Ganoderma Lucidum Spore Breaking,Extraction Of Polysaccharide And Glutathione,Separation And Purification Of Polysaccharide And Its Activity

Ganoderma lucidum is a precious Chinese herb belonging to the family of Ganoderma lucidum,and has been known as the"King of Herbs"for over 2,000 years in China.Ganoderma lucidum spore powder is the seeds ejected from Ganoderma lucidum during maturation,which has all the genetic information and similar functional components of Ganoderma lucidum.Ganoderma spore powder can regulate the immune system,lower blood lipids,anti-allergy,anti-fatigue,antioxidant,anti-tumor,anti-inflammatory and many other functions.Therefore,in this study,Ganoderma lucidum spore powder was used as the raw material,firstly,the spore powder was broken by ultra-micro crushing to determine the best breaking conditions,and then the changes of spore powder before and after breaking were compared by electron microscopy and infrared scanning,and the effect of the breaking effect on the polysaccharide yield.Then the polysaccharides were extracted from the broken spore powder to determine the best extraction conditions.The crude polysaccharides obtained by the optimal extraction conditions were separated and purified to obtain homogeneous polysaccharides,and the antioxidant activities of the crude and purified polysaccharides were determined by antioxidant tests.The by-product protein produced during the extraction of Ganoderma lucidum spore powder was further utilized to extract glutathione from it,and the content and yield of glutathione obtained were measured,and the test results were as follows:（1）Ganoderma spore powder wall-breaking test:Ganoderma spore powder was coarsely crushed,then high-pressure homogenized and freeze-dried at a certain concentration,and then wall-breaking was carried out in an ultra-micro crusher,with spindle speed and crushing time as single factors,and the best process conditions were finally determined as spindle speed 2200 r/min and crushing time 20 min,when the wall-breaking rate reached about 98%.Through the observation of appearance,color,odor,taste and texture,the differences between before and after breaking the wall of Ganoderma lucidum spore powder were compared;through the comparison of before and after electron micrographs and infrared spectral analysis,it was found that the active ingredients were fully released after breaking the wall,and the active ingredients in Ganoderma lucidum spore powder did not change significantly;through the comparison of the polysaccharide yield of Ganoderma lucidum spore powder,it was found that the polysaccharide yield of Ganoderma lucidum spore powder before breaking the wall was0.83%,and after breaking the wall was The polysaccharide yields were positively correlated with the wall breaking rate of Ganoderma spore powder.（2）The extraction process of Ganoderma lucidum spore powder polysaccharide was optimized by using ultrasonic-assisted hot water extraction method with ultrasonic time,water bath temperature,material-to-liquid ratio,ultrasonic power and extraction times as single factors.The results showed that the order of the influencing factors on the polysaccharide yield was material-to-liquid ratio>ultrasonic power>ultrasonic time>water bath temperature.The optimum extraction process conditions were 1:30（g/m L）,450 W ultrasonic power,1.5 h ultrasonic time,50°C water bath temperature,and 2 times of extraction,and the polysaccharide yield was 6.04%.The average decolorization rate of polysaccharide was 86.55%and the average retention rate of polysaccharide was 80.35%by the resin adsorption method;the average decolorization rate of polysaccharide was 67.78%and the average retention rate of polysaccharide was 72.09%by the activated carbon adsorption method.Then GLSP-1was purified by DEAE-52 cellulose and Sephadex G-100 gel column chromatography to obtain the pure polysaccharide of Ganoderma lucidum spore powder（GLSP60A-1）with homogeneous components and high purity.By UV full-band scanning,GLSP60A-1 had no absorption peaks at 260 nm and 280 nm,indicating that the separated and purified polysaccharide did not contain nucleic acid and protein;the results of IR spectral scanning showed that GLSP60A-1 contained the characteristic absorption peaks typical of polysaccharides,and the structure containedɑ-type glycosidic bonds with pyranose ring structure.（3）Comparing the three methods for GSH extraction,it can be seen that the yield of GSH replacement by copper salt method was 71.29%,the highest yield of LX-18 resin extraction was 79.35%among the three different types of resin adsorption GSH,and the highest yield of AOT-isooctane extraction was 82.07%among the three reverse micellar extraction methods for GSH extraction.Finally,the AOT-isooctane reverse micelle extraction method with uncomplicated operation,less error factors and the highest GSH yield was selected for GSH extraction,and single-factor and response surface optimization tests were conducted to finally obtain the optimal process conditions for GSH extraction with AOT-isooctane concentration of 36.96 g/L,W₀25.4 and K⁺concentration of 0.53 mol/L,when the highest GSH yield was obtained.The average value was 89.583%.（4）In the DPPH free radical scavenging experiment,the scavenging ability of DPPH free radical was VC>GLSP-1>GLSP60A-1.The EC₅₀ of GLSP-1 and GLSP60A-1samples were 0.38 mg/m L and 4.22 mg/m L,respectively.In the ABTS free radical scavenging test,the scavenging ability of ABTS free radical was VC>GLSP60A-1>GLSP-1.The EC₅₀ of GLSP-1 and GLSP60A-1 samples were 1.20 mg/m L and 0.19mg/m L,respectively.In the OH radical scavenging test,the scavenging ability of OH radical was VC>GLSP-1>GLSP60A-1.The EC₅₀ of GLSP-1 and GLSP60A-1 samples were 0.18 mg/m L and 4.51 mg/m L,respectively.