Preliminary Study On The Selenium Collecting Properties And Selenium-containing Active Substances Of Ganoderma Lucidum

Objective:Liquid selenium-enriched deep fermentation is safe to operate and can obtain high biomass of selenium-enriched active substances in a short period of time.By studying the liquid selenium-rich deep fermentation of Ganoderma lucidum,exploring the physiological and biochemical indexes of its mycelium,the activity of selenium-containing active substances and the binding mode of selenium and macromolecules,understanding the selenium collecting properties of Ganoderma lucidum,expanding the way for the utilization of selenium resources,and laying the foundation for scientific selenium supplementation and the development of selenium economy and selenium industry.Methods:In the experiment,sodium selenate,sodium selenite and selenomethionine were used as selenium sources and mycelial biomass was used as indicators to screen the selenium sources for Ganoderma lucidum liquid selenium enrichment fermentation and to optimize their fermentation conditions by response surface.The T-SOD,POD and PPO activities and MDA and PRO contents of Ganoderma lucidum mycelium were determined by using the kit method.The polysaccharides were extracted by ultrasound-assisted alkaline extraction and water extraction,initially purified by Sevage method,and the polysaccharide content was determined by anthrone sulfate method.The crude extracted soluble proteins were obtained by using phosphate buffer as solvent,ammonium sulfate for precipitation and dialysis,and their protein contents were determined by using the KOMAS Brilliant Blue G250 method.Analysis was performed to compare the antioxidant activity of the active substances.Microwave digestion and atomic fluorescence spectrometry were used to determine the total selenium and the selenium content of the active substances.Due to the different solubility of proteins,water-soluble,salt-soluble,alcohol-soluble and alkali-soluble proteins were extracted sequentially by continuous extraction method and their selenium contents were determined;the effect of selenium on the amino acid types and contents of water-soluble proteins was studied by amino acid auto-analyzer,and their selenoamino acids contents were analyzed qualitatively and quantitatively by LC-MS.The CCK-8 method was used to determine the effects of selenium-rich Ganoderma lucidum selenopolyaccharides and selenoproteins on the effects of HCCLM,A549and The CCK-8 method was used to determine the inhibitory effects of selenium-enriched Ganoderma lucidum selenopolyaccharides and selenoproteins on HCCLM,A549 and HSC-3 tumor cells,while the unenriched group was used as a control.Results:1.The best selenium source for the liquid fermentation of selenium-enriched Ganoderma lucidum was sodium selenite,and the response surface optimization results were selenium concentration of 95?g/m L,fermentation temperature of 30?,fermentation period of 11 d,initial fermentation solution p H natural,the biomass of selenium-enriched Ganoderma lucidum mycelium reached the maximum of 5.6597 g/L,which was lower than the theoretical value of 5.727 g/L,and the utilization rate of selenium source of mycelium transforming sodium selenite into organic selenium was 4.7942%;2.the POD,T-SOD activity and PRO content of the selenium-added group were lower than the control group,and the PPO activity and MDA content were higher than the control group,at the selenium concentration,the PPO activity was promoted,but the POD and T-SOD activities were inhibited,and the mycelial resistance was decreased;3.the polysaccharide content of ultrasonic-assisted alkaline extraction was higher,which could better promote the precipitation of polysaccharides,and the selenopolyaccharide and selenoprotein content of the selenium-added group were higher than the control group.4.The scavenging rate of DPPH radicals reached the maximum at the concentration of selenopolyaccharide and selenoprotein amount of 5 mg/m L,and the selenium-added group was higher than that of the group without selenium;the scavenging rate of iron reducing power and ABTS radicals reached the maximum at the concentration of selenopolyaccharide and selenoprotein amount of 2 mg/m L,and the selenium-added group was higher than that of the control group.The selenoprotein had strong antioxidant effects on DPPH radicals,reducing power and ABTS radicals,and the scavenging power of alkaline extracted polysaccharide on DPPH radicals was better than that of water extracted polysaccharide.5.Compared with the control group,the selenium-added group of selenium polysaccharide and selenoprotein was higher in the the selenium content,indicating that the addition of inorganic selenium was beneficial to the accumulation of selenium polysaccharide and selenoprotein;the selenium content of selenium polysaccharide extracted by alkali was higher than that of selenium polysaccharide extracted by water,indicating that alkali extraction was more beneficial to the accumulation of polysaccharide selenium.Comparing the different soluble proteins in the mycelium of selenium-added and without selenium groups,we found that the selenium content in water-soluble proteins was the highest,419.2916 and 11.4083?g/g,respectively;6.There were 17 types of amino acids hydrolyzed in Ganoderma lucidum mycelium,among which aspartic acid,glutamic acid and leucine were higher,and cystine and methionine were lower;selenomethionine and selenocystine were the selenium-enriched Ganoderma lucidum selenomethionine and selenocystine are the main forms of organic amino acids present in the mycelium of selenium-enriched Ganoderma lucidum;7.The inhibitory proliferation effect of selenopolysaccharide in the selenium-added group on HCCLM,A549 and HSC-3 tumor cells was better than that in the control group,and its IC₅₀on HCCLM,A549 and HSC-3 tumor cells were3.765,3.23 and 2.267 mg/m L,respectively;the selenoprotein in the selenium-added group on The IC₅₀of selenoprotein on HCCLM,A549 and HSC-3 tumor cells were636.987,506.97and 555.598?g/m L,respectively,which inhibited proliferation less effectively than the control group,probably because the protein activity was inhibited by the higher selenium content in selenoprotein.Conclusion:Ganoderma lucidum mycelium has strong selenium tolerance,and the selenium collection by liquid selenium-enriched deep fermentation has good effect,and the combination of selenium and macromolecules in mycelium are selenopolysaccharide,selenoprotein,selenocysteine and selenomethionine,which not only provide some reference for the deep research of selenium-enriched Ganoderma lucidum at biological and molecular levels,but also provide theoretical and technical support for the utilization of selenium resources.