Heat-induced Ovalbumin Fibrils As The O/W Pickering Emulsion Stabilizer: Self-assembly Mechanism And Emulsion Properties

As the main component protein of egg white,Ovalbumin has a wide range of application prospects in the food processing industry because of the good emulsifying and gelling properties.Heat treatment is a necessary part of food processing,which can cause physical and chemical changes of food components,thereby affecting the properties and potential usage of foods.Ovalbumin exhibits migration,degradation,and combination during the heat treatment process,which caused not only new chemical reactions,but also new physical structures of new compounds.It is a well-known phenomenon that food components would have nanostructures after heat treatment,but there is still a lack of scientific understanding on the question of“how proteins self-assemble to form micro/nano structures”and“what are the uses of nanostructures”.Therefore,it is supported by great theoretical basis and significant application prospects to study the molecular properties of thermally polymerized ovalbumin self-assembly and developing protein fibril stable emulsions.Studies have shown that protein can undergo thermally induced self-assembly behavior during heat treatment to form micro/nano-particles and take on new structural characteristics,which can prepare stable Pickering emulsions.In this study,ovalbumin was used as the main raw material to prepare protein self-assembled fibrils by heat treatment for different time,then Pickering emulsion was prepared by high-pressure microjet technology to explore the emulsification properties of thermally polymerized protein self-assembled fibrils.In this study,a systematic research was conducted on the fibril molecular characteristics,tructural characteristics,interface properties and emulsion stability.Besides,the influence of heat treatment on ovalbumin self-assembled fibrils as well as the molecular characteristics were determined.This study also explored the changing trends of molecular properties and the formation mechanism of protein fibrils during heating,revealing the relationship between the microstructure of ovalbumin self-assembled fibrils and the macroscopic morphology of aggregates,and provided a theoretical basis for the use of protein fibrils to stabilize emulsion applications and practical reference.The main research contents and results are as follows:1.Ovalbumin was used as raw material to explore the regulating effect of heat treatment on the process of protein self-assembly fibrils.Fluorescent labeling and dynamic light scattering were combined to study the macroscopic size and molecular morphology of ovalbumin self-assembled fibril,the study showed that the protein fibrils heated for 12 hours changed the most.The fluorescence intensity and surface hydrophobicity of Thioflavin T increased to 120.42 a.u.and 3958.8 a.u.respectively（p<0.05）.There were substances with a particle size greater than 10,000 nm in the heated protein sample,and the PDI value of the protein fibril heated for 12 hours was 0.62,the zeta potential value was 43.6 m V,showing the highest stability.In addition,the results of SDS-PAGE and transmission electron microscopy showed that protein fibrils were formed by small peptides that were proteolyzed.Prolonging the heating time appropriately would improve the efficiency of fibril formation.2.Circular dichroism,infrared and ultraviolet spectroscopy were used to comprehensively analyze the structural information of self-assembled fibrils,and explored the influence of heating time on the conformation of ovalbumin self-assembled fibrils and the microenvironment of functional groups.The results showed that the secondary structure of the protein fibril heated for 12 hours was:α-helix 16.4%,β-sheet 3.2%,β-turn 74.6%,and random curl 5.8%.β-turn content increased for 15.8%compared with unheated protein.In addition,he wavelengthλ_max corresponding to the maximum intensity of intrinsic fluorescence was red-shifted from 333.1 nm（0 h）to336.9 nm（24 h）.It could be inferred that some tryptophan residues in the protein are exposed to polar environments binding hydrophobicity surface;The peak-to-valley ratio"r"value of UV second derivative decreased from 1.73（0 h）to 1.41（24 h）,indicating that the internal structure of the protein had unfolded and changed.Compared with unheated protein,heat treatment not only caused the exposure of amino acid residues and hydrophobic groups within the protein,but also increased the polarity of the microenvironment in the protein fibril sample.During the self-assembly process,the protein was hydrolyzed into polypeptides,thus forming tightly structured protein fibrils.3.The Pickering emulsion was prepared by the microfluidizer method to analyze the emulsification activity and interface characteristics of self-assembled protein fibrils.The influence of self-assembled protein fibrils on the stability of the emulsion was also explored.The results showed that the protein fibrils heated for 12 h could improve the water-oil interfacial tension（decreased from 22.82 m N/M to 18.10 m N/M）,and the absolute value of zeta potential increased from 6.24 m V（0 h）to 21.04 m V（0 h）（p<0.05）;the gas-liquid interfacial tension of the Pickering emulsion decreased from 56.25m N/M（0 h）to 51.2 m N/M（12 h）.The adsorption capacity and the concentration of interface protein of emulsion were improved from 78.96%and 3.94 mg/m² to 98%and4.89 mg/m²,respectively.The average particle size of the emulsion stabilized by protein fibrils stabilized was 475nm,which was smaller than the average particle size（550nm）of the emulsion stabilized by unheated ovalbumin.The changes in the surface properties of the protein fibrils enhanced the formation of the 3D network structure of the emulsion,improving the strength of interaction between emulsion droplets and helped the system to resist droplet aggregation during storage.4.The changes in the microstructure of stable emulsions during storage and the oxidative stability in emulsions were explored.The results showed that the emulsion stabilized by 12 h protein fibrils exhibited the best stability.The Zeta potential decreased from 43.6 m V to 23.9 m V,the particle size of the emulsion increased from475 nm to 912 nm with a relatively uniform oil droplet distribution.The interface protein adsorption capacity as well as the interface protein content decreased from 98%and 4.89 mg/m² to 64%and 2.78 mg/m²,respectively.The flocculation index and polymerization index increased from 3.05 and 3.55 to 9.04 and 23.90.and the fat floating rate was 7.22,indicating that the emulsion had great storage stability.In addition,the POV value and TBARS value of the emulsion indicated that the protein fibril inhibited the formation of oxidation products during the storage of the emulsion and had good antioxidant capacity.