| The mixed use of high-quality animal protein and plant protein has nutritional advantages,which is in line with the development strategy of "Dual-protein Project" that has been put forward in China.At present,the dual-protein is mainly mixed directly during processing.The synergistic effect of the two heterologous proteins is not significant,and even has an antagonistic effect,which severely limits the application of the dual-protein in the food industry.Domestic and foreign scholars also focus on the mixing ratio,processing conditions,etc.Still,there is a lack of systematic research on the effects of different preparation methods on the structure and function of the dual-protein.In this paper,pea and grass carp with high quality and low price were selected as raw materials.The isoelectric solubilization/precipitation(ISP)method was used to complete dissolution and precipitation in the same dispersion system to obtain co-precipitated dual-protein(Co).Pea protein isolate(PPI)and grass carp protein isolate(CPI)were prepared by the ISP method,and the two single protein powders were mechanically mixed to obtain blended dual-protein(BL).With PPI,CPI for comparison,the structure of Co and BL characterization and nutrition,emulsification,gel and other features a comprehensive comparative analysis,focus on evaluating the functional characteristics of Co,analyzing the structure-activity relationship,trying to proven Co relative advantage,revealing the effect mechanism of co-precipitation on the structure and function of dual-protein,exploring the Co in the application of the minced fish system,and providing a scientific basis for expanding its application in the food industry.The specific research results are as follows:1.Optimization of parameters for preparing co-precipitated dual-proteins by the ISP method.The optimal p H for co-precipitation of pea and grass carp protein was 5.0,which was in the middle of the isoelectric point of pea and grass carp protein,which was favorable for the interaction of heterologous proteins.The subunit composition,basic composition,and functional characteristics of Co prepared by five kinds of p H extraction were compared and analyzed.It was found that the alkali method(Co9,Co10,Co11)was superior to the acid method(Co2,Co3).The ratio of Vicilin /Legumin α+β of the Co10 subunit was the highest,and its solubility,emulsification and the gelation properties were generally superior.The optimal extraction p H was10.0.2.Study on the structure characterization and nutritional properties of the co-precipitated dual-protein.The protein composition was characterized by Size-exclusion chromatography-multi-angle laser light scattering,transmission electron microscopy,and two-dimensional electrophoresis.It was found that Co had more aggregated components,with Mw of ～1200 k Da accounting for 18.4%,and the microscopic morphology showed "clustered grafting" characteristics,with 86 different protein spots compared with BL.Further,through spectroscopy characterization of secondary and tertiary structure and reduced and non-reduced SDS-PAGE analysis of quaternary structure,show co-precipitation causing secondary structure β-sheet toα-helix,tertiary structure non-covalent cross-linking degree more tend to the folding state,quaternary structure occurred more disulfide bond covalent cross-linking,generate more soluble aggregates and concentrated vicilin,and the value of vicilin /legumin α+β were 2.82 times that of BL.These differences suggest that the functional properties of Co may surpass those of PPI and CPI.Analysis of amino acid composition showed that the Met+ Cys-S and Lys contents of Co were significantly higher than that of BL,and the amino acid score of digestibility modification was 1.00(adult)and 0.87(child).Correlation analysis showed that Co digestibility was attributed to increased α-helix/β-sheet and decreased Arg/Lys ratio by co-precipitation.3.Study of the properties of emulsifying property of co-precipitated dual-protein.Four kinds of proteins were used as emulsifiers to prepare O/W emulsions,respectively.The emulsifying ability indexes such as solubility,surface hydrophobicity,EAI,ESI,and physicochemical indexes such as particle size distribution,zeta-potential,interfacial protein adsorption percentage and content,and confocal laser scanning of the emulsions were measured.The results showed that Co and BL were generally better than PPI and CPI,showing the synergistic effect of dual-protein,and Co was significantly better than BL.The secondary structure of the interface protein was determined by circular dichroism spectroscopy,and it was found that the α-helix/β-sheet ratio of Co was significantly higher than that of BL(p<0.05).The emulsion stability was characterized through the creaming index test,multiple light scatterometers,cryo-scanning electron microscope,dynamic shear rheometer,etc.It showed that Co emulsion was more stable than PPI,CPI,and BL.Correlation analysis showed that Co emulsifying characteristics were dominated by solubility and net charge,which were attributed to the higher adsorption percentage and α-helix ratio of interfacial protein.4.Study of the properties of co-precipitated dual-protein heat-induced gels.By analyzing the free sulfhydryl,surface hydrophobicity,and thermal stability of the protein,as well as the secondary structure of the protein,intermolecular force,water holding capacity,texture,and microstructure of the gel,the results showed that the heat-induced properties of PPI and CPI were between BL and Co.PPI and CPI had significant thermal denaturalization temperature differences and different gel network structures,which resulted in severe steric hindrance,many holes,and loose structure in gel of BL,showing an obvious antagonistic effect.Co had the highest free sulfhydryl content(p<0.05),making disulfide bond the most contributing factor of gel intermolecular force(p<0.05);The gel of Co has the largest β-sheet content,and has significant advantages in water holding capacity and hardness,which shows synergistic effect.Correlation analysis results showed that the advantage of Co gel was explained by the high content of β-sheet and the high contribution ratio of the disulfide bond.5.Effect of co-precipitation dual-protein pre-emulsification on gel properties of surimi.In order to understand the actual performance of the advantages of Co’s emulsification and gelation properties in complex food systems,Co pre-emulsified soybean oil instead of fat was used to prepare fish sausage,and the characteristics of fish sausage gel were analyzed.The results showed that the quality of Co fish sausage gel was comparable to CPI on the whole,and the sensory score was slightly lower than CPI,but significantly higher than BL and PPI.The plant protein in Co fish sausage accounts for 3.16% of the total weight,which meets the requirements of dual-protein food,caters to the trend of healthy consumption,and has commercial promotion value. |
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