Changes Of Nanocomposite Packaging Flammulina Filiformis On The Postharvest Mitochondria Energy Metabolism

Fresh Flammulina filiformis（F.filiformis）has high water content,and it has strong respiration and energy metabolism after harvest,which is prone to quality deterioration.In order to solve this problem and extend the shelf life of F.filiformis,the laboratory prepared a nano packaging bag in the early stage,which can effectively inhibit the deterioration of the quality of F.filiformis.This study firstly compared the discontinuous density gradient centrifugation method（DDGCM）,yeast mitochondrial extraction kit method（YMEKM）and improved Pulilai kit method（IPKM）to determine the optimal extraction method for F.filiformis mitochondria.F.filiformis were packaged in nano packaging material（Nano-PM）and normal packaging material（Normal-PM）.The postharvest oxidative damage,energy level and energy metabolism pathway indicators of F.filiformis mitochondria were measured.And mitochondrial proteomics analysis was used to screen the differentially expressed proteins of mitochondria,clarifing the law of energy changes in mitochondria and the regulation pathways of differential proteins related to energy metabolism.The analysis reveals the molecular mechanism of Nano-PM on the preservation of F.filiformis from the perspective of mitochondrial energy metabolism.More information of each research chapter was shown as follow:1.Mitochondrial membrane integrity and functional activity indicators were measured.The alcohol dehydrogenase activity in mitochondria and the cytochrome C oxidase activity in cytosol were lowest and the number of complete mitochondria was most in samples which were extracted by IPKM,demonstrating the IPKM obtained mitochondria had the best membrane integrity.The mitochondrial superoxide dismutase activity extracted by IPKM was the highest,which was 7.31%and 25.59%significantly higher than that extracted by DDGCM-1 and the YMEKM,respectively.And their mitochondrial respiration rates were highest.Janus green B staining test had the largest number of active mitochondria,demonstrating the IPKM obtained mitochondria had the best functional activity.2.The related indicators of postharvest aging and oxidation damage were observed.On the 21st day of storage,Nano-PM effectively reduced the condensation of water vapor,inhibited the elongation,umbrella opening,and browning of F.filiformis,and the content of hydrogen peroxide and superoxide anion were 22.86%and 25.00%significantly lower than Normal-PM at the 18th days,respectively,and maintaining antioxidant enzyme activity.The energy level was assessed,it was showed that Nano-PM can also delay the decrease of F.filiformis mitochondrial membrane potential during storage,thereby inhibited the decline of ATP levels and maintained a high level of energy charge value.The ATP content of Nano-PM groups were still 2.10μg·g^-1FW significantly higher than that of Normal-PM.Through the measurement of energy metabolism-related indicators,it was found that Nano-PM can inhibit respiratory metabolism by inhibiting the activity of enzymes related to the tricarboxylic acid cycle,thereby inhibiting the senescence of F.filiformis caused by excessive respiration rate.And Nano-PM can maintain the enzyme activity of the electron transport chain complex and the expression level of its coded genes,maintain oxidation phosphorylation and ATP levels.And delay cell senescence caused by damage to the inner mitochondrial membrane.3.The biological information analysis was combined with mitochondrial proteomics,different packaging F.filiformis had significant differences proteins of the mitochondria after harvesting.Compared with Normal-PM,a total of 122 and 134significantly different proteins were detected on the 9th and 21st days of storage in Nano-PM F.filiformis.The energy metabolism-related biological processes of Nano-PM and Normal-PM differentially expressed protein enrichment during storage included carbohydrate catabolism,cellular aldehyde metabolism,glutamate catabolism,and glutamate biosynthesis.During the process,the corresponding molecular functions included aldehyde dehydrogenase（NAD）activity,aldehyde dehydrogenase（NAD（P）+）and glutamate dehydrogenase（NADP+）activities.The main energy metabolism pathways of differentially expressed proteins of F.filiformis in different packages during storage in the mitochondria included amino acid metabolism pathway,tricarboxylic acid cycle pathway and pyruvate metabolism pathway.Nano-PM maybe can inhibit enzymes related to energy metabolism pathways,inhibit ATP consumption and respiratory metabolism,thereby maintaining the energy supply of F.filiformis during storage,and can maintain a high level of NADPH by maintaining the pentose phosphate pathway to provide sufficient reducing power for cells,thereby delaying browning and aging.