Study On The Polysaccharide And Its Biochemical Activity Of Poria Cocos Based On Fermentative And Enzymatic Conversion

Poria cocos is a kind of medicinal and edible fungus that has multiple physiological functions.The main functional component of Poria cocos is?-pachyman,which accounts for about 84.2%of the dry Poria cocos.Modern studies have confirmed that?-pachyman are ?-?1-3?-D-glucans containing a small amount of ?-?1-6?glucan branched chains,which is high molecular weight,compact structure,poor water solubility,and low biological activity.It is one of the key ways to more efficiently develop and utilize water-soluble polysaccharides by useing microbial fermentation or enzymatic hydrolysis or other degradation technologies.Through effective conversion of polysaccharide macromolecules,these water-soluble would be more higher bioavailable and smaller molecule.In this study,in order to obtain conversion process of water-soluble polysaccharides?WSP?with higher content and better utilization of biological activity,microbialfermentation,?-glucanaseenzymatichydrolysisand co-degradation by fermentation-enzymatic hydrolysis were used to transformed polysaccharides.The microstructure and infrared spectrum characteristics of three differently converted Poria Cocos were studied,and their water-soluble polysaccharides were determined.The physicochemical properties,antibacterial properties,in vitro antioxidant activity,and immunomodulatory effects were evaluated comprehensively to obtain a higher content and more active water-soluble polysaccharide conversion process.The main results were as follows:1 Screening,separation and identification of suitable strains of Poria cocos fresh samples,and comparing the differences of polysaccharides fermented with different strains of Poria cocos polysaccharides,screening and determining the dominant strains of microbial fermentation and transformation of Poria cocos polysaccharides.The results were as follows:Two strains of suitable strains were screened and isolated from the fresh samples.According to the morphological and molecular biological methods,they were identified as Fusarium solani and Penicillium chrysogenum.The content of water-soluble polysaccharides was used as the evaluation index,and the differences in Ma,Mc,and Aspergillus niger fermentation in the laboratory were compared.The content of water-soluble polysaccharides after fermentation in the Ma,Mc,and An groups was 0.69%,0.92%,and 2.66%?CK group 0.58%?,An group compared with CK group increased 4.58 times,compared with Ma,Mc group also significantly increased?P<0.05?.Therefore,it was proposed to screen and identify the dominant strains of Aspergillus niger for subsequent transformation fermentation experiments.2 Degradation and transformation of Poria Cocos polysaccharides were carried out in three ways:fermentation with Aspergillus niger,?-glucanase digestion,and fermentation-enzymatic hydrolysis.The optimum technical parameters of the three processes were obtained by orthogonal and response surface optimization using the content of water-soluble polysaccharides as the evaluation index.?1?An Aspergillus niger fermentation conversion process:the ratio of material to water was 1:1.16 g/mL,fermentation temperature at 31.6?,the fermentation time was 9.02d?216.5h?,the initial pH value was 5.5,and the inoculum amount was 4%.The content of water-soluble polysaccharide after conversion was up to 4.54%,which was 7.83 times higher than that of CK group.?2??-glucanase enzymatic conversion process:?-glucanase activity is 50 U/mg,enzyme dosage was 9%?w:w?,reaction liquid pH was5.5,reaction temperature was 55?,the reaction time was 150 min,and the content of water-soluble polysaccharide was up to 9.20%,a significant increase of 15.87 times.?3?Fermentation-enzymatic hydrolysis synthesizing process:under the optimum process parameters?1?above,the fermented mash substrate was obtained,and the system was added with 7%enzyme activity of 50u/mg of?