Study Of The Effect Of Cathepsins From Blue Scad(Decapterus Maruadsi) And Kininogen On Gel Strength Of Surimi

During the manufacturing process of surimi, the phenomenon of modori is an importantfactor that affects the final quality of the products. According to current studies, endogenouscathepsins in the lysosome, especially cathepsin L play significant role in the degradation ofmyofibillar proteins which led to serious phenomenon of modori. Thus, addition of cysteineproteinase inhibitors is likely to increase the elasticity and improve the quality of surimi.In the present study, cathespin L and B were purificated to homogeneity from the muscle ofBlue scad (Decapterus maruadsi) by ammonium sulfate fractionation and columnchromatographies. Experimental results showed that, the yield of cathepsin L and B was1.5%and3%, and the purification folds was1638.0and2619.3respectively. The result of SDS-PAGEindicated that the molecular mass of cathepsin L was30kDa and that of cathepsin B was27kDa。Peptide mass fingerprinting revealed that the enzyme from Blue scad (Decapterusmaruadsi) had a high similarity (44%) in108amino acid sequences compared with cathepsin Lfrom lates calcarifer. The enzyme activity of Cathepsin L was effectively inhibited by E-64ofcystatin proteinases inhibitor, but was not obvious affected by other proteinase inhibitors.Cathepsin L and B both only hydrolysed the substrates for cathepsins. The main differencesbetween these two enzymes were that cathepsin L only hydrolyses the substrate ofZ-Phe-Arg-MCA while cathepsin B not only hydrolyses Z-Phe-Arg-MCA but alsoZ-Arg-Arg-MCA. Using Z-Phe-Arg-MCA as substrate, the kinetic constants of cathepsin L fromBlue scad (Decapterus maruadsi) were determined with Km2.77μmol/L and Vmax0.04μmol/Land kcat22.76S-1.On the other hand, in this study, two types of cysteine proteinase inhibitor (kininogenⅠandⅡ) were purified to homogeneity from pig plasma by ammonium sulfate fractionation andcolumn chromatographies. Experimental results showed that, the yield of kininogenⅠandⅡwas0.7%and24.1%respectively, and the purification folds was613.7and295.7respectively. Theresult of SDS-PAGE indicated that the molecular mass of kininogenⅠwas58kDa and kininogenⅡ was116kDa. The kininogenⅠand Ⅱ had similar characteristics of inhibiting effect to papain,and the IC50of kininogenⅠand Ⅱ was3μg/mL and3.5μg/mL, respectively. Purifiedkininogens revealed inhibitory effect on the degradation of myofibillar proteins of freshwaterfish silver carp, especially the myosin heavy chain. However, the inhibition effect varies with season for the degradation of myofibillar proteins of marine fish.In the preparation of fish surimi, when the addition of kininogen Ⅱ reached0.03%of theminced fish meat, a24%increase of gel strength was detected. Thus, the gel strength of fishsurimi can be enhanced significantly by addition of kininogens.