Preparation Of Protein-polyphenol-polysaccharide Conjugate And Its Delivery To Beta-carotene

Food functional factors are widely used in food systems as a class of active substances with functions such as antioxidant,antimicrobial,antiviral,and enhancing body immunity.However,some lipid soluble functional factors,such asβ-carotene,suffer from poor water solubility,easy oxidative degradation and low bioavailability,which limit their application in the food field.In recent years,the emergence of emulsion delivery system based on food raw materials such as protein and polysaccharide has broken the bottleneck in the delivery of lipid soluble functional factors.First,SPI-TA conjugate was prepared by the covalent reaction between soy protein isolate（SPI）and tannic acid（TA）.Then,the binary conjugate was covalent to carbohydrate（maltodextrin/polydextrose）through Maillard reaction to form ternary conjugate.The ternary physical mixture of SPI-TA and MD/PD were prepared under the same conditions for comparison,and the effects of covalent grafting of polyphenols（TA）and polysaccharides（MD/PD）on the structure,physicochemical properties and functional properties of SPI were investigated by modern analytical tools.After that,β-carotene emulsions were prepared by proteins and its conjugates as emulsifiers,and the effects of different environmental stresses on the stability of emulsions stabilized by different emulsifiers（freeze-thaw stability,salt ion stability,heating stability,p H stability）were investigated by creaming index;Using the retention ofβ-carotene as an indicator to investigate the effect of different storage conditions（light stability,storage temperature stability）on the chemical stability ofβ-carotene emulsions stabilized by different emulsifiers;and to evaluate the antioxidant activity of different groups ofβ-carotene emulsions by radical scavenging ability and Fe³⁺reduction ability.Then,the oxidative stability,in vitro simulated digestive stability and gastrointestinal bioaccessibility of different groups of emulsions were evaluated,and the emulsion groups with higher bioaccessibility were selected to evaluate the cellular transport ofβ-carotene in the different groups of emulsion digestive juice by Caco-2 monolayer cell model.The results of this paper are as follows:（1）Preparation and characterization of conjugateThe reduction of sulfhydryl thiol and free amino group contents in SPI and the appearance of high molecular weight bands in the SDS-PAGE images confirmed the formation of covalent bonds in SPI-TA and SPI-TA-MD/PD conjugates.The amount of TA grafted in SPI-TA was35.56±1.32μmol/g,and the degree of grafting of MD and PD in SPI-TA-MD/PD ternary conjugates was 46.54%and 32.26%,respectively.The results of UV-Vis absorption spectroscopy and FTIR spectroscopy analysis of SPI,its conjugates and physical mixtures showed that the covalent grafting of TA and MD/PD changed the structure of SPI,resulting in a decrease ofβ-folding content and an increase ofα-helix,β-sheet and random coil content of SPI.Compared with SPI,SPI-TA and ternary physical mixtures;the SPI-TA-MD/PD ternary conjugates exhibited more excellent solubility,emulsification,antioxidant activity,foaming capacity,surface hydrophobicity and thermal stability;the two ternary conjugates had more similar structural and functional properties;however,the SPI-TA-PD covalent The two ternary conjugates have similar structural and functional properties;however,the foaming capacity of SPI-TA-PD conjugate are significantly better than those of SPI-TA-MD conjugate.（2）Preparation ofβ-carotene emulsions and their stabilityThe emulsions prepared with SPI-TA-MD as emulsifiers exhibited better physical stability to ionic strength,heating treatment,and freeze-thaw treatment,as evidenced by smaller creaming index.During long-term storage at different p H conditions,droplets encapsulated in SPI-TA conjugates and SPI-MD conjugates and SPI-TA-MD ternary conjugates exhibited smaller particle size and better stability against droplet aggregation than droplets encapsulated in SPI and SPI control groups.The ternary conjugates can effectively reduce the droplet size ofβ-carotene emulsions near the isoelectric point;they effectively inhibit the degradation ofβ-carotene in emulsions under Simulated sunlight and different storage temperature conditions and protect the encapsulatedβ-carotene.Ternary conjugate-stabilizedβ-carotene emulsions have better free radical scavenging and reducing ability;However,the SPI-TA-MD ternary conjugate was slightly weaker than SPI-TA in inhibiting lipid oxidation.（3）Digestive properties ofβ-carotene emulsionsThe results of different groups ofβ-carotene emulsions simulating gastrointestinal digestion showed that the ternary conjugate group of emulsions had the best gastrointestinal stability and the highest gastrointestinal bioaccessibility ofβ-carotene at 22.89%.The safety of SPI and its conjugates was verified by cellular experiments.The digestive juice ofβ-carotene emulsions stabilized by SPI and its conjugates SPI-TA and SPI-TA-MD were non-cytotoxic at higher dilutions（dilutions greater than 8）and showed low toxicity at lower dilutions.The cell antioxidant activity of SPI-TA and SPI-TA-MD conjugates was significantly higher than SPI,which is consistent with the results of antioxidant capacity measured by in vitro chemical method in Chapter 2.Caco-2 cells showed transmembrane resistance values greater than 1000Ω-cm²after 24 days of culture,and their apical enzyme line alkaline phosphatase activity remained stable,indicating that they had completed differentiation to form monolayer.The apparent permeability coefficient P_app ofβ-carotene in emulsion digestive juice stabled by SPI,SPI-TA and SPI-TA-MD was greater than 1×10^-6,The cellular transport efficiency ofβ-carotene in the emulsion digestive juice of the SPI,SPI-TA and SPI-TA-MD groups was 16.30%,20.50%and26.60%,respectively.In summary,SPI-TA-MD conjugates can be used as emulsifiers to prepare emulsion delivery systems and to improve the physicochemical stability,bioaccessibility and cellular transport rate ofβ-carotene in emulsions.The above results can not only provide theoretical basis for improving the physicochemical properties and functional properties of soybean protein isolate,but also provide new food-grade raw materials for the construction of food functional factor delivery systems.