| This research is one of the main research contents of the the National Natural Science Foundation of China.The method of extraction of mycelium protein from Grifola frondosa was optimized by orthogonal design.The interaction between the active ingredient of Gastrodia elata Bl.and the fermentation of Grifola frondosa was studied,The effects of the active ingredients of Gastrodia elata Bl.on the growth of mycelia and the synthetic quality of extracellular proteins of Grifola frondosa was studied.To study the effect of ethanolic extracts of Gastrodia elata Bl.on the antioxidant activity of mycelium proteins of G.frondosa.The differential proteins were identified by 2-dimensional gel electrophoresis(2-DE)and mass spectrometry(MS)and bioinformatics analysis.Use the successful identification of differential proteins at the proteomics level to explain a variety of physiological and biochemical indicators of the fermentation process.In order to obtain high extraction rate of protein from G.frondosa mycelium,the G.frondosa mycelium protein was extracted by ultrasonic wave with the G.frondosa mycelium as raw material.On the basis of single factor experiments,with the extraction rate of protein as the evaluation index,the extraction processing was optimized by orthogonal experiments.The results showed that the optimum extraction conditions were liquid-material ratio 95∶1(m L∶g),ultrasonic power 600 W,ultrasonic temperature 35 ℃,ultrasonic time 3 min and p H 10.5.Under the conditions,the protein extraction rate was(5.10±0.04)%.Ethanolic extracts of Gastrodia elata Bl.was added into the system of G.frondosa submerged fermentation,and then supplementation of ethanolic extracts of Gastrodia elata Bl.were optimized according to the biomass and the synthetic quality of extracellular proteins in G.frondosa,The change components in ethanolic extracts of Gastrodia elata Bl.before and after sterilization,including gastrodin 、p-hydroxybenzyl alcohol、 p-hydroxybenzaldehyde and parishin,were analyzed using HPLC,Moreover,the relationship of this changes between ethanolic extracts of Gastrodia elata Bl.and in G.frondosa fermentation system were also investigated.The result revealed that 7%(v/v)ethanolic extracts of Gastrodia elata Bl.could facilitate G.frondosa to grow and produce extracellular protein,and the content of two experimental indexes reached the maximum at the 11 th day after the submerged fermentation of G.frondosa.It was found that the content of parishin in ethanolic extracts of Gastrodia elata Bl.decreased after sterilization,while the content of gastrodin increased obviously.Furthermore,the ethanolic extracts of Gastrodia elata Bl.were added into the system of G.frondosa submerged fermentation and then they were incubated for 14 days.Finally,the contents of p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde decreased,nevertheless parishin increased by 161.11%.To study the effect of ethanolic extracts of Gastrodia elata Bl.on the antioxidant activity of mycelium proteins of G.frondosa.The experimental group(7%(v/v)Gastrodia elata Bl were added into the fermentation of G.frondosa)and the control group(not added)for 11 days.The mycelium protein content of the G.frondosa in the experimental group and the control group was 26.76 ± 0.56 g / 100 g and 27.52 ± 0.76 g / 100 g.The mycelium protein of the experimental group was slightly lower than the control group.The antioxidant capacity(DPPH scavenging ability,hydroxyl radical scavenging ability and reducing power)of the experimental group and the control group were lower than the Vc group.The concentration was 1.0 mg/ml,the elimination ratio of DPPH scavenging in the experimental group was 15.51% higher than the control group(P<0.05).The concentration was 0.8 mg/ml,the experimental group and the control group had a certain degree of hydroxyl radical scavenging ability and the experimental group was 23.96% higher than the control group(p <0.05).The concentration was 0.8 mg/ml,the experimental group and the control group had a certain degree reducing power and the experimental group was 12.50% higher than the control group(p<0.05).This indicated that ethanolic extracts of Gastrodia elata Bl.could promote the antioxidant energy of mycelium protein of G.frondosa.Ethanolic extracts of Gastrodia elata Bl.was added into the system of G.frondosa submerged fermentation,screening and identification of differentially inverogenic proteins of G.frondosa mycelium based on 2-DE and MS.The electrophoretic images were compared with each other,and the differential protein was screened.The T-test was performed on the three replicates.The protein with the p-value less than 0.05 was significant.The standard of the difference protein spots was more than 2 or Less than 0.5 was considered a differential protein.There were 68 differentially expressed protein spots in the experimental group compared with the control group,of which 28 protein spots were up-regulated and 40 protein spots were down-regulated.MALDI-TOF / TOF was selected for representative 18 differences.The obtained PMF data and peptide sequence data were searched by Mascot in the NCBInr database and compared with the known protein peptide mass data in the database.13 protein spots were identified,and 9 up-regulated protein spots were identified(8 proteins were successfully identified),and 4 down-regulated protein spots were identified(4 proteins were successfully identified).The up-regulated proteins were Myosin regulatory light chain cdc4,Glucose-regulated protein,ATP synthase subunit delta,Cytochrome b5,Heat shock protein HSS1,Heat shock protein,A0H81-11392 protein and putative peroxiredoxin,The down-regulated proteins were arginase,Phosphomutase-like protein 3,Aldehyde dehydrogenase and Enolase. |
PDF Full Text Request