Inhibitory Effect And Mechanism Of Protein Stabilizers For Thermal Denaturation And Aggregation Of Tilapia Myosin

In order to better understand and control aquatic protein thermal aggregation,improve the thermal stability of aquatic protein,myosin were extracted from tilapia meat and the heated treatment of myosin in water bath was also tested in this study.Under different p Hs,ionic strengths,heating temperature and time conditions,changes in the solubility and conformation of tilapia myosin subjected to thermal treatment were detected at protein concentration of 2.0 mg/mL.Meanwhile,the inhibition effect on the heat aggregation of myosin was also investigated by adding protein stabilizers.Moreover,the inhibitory mechanism of SDS and arginine on the thermal denaturation and aggregation of tilapia myosin was analyzed.The research results provided a theoretical basis for improving the thermal stability of aquatic protein and the rational development of aquatic protein resources.The main results are as follows:1.Effect of pH values(5.5-8.0)and salt concentration(1-600 mmol/L KC l)on the thermal stability of tilapia myosin were obvious.Under low salt concentration or ac idic pH conditions,the thermal stability of myosin was poor,but well at high salt concentration or alkaline.In the process of heat treatment(40-90?,1?/min),myosin molecules was unfolded and its ANS-S0 increased(p<0.05)at acidic pHs.Especially from the 50 to 70?,solution turbidity increased significantly and aggregates size increased(p<0.05),while the solubility,?-helix content and total sulfhydryl content of myosin significantly decreased(p<0.05).Moreover,electrophoretic analysis revealed significant degradation of myosin molecules.At neutral p Hs,the solubility of myosin decreased significantly(p<0.05)after heat treatment,and secondary or tertiary structure of molecule had obvious changed,but under acidic conditions,the conformation of myosin was less changed.The heat treatment had little effect on the turbidity and solubility of myosin at p H 8.0,the particle size of aggregates significantly had no significant change(p>0.05),the myosin heavy chain was linked to the formation of the polymer,and the total sulfhydryl content decreased;In addition,in low ionic strengths(1-150 mmol/L KC l),the myosin was filaments,so the turbidity was higher and solubility was lower.After heating treatment,the turbidity of myosin system increased,the solubility and ?-helix content decreased significantly(p<0.05).At higher ionic strengths(300-600 mmol/L KC l),the protein present the monomeric state,molecular structure of myosin was stretched,so the solubility of myosin was higher and the ANS-S0 and ?-helix content were increased gradually.C hanges in the solubility and conformation of myosin were also obvious after heat treatment,but compared with the low ionic strength,the changes were less.2.Effect of heating preservation(30-80?,5-60 min)on the solubility and molecular structure of myosin was related to the heat temperatures and times.The obvious changes of turbidity and solubility of myosin dispersion were observed at 30-40?,but ANS-S0 increased significantly and ?-helix content decreased(p<0.05)with increasing heating time.In the range from 50? to 80?,5 minutes heating treatment led to obvious flocculation and precipitation of myosin dispersion,surface hydrophobicity of myosin molecule significantly increased(p<0.05),and the solubility and ?-helix content decreased.With the prolongation of holding time(>5 minutes),the solubility and structure of myosin molecules still had slight changes,but not significant(p>0.05).3.In order to further study on the improvement of the thermal stability,sucrose,arginine(L-Arg),sodium dodecyl sulfate(SDS),carrageenan was respectively added to myosin dispersion at three ionic strengths(1,150,600 mmol/L KCl)conditions,the changes in turbidity and solubility of myosin were investigated during the heating treatment(50?,30 min),and the inhibitory effect of protein stabilizer on thermal aggregation of myosin was analyzed.Comparatively,at 1 mmol/L and 150 mmol/L KC l,with the increase of the amount of L-Arg and SDS(0-3 mmo/L),the turbidity of myosin dispersion decreased significantly(p<0.05),the solubility increased,and the inhibition effect of thermal aggregation was obvious.While,with the increase of additive amount(3-10 mmo/L),the changes of turbidity and solubility were not obvious.Sucrose or carrageenan had no influence on solubility and turbidity of myosin during heat treatment(p>0.05),and their effect of inhibition was not obvious.At 600 mmol/L KCl,the addition of L-Arg could obviously inhibit thermal aggregation of myosin molecules.However,thermal aggregation of myosin dispersion was more obvious by adding sucrose,SDS or carrageenan.Therefore,SDS and arginine would be studied in further study.4.At various pHs(pH 6.0,7.0 and 8.0)and ionic strengths(1-150 mmol/L KCl),the effects of heat treatment(50?,30 min)on turbidity,solubility and molecular structure of myosin were studied by adding 3 mmol/L SDS.The inhibitory effect and mechanism of SDS on the thermal denaturation and aggregation of myosin were also analyzed.In the experimental range,at ac idic and neutral pHs,heat treatment resulted in apparant aggregation and precipitation of myosin dispersion.Heat treatment after the addition of SDS,the turbidity and particle size aggregates of myosin significantly decreased(p<0.05),surface hydrophobicity and solubility increased significantly(p<0.05),and ?-helix content also increased.At alkaline pHs,the myosin dispersion containing 3 mmol/L SDS was still clear after 30 min heating treatment at 50 ?,the solubility significantly increased,and the obvious inhibitory effect of SDS on thermal denaturation and aggregation was obtained.Preliminary analysis indicated that the above results might be related to the electrostatic interaction and hydrophobic interaction between SDS and protein molecules.5.At various pHs(6.0,7.0 and 8.0)and ionic strengths(1-600 mmol/L KC l),the effects of heat treatment(50?,30 min)on turbidity,solubility and molecular structure of myosin were studied by adding 3 mmol/L L-Arg,the inhibitory effect and mechanism of L-Arg on the thermal denaturation and aggregation of myosin were also analyzed.The results showed that L-Arg could increase the solubility of myosin at low ionic strengths(p<0.05),inhibit the aggregation of protein molecules,decrease the turbidity of the solution,and reduce the size of the aggregates.Compared to heating groups without L-Arg,3 mmol/L L-Arg could decrease the turbidity and increase the solubility of myosin after heating treatment and the inhibition effect was obvious.Especially,the inhibitory effect was more significant at acid p Hs and low ionic strengths(p<0.05).Meanwhile,surface hydrophobicity of myosin with L-Arg increased significantly(p<0.05),?-helix content showed a decreasing trend.Preliminary analysis indicated that the inhibition effect might be related to the solubilization of arginine and the electrostatic interaction with myosin molecules,and the related mechanism should be further studied.