Preparation Of Thymol-Embedded-Polysaccharides Complex Coating Agent And Its Inhibitory Effect And Mechanism On Aspergillus Niger Of Postharvest Blueberry

Blueberries are a uniquely flavored,nutrient-rich,high-value small fruit that are extremely prone to spoilage and deterioration after harvest,and existing technologies cannot keep up with the strong demand for high-quality fresh fruit in the market.A.niger on blueberries after harvest has long caused huge post-harvest losses.Previous research has demonstrated that thymol,a plant essential oil component with broad-spectrum antifungal properties,can effectively inhibit the growth and reproduction of A.niger.However,the unstable nature and strong volatility of thymol severely limit its application in the fresh food logistics field.The study developed a stable and well-encapsulated thymol-chitosan complex coating agent（TKL）and verified its efficacy through low-temperature storage experiments.The study further investigated the effect of TKL on the physiological and biochemical characteristics of A.niger and explored its antibacterial mechanism through bioinformatics tools.The findings provide a new approach for the development of a new green preservation technology for blueberries and offer important theoretical and technical support for the development of natural antifungal post-harvest treatment technology.The main results are listed as below.（1）By optimizing the key process parameters in the preparation of TKL,the process for the preparation of thymol beta-cyclodextrin sustained-release microcapsules（TMs）,the proportion of KGM/LAG in the film-forming matrix（KL）,and the optimum amount of TMs in KL were determined,and then the optimum TKL was structurally characterized.The results showed that under the best process conditions,the encapsulation efficiency of TMs was 91.40±0.74%,and the actual drug loading was 26.10±0.49 mg/g.The TMs had the advantages of small particle size,good thermal stability,good water-based wetting property and high odor threshold and exhibited sustained release effect and broad-spectrum antifungal activity.The total polysaccharide content was 0.075%（w/v）,and the KL with a KGM/LAG ratio of 1:2 could effectively wet the blueberry epidermal wax and spontaneously form a gas-adapted package after air-drying film formation,with excellent barrier properties.The best comprehensive antibacterial TKL formula（TKL60）was 0.22%TMs+0.025%KGM+0.05%LAG（w/v）,and the film-forming agent had good interaction among its components,with the newly formed polymer being able to combine well with the blueberry epidermal wax,filling the original wax defect area,forming a white film layer of about0.39±0.15μm,and significantly improving the appearance quality of the fruit.Therefore,TKL60 is a promising post-harvest treatment technology for blueberries.（2）CK and KL were used as controls,and the effect of TKL60 on the storage quality,fruit peel physicochemical properties,and epicuticular wax of blueberries during cold storage at 2℃was evaluated to explore the application effect of TKL60 in postharvest preservation of blueberries.The results showed that,compared with the control group,TKL60 significantly reduced the weight loss rate,decay rate,and total number of fungal colonies on the fruit surface,inhibited fruit respiration and ethylene release,delayed fruit softening process,and decreased the degradation rate of titratable acid（TA）,L（+）-ascorbic acid（L-VC）,and anthocyanins,improved the sensory quality of the fruit after long-term refrigeration,thereby extending the shelf life of blueberries to 49 d.In addition,TKL60could maintain the activity of antioxidant enzymes in the fruit peel tissue,inhibit the accumulation of reactive oxygen species（ROS）,alleviate the degree of membrane lipid peroxidation in the fruit peel,and maintain the integrity of the fruit peel cell structure.Meanwhile,TKL60 significantly increased the total wax content of blueberries during cold storage,which could help to protect the crystal structure of epicuticular wax,delay the conversion of wax components,and improve the activity of key enzymes involved in the synthesis pathway of epicuticular wax in the peel of blueberries during the mid and late stages of storage.Correlation analysis showed that the total number of fungal colonies on the fruit surface had the most significant influence on the physicochemical properties of blueberries,followed by the total wax content.In conclusion,TKL60 is a good coating agent for post-harvest preservation of blueberries,and its antifungal activity is a key factor in extending the shelf life of blueberries.（3）The relationship between the release of THY by TMs in TKL and the bacteriostatic effect was analyzed by in vitro antibacterial experiments,and the effect of TKL60 on the growth of A.niger mycelium was investigated.The results indicated that KL had no significant effect on the growth of A.niger,and there was a positive correlation between the THY released by TMs in TKL and the antifungal effect.The half-maximal effective concentration(EC₅₀^TKL)of THY on A.niger in the TKL-encapsulated state was 99.49±2.80mg/L,and the toxic dose of TKL60 on the target fungi was about 3/5 EC₅₀^TKL.Further testing of physiological and biochemical indicators of A.niger mycelium revealed that although TKL60 did not cause significant damage to cell membranes,it significantly interfered with the normal physiological and metabolic activities of