| Sea cucumber is a kind of aquatic product with high economic value in China.In 2018,its total Chinese output was over 174 thousand tones,and its industrial output value was over RMB 40 billions Yuan by estimated.Sea cucumber has a variety of processed products,among which ready-to-eat and fresh sea cucumber can reduce the loss of a large number of nutrients by avoiding the drying process,and it is convenient to eat and has a good taste.Among many indexes to evaluate the quality of ready-to-eat sea cucumber,tenderness is more important.More than 70% of the protein in the body wall of sea cucumber is collagen,and in natural state,collagen cross-linked and aggregated into collagen fiber network structure with strong mechanical properties,so the tenderness of its processed products is difficult to control.Lowtemperature tenderizing technique can improve palatability of the body wall of sea cucumber.However,it is still unclear which endogenous enzymes are involved in the low-temperature tenderizing process of the body wall of sea cucumber,how these endogenous enzymes are activated and which biomolecular signaling mechanisms are involved.Therefore,this paper focused on explaining the above problems and revealed the mechanism of endogenous enzymes involved in regulating the tenderizing process of sea cucumber(Apostichopus japonicus,A.japonicus),as well as the molecular biological mechanism of the activation of endogenous enzymes during the tenderizing process,so as to provide theoretical basis for the quality control of the tenderizing process of A.japonicus.(1)Texture profile analysis,rheological analysis,differential scanning calorimetry(DSC),light mirror-histochemistry,scanning electron microscope(SEM)and electron paramagnetic resonance spectroscopy(EPR)were used to characterize the physicochemical properties of the A.japonicus body wall(AJBW)during low-temperature tenderization.The changes of the first structure of the protein were characterized by the determination of the contents of carbonyl group,sulfhydryl group and amino group,protein distribution,and the dissolution of heatsoluble collagen.The changes of the secondary and tertiary structure of the protein were characterized by the determination of circular dichroism(CD)and surface hydrophobicity.The results showed that the hardness,chewiness and springness of the AJBW decreased with the time of tenderizing.The thermal stability of collagen decreased,and collagen fibrils dissociated in microstructure.In the study of water-soluble proteins,the amino acid side chain modification of proteins,the unstable transformation of the second and third structure of proteins,and the large amount of free radicals were found,indicating the occurrence of protein oxidation.At the same time,free amino release and structural protein degradation were found,indicating the occurrence of protein degradation.It was revealed that the essence of tenderizing in AJBW was the damage of collagen fiber structure,accompanied by protein oxidation and protein degradation,which laid a foundation for the follow-up research on tenderizing mechanism.(2)The endogenous enzyme activity in AJBW during tenderization was determined by spectrophotometry.Then on the one hand the AJBW was treated with exogenous collagenase,on the other hand the AJBW was treated with protease inhibitor,respectively.The degradation of protein was illustrated by measuring the changes of TCA soluble oligopeptide,hydroxyproline content and protein pattern.The results showed that the activities of cathepsin L,caspase-3 and MMPs were improved in AJBW during tenderization.The AJBW could be degraded by the collagenase,and the protein pattern was consistent with that of protein after tenderization by low-temperature treatment.The inhibitors of cysteine protease,metalloproteinase and serine protease could inhibit the degradation of small molecular proteins to different degrees,and the latter two had obvious effects on the degradation of large molecular proteins.It was confirmed from three different angles that endogenous enzymes,mainly including of cysteine proteinases,metalloproteinases and serine proteinases,were involved in protein degradation during tenderization.(3)The proteomics technique of iTRAQ(isobaric tags for relative and absolute quantitation)was used to study the expression of differential abundant proteins(DAPs)in the AJBW during tenderization by low-temperature.Then the biological function and signal pathway annotation of DAPs were performed by bioinformatics analysis.The results showed that the protein expressions of cathespins,calpains,caspases,proteasomes and MMPs were significantly up-regulated.Bioinformatics analysis proved that the tenderization process mainly involves oxidative stress,innate immune response,apoptosis,cytoskeleton protein and extracellular matrix(ECM)protein reorganization and so on.Moreover,the biological processes interacted with each other.The molecular mechanism of endogenous enzyme-mediated tenderization was revealed from protein expression level.(4)The transcriptional sequencing(RNA-seq)technology was used to study the expression of differentially expressed genes(DEGs)in in the AJBW during tenderization by lowtemperature.Then biological function and signal pathway annotation of DEGs were performed by bioinformatics analysis.Quantitative real-time fluorescence reverse transcriptional polymerase chain reaction(RT-qPCR)technology was used to verify the accuracy of RNA-seq by detecting changes in the expression levels of characteristic genes.The results showed that the changes in the expression levels of the 10 genes analyzed by RT-qPCR were similar to the changes in the RNA-seq results,which verified the repeatability and reliability of the RNA-seq results.The expression levels of MMP 2 and MMP 16 by RNA-seq were significantly upregulated.Bioinformatics analysis has proved that the tenderization process mainly includes oxidative stress,innate immune response,apoptosis,cytoskeleton protein and ECM protein reorgnization and so on.The biological processes interacted with each other.The molecular mechanism of endogenous enzyme-mediated tenderization was revealed from gene expression level.(5)The A.japonicus collagen fiber was taken as the model to conduct oxidation,thermal treatment and the combined action of the two.The dissolution and degradation of protein and glycosaminoglycan(GAG)in the dissolved products were studied by colorimetry,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and high performance liquid chromatography(HPLC).Then electron paramagnetic resonance(EPR),Fourier transform infrared spectroscopy(FTIR),DSC and SEM were used to characterize the structural changes of collagen fibers.Finally,the model group of thermal treatment was treated with anti-oxidation by adding epigallocatechin gallate(EGCG),and the participation of oxidation in the thermal treatment process was judged after the above test and analysis.The results showed that oxidation and thermal treatment could lead to the dissolution of protein and glycosaminoglycan,and oxidation could more easily promote the dissolution of small molecule protein and degradation of proteoglycan.Oxidation and thermal treatment could both lead to the oxidation of collagen,promote the disaggregation of collagen fibrils,the conversion from ?-helix to ?-folding,the reduction of thermal denaturation temperature,and the reduction of the characteristic decomposition temperature of peptide bond fracture.When the EGCG addition amount reached 125 ?g/mL,it could significantly and effectively inhibit the structural changes of proteoglycan and collagen caused by thermal treatment,and improve the stability of collagen fibers.It was revealed that oxidation played an important role during tenderization.Thermal treatment could produce numerous free radicals,trigger protein oxidation,and directly lead to the destruction of collagen fiber structure,which was shown as the improvement of tenderness.To sum up,low-temperature tenderization in AJBW was a complicated biological process,involve ng the participation of endogenous enzymes cathespins,calpains,caspases,proteasomes and MMPs,as well as oxidative stress,innate immune response,cell apoptosis,cytoskeletal and ECM protein reorgnization.Oxidation was not only crucial to the activation of biological signals,but also can directly damage the structure of collagen fibers. |
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