-glucanase and reaction.At pH 5.5,temperature 50?and reaction time 120 min,15.20%of water-soluble polysaccharides could be harvested after synergistic transformation.The highest water-soluble polysaccharide content was obtained from the three conversion methods,which was 26.21 times that of CK group,and the variety of the process was significantly different?P<0.05?.However,the changes in the activity of water-soluble polysaccharides and the amount-effect relationship during the transformation process remain to be studied.3 Microstructure and infrared spectrum analysis of three kinds of transformants,and their antibacterial properties,in vitro antioxidant activity,and immunomodulatory effects of water-soluble polysaccharides.The results were as follows:?1?After drying,the fermentation,menzymolysis,fermentation-enzymatic hydrolysis of the synergistic transformation between the dried Poria Cocos and the CK group,there were significant differences in the microstructure.After the transformation,the Poria Cocos particles were broken,the surface was rough,and the shape was irregular.After conversion,the infrared spectrum of the Poria Cocos shows that it still has the characteristic structure of the general polysaccharides.?2?The water-soluble polysaccharides obtained from three kinds of technics have a certain antibacterial effect on Staphylococcus aureus,Escherichia coli and Bacillus subtilis,and they were significantly different from those of CK group?P<0.05?.The fermentation-enzymatic hydrolyzed water-soluble polysaccharide?F-EWSP?group had the strongest bacteriostatic effect against Staphylococcus aureus and Escherichia coli,and its inhibitory zone diameters were 14.67 mm and 11.68 mm,respectively.There was no antibacterial activity against Bacillus subtilis.effect.Among the three water-soluble polysaccharides after transformation,only the FWSP had inhibitory effect on Bacillus subtilis.The diameter of inhibition zone was 6.02 mm.It was shown that the antibacterial properties of water soluble polysaccharides against Gram-negative bacteria and non-spores bacteria after transformation were strong.?3?In the test of in vitro antioxidant activity,the three water-soluble polys accharides of the transformed fleas showed dose-effects on the clearance rates of O₂^-·,DPPH·,·OH,and ABTS⁺·.The antioxidant effect of the polysaccharid e group was stronger than that of the original water soluble polysaccharide gro up?WSP?.At the concentration of 4mg/mL,the fermentation-enzymatic hydroly zed water-soluble polysaccharide?F-EWSP?group had the best antioxidant activ ity,O₂^-·clearance rate reached 42.37%,DPPH·clearance rate reached 46.53%,ABTS⁺·clearance rate reached 49.54%.·OH removal rate was 45.57%.The strength of anti-oxidation was fermentative-enzymatic hydrolysis water-soluble p-olysaccharide?F-EWSP??enzymatic hydrolysis water-soluble polysaccharide?E-WSP??fermentation water-soluble polysaccharide?F-WSP?.?4?Construction of cyclophosphamide-induced mouse immunocompromise model,quantitative gavage conversion of water-soluble polysaccharides,and inv estigation of three water-soluble polysaccharides of transterpenoids on mouse or gan index,ear swelling rate,serum hemolysin level,IgG and IgM content,ma crophage phagocytosis rate and other immune functions,the results show that t he three water-soluble polysaccharides can improve the above-mentioned indicat ors of the model mice,and there was a dose-effect relationship between the in tensity of the immune effect and polysaccharide concentration.The high dose o f F-EWSP at 100 mg/kg BW had the highest immunomodulatory effect on mic e:spleen and thymus index were 87.96 mg/g and 50.61 mg/g,respectively,wh ich were 1.99 times and 1.62 times higher than those in the model group,resp ectively;Ear swelling rate was 109.37%,which was 1.69 times higher than tha t of the model group.The level of hemolysin was 0.49,which was 2.33 times higher than that of the model group.The content of Ig G and Ig M was 11.21mg/mL and 1.94 mg/mL,which was 1.59 times and 1.64 times higher than t-hat of the model group,respectively.The phagocytosis rate of macrophages wa s 48.93%,which was 1.56 times higher than that of the model group.There w as a significant difference between each index and the model group?P<0.05?.The immunomodulatory effects of three kinds of water-soluble polysaccharides i-n transformation on immune-lowering mice induced by cyclophosphamide were F-EWSP>EWSP>FWSP.In summary,the fermentation-enzymatic synergistic conversion process of the first enzymatic hydrolysis after fermentation in the three different conversion processes has the best effect,and the content of the water-soluble polysaccharide in the trans-terpene is as high as 15.20%,which is a single fermentation transformation or an enzymatic hydrolysis transformation.3.35 times and 1.65 times the content of the polysaccharide.In the antibacterial properties,in vitro antioxidation,and immunomodulatory effects in mice,fermentation-enzymatic hydrolysis of the water-soluble polysaccharides of Poria Cocos showed better results than that of other two processes.