| Curcumin(Cur)is a typical hydrophobic polyphenol compound with multiple pharmacological activities such as antioxidant,anti-inflammatory,anti-cancer,and antitumor effects.However,the application of Cur is still limited by its solubility,stability,adsorption,and bioavailability.Using natural biopolymers such as proteins and polysaccharides to construct composite matrices as bioactive ingredient encapsulation and delivery carriers is an effective way to improve their bioavailability and maintain their bioactivity.Due to its good gelation,amphiphilicity,nutritional,and biological activities,ovalbumin(OVA)has gradually become an ideal carrier for the preparation of various functional ingredients,and has broad application prospects in the food and pharmaceutical industries.However,in the actual delivery application,OVA still has the drawbacks of poor emulsification and stability,and its excellent performance has not been fully utilized.Therefore,this study focuses on the low emulsification and stability of OVA,and based on the enzymatic modification of OVA with chitosan oligosaccharides(OCS)and molecular cross-linking,constructs glycosylated protein cross-linked products.Based on this,using Cur as a model of bioactive ingredient,sugar-modified OVA is used to form stable thermal aggregates,and cold-cured OVA emulsion gels induced by glucono-delta-lactone(GDL)were prepared.The simulated in vitro digestion process of Cur-loaded emulsion gels in the oral cavity,stomach,and intestine was used to elucidate its slow-release and stabilization mechanisms and provide a theoretical basis and technical support for the application of enzymatic glycosylation modification in protein function enhancement and delivery carriers.The main results are as follows:(1)Construction of TGase-mediated OCS-modified OVA complexes,and exploration of the impact of glycosylation modification on the physicochemical,structural,functional,and rheological properties of OVA.The results of physicochemical analysis showed that the physical mixture of OVA and OCS(OVA+OCS)led to a significant increase in particle size and polydispersity index(PDI)of protein particles,making the mixture system unstable.Further TGase treatment resulted in the formation of sugar-modified cross-linked OVA(GCOVA),and the particle size and PDI data showed that its stability was enhanced.The changes in free amino acid content under enzyme treatment for 4 hours reflected that the glycosylation reaction and cross-linking reaction were close to saturation.Through analysis of the primary,secondary and tertiary structure of GC-OVA and its microstructure,it was found that GC-OVA modified with OCS formed large molecular aggregates between 40～180 kDa;compared with untreated OVA and OVA+OCS systems,GC-OVA formed a dense network structure inside,with an increased number of hydrogen bonds and a more open and disordered structure.At 4 hours of enzyme treatment,GC-OVA emulsification properties and DPPH activity were the best(p<0.05),and excessive cross-linking would cause spatial steric hindrance and affect the binding sites of glycosylation.In addition,compared with untreated OVA and OVA+OCS systems,GC-OVA had a larger apparent viscosity G’/G",as well as stronger gel hardness and cohesion characteristics.The above research results show that glycosylation modification effectively enhances the emulsification,gelation,and rheological properties of OVA,and its enhancement effect is mainly achieved by affecting the physicochemical properties of OVA such as particle size,potential,and free amino acid content as well as protein particle microstructure and microenvironment,and its effect is closely related to the degree of glycosylation modification.(2)Construction of GC-OVA thermal aggregates and study on the influences of heat treatment time and glycosylation modification on the thermal aggregation behavior and rheological properties of OVA.The degree of glycosylation modification significantly affected the changes in free thiol content during the heating process of GC-OVA samples,promoting partial denaturation of proteins and the formation of hydrophobic interactions.The rheological results showed that the higher the degree of glycosylation modification,the larger the final G’ value of the protein thermal aggregates,and the frequency-dependent properties of the glycosylated cross-linked products were similar at 25℃;glycosylation modification markedly increased the viscoelasticity of GC-OVA thermal aggregates.By analyzing the solubility of thermal aggregates in different solutions,it was found that the intermolecular interactions between thermal aggregates were mainly hydrophobic interactions,followed by disulfide bonds and hydrogen bonds.The degree of glycosylation cross-linking modification was closely related to the thermal aggregation behavior of OVA.Low degrees of glycosylation modification can promote rapid formation of thermal aggregates,while products with higher degrees of modification can delay the formation of thermal aggregates.The initial low degree of aggregation can provide favorable conditions for protein denaturation,while the higher degree of aggregation in the later stage is conducive to the formation of stable thermal aggregates.The products with higher degrees of glycosylation modification can improve the viscoelasticity,stability,and rheological properties of OVA thermal aggregates,thereby promoting the formation of gels.(3)Construction and characterization of GDL-induced cold-cured emulsion gel carrier system,and study on the influence of glycosylated OVA emulsion gel on Cur embedding and in vitro digestion behavior.GC-OVA thermal aggregates were used as the water phase and sunflower seed oil as the oil phase to form a cold-cured emulsion gel induced by GDL.Cur was used as a model of bioactive ingredient to simulate the process of in vitro digestion of Cur-loaded emulsion gels in the oral cavity,stomach,and intestine,in order to elucidate its slow-release and stabilization mechanisms.During the preparation of the cold-cured emulsion gel,the GDL content significantly affected the pH change of the emulsion gel,and a GDL addition of 0.4%could result in an emulsion gel with a higher water holding capacity.Texture and confocal laser scanning microscopy(CLSM)data showed that the oil phase volume fraction was closely related to the gel strength,and the gel network structure gradually aggregated under the condition of φ=15%.At this time,the oil droplets were relatively uniform in shape and size,and the particle size and potential were stable.In contrast,in high oil phase(75%),the oil droplets and