Fermentation Preparation Of Auricularia Heimuer Melanin And Its Antioxidant Effects

Melanin is a class of polymers formed by the oxidation of polyphenols and indoles and other compounds with biological activities such as antioxidant,anti-radiation and antibacterial,which are widely used in food and medicine.Auricularia heimuer is an important black food,however,the melanin yield is relatively low,which limited its application in food and medicine.Therefore,it is important to improve the yield of A.heimuer melanin,explore the regulatory mechanism of melanin metabolism,and study the antioxidant effect of melanin for the production and application of A.heimuer melanin.In this study,the structural characterization,stability and in vitro antioxidant activity of melanin from fruiting bodies,mycelium and fermentation of A.heimuer were compared.The conditions of liquid fermentation for A.heimuer melanin production were optimized.The mechanism of temperature regulation on melanin metabolism in A.heimuer based on transcriptomics was studied.The extraction parameters of microwave-assisted extraction of melanin in fermentation were optimized.The protective effects of melanin on H₂O₂oxidative damage in Hep G2 cells and D-galactose oxidative damage in mice were studied.The main findings are as follows.（1）Comparative analysis of structural characterization and in vitro antioxidant activity of three A.heimuer melanins.The melanin was extracted from the fruiting bodies,mycelium and fermentation of A.heimuer by alkali solubilization and acid precipitation method.The structural characterization,solubility,stability and in vitro antioxidant activity of three melanins were compared and analyzed.The UV-Vis results showed that the maximum absorption peak of three melanins was at 210 nm.FTIR results showed that the three melanins had absorption peaks at 3500～3300cm^-1,1620～1600 cm^-1and 1150～1000 cm^-1.The SEM results showed that all three melanins showed blocky,irregular surface crystalline structures.The HPLC results showed that three melanins had only one absorption peak with a retention time of about 2.410 min.All three melanins were soluble in alkali solution,insoluble in water and the organic solvent of this study.The three melanins have good temperature stability and light stability,but are unstable to Fe²⁺and Cu²⁺.The three melanins had strong scavenging ability for DPPH·,ABTS and·OH,among which the melanin from fermentation had the strongest scavenging ability for DPPH·,ABTS and·OH with IC₅₀of 3.82 mg/m L,2.06 mg/m L and 144.20 mg/m L,respectively.（2）Optimization of medium formulations for high melanin yield of A.heimuer fermentation.A single-factor method and response surface method were used to optimize the formulation of A.heimuer fermentation medium for melanin production.The results showed that glucose,yeast extract and salicylic acid had significant effects on melanin yield（P<0.05）.The optimal medium formulation of A.heimuer fermentation for high melanin was:potato 200g/L,glucose 24.1 g/L,yeast extract 4.2 g/L,salicylic acid 120.3 umol/L,Ca Cl₂1.0 g/L,KH₂PO₄2.0 g/L,Mg SO₄0.5 g/L,Vitamin B₁1 mg/L.The melanin yield under these conditions was 0.318%,which was 22.3%higher than that before optimization.（3）The regulation mechanism of temperature on melanin metabolism was studied using transcriptomics.The effect of temperature on the growth rate of A.heimuer mycelium and melanin yield in fermentation was investigated,and the DEGs,GO function,and KEGG pathway of temperature regulation on A.heimuer melanin metabolism was studied based on transcriptomics.The results showed that the growth rate of A.heimuer mycelium was the fastest at 30℃,normal growth at 35℃and stopped at 38℃.The A.heimuer mycelium in solid medium produced the highest melanin after stressed at 44°C for 2 h.The A.heimuer fermentation was induced by 44℃for 2 h,and the melanin yield was increased by 13.8%.Transcriptome sequencing at 25°C,35°C and 44°C yielded a total of 10879 DEGs,including2681 DEGs in the T35VST25 group;4188 DEGs in the T44VST25 group;and 4010 DEGs in the T44VST35 group.GO functional enrichment