A.niger,leading to an increase in the number and size of mitochondria and lipid droplets,the formation of a large number of autophagic vacuoles,a significant increase in total lipid content,and a significant decrease in soluble protein and ATP content.The ROS level and mitochondrial membrane potential of the TKL60 group decreased by 8.83%and 17.11%,respectively,compared to CK,and the enzyme activities of citrate synthase（CS）,isocitrate dehydrogenase（ICDH）,ATP synthase（ATPase）,hexokinase（HK）,6-phosphofructokinase（PFK）,and pyruvate kinase（PK）decreased by more than 10%.The only exception was alpha-ketoglutarate dehydrogenase（α-KGDH）,whose enzyme activity increased by 31.75%.Overall,TKL60 has some antifungal activity against A.niger,which may be related to its induction of metabolic derangements and changes in energy status.（4）A non-targeted metabolomics approach was performed to study the metabolic characteristics of A.niger after TKL60 treatment for 48 h.A total of 204 significantly different metabolites（DMs）were identified,of which 70 were up-regulated and 134 down-regulated.Organic acids and their derivatives,lipids and lipid-like molecules showed the most significant changes in expression levels.Based on the variations of key secondary metabolites in the TKL60 group,the expression levels of 1,2-dioleoyl-sn-glycero-3-phosphoglycerol,glutamine,and DI-malic acid were up-regulated 75.87-fold,1.70-fold,and1.96-fold,respectively,while the levels of acetyl-Co A,acetate,and NAD+were decreased to 31.07%,39.37%,and 30.27%of normal levels.KEGG pathway enrichment analysis revealed that TKL60 significantly affected amino sugar and nucleotide sugar metabolism,pantothenate and Co A biosynthesis,pyruvate and dicarboxylate metabolism,and D-glutamine and D-glutamate metabolism in the fungus.Therefore,the changes and flow of metabolites in A.niger after TKL60 treatment were closely related to lipid,amino acid,and carbohydrate metabolism.TKL60 may inhibit the growth of A.niger mycelium by interfering with lipid and NAD+-related core metabolic pathways.（5）TMT proteomics technology was used to investigate the protein expression characteristics of A.niger after TKL60 treatment for 48 h.743 differentially expressed proteins（DEPs）were identified,of which 338 were up-regulated and 405 were down-regulated.Subcellular localization showed that 86.54%of the DEPs were concentrated in the cytoplasm,nucleus,and mitochondria.GO functional analysis revealed 489,642 and 681DEPs involved in biological processes,molecular functions and cellular components,respectively.Among these proteins,the highest frequency of antioxidant-related GO classification was found in A0A117DWC4 protein of peroxidase oxidoreductases（kat G）,A0A117DVZ9 and A0A100I1U7 protein of catalase（CAT）.In the TKL60 group,A0A117DWC4 expression was 1.24-fold higher than that of CK,while A0A117DVZ9 and A0A100I1U7 protein expression levels were downregulated to 78.03%and 71.82%,respectively,compared to the original level.KEGG pathway analysis revealed that the enrichment of glyoxylate and dicarboxylate pathways showed the highest significance,and overall showed a significant downregulation trend.Compared with CK,the expression levels of A2R4H2 protein of formaldehyde dehydrogenase（FDHI）,A2QJ30 protein of acetyl-Co A synthetase（ACS）,A0A117E0R2 and A2R4W7 proteins of malate synthase（MLS）,and A0A100I3Y5 protein of isocitrate lyase（IL）were significantly downregulated in the TKL60group.In summary,TKL60 may inhibit the growth of A.niger by impairing its antioxidant defense function,leading to a decrease in NAD+levels in the cell and disrupting carbohydrate metabolism.（6）Using metabolomics and proteomics correlation analysis,this study investigates the potential drug targets and antifungal mechanism of TKL60 against A.niger.The results showed that DEPs and DMs were associated with 69 KEGG pathways.TKL60 interfered with lipid metabolism and antioxidant defense system-related pathways,leading to a significant down-regulation of carbohydrate and energy metabolism pathways.By screening important KEGG pathways and focusing on interrelated DMs and DEPs,the results suggested that the A0A124BXR8 protein of 3-oxoacyl-[acyl-carrier-protein]reductase（Fab G）and the A0A117DWC4 protein of kat G enzyme could be the drug targets of TKL60to inhibit A.niger hyphal growth.Validated results showed that,compared to CK,treatment with TKL60 led to a 13.79-fold and 6.62-fold increase in activity of Kat G and Fab G enzymes of A.niger respectively.Relative expression levels of both the kat G and At WU＿09702 genes in TKL60 group were significantly upregulated by more than 12-fold,while the ratio of NADH/NAD+and the level of palmitic acid increased by 4.29-fold and 134.29%,respectively.But the H₂O₂ content in A.niger treated with TKL60 decreased to 32.06%of the normal state.In summary,the mechanism of TKL60’s inhibitory effect on A.niger may be that the coated film releases THY at a dose lower than EC₅₀^TKL,which binds to A0A124BXR8 and A0A117DWC4 proteins to promote the overexpression of kat G and Fab G,thereby stimulating the cellular antitoxin response.Therefore,A.niger mycelial cells produced large amounts of TAG to form LDs to phagocytose THY,while significantly increasing the intracellular ROS clearance rate,causing a large amount of NAD+depletion,resulting in disruption of cellular carbon metabolism and imbalance of energy homeostasis,and limiting the growth of A.niger mycelium.