| Schizophyllan（SPG）is a neutral polysaccharide extracted from the fruiting body or fermentation broth of Schizophyllum commune（S.commune）.It has good activities in anti-tumor,immune regulation,antioxidant,anti-inflammatory,moisturizing,and so on.As a medicinal and food homologous plant,Radix Puerariae（RP）is rich in active ingredients such as polysaccharide,puerarin,isoflavones,saponins,and other ingredients,and has the effects of antioxidant,anti-inflammatory,hypoglycemic,detoxification and liver protection.In this paper,the liquid bidirectional fermentation technology was used,with S.commune as fermentation strain and RP as a nutrient substrate,to increase the production of Schizophyllan,while promote the increase of anti-oxidative and anti-aging activities of fermentation broth,which provides an experimental and theoretical basis for the comprehensive utilization of S.commune.（1）Optimization of fermentation conditions of Radix Pueraria fermented by Schizophyllum commune.Taking the biomass of S.commune and the yield of exopolysaccharide as evaluation indicators,the optimal nutrient substrate RP was screened from 4kinds of medicine and food homologous plants.The initial pH was 6.0,the addition concentration of RP was 10.00 g/L,the inoculum size was 5.0%,and the fermentation was carried out 160 r/min for 6.0 d at 28°C,and the yield of the exopolysaccharide of fermentation product was 4.86±0.11 g/L.Ultra violet spectra and fourier transform infrared spectroscopy showed that the addition of RP extracts didn’t affect the chemical structure of SPG.Through the optimization of the fermentation conditions,the yield of SPG reached 8.41±0.12 g/L when the initial pH of the medium was 5.40,the addition concentration of RP was 12.80 g/L,and the inoculation size was 10.00%.Compared with the blank control group without RP addition（6.76±0.52 g/L）,the yield of SPG increased by 1.24 times.（2）Determination of active components in the fermentation products of Radix Puerariae fermented by Schizophyllum commune and identification of the antioxidant active components.Schizophyllum commune fermented supernatant group cultured from regular medium（SC）was used as the blank control group,and Schizophyllum commune fermented supernatant group cultured from Radix Puerariae supplemented medium（SC-RP）were set as the experiment group.The composition and antioxidant activity of both SC and SC-RP were studied.The results showed that when the concentration of SPG were 6.76 g/L,the content of total protein in SC-RP was 4.66±0.05 g/L,and the content of total phenolics was 3731.56±54.07 mg GAE/100 g,FW and total flavonoids was 27.97±0.41mg CE/100 g,FW,which were significantly higher than those in the blank control group（p<0.05）,and the contents of total phenolics and total flavonoids increased to 12.94 and 3.30 times compared to the blank control group,respectively.The total antioxidant capacity of SC-RP or SC were identified by 4different chemical antioxidant evaluation systems in vitro（The methods of Fenton,ABTS+,FRAP and ORAC）.The results showed that SC-RP’s antioxidant capacity was significantly enhanced（p<0.05）,and the FRAP value and ORAC value of SC-RP were respectively increased to 14.69 times and 11.56times of the blank control group.Correlation analysis showed that the in vitro antioxidant capacity of SC-RP was highly correlated with total phenolics and flavonoids（r>0.90）.The active components of SC-RP were analyzed by HPLC-MS/MS technology,and 8 key antioxidant active substances compared to the SC were identified as:puerarin,genistin,genistein,coumestrol,resveratrol,isorhapontigenin,esculetin and diosmetin.Puerarin,genistin,genistein,and coumestrol were derived from RP,and resveratrol,isorhapontigenin,esculetin,and diosmetin were the new active components generated by biotransformation in the process of bidirectional fermentation.（3）The research of anti-aging activity in vivo of SC-RP.The Caenorhabditis elegans（C.elegans）aging models were used to detect the anti-aging activities of SC-RP in this study,and the mean lifespan of the blank control group without SC-RP was 16.27±1.34 d.The middle-dose group（10μg/mL）and the high-dose group（20μg/mL）of SC-RP could significantly prolong the mean lifespan of nematodes to 20.32±0.88 d and 20.38±1.30 d,respectively,and the lifespan extension rates were 24.89%and 25.26%,respectively（p<0.05）.SC-RP can also reduce the accumulation of lipofuscin in nematodes,improve the physical ability of nematodes in middle and late age,and also improve the stress ability of nematodes under stresses such as H2O2-induced oxidative stress,paraquat-induced oxidative stress,and heat stress.In addition,SC-RP can significantly increase the activities of antioxidant enzymes SOD and CAT in nematodes and reduce the accumulation of oxidative by-products TBARS and ROS（p<0.05）,showing excellent anti-aging activity.（4）The research of anti-aging mechanism of SC-RP.The anti-aging mechanism of SC-RP was further explored through the mutant nematode（daf-16,age-1,clk-1,mev-1,skn-1）and transgenic nematode（LD1,CF1553 and TJ356）models.The middle dose SC-RP（10μg/mL）significantly up-regulated the expression levels of anti-aging key genes daf-2,gst-4 and sod-3 genes,while that the expression levels of daf-16,skn-1 and clk-1genes were significantly down-regulated（p<0.05）.SC-RP also promoted the nuclear translocation of DAF-16 and SKN-1 and the expression of antioxidant-related protein SOD-3（p<0.05）.Compared to the control group,SC-RP had no significant effect on the mean lifespan of daf-16,skn-1 and clk-1nematodes（p>0.05）,indicating that daf-16,skn-1 and clk-1 genes may be the key genes affecting the anti-aging activity of SC-RP.The above results indicate that SC-RP exerts the antiaging effect through insulin/insulin-like growth factor signaling（IIS）pathway,SKN-1/Nrf2 pathway and mitochondrial metabolic pathway. |
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