Preservation Effect And Mechanism Of Direct Immersion Freezing For The Frozen Storage Of Grass Carp(Ctenopharyngodon Idell)

Grass carp(Ctenopharyngodon idellus)is the cash aquatic in China,accounting for 1/5 of the production of freshwater farmed fish,and has a huge consumer market.At present,grass carp processing was mainly focused on frozen primary processing.However,protein denaturation was easy to occur during freezing and frozen storage using traditional air blast freezing,which had a great impact on the quality of grass carp.The liquid nitrogen freezing process could cause the product to crack,and the cost of liquid nitrogen was high and the consumption was also large,so it was not suitable for large-scale grass carp frozen processing.Therefore,the development of new freezing technology is of great significance to the processing of aquatic products.Immersion freezing has a higher freezing rate and can better maintain the quality of products.It has been used in vegetables,fruits,pork and large pelagic fish,but there were few reports on the application of freshwater fish,and its fresh-keeping effect needs to be further studied.Therefore,this study took grass carp as the research object,and used edible freezing medium to evaluate the fresh-keeping effect of direct immersion freezing on grass carp frozen storage.In addition,the mechanism of improving the quality of frozen grass carp by direct immersion freezing was expounded from the aspects of water characteristics and protein denaturation.The biochemical process and mechanism of quality change at the molecular level were discussed by proteomics.The main contents were as follows:(1)The freezing rate of direct immersion freezing was measured,and the effect of direct immersion freezing on the fresh-keeping of frozen grass carp was studied.The quality of frozen grass carp was evaluated by the indicators of surface quality,texture characteristics,microstructure and water retention.The results showed that the freezing rate of immersion freezing was 8.9 times that of air blast freezing,and the phase transition time was 1/30 that of air blast freezing.After 24 weeks of frozen storage,the whiteness of the fish in the immersion freezing group,the liquid nitrogen freezing group and the air blast freezing group increased by 27.57%,91.79%and 64.17%,respectively,compared with the fresh group.The fish surface color in the immersion freezing group was the closest to that in the fresh group,and the surface color of the fish in the liquid nitrogen freezing group deteriorated seriously.At the same time,the microstructure analysis showed that,compared with the air blast freezing,the immersion freezing had less damage to the muscle,which was similar to the liquid nitrogen freezing group.The thawing loss and cooking loss of the immersion freezing group were 8.29%and 6.82%lower than those of the air blast freezing group,and 1.59%and 3.16%lower than those of the liquid nitrogen freezing group,respectively.In summary,the immersion frozen grass carp had acceptable surface quality and good internal quality during frozen storage.(2)The effect of direct immersion freezing on the water characteristics of frozen grass carp muscle was studied.The growth of ice crystals after immersion freezing and during frozen storage was analyzed.The water migration law of grass carp muscle during frozen storage was studied,and the change of unfrozen water content was determined.The results showed that the immersion freezing was similar to the liquid nitrogen freezing in the control group,forming smaller intracellular ice crystals.The increase of equivalent diameter and area of ice crystals in immersion freezing group was smaller than that in air blast freezing group,which indicated that immersion freezing can effectively slow down the growth of ice crystals during frozen storage.The results of LF-NMR showed that after 16 weeks of frozen storage,the T21 and T22 of the immersion freezing group were smaller than those of the control air blast freezing group and the liquid nitrogen freezing group,indicating that the immersion freezing could delay the transformation from immobilized water to free water in grass carp muscle.Before 12 weeks,there was no significant difference in the unfrozen water content between the immersion freezing group and the control liquid nitrogen freezing group,and both were greater than the air blast freezing group.After 12 weeks of frozen storage,the unfrozen water content in the grass carp muscle of the immersion freezing group was significantly higher than that of the liquid nitrogen freezing group and the air blast freezing group,indicating that the immersion freezing can effectively maintain the unfrozen water content in the grass carp muscle.On the whole,immersion freezing can slow down the water migration and loss of grass carp during frozen storage,reduce the loss of unfrozen water,and effectively slow down the quality deterioration during frozen storage.(3)The effect of direct immersion freezing on the protein properties of frozen grass carp was studied.The changes of AG enzyme,NAG enzyme and cathepsin activities during frozen storage were measured.The degradation degree of myofibrillar protein(MFI,TCA-dissolved peptide,SDS-PAGE)and oxidation degree(carbonyl group,dimeryl tyrosine,sulfhydryl group,active sulfhydryl group and disulfide bond)were analyzed.The results showed that the activities of AG enzyme and NAG enzyme in the immersion freezing group were significantly lower than those in the air blast freezing group,indicating that the immersion freezing had better tissue integrity than the air blast freezing.The activities of cathepsin B and B+L were inhibited by low temperature in the first 8 weeks,but the activitied of cathepsin B and B+L were no longer affected by low temperature after 8 weeks.At 24 weeks of frozen storage,the activities of cathepsin B,cathepsin B+L and cathepsin D had no significant difference between the immersion freezing group and the liquid nitrogen freezing group,and were significantly lower than those of the air blast freezing group.These results indicated that immersion freezing could effectively inhibit the activity of cathepsin.In addition,with the extension of frozen storage time,the contents of MFI and TCA-dissolved peptide gradually increased,indicating that the muscle proteins of grass carp were degraded to different degrees.Compared with the air blast freezing,the protein degradation degree of the fish in the immersion freezing group was less severe.The contents of carbonyl group,dimeryl tyrosine and disulfide bond increased gradually,while the contents of sulfhydryl group decreased gradually,and the oxidation protection of grass carp myofibrillar protein in the immersion freezing group was more significant.Therefore,immersion freezing slowed down the quality change of frozen grass carp by maintaining the integrity of muscle cells,reducing cathepsin activity and slowing down the degree of protein degradation and oxidation.(4)The changes of protein structure during direct immersion freezing of grass carp were studied.The protein composition,protein structure,chemical force,particle size and Ca2+ATPase activity of grass carp muscle during frozen storage were determined.The results showed that with the extension of frozen storage time,the content of water-soluble protein increased and the content of myofibrillar protein decreased.At 24 weeks of frozen storage,the content of myofibrillar protein in the immersion freezing group was significantly higher than that in the control air blast freezing group and liquid nitrogen freezing group.In addition,theα-helix,β-sheet,fluorescence intensity,chemical bond content and Ca2+-ATPase activity in immersion freezing group and liquid nitrogen group were significantly higher than those in the air blast freezing group,which indicated that it was more beneficial to maintain the stability of protein structure.In general,compared with air blast freezing,the stability of protein structure was stronger and the degree of protein denaturation was lower,which was more conducive to maintaining the quality of frozen products.(5)TMT-labeled proteomics technology was used to study the changes of muscle proteomics in directly impregnated frozen grass carp during frozen storage,and to explore the biochemical processes and mechanisms that cause quality changes at the molecular level.The results showed that there were 156 differential abundance proteins in the muscle protein of grass carp after 12 and 24 weeks of frozen storage compared with the fresh group.These differential abundance proteins were mainly involved in biological processes(BP)and cell composition(CC).The overall functional enrichment of differential abundance proteins showed that the biochemical processes related to mitochondria in grass carp muscle cells were significantly affected with the extension of frozen storage time.KEGG pathway analysis showed that differential abundance proteins were mainly involved in glycolysis/gluconeogenesis,HIF-1 signal pathway,P13K-Akt signal pathway and citric acid cycle during frozen storage.The correlation analysis between differential abundance proteins and quality indexes showed that 5,5,1 and 5 proteins were closely related to whiteness,shear force,hardness and thawing loss,respectively.In the present study,it was clarified that direct immersion freezing could effectively slow down the quality deterioration of frozen grass carp.The mechanism of improving the quality of immersion freezing was elucidated from the aspects of microstructure,water characteristics and protein characteristics.The biological process and mechanism of quality change were discussed at the molecular level by proteomics technology,and 7 differential indicator proteins related to quality change were screened out.This study comprehensively revealed the effect and mechanism of immersion freezing on the quality of frozen grass carp,provided a theoretical basis and reference for the frozen preservation of freshwater fish,and had important practical significance for promoting the quality improvement and industrial development of freshwater fish processing products.