Quality Changes And Mechanism Of Crisp Grass Carp(Ctenopharyngodon Idellus C.et V) During Crispness Formation Process

Crisp grass carp(Ctenopharyngodon idellus C.et V)is obtained by feeding faba bean to grass carp to make the muscle be crisp and firm.The price of crisp grass carp is 2 to 3times higher than that of ordinary grass carp,and the muscle textural characteristic such as hardness,springiness,and chewiness have been significantly improved.At present,crisp grass carp is a novel aquaculture species in southwest China,facing problems such as lack of scientific basis,unknown muscle quality formation law,and unclear mechanism during the feeding process.Previous studies have attempted to reveal the flesh quality formation mechanism of crisp grass carp from muscle quality changes,component changes,and protein molecular structure changes but they were not comprehensive enough.Grass carp that has been feeding with faba bean for different days is used as the research object in this study.Firstly,the law of quality change during the culture of grass carp was studied from nutrient composition,texture and flavor.On this basis,the optimal culture cycle was determined.Then,the reasons for muscle quality formation were analyzed by studying factors such as oxidative stress,protein cross-linking,and apoptosis during the culture process of crisp grass carp.Finally,the Tandem mass tag(TMT)quantitative proteomics technology was used to further reveal the crispness formation mechanism of grass carp from the perspective of proteomics.The specific research contents and conclusions are as follows:(1)The quality change law of crisp grass carp during culture(0～150 d)was studied,and the better culture cycle was determined through correlation analysis.The results showed that with the increase of culture days,the nutritional components(protein,water,fat,ash)in the muscle of grass carp did not change significantly.The relative content of saturated fatty acids gradually increased in 0～90 d,while polyunsaturated fatty acids gradually decreased.The hardness,chewiness,springiness,gumminess and resilience of muscle increased significantly.The hardness was stable at 30 days,and the chewiness,gumminess,recovery were stable at 60 days.The water-holding capacity of grass carp muscle gradually enhanced after faba bean feeding and reached a maximum at 90 days.There was a large difference between the odor of crisp grass carp and that of grass carp at the early stage.The content of hexanal,heptanal,nonanal,1-octen-3-ol and other fishy smell substances reached the lowest at 90 days.In summary,the optimal feeding cycle of crisp grass carp is the 90 th day.(2)The reasons for the formation of grass carp flesh quality in the optimal period of culture were studied.The results showed that grass carp had oxidative damage in the early stage of the culture period,and the adaptation to oxidative environment was enhanced and oxidative damage was weakened in the later stage of the culture period.The total antioxidant capacity showed a trend of decreasing and then increasing(the minimum value at 60 d).The content of malondialdehyde(MDA)showed a trend of increasing(0～30 d)and then stabilizing.Oxidative damage did not increase the degree of muscle myofibrillar fragmentation index(MFI)but increased the cross-linking degree of myofibrillar protein within the first 30 days after faba bean feeding,which showed that the content of dityrosine increased significantly(P<0.05).This may be the main reason for the change of flesh quality in the early stage of the culture period(0～30 d).TUNEL staining showed that the degree of apoptosis in grass carp muscles increased on the 30 th day,and the change in the degree of apoptosis was not obvious after that;The m RNA expressions of pro-apoptotic genes CTSB,Bax and caspase-3 were up-regulated on the30 th day,and the m RNA expression of anti-apoptotic gene Bcl-2 was down-regulated;The m RNA expression of pro-apoptotic genes CTSB,Bax and caspase-3 decreased on the 90 th day,and the m RNA expression of anti-apoptotic gene Bcl-2 increased.Collagen content and the degree of cross-linking increased after 30 days.Masson staining showed that collagen appeared around myofibrils and replaced them.Apoptosis led to an increase in the gap between muscle fibers and a decrease in the diameter of muscle fibers,which resulted in the replacement of muscle fibers by collagen.In addition,with the further extension of the faba feeding days,the cross-linking of proteins increased,which in turn changed the textural properties of grass carp muscle and further increased its hardness.(3)The dynamic changes of proteins in grass carp muscle at the critical culture days(0,30 and 90 d)were analyzed by TMT proteomic technology,and the mechanism of crisp grass carp was explained from the molecular point of view.The results showed that 3280 proteins were identified at three groups of grass carp muscle cultured for different days.There were 135 differentially expressed proteins(DEPs)between the muscle of grass carp cultured on the 30 th day(CGC30)and common grass carp(GC).The Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis indicated that a total of 157 pathways were enriched in the CGC30 VS GC group,among which 12 pathways were significantly enriched,involving PPAR signaling pathway(related to fat deposition),glycolytic pathway(related to energy metabolism),and Hippo signaling pathway(related to growth and apoptosis).The down-regulation of the glycolytic pathway and β-oxidation pathway indicated that the energy demand of grass carp decreased in the early stage of faba bean feeding(0～30 d);Oxidation-related proteins SOD2 and UQCRC2 were down-regulated,suggesting that grass carp suffered oxidative damage in the early stage of faba bean feeding;the pro-apoptotic protease CTSB was up-regulated and cytoskeletal proteins were down-regulated on the 30 th day,which proved the occurrence of apoptosis to a certain extent.There were 173 DEPs between the muscle of grass carp cultured for 90days(CGC90)and CGC30.The KEGG pathway analysis indicated that a total of 153 pathways were enriched in CGC90 VS CGC30 group,among which 21 pathways were significantly(P<0.05)enriched.The tricarboxylic acid cycle and β-oxidation pathway were enhanced,indicating that the energy demand of grass carp increased in the later stage of faba bean feeding(30～90 d).The expression of oxidation-related proteins SOD2 and UQCRC2 were up-regulated at 30～90 days,and the expression of pro-apoptotic protease CTSB was down-regulated,indicating that grass carp gradually adapted to the oxidative stimulation of faba bean and the apoptosis was controlled.Compared with GC,the expression of collagen type XII(COL12A)in the muscle of grass carp after crisping was up-regulated,indicating that the change of the hardness of crisp grass carp was related to the increase of collagen.