The Study On Effect Mechanism Of O₂/CO₂ Controlled Atmosphere Pretreatment On Wound Healing Of Fresh Cut Agaricus Bisporus

White mushrooms（Agaricus bisporus）after fresh-cutting usually lead to a series of physiological and biochemical changes,thus shortening the shelf life.The formation of wound healing on the surface of fresh-cut fruits and vegetables can effectively reduce water loss and pathogen invasion which prolong the storage period of fruits and vegetables.The O₂/CO₂ treatment is widely used in the preservation of fruits and vegetables due to its unique advantages.However,there are fewer studies on the preservation effect and wound healing mechanism of fresh-cut white mushrooms after O₂/CO₂ pretreatment.The theoretical mechanism of O₂/CO₂ pretreatment on wound healing regulation of fresh-cut white mushrooms was revealed through combination of physicochemical analysis and proteomics analysis.The main research results and conclusions of this paper are as follows:（1）To analyze the effect of different pretreatment time on the quality of fresh-cut white mushrooms,80%O₂+20%CO₂ was selected as a gas conditioning treatment concentration and the pretreatment time was 0,3,6,9 h,respectively.The effect of different pretreatment time on the preservation quality of fresh-cut white mushrooms were explored.The results showed that O₂/CO₂ pretreatment had obvious effects on the quality of fresh-cut white mushrooms.Among those,3 h treatment could effectively reduce the browning of fresh-cut white mushrooms during storage.At the same time the total antioxidant capacity and total phenols content were improved.The MDA level and the membrane permeability were reduced,which improved the stress resistance.Meanwhile,based on determining the optimal O₂/CO₂ pretreatment time,O₂/CO₂ pretreatment before and after cutting of white mushrooms was carried out respectively.Results showed that O₂/CO₂ pretreatment after cutting could effectively improve the total phenols content and antioxidant capacity,which improved the stress resistance and maintained the color,protein content of fresh-cut white mushrooms.The O₂/CO₂ pretreatment after cutting could not only maintained the quality of fresh-cut white mushrooms,but also promoted the components synthesis of wound healing.（2）In order to study the effect of O₂/CO₂ pretreatment on wound healing of fresh-cut white mushrooms,the wound healing mechanism was explored from the perspective of phenolic accumulation,phenylpropanoid metabolism and water status and distribution.The results indicated that O₂/CO₂ pretreatment after cutting increased the content of total phenols,flavonoids and lignin.The pretreatment after cutting could stimulate the phenylpropanoid pathway,induce the levels of individual phenols.Additionally,with a longer storage time for different fresh-cut white mushrooms,the results of low-field nuclear magnetic resonance（LF-NMR）indicated that the amplitude of free water significantly decreased while the amplitude of immobilized and bound water increased in all samples.The moisture content in fresh-cut white mushrooms mainly depended on the free water content.O₂/CO₂ pretreatment after cutting maintained the level of free water inside while decreased the level of free water on the surface of fresh-cut white mushrooms.In conclusion,O₂/CO₂ pretreatment after cutting effectively enhanced wound healing by promoting the loss of surface water.Moreover,the wound healing on the surface inhibited the water mobility and transformation inside white mushroom slices,thus maintaining the quality of fresh-cut white mushrooms.（3）In order to study the effect of O₂/CO₂pretreatment on wound healing of fresh-cut white mushrooms,the relationship between reactive oxygen species（ROS）metabolism and wound healing mechanism was analyzed.Results showed that O₂/CO₂pretreatment after cutting could induce the production of hydrogen peroxide（H₂O₂）and superoxide anion(O₂^-.)in the early stage,as well as the NADPH oxidase（NOX）activity.Enzymes and endogenous antioxidants involved in ROS scavenging were enhanced by O₂/CO₂pretreatment after cutting during the whole storage.Besides,O₂/CO₂pretreatment after cutting maintained high level of phenylalanine ammonia-lyase（PAL）activity,enhanced the content of total phenols and lignin,accelerated the As A-GSH cycle.Therefore,it could be concluded that O₂/CO₂ pretreatment after cutting induced defense response by increasing ROS content in the early stage.Thereby,it stimulated the activity of ROS scavenging enzyme and enhanced the defense and resistance ability to injury stress,which also proved that ROS mediated the accumulation of total phenol and lignin and other components of wound healing.In conclusion,the ROS could be a second messenger for activating phenylpropanoid metabolism and inducing the accumulation of phenolic compounds and lignin,which was a consequence for promoting wound healing of fresh-cut white mushrooms.