analysis revealed that 1204 DEGs were enriched to BP,446 DEGs were enriched to MF,and 524 DEGs were enriched to CC in the T35VST25 group.1212 DEGs were enriched to BP,820 DEGs were enriched to MF,and 1152DEGs were enriched to CC in the T44VST25 group.In the T44VST35 group,1896 DEGs were enriched in BP,764 DEGs were enriched in MF,and 878 DEGs were enriched in CC.KEGG pathway enrichment analysis revealed that the significantly enriched pathways common to all three groups were starch and sucrose metabolic pathway,carbon metabolic pathway,ethoxylate and dicarboxylate metabolic pathways.The significantly enriched pathways in both groups were cytochrome P450 metabolism pathway and phenylpropanoid biosynthesis pathway.The significantly enriched pathways in the single group were melanin synthesis pathway and tyrosine metabolism pathway.RT-PCR results indicated that the transcriptome sequencing results were reliable.（4）Optimization of extraction parameters for microwave-assisted extraction of melanin in A.heimuer fermentation.The microwave-assisted extraction parameters of melanin in fermentation were optimized by single-factor method and response surface method.The results showed that the effects of alkaline dissolution p H,acid precipitation p H and microwave time on melanin yield were significant（P<0.05）,while the effects of microwave power on melanin yield were not significant（P>0.05）.The optimum extraction parameters for microwave-assisted extraction of melanin from A.heimuer fermentation were:alkaline dissolution p H of 12.3,acid precipitation p H of 3.1,and microwave time of 53 s.The melanin yield under these conditions was 0.406%,which was 12.2%higher than that before optimization.（5）Protective effects of A.heimuer melanin on oxidative injury in Hep G2 cells induced by H₂O₂.The model of H₂O₂-induced oxidative damage in Hep G2 cells was established to study the protective effect of melanin on oxidative injury in Hep G2 cells induced by H₂O₂.The results showed that 10～500μg/m L melanin did not exhibit toxicity to Hep G2 cells.The model of H₂O₂-induced oxidative damage in Hep G2 cells was successfully established after treatment with 800 umol/L H₂O₂for 2 h.The incubation of 300～500μg/m L A.heimuer melanin for 2h～4 h or 200～500μg/m L A.heimuer melanin for 6 h significantly increased the survival rate of H₂O₂-induced Hep G2 cells（P<0.05）.A.heimuer melanin protected Hep G2 cells damaged by H₂O₂oxidation by decreasing MDA content,ROS and LDH activity,and increasing SOD and GSH-Px activity in Hep G2 cells.（6）Protective effects of A.heimuer melanin on oxidative injury in mice induced by D-galactose.The model of oxidative damage in mice was established using D-galactose,and the protective effect of A.heimuer melanin on D-galactose oxidative injury mice was investigated.The results showed that the mice in the A.heimuer melanin-treated group were in better mental condition compared with the model group.The weight gain rate of the mice was higher than that of model group.The organ indices of heart,liver,stomach and kidney were higher in the A.heimuer melanin-treated mice,with a melanin concentration dependence,and the difference was significant（P<0.05）.The liver cells in the A.heimuer melanin-treated group of mice were more neatly arranged,with clearer nuclei and more uniform cytoplasm.A.heimuer melanin decreased MDA content,ALT and AST activities,and increased SOD,GSH-Px,CAT and T-AOC activities in serum and liver tissue homogenates of oxidatively damaged mice.A.heimuer melanin treatment increased the abundance and diversity of gut microbiota,improved the gut microbiota composition in mice,and maintain the stability of the gut microbiotain mice.Antioxidant enzyme activities in mouse serum and liver tissue homogenates were significantly positively correlated,and abundance and diversity of mouse gut microbiota were positively correlated with serum and liver tissue homogenate antioxidant enzyme activities.The significant enrichment of Genus Prevotellaceae in mouse intestine was correlated with the antioxidant enzyme activities in serum and liver tissue homogenates.