BYG)was linked by β-1,3-glucoside bonds as backbone,β-1,6-glucoside bonds as side chain.The results of FT-IR and XRD showed that compared with the commercial β-glucan(Glucan 300)prepared by acid-alkali method,the structure ofβ-glucan(BYG)prepared by GluM enzymolysis has lower degree of crystallization,looser aggregation and well-maintenance of the triplex glucan structure.The results of SEM and AFM showed that β-glucan(BYG)has rod-like structure with a size of ca.1×5μm in dimension and nanoparticle-like substructures with a length of about 100 nm.The average relative molecular weight(MW)of mannoprotein was 112 kDa.The monosaccharide composition of mannoprotein contained mannose and glucose,and the content of mannose accounted for 98.7%of the total carbohydrate.It contained 16 common amino acids,most of which were hydrophilic amino acids.Protein and polysaccharide were connected with glycoside bonds in mannoprotein,and the backbone moiety of mannoprotein was mainly composed α-1,6-Manp,and α-1,2,α-1,3,α-1,2/6-Manp residues with the side chain.The results of FT-IR and SEM showed that mannoprotein has typical polysaccharide and protein structure with the fibrous morphology.3.Evaluation and mechanism of immunomodulation activity of β-glucan prepared by enzymolysis of GluMThe immunomodulation activity of β-glucan(BYG)prepared by GluM enzymolysis was evaluated in colitis mice model induced by DSS.The results showed that BYG significantly reduced the disease index of colitis mice(p<0.05),including the amelioration of weight loss,diarrhea,fecal occulted blood;attenuated symptoms of shortened colon and enlarged spleen;protected the colonic mucosal tissue of mice,and reduced the infiltration of inflammatory cells.In terms of oxidative stress,BYG significantly reduced the production of radical NO,the content of MDA and inhibited the activity of MPO in colonic tissues of mice(p<0.05),which inhibited the oxidative stress response in colonic tissues of mice with colitis.In terms of cytokine secretion,β-glucan significantly down-regulated the expression of pro-inflammatory cytokines IL-1,IL-6,TNF-and IFN-γ in the serum of mice with colitis,and up-regulated the expression of anti-inflammatory cytokines IL-10(p<0.05),thus promoted the balance between pro-inflammatory cytokines and anti-inflammatory cytokines in the serum of mice with colitis.BYG significantly down-regulated the expression of iNOS,COX-2 and NLRP3 inflammasome in mice with colitis(p<0.05).In terms of intestinal mucosal integrity,BYG increased the expression of ZO-1,Occludin-1 and Caludin-1 in colonic epithelial mucosa of colonic mice(p<0.05),which protected the physical barrier of colonic mucosal layer.In addition,BYG significantly increased the production of SCFAs in the feces of mice with colitis(p<0.05),mainly increasing the content of acetic acid,n-butyric acid and total short-chain fatty acids.Compared to commercial β-glucan(Glucan 300)prepared by acid-alkali method,BYG prepared by GluM enzymolysis could significantly reduce oxidative stress occurs in colon and pro-inflammatory cytokines secretion,and improve the intestinal mucosa in tight binding protein expression and decrease inflammatory protein expression(p<0.05),but faeces SCFAs levels were not significantly different(p>0.05).The above results demonstrated that compared with Glucan 300,BYG had better immunomodulation activity to alleviate more effectively symptoms of inflammation in mice with colitis induced by DSS.4.The physicochemical properties and application of mannoprotein prepared by enzymolysis of GluMThe rheological properties,emulsification stability and application in food process of mannoprotein prepared by enzymolysis of GluM were studied.Mannoprotein solutions showed a non-Newtonian pseudoplastic behavior at all concentrations and apparent phenomenon of shear-thinning.In dynamic viscoelastic properties,it was always existed storage moduli G’>loss moduli G",which showed an elastic behavior.The increase of temperature led to the decrease of the viscosity of mannoprotein solutions.The viscosity of mannoprotein solutions remained stable in the pH range of 5.0-13.0,during freezing and thawing process,and in presence of salts.The effect of mannoprotein on the stability of sodium caseinate-olive oil emulsion during storage time were evaluated.When the concentration of mannoprotein was not lower than 6.0%in the emulsion,it had good emulsification stability and remained group stable for 7 days.Due to smaller and more uniform of particle size of the emulsion drops,the emulsion system was more stable.Mannoprotein emulsion can significantly improve the WHC and strength of blended myofibrillar protein gel(p<0.05),by binding free water into the three-dimensional network structure of protein gel to form a dense network structure.Effects of mannoprotein emulsion as substitute of pork back fat(25%,50%,75%and 100%)on chemical composition,texture,fatty acid profile and lipid oxidation of the low-fat emulsified sausage were evaluated.Compared with the control group,the content of protein and water in the low-fat emulsified sausage in treatment groups were significantly increased,the content of fat was significantly decreased,the PUFA and PUFA/SFA were significantly increased and the oxidation degree,TBARS value were significantly decreased(p<0.05).The hardness and chewiness of the low-fat emulsified sausage increased significantly(p<0.05)with mannoprotein emulsion above 50%.Therefore,mannoprotein emulsion as substitute of pork back fat in the low-fat emulsified sausage could effectively reduce the fat content and improve the textural properties,PUFA content and oxidation stability,hence improve nutritional and edible quality of emulsified sausage.In the study,the technical parameters of EGB large-scale liquid fermentation were established,and a novel,green and efficient extraction method to prepare β-glucan and mannoprotein based on enzymolysis of GluM was first proposed.It proved that β-glucan(BYG)prepared by enzymolysis of GluM had better immunomodulation activity,compared to commercial β-glucan(Glucan 300)prepared by acid-alkali method.It provided theoretical basis of further exploiting yeast resources and intensive processing of yeast to prepare high added value products.