Effect Of Legume Bean Trypsin Inhibitor On Fish MBSP

The production of surimi products is one of the important ways for effective utilization of fisheries resources.Studies have shown that myofibril-bound serine proteinase?MBSP?is a key enzyme leading to gel degradation during surimi preparation,while there is still no effective mean to prevent degradation action of MBSP during processing of surimi products.Beans are rich sources of trypsin inhibitors and are generally safe for consuming.Therefore,in this study,the author intended to prepare natural trypsin inhibitors?TIs?from soybean,mung bean and black soybean,and efficiently expressed recombinant crucian MBSP.The in vitro interaction of TIs with MBSP and their effects on the quality of surimi was investigated with a purpose to effectively suppress the modori phenomenon during surimi processing.High-purity soybean trypsin inhibitor?STI?,mung bean trypsin inhibitor?MBTI?and black soybean trypsin inhibitor?BSTI?were obtained by biochemical separation techniques such as sulfuric acid digestion,heat treatment precipitation,pH precipitation,ammonium sulfate salting out and column chromatography.The analysis of properties showed that STI,MBTI and BSTI were stable in the range of pH 2-11;STI and BSTI showed higher inhibitory activity at 60?,while MBTI even had a higher inhibition property at 90?.The effect of metal ions on TIs inhibitory activity showed Fe²⁺could significantly reduce the activity of STI,MBTI and BSTI.Circular dichroism analysis indicated STI contained 40.6%?-sheet,21.7%?-turn,31.3%random coil,7.8%?-helix;MBTI contained?-fold 69.6%,?-turn 8.6%,random coil 22.3%,?-helix2.2%;and BSTI contained?-fold 43.4%,?-turn 21.2%,random coil 29.0%,?-helix 8.1%.By scanning the secondary structure changes of STI,MBTI and BSTI at different temperatures,the denaturation temperature of the three inhibitors were 64±0.1?,91±0.6? and 61±0.9?,respectively.The inhibitory effect and inhibition kinetics of MBSP by STI,MBTI and BSTI were analyzed using the fluorescent substrate Boc-Gln-Arg-Arg-MCA.The results indicated STI,MBTI and BSTI could inhibit MBSP,and the inhibition of MBSP by STI and MBTI was competitive inhibition,while BSTI was non-competitive inhibition.The inhibition constants K_i of the three TIs were 0.32 nmol/mL,0.077 nmol/mL,and 1.42 nmol/mL,respectively.STI,MBTI and BSTI all showed a high affinity for MBSP.Protein interaction analysis showed that the affinity constants K_D of three kinds of bean inhibitors and MBSP were 1.07×10^-77 mol/L,1.257×10^-88 mol/L,and 1.797×10^-77 mol/L,respectively.Myosin heavy chain?MHC?would be strongly degraded by MBSP at 55?.The results of this study indicated that STI,MBTI and BSTI with a final concentration of 1?g/mL could effectively protect MHC from degradation.The effect of STI,MBTI and BSTI on the quality of surimi were further analyzed.It was found that these three legume trypsin inhibitors could improve the gel strength of surimi to some extent.The results of simulated gastrointestinal fluid digestion showed that the STI,MBTI and BSTI had no adverse effect on the digestive properties of the surimi at an addition of 0.1%.In summary,all three legume trypsin inhibitors could inhibit MBSP and suppress the modori phenomenon of surimi caused by MBSP,suggesting the application feasibility of TIs in surimi processing.