Effect Of Modified Konjac Oligo-glucomannan On Glycation Of Silver Carp Myosin

Fish protein has rich nutritional value,and its main myosin(Ms)has good functional properties such as gelation,water holding capacity and emulsifying properties.However,due to the soft and delicate protein structure of the natural fish muscle,it is highly prone to denaturation,resulting in a sharp decline in functional properties,which greatly limits the processing and application of fish myosin.In order to improve the functional properties of myosin,it can be modified.Glycation is based on Maillard reaction mechanism and it is green and effective among protein modification methods.It has the advantages of simple operation,free of chemicals addition,and significant improvement of protein functional properties.In this study,the glycoconjugates of myosin-modified konjac oligo-glucomannan(Ms-MKOG)were prepared by glycation using silver carp myosin(Ms)and konjac oligo-glucomannan(KOG)as main raw materials.KOG was modified by deacetylation,carboxymethylation and phosphorylation to obtain modified konjac oligo-glucomannan(MKOG).The physicochemical,structural and functional properties of glycoconjugates were systematically studied in order to study the effect of the structural changes of sugar donors on myosin glycoconjugates.The structure-activity relationship between the structure of sugar donors and the functional properties of glycoconjugates is constructed.The present study would enrich and improve the theory of glycation,and provide a theoretical basis for the modification of food proteins.Three groups of MKOG and myosin were glycated to prepare glycoconjugates.The physicochemical properties of glycoconjugates were determined,including analysis of grafting degree,free amino acid,SDS-PAGE,isoelectric point,total sulfhydryl group,and amino acid composition.The results showed that the content of free amino acids decreased significantly(P<0.05),in which the content of free amino acids in Ms-DKOG decreased gradually from Da0 to Da5,while the content of free amino acids in Ms-CMKOG and Ms-PKOG was the lowest at CM1 and PK0,respectively.The relative content of lysine and arginine in the three groups of glycoconjugates decreased significantly(P<0.05),and the decreasing trend was the same to that of free amino acids.After glycation,the isoelectric point of glycoconjugates decreased in different degrees,and the isoelectric point of Ms-PKOG decreased slightly.The total sulfhydryl reduction was significant relative to Ms,in which the total sulfhydryl groups of Ms-DKOG gradually increased from Da0 to Da5,while the total sulfhydryl groups of Ms-CMKOG and Ms-PKOG were highest at CM1and PK0,respectively.SDS-PAGE analysis showed that high molecular weight aggregates were formed,and their molecular weight was closely related to the grafting degree of glycoconjugates.In addition,Fourier transform infrared spectroscopy(FT-IR)demonstrated that sugar molecule was attached to Ms in the form of covalent bonds.The apparent structure,primary structure,second structure,denaturation temperature and tertiary/quaternary structure of glycoconjugates were characterized by scanning electron microscopy(SEM),atomic force microscopy(AFM),infrared spectroscopy,circular dichroism(CD),differential scanning calorimetry(DSC),endogenous fluorescence spectroscopy(IFS)and surface hydrophobicity.The results showed that the surface hydrophobicity of glycoconjugates was significantly lower than that of Ms(P<0.05),and the surface hydrophobicity of Ms-DKOG was lower than that of Ms-CMKOG and Ms-PKOG,respectively.The relative content of?-helix,?-strand and?-turn in secondary structure decreased,especially in Ms-CMKOG and Ms-PKOG,where the relative content of?-helix decreased significantly.The denaturation temperature of glycoconjugates by different sugar donors increased continuously after glycation.The results of SEM and AFM showed that protein dissociation and protein aggregation occurred simultaneously,but mainly in the forms of aggregation.The three glycoconjugates had some changes in the microstructures,among which,the microstructure of Ms-DKOG changed significantly.Endogenous fluorescence spectra showed that the maximum emission wavelength of glycoconjugates would be blue-shifted.The fluorescence strength of Ms-CMKOG and Ms-PKOG decreased due to the presence of quencher,which indicated that glycation can change the structure of glycoconjugates and further improve the functional properties of proteins.By studying functional properties,it is found that the solubility of glycoconjugates(especially in high ionic concentration solution)was significantly higher than that of Ms(P<0.05).The solubility of Ms-DKOG was higher than that of Ms-PKOG,but the change of Ms-PKOG was not obvious.The thermal stability of Ms-DKOG was also significantly increased as compared with Ms(P<0.05),and the increase in thermal stability of Ms-DKOG was the most obvious.The emulsifying properties(including EAI and ESI)of the glycoconjugates were significantly improved,and the emulsifying properties of Ms-DKOG were the best.A prediction model of myosin digestion in vitro was established based on artificial neural network(ANN).ANN model is built by using nftool in the NET toolkit of MATLAB.Ten hidden neurons were set up in the experiment,and Levenberg-Marquardt training algorithm was used to calculate iteratively.The results showed that the average error between the test value and the output value is small,and the overall R~2 of fitting regression is 0.9687 and the R~2 of the test set is 0.99232.The results showed that the fitting effect and prediction ability of the neural network are good,and the target of predicting hydrolysis degree could be well fulfilled.The error between the experimental results of the selected test group and the output value of the neural network is within(±7%),which showed that the neural network prediction model has high prediction ability and accuracy.