Optimization Of Chitosan-polyphenol Composite Coating Preservation Technology And Its Effect On The Stability Of Flesh Protein Components

Freshwater fish is a daily ingredient for consumers and an important supplier of high quality intaking protein.In recent years,the consumption of pre-prepared dishes has become increasingly popular,contributing to a shift in the traditional mode of selling fresh fish towards diversification and convenience.However,fish is highly susceptible to spoilage by internal and external factors,and its preservation has always been a challenge in practical sales.Numerous studies have shown that chitosan coating can inhibit deterioration of fish quality and effectively extend the shelf life.However,there are still problems in the existing studies,such as the lack of systematic preparation of chitosan coating solution,the freshness preservation effect still needs to be improved and the mechanism of action is unclear.Therefore,this paper took grass carp（Ctenopharyngodon idellus）as the research object,analysed the influence of two factors,chitosan molecular weight and acid dissolution,to optimise the chitosan coating solution,screened the suitable chitosan and polyphenol composite coating system to further improve the preservation effect,and studied the effect of chitosan-based coating on fish quality maintenance with protein change,so as to lay a theoretical foundation for the improvement and practical application of chitosan-based coating preservation technology for aquatic products.Firstly,the effects of different H⁺soluble systems（acetic acid,propionic acid,lactic acid and mixed acid systems）and molecular weights（50,200 and 500 k Da）of chitosan coating films on the freshness of refrigerated fillets were analysed by comparing the sensory quality,microbiological and physicochemical parameters of the samples in different groups during storage.The results of inhibition circles and microbial counts showed that the inhibition effect of chitosan coating films under different H⁺environments varied significantly,with the acetic-propionic acid coating group being the most effective,with a reduction of 1.17 log₁₀cfu/g in Pseudomonas counts on day 6 of storage compared to the control group;meanwhile,the 200 k Da coated fillets maintained the best sensory quality during storage and extended the shelf life 3 days compared to the control group.At the end of storage,the shear force of the fillets increased by 126.7%,the volatile basic nitrogen（TVB-N）decreased by nearly 20%,and the juice loss rate,total viable counts（TVC）and putrescine content were better than other groups.In conclusion,200 k Da molecular weight chitosan dissolved in 1%acetic-propionic acid system is recommended as the preferred coating base for fresh fillets preservation.On this basis,the freshness of fillets was further improved by compounding polyphenol extracts（grape seed,pomegranate peel and green tea）with chitosan coating.The results showed that after 12 days of refrigeration,the quality of fillets was better in coated groups compared to the control group,with a decrease of 38.9%～70.3%and 10%～16.5%in TVB-N and K value,respectively,and an increase of 75.6%～250.2%in shear force value.High-throughput sequencing results showed that Pseudomonas and Aeromonas were specific spoilage organisms（SSOs）for refrigerated grass carp,with the relative abundance of both reaching 79.5%and 10.3%,respectively,in the control fillets after 12 days of refrigeration.In contrast,the relative abundance of SSOs in the fillets of the coated group was significantly lower than that of control group,indicated the coating had an inhibitory effect on the growth of SSOs.In addition,the combination of chitosan and polyphenol extracts synergistically improved the overall effect on the preservation of fillets.After 12 days of refrigerated storage,the thiobarbituric acid-reactive substances（TBARS）,TVC and juice loss rate of the samples in the composite coated group decreased by 10%～34.5%,5.3%～13.9%and 12.7%～34.7%,respectively,compared with those in the single chitosan group.The chitosan-green tea extract（CH-GTE）coated fish fillets showed the slowest increase in TVB-N,TBARS,TVC and K value during storage and the smallest decrease in shear,indicated that this coating was more effective than other groups in preserving the freshness of fish fillets.Finally,the effects of CH-GTE coating on the stability of protein fractions of refrigerated fillets were investigated by using protein chemistry,proteomics and endogenous enzyme activity evaluation.The results showed that the CH-GTE coating film inhibited protein degradation and endogenous enzyme activation in the refrigerated fillets,and the whole protein electrophoresis bands of the fillets in the CH-GTE group changed less than those in the control group during storage.The maximum activation values of Cathepsin B,L and calpain were only 60.4%～71.4%of those in control.Label-free proteomics analysis showed that after 12 days,the number of differential proteins between the fresh fillets and control group was 1.21 times higher than their corresponding values with coated group;combined with PCA and hierarchical clustering analysis,the protein composition of the fillets with coated had higher similarity to the fresh sample.In addition,46 identified differential functional proteins（tissue structure,energy metabolism and protein turnover）were associated with changes of quality of refrigerated fillets during storage.Comprehensive analysis showed that the chitosan-based coating could prevent the deterioration of quality especially the softening of flesh texture by inhibiting the degradation of structural proteins such as myosin and troponin and delaying the decrease in protein phosphorylation.