| Heat-induced aggregation of myosin is a key factor to determine the surimi gel and its product quality, including the texture, taste and water holding capacity. Heat treatment is essential to the processing of gel products, and it is also a common method of protein denaturation. In this study, we investigated the protein structure and gel properties of bighead carp myosin at different heating temperature by the methods of low-field nuclear magnetic resonance(NMR), Raman spectroscopy, scanning electron microscopy(SEM), differential scanning calorimetry and circular dichroism. The characteristics of gel water distribution, the rules of myosin denaturation and aggregation and the formation of the three-dimensional gel network structure are described clearly. The aim is to improve the surimi gel product quality and enrich the theory of gel-forming. The detailed research contents and results are as follows:1. The changes of moisture distribution and migration changes of heat-induced bighead carp myosin gelation are studied by low field nuclear magnetic resonance technique. The results show that the T2 distribution fitting curves appeared three peaks for myosin gel at different temperatures. The relaxation time and the corresponding water are T21, 1-10 ms(with water), T22, 100-600 ms(immobile water) and T23, 1000-3000 ms(free water), respectively. With the increase of temperature, the relaxation time of T21 increased first and then decreased. T22 relaxation time and the corresponding water content decreased significantly. T23 relaxation time increased from 932.6 ms to 2154.43 ms, and remained constant at temperatures above 50 °C, while the moisture content increased from 13.84% to 28.85%. The principal component analysis(PCA) was used to reduce the number of parameters of LF-NMR by dimension-reduction method. The cumulative variance of the first two principal components was 95.09%. The first principal component was negatively correlated with the first and second peak parameters, positively with the third peaks. Whereas the second principal component was only positively correlated with the first peak parameters. The principal component scores showed different relaxation characteristics between samples. The gelation process could convert at a certain temperature between 50-60 °C.2. The effect of temperature on the secondary structure and three-dimensional network structure of bighead carp myosin gel was studied by Raman spectroscopy and scanning electron microscopy(SEM). With the increase of heating temperature, the α-helix content showed a linear decline, from 39.44% to 24.22%, and the content of β-folding structure increased significantly. Meanwhile, the content of random coil and β-corner structure increased respectively from 27.95% to 39.46% and from 10.56% to 14.44%. From the SEM pictures it was observed that the compact and uniform threedimensional network structure of myosin gel gradually formed between 40 and 60 °C, while the gel fractal dimension increased from 2.841 to 2.89 and aperture equivalent diameter decreased from 0.105 μm to 0.080 μm. When the temperature rose to 70 °C and 90 °C, the gel network structure became coarse, and the partial rupture phenomenon was occurred frequently. At 70 °C and 90 °C, the gel fractal dimension reduced to 2.875 and 2.871 and pore average equivalent diameter increased to 0.121 μm and 0.128 μm.3. The changes of myosin secondary and tertiary structure as a function of heating temperature were explored by detecting the turbidity, solubility, intrinsic fluorescence and circular dichroism. The results showed that the turbidity of myosin increased at first and then decreased with the increase of heating temperature, finally reached the maximum value at 45 °C. Changes of tertiary structure of bighead carp myosin with the increase of heating temperature was as follows: below 45 °C the tertiary structure showed high stability; then as the temperature increased from 45 °C to 65 °C, the stability of protein spatial structure decreased sharply. The denaturation of tertiary structure was spontaneous, and the unfolding rate was rapid. As the temperature increased to 85 °C, tertiary structure denatured absolutely. Changes of myosin secondary structure with the increase of heating temperature were as follows: with the increase of temperature, α-helix content decreased significantly while β-sheet and β-turn content increased significantly. Random coil content also increased but no significance. At different temperatures the change degree of α-helix and β-sheet structure was exactly same and the change rate was consistent with each other, which suggested that the α-helix structure transformed to β-sheet structure during heat treatment. The changes in β-turn structure were higher than the α-helix and β-sheet structure, which indicated that the temperature had a greater impact on the β-turn structure of the myosin.To sum up, at different heating temperatures, the content of myosin secondary structure existed differences. Tertiary structure expanded in segments, which resulted in the different aggregation at low or high heating temperature. The gel network structure also showed big difference, which changed the gel water distribution and migration. In consequence, heat treatment is an important factor to effect the myosin gel structure and internal moisture. The appropriate temperature is necessary for good gel forming. |
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