Behavior Of Heat-induced Denaturation And Aggregation Of Protein From Tilapia Muscle And Its Inhibition

Heat treatment is a essential process on the food processing, is also a common method of protein denaturation. Water-soluble protein, salt-soluble protein and myosin was extracted from tilapia white meat, and heating treatment in water bath was tested in this study. Changes of turbidity, solubility, total sulphur content, surface hydrophobicity, tryptophan fluorescence, SDS-PAGE profile of the protein at different heating condtion and pH of solution were determined, and then thermal denaturation kinetics was analyzed and the mechanism of thermal denaturation and aggregation was studied. The inhibition effect of addition SDS and β-CD on thermal denaturation and aggregation of myosin was experimented, the improvement of artificial molecular chaperon-mediated thermal stability of myosin in the liquid system was discussed. The main purpose was to control the denaturation and aggregation in an acceptable range, and provided the theory basis for the effective use of fish resources and processing and development of related products. The main results were as follows:(1) The studies on the thermal denaturation rate(30~90℃, 0~30min) of water-soluble protein and salt-soluble protein and denaturation kinetics analysis showed that, the D and Z values of water-soluble protein were lower than that of salt-soluble protein at various temperatures. The denaturation reaction progression of water-soluble protein and salt-soluble protein were 1.2 and 1.1, and thermal denaturation reaction activation energy Ea were 59.97 and 9.81 kJ/mol, respectively. The turbidity changes and aggregation rate of water-soluble protein were greater than that of salt-soluble protein at the same conditions. the reduction and unreduction SDS-PAGE analysis of soluble protein by heat treatment showed the band became with sight shallower. Therfore, the heat resistance of salt-soluble protein was better than that of water-soluble protein.(2) Actomyosin was prepared from tilapia muscle and the heating treatment(30~80℃, 1℃/min) was carried at protein concentration of 2mg/mL. Heat-induced dynamic rheology study showed that of heat-induced gel of tilapia actomyosin was an unstable dynamic rheological change process. The elastic modulus(G’) increased slowly within 30℃ with increasing temperature and reached its lowest point at 46℃ following the rapidly increase at higher than 80℃. Turbidity increased by S-shape curve an the surface hydrophobicity increased gradually, especially between 40~60℃(p<0.05). Solubility and total sulfhydryl group content declined gradually, fell the most rapidly between 30~50℃, and the value of tryptophan fluorescence decreased rapidly after 60℃. SDS-PAGE profile analysis showed that the formation of disulfide group at temperature higher than 45℃, which indicated conformation changes and aggregation denaturation of actomyosin during heat-treatment. Combining with the maximum aggregation rate of actomyosin at 45℃ and the lowest energy G’ at 46℃, the denaturation temperature of tilapia actomyosin was estimated presumedly at 46℃.(3) Myosin was extracted, and effect of heating treatment(30~90℃, 1℃/min) on thermal denaturaion and aggregation of myosin was determined, at various pH values(pH 2.0, 5.0, 6.0, 7.0, and 11.0) and 2mg/mL of protein concentration. Comparatively, changes of the turbidity and solubility was not obvious during heating treatment(p>0.05). At pH 7.0, the turbidity increased and the solubility decreased with increasing heating temperature, and a larger change in the molecular structure of myosin was observed. The apparent activation energy Ea of tilapia myosin were 166.51 kJ/mol and 164.63 kJ/mol the basis of the change of Ca2+-ATP enzyme activity at pH 7.0. Under the acid condition, the thermal stability was poor, and aggregation strength of myosin was pH 6.0 < pH 2.0 < pH 5.0. The obvious changes of myosin was obtained at pH 6.0 and higher than 60℃, but no obvious aggregation and changes of particle diameter was observed(p>0.05). According to changes of thermal denaturation rate, reaction order were calculate as 1.3 and 1.4, and the apparent activation energy Ea were 102.92, 286.67 kJ/mol at pH 2.0 and pH 5.5, respectively.(4) The inhibition effect of addition of SDS and β-CD on the thermal denaturation and aggregation of myosin was studied by turbidity and solubility changes. Single factors analysis showed that, in the range of experiment, 5mmol/L SDS was added to 2mg/mL myosin at pH 6.0, and then the sample was heated at 50℃ 30 min followed addition of β-CD(SDS and β-CD ratio of 1:2), which could effectively inhibit thermal denaturation and aggregation of myosin. The inhibition mechanism analysis of thermal denaturation and aggregation at above conditions showed that effect of addition of SDS and SDS-β-CD on the solubility and turbidity(p>0.05) in the process of heating treatment(30~80℃, 30min) was effectively inhibited. Destructive effects of heat-treatment on sulfydryl and tryptophan residues of myosin molecules was inhibited, and thus decreased interactions between protein molecules resulted in the inhibition of thermal denaturation and aggregation of myosin. All results indicated that thermal stability of the system was improved by addition of SDS-β-CD.