Study On The Aggregation Behavior And Mechanism Of Bighead Carp(Aristichthys Nobilis) Myosin Affected By Heating Temperature

Bighead carp(Aristichthys nobilis) is one of the edible freshwater fish for cooking,which has high protein and low cholesterol. Surimi processing could improve the economic value of bighead carp. Freshwater fish and marine fish are quite different because of the living environments, leading to the difference in muscle protein gel properties. Therefore, the gel properties of freshwater fish were deeply detected. At present heat-induced gelation of bighead carp muscle protein has been reported, whereas during the protein thermal denaturation process the formation of protein clusters and their impact on the final gel properties are less reported. The topic regarded bighead carp myosin as research object, using rheometer, circular dichroism, dynamic light scattering, scanning electron microscopy and atomic force microscopy technologies to study the influence of heating temperature on myosin structure and aggregation behavior. The rheological behaviors at different heating temperatures were verified. The influence of temperatures on the denaturation rate and aggregation rate of myosin was explored. The effect of the difference in the myosin clusters formed during the gelation process at different temperatures on the gel properties were elaborated. Therefore, this research was aimed to provide a theoretical basis for bighead carp thermal processing and to promote the development of freshwater fish surimi industry. The main results are as follows:1. Circular dichroism and biochemical methods were applied to determine structure and physicochemical properties of bighead carp myosin under different heating temperatures, and the differences in denaturation rate and aggregation rate of myosin were analyzed. The results indicated that the melting temperature of myosin was around 40 °C. The myosin occurred a higher degree of unfolding at 90 °C than that of at 50 °C, indicating that the denaturation rate of myosin was faster at 90 °C and myosin conformation was more unstable at high temperature. The maximum of surface hydrophobicity was obtained at 50 °C for 30 th min and at 90 °C for 15 th min. Furthermore, the higher surface hydrophobicity was gained at 90 °C compared to that of at 50 °C. In the process of heating, the change of turbidity was caused by the aggregating of myosin. Turbidity of the myosin solution heated at 50 °C gradually increased with increasing heating time. However, turbidity increased to the maximum at 6th min, and then decreased with prolonged heating at 90 °C, which was caused by the precipitation of myosin aggregates. The results indicated an increase in turbidity resulted from the clusters in solution. However, the protein clusters at two temperatures were different. The protein clusters grew slowly at 50 °C and difficult to precipitate, while myosin clusters grew quickly at 90 °C and tended to settle. This may be caused by its faster aggregation rate at 90 °C than that of at 50 °C.2. The rheological behavior of myosin was measured by rheometer at different heating temperature treatment. The results showed that key points of storage modulus(G’) of myosin at 50 °C and 90 °C was different. G’ has two peaks at 50 °C, but only one peak at 90 °C. Furthermore, with the extension of preheating time, G’ of myosin heated at 50 °C increased, but decreased at 90 °C, which was due to the different state of protein clusters formed at intermediate stage at two temperatures. Heating at 50 °C, the unfolding rate of myosin molecules were relatively high and aggregation rate was rather low, which caused the myosin head region combining and myosin tail extending outwardly to form open-type protein clusters. Therefore, G’ increased with the extending of heating time and the ability of forming a gel increased. Heating at 90 °C, the unfolding rate of myosin molecules were relatively high and aggregation rate was also quite high. Myosin could combine with each other through head region, head-tail and tail, forming closed-type protein clusters. Only little tail extended outwardly and reduced with the increasing heating time. Therefore, a decrease in G’ was obtained with the extension heating time, which led to the gel forming ability decreased. Combined with SDS-PAGE electrophoresis analysis, myosin heavy chain was not hydrolyzed at 50 °C, however, it was hydrolyzed at 90 °C. As the preheating time extended, the degree of degradation of MHC was greater. The heavy chain of myosin played a key role in gel formation, which may be another reason for poor gel properties at 90 °C.3. Dynamic light scattering(DLS), scanning electron microscope(SEM) and atomic force microscope(AFM) technology were used to study the aggregation behavior of bighead carp myosin. The DLS results indicated that the diameter of myosin clusters was uniform at two temperatures. Myosin gathered at a rather slow rate at 50 °C, resulting in the small diameter of protein clusters. The aggregation rate of myosin was quite fast at 90 °C, leading to the large diameter of protein clusters. The DLS results were corresponding with the results of turbidity and G’. The AFM results showed that myosin was evenly distributed in a monomer or filament(oligomer) form. Compared to the treatment at 50 °C, aggregation behavior of myosin was more intense at 90 °C and the volume of aggregates increased more rapidly with the extension of the heating time. Meanwhile, more irregular clusters formed in mica and distributed unevenly. SEM results showed that myosin at high concentration could form relatively delicate uniform gel structure at 50 °C. The myosin filaments were visible, owning dense filamentous network structure and small aperture. However, the myosin gel formed large hole structure at 90 °C and there were no apparent myosin filaments. Protein aggregates were compacted form, meanwhile fracture occurred.