The Role Of Exogenous L-arginine In The Heat-induced Aggregation Of Bighead Carp Myosin:Machanism And Application

With the rapid development of the aging society in China,more and more people will have difficulty in chewing.So it is necessary to develop the convenient foods that are high in protein,low in fat and calories,easy to digest and eat.Fish meat is one of the best sources of protein for the elderly.However,current fish meat products are still dominated by surimi products with high elasticity,hardness,and chew resistance.Animal muscle proteins are easy to denatured and solidified during heating,which limits the development of new types of fish protein products such as liquid protein products.The author found that adding L-arginine(Arg)can significantly suppress the heat-induced aggregation behavior of myosin,the most dominant protein in fish myofibrillar protein.Therefore,the addition of Arg to myosin shows the potential to develop new types of fish protein products(such as liquid fish protein products)and expand the diversity of fish products.However,the mechanism of Arg in the heat-induced aggregation of myosin has not been clearly elucidated.Therefore,this dissertation chose fish meat as the raw material to carry out research on the role of exogenous Arg in the heat-induced aggregation of bighead carp myosin: machanism and application.The main research contents and results are as follows:(1)The influence of Arg on the phase behavior of myosin during heat-induced aggregation.Differential scanning calorimeter,dynamic rheometer,dynamic light scattering,scanning electron microscope(SEM),atomic force microscope(AFM)and other techniques were used to investigate the heat-induced phase behavior of myosin containing Arg,and the phase diagram was drew.The results showed that:(?)Arg increased the soluble protein content of myosin from 26.25% to 50.50% after two-stage heating(P < 0.05),which significantly weakened the heat-induced phase separation phenomenon of myosin.(?)When it is unheating,the image of “Y”-shaped myosin monomer was clearly observed by adding40 m M Arg.(?)After two-stage heating,the degree of protein aggregates dispersion increased with the increase of Arg concentration.(?)The increase in p H value induced by Arg and special structures of Arg played important roles in inhibiting the heat-induced aggregation behavior of myosin.(?)Arg suppressed the heat-induced aggregation of myosin by inhibiting tail-to-tail cross-linkings between denatured myosin molecules.(2)The effects of Arg on the molecular structures of myosin during heat-induced aggregation.The effect of Arg on the molecular conformations of myosin at different heating stages was studied by measuring endogenous fluorescence,surface hydrophobicity,circular dichroism,and secondary structure contents.The results showed that:(?)During the heating process,the addition of Arg significantly changed the secondary structures of myosin,and resulted in a decrease in the content of ?-helical structure(P < 0.05)and an increase in the content of random coil structure(P < 0.05).(?)Arg induced the absence of ordered-secondary structures of intramolecular myosin.(3)The mechanism of the interaction between Arg and myosin.Three myosin-guanidine group mixtures(Arg,guanidineacetic acid,guanidinium sulphate)were prepared to investigate the effects of guanidine groups on the turbidity,particle size,morphology,and hydrogen bonds during the heat-induced aggregation of myosin.Furthermore,molecular docking technology was used to introduce the molecular mechanism of the interaction between Arg and myosin.The results showed that:(i)Guanidine group,as a special structure of Arg,was a key factor in inducing the the absence of ordered-secondary structures of myosin,but it is not the only functional structure on the Arg molecule.(?)Arg disturbed the existed hydrogen bonds of myosin and formed new hydrogen bonds with myosin molecules,which is the fundamental reason for the absence of ordered-secondary structures of myosin molecules.(?)The most possible binding sites of Arg and myosin molecules were as follows: The carboxyl of Arg formed hydrogen bonds with the sidechain of Arg147 and backbone of Cys122.The amino of Arg formed hydrogen bond with the backbone of Leu120.The guanidine group of Arg formed hydrogen bonds with the sidechain of Thr177 and backbone of Cys122 and Leu175.(4)Application research based on the Arg-myosin interaction mechanism 1:Combined effects of Arg and ultrasonic treatment on the physical stability of O/W emulsions stabilized by fish myosin.Raman spectroscopy,ultraviolet spectroscopy,circular dichroism,and confocal laser scanning electron microscopy,particle size analyzer and profile analysis tensiometer were used to evaluate the combined effects of Arg and ultrasonic treatment on the physical stability of O/W emulsions stabilized by fish myosin.The underlying mechanisms were as follows:(i)Arg(40 m M)and ultrasound(5%,10%,and 20% amplitude)synergistically promoted the unfolding of natural myosin by inducing the exposure of buried aromatic and aliphatic amino acid residues.(?)The increase in ?-helix and ?-sheet content of interface myosin was beneficial to promote the cross-linking of adjacent interface myosins through intermolecular interactions.(?)A stable protein gelation network formed at the O/W interface,which helped protect the inner soybean oil droplets from unstable factors and improved the physical stability of the emulsion.(5)Application research based on the interaction mechanism of Arg and myosin2: Arg improved the effects of endogenous transglutaminase on the properties of low-salt surimi gel under microwave heating.Low salt-Arg-TGase surimi gel samples induced under different microwave time were prepared,and the gelling properties,microstructures,gel qualities,moisture status and protein properties of the Low salt-Arg-TGase surimi gels were compared with the traditional two-stage water bath high/low salt surimi gel and the microwave-induced low-salt surimi-TGase composite gel without Arg.The results showed that:(i)Compared with the traditional two-stage high-salt surimi gel,TGase alone significantly reduced the gel strength and water holding capacity of low-salt surimi(P<0.05)and increased the cooking loss(P<0.05)of low salt-surimi gels under microwave heating conditions.(?)After 4 minutes of microwave heating,the gel quality of the low salt-Arg-TGase composite surimi gel reached an equivalent level to that of the traditional two-stage high-salt group(P>0.05).(?)Microwave heating ameliorated the unfriendly flavors produced in the traditional two-stage water bath process,and the effect of adding the Arg group was better.