Study On Fermentation Of Polygonatum By Ganoderma Lucidum And Its Protective Effect On Liver Injury Induced By Carbon Tetrachloride In Mice

Using the catalysis of enzymes in the metabolic system of edible fungi,various components in the substrate of traditional Chinese medicine were decomposed and transformed to improve the efficacy of traditional Chinese medicine,providing a new idea for the development of traditional Chinese medicine.In this study,Ganoderma lucidum strain was used as the starting strain and Polygonatum odoratum was used as the substrate to explore the changes in the content of water-soluble polysaccharides during liquid fermentation.The polysaccharides after fermentation were separated and extracted.The antioxidant activities in vitro and in vivo of the water-soluble polysaccharides after fermentation were explored by comparing Polygonatum odoratum polysaccharides with Ganoderma lucidum polysaccharides.Polygonatum odoratum was fermented by Ganoderma sanensee,Cordyceps militaris,Ganoderma lucidum and Ganoderma lucidum in Hunan.The polysaccharide production ability of each fermentation system was detected at different time periods,and screen the Ganoderma sanensee was the best strain.It highest polysaccharide content was 4.164±0.206 mg/m L on the fourth day.The effects of nitrogen source,soybean meal addition,Polygonatum odoratum addition,potassium dihydrogen phosphate content,magnesium sulfate heptahydrate content,inoculation amount,time,temperature and other factors on the polysaccharide yield of fermented Polygonatum odoratum were investigated by single factor experiment.The Plackett-Burman experiment was used to screen the factors,and three significant factors affecting the polysaccharide improvement rate were determined from seven factors,which were the addition amount,time and inoculation amount of Polygonatum odoratum.Through the response value design response surface experiment,the mathematical model of polysaccharide improvement rate of fermented Polygonatum odoratum was discussed.The results showed that the optimal fermentation conditions determined by Box-Behnken experiment were as follows:bamboo 6%,soybean meal 2%,potassium dihydrogen phosphate0.15%,magnesium sulfate heptahydrate 0.15%,VB₁ 0.005%,inoculum 8.7%,fermentation time 4 d,and temperature 30℃.Under these conditions,the polysaccharide content of the fermentation broth reached 5.91 mg/m L,which was 73.48%higher than that of the unfermented medium 3.41 mg/m L.According to the optimized fermentation process,the fermentation broth was collected,the impurity protein was removed by Sevage reagent,the pigment was removed by AB-8 macroporous resin,and the dialysis bag was 8000–14000 Da for 48h.The FOP was obtained by freeze-drying.Polygonatum odoratum polysaccharide POP and Ganoderma lucidum polysaccharide GOP were obtained in the same step,and the antioxidant activities of FOP,POP and GOP in vitro were determined.The scavenging rates of FOP on ABTS free radical,DPPH free radical and hydroxyl free radical were37.55±2.91%,38.26±1.03%and 89.40±0.73%,respectively.The ABTS,DPPH and hydroxyl radical scavenging rates of POP were 21.13±1.77%,23.46±1.244%and65.37±6.26%,respectively.The ABTS radical,DPPH radical and hydroxyl radical scavenging rates of GOP were 52.62±0.62%,52.77±3.14%,79.91±0.19%,respectively.The results showed that the antioxidant capacity of FOP and GOP was significantly stronger than that of POP.ABTS scavenging rate of GOP was significantly stronger than that of FOP,DPPH scavenging rate of GOP was stronger than that of FOP but not significantly indigenous,hydroxyl radical scavenging rate of FOP was stronger than that of GOP but not significantly indigenous.Through the establishment of carbon tetrachloride-induced liver injury model in mice,the in vivo antioxidant effects of different polysaccharides and their protective effects on liver injury were studied.After adaptive culture for 7 days,50 KM mice were randomly divided into 5 groups with 10 mice in each group.They were control（negative contro,NC）group,CCl₄ group,Polygonatum odoratum polysaccharide（POP）group,Ganoderma lucidum polysaccharide（GOP）group,and fermented polysaccharide（FOP）group,respectively.POP group,GOP group FOP group were gavaged with 100mg/kg FOP 100 mg/kg POP,100 mg/kg GOP,model group and control group were gavaged with the same dose of normal saline for two weeks.After 14 days of intragastric administration,0.2%CCl₄olive oil solution（10 m L/kg）was intragastrically administered to CCl₄ group,FOP group,POP group and GOP group for one hour.NC group mice were gavaged with the same volume of olive oil.All mice were fasted without water.After 20 h,blood was taken from all mice eyeballs.HE staining was used to detect liver histopathology and the activities of various antioxidant enzymes in the liver.The results showed that fermented polysaccharides,Ganoderma lucidum polysaccharides and Polygonatum odoratum polysaccharides could enhance liver antioxidant capacity and reduce lipid peroxidation,and their hepatoprotective effect may be closely related to enhancing liver antioxidant and inhibiting oxidative stress.