（4）In order to study the mechanism of cell wall metabolism on wound healing,the changes of cell wall composition,microstructure and enzyme activities related to cell wall metabolism were further analyzed.The results showed that the O₂/CO₂ pretreatment after cutting could effectively inhibit the degradation of cell wall components such as chitin,protein,polysaccharide,cellulose and increased the attachment of lignin in cell walls.Meanwhile it inhibited the activities of chitinase,β-1,3-glucanase,cellulase andβ-galactose glucoside enzyme.The lower enzyme activities of cell wall decompression reduced the breakdown of cell wall components,maintained the structural integrity of the cell wall,and reduced the occurrence of softening.Therefore,O₂/CO₂pretreatment after cutting could improve the wound healing of white mushrooms,which was related to the accumulation of cell wall components and the attachment of lignin to the cell wall.（5）In order to prove and explore the metabolic pathway related to wound healing of fresh-cut white mushrooms,proteomic analysis was performed on Air and O₂/CO₂pretreatment white mushrooms which were stored for 0 and 4 days.The proteomics results showed that a total of 3276 proteins were identified in the proteomics of three samples.The proteomics annotated with>1.5-fold differential changes were counted which 65proteins were up-regulated and 56 ones were down-regulated in 4d Air vs.0d;51 proteins were up-regulated and 32 ones were down-regulated in 4d O₂/CO₂ vs.0d;54 proteins were up-regulated and 40 ones were down-regulated in 4d O₂/CO₂ vs.4d Air.These differentially expressed proteins involved a total of 30 metabolic pathways.Among them,the phenylpropanoid metabolism,secondary metabolite anabolic pathway,amino acid metabolism were related to cell wall metabolism,sugar metabolism,lipid metabolism,and pentose phosphate pathway（PPP）involving more differential proteins.The results showed that chorismate mutase,prephenate dehydratase and histidinol-phosphate aminotransferase were up-regulated by O₂/CO₂ pretreatment after cutting which indicated that O₂/CO₂pretreatment after cutting could increase the production of phenylalanine,providing sufficient prerequisite substances for the phenylpropanoid metabolism.And it also increased the activity of enzymes in phenylpropanoid metabolism like histidinol-phosphate aminotransferase,ring-1,2-phenylacetyl-Co A epoxidase subunit Paa A,2-（1,2-epoxy-1,2-dihydrophenyl）acetyl-Co A isomerase and catalase-peroxidas,thus promoting the production of wound healing components.In addition,O₂/CO₂ pretreatment after cutting improved their resistance to cutting damage,the synthesis ability of related enzymes and structural proteins,and the synthesis ability of inhibitory compounds by regulating the synthesis and metabolic pathways of amino acids to improve resistance of white mushrooms,for example,the up regulation of the ring-1,2-phenylacetyl-Co A epoxidase subunit Paa A.At the same time,α-galactosidase,α-glucosidase and chitinase were significantly downregulated under the O₂/CO₂ pretreatment after cutting,indicating that it could reduce cell wall hydrolysis by promoting healing and maintaining the morphological structure of the cell wall.It demonstrated that the wound healing of fresh-cut white mushrooms was influenced by cell wall metabolism.Finally,the O₂/CO₂pretreatment after cutting regulated the glycolysis（EMP）and PPP pathways like ribose5-phosphate isomerase A and fructose-bisphosphate aldolase,providing important precursors for the shikimic acid pathway and sufficient energy for the wound healing process.Therefore,the wound healing of fresh-cut white mushrooms might be related to the regulation of phenylpropanoid metabolism by PPP pathway.（6）To verify the relationship between wound healing and phenylpropanoid metabolism regulated by PPP pathway,the effects of O₂/CO₂ pretreatment after cutting on the phenylpropanoid metabolism and NADPH level produced via the PPP pathway in white mushroom slices were analyzed.The results showed that O₂/CO₂ pretreatment after cutting enhanced phenylpropanoid metabolism,which was demonstrated by higher total phenol,flavonoid,lignin content and the expression of enzyme genes related to the phenylpropanoid metabolism.O₂/CO₂ pretreatment after cutting also increased PPP pathway and tricarboxylic acid cycle（TCA）rates.However,it reduced the total respiratory rate and EMP rates,and inhibited the decline in ATP content and improved NAD⁺kinase（NADK）activity.Moreover,compared with Air pretreatment after cutting the NAD⁺and NADH contents decreased,whereas NADP⁺and NADPH contents increased in O₂/CO₂pretreatment.To further investigate the relationship between respiratory metabolism and phenylpropanoid metabolism,whole white mushrooms were immersed in 0.1 mol L^-1Na₃PO₄（a PPP inhibitor）as the experimental group and were immersed in distilled water as a control group,respectively,and then the slices were pretreated with 80%O₂+20%CO₂for 3 h.Meanwhile,another group of whole white mushrooms were immersed in distilled water and treated with air as a blank group.The results showed that the activity of enzymes and metabolites related to phenylpropanoid metabolism of white mushrooms treated with0.1 mol L^-1 Na₃PO₄ were lower than those of the blank group.The above results indicated that O₂/CO₂ pretreatment could effectively induce the phenylpropanoid metabolism by stimulating PPP metabolism and increasing NADPH levels.In conclusion,PPP metabolism played an important role in the phenylpropanoid metabolism of fresh-cut white mushrooms.