Application Of Asymmetrical Flow Field-flow Fractionation For Separation And Characterization Of Thermal Aggregation Behavior Of Soybean Protein Isolate

Soybean protein isolate?SPI?produced from low-temperature defatted soybean meal is a high-quanlity plant protein which is rich in nutritional value and is widely used in the field of food processing.Heat treatment is an inevitable process of food processing,which leads to the accumulation of proteins in food.Protein thermal aggregation has a significant effect on the nutritional and functional properties of food.In this paper,asymmetrical field flow-field fractionation?AF4?coupled with UV-Visible light detector?AF4-UV/Vis?was used to separate and characterize SPI for the first time.In the first part?i.e.,Chapter 2?,SPI was extracted from defatted soybean meal by alkaline extraction-acid precipitation method.Efects of different dissolution conditions?solvent type,solvent pH,solvent concentration?on SPI were studied by AF4-UV/Vis.At the same time,the effects of AF4 analysis conditions?i.e.injection mass,cross-flow rate,carrier ionic strength,membrane type,and channel thickness?on SPI separation and characterization were investigated.And the repeatability of the experimental method was verified to ensure the applicability of the system.The optimum separation conditions the detector flow rate is 1mL/min,and the cross-flow flow rate was 1.5 mL/min;the carrier liquid was 10 mM phosphate buffer saline?PBS?solution.And 5 mM NaCl is added to adjust the ionic strength and 3 mM NaN₃ is used for bacteriostasis;The type of ultrafiltration membrane is regenerated cellulose?RC?membrane;Thickness of spacer is 350?m.In addition,the repeatability of AF4-UV/Vis was verified to ensure the applicability of the AF4 system under the optimum conditions.The results showed that AF4 technology has good reproducibility for analyzing SPI.The second part?i.e.,Chapter 3?,thermal aggregation behavior of SPI was investigated by AF4-UV/Vis.Effects of heat treatment temperature,heat treatment time,protein concentration,salt ion strength and type on the thermal aggregation behavior of SPI were studied.The results showed that when the heat treatment temperature increases to 75?,the SPI is denatured,and the elution peak of the larger sample appears in the AF4-UV/Vis fractograms.Meanwhile,the dissociation and reaggregation of SPI subunits during thermal aggregation were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.Different heat treatment time affect the degree of protein denaturation,thus there are different changes in the relative content of thermal aggregates.The increase of protein concentration promotes the cross-linking rate between protein molecules,which is beneficial to the occurrence of protein aggregation.Salt ions mainly affect the surface charge distribution of protein molecules.And it will weaken the interaction between protein and water molecules,promoting the cross-linking between protein molecules.Effect of type of salt on SPI thermal aggregation was consistent with the hofmeister sequence.In this study,the SPI separation conditions and the factors affecting thermal aggregation was investigated by using AF4-UV/Vis technology under approximately in-situ conditions.AF4-UV/Vis was proved to be a powerful tool for studying thermal aggregation behavior of SPI.The results obtained in this paper can provide basic data support for the food industry containing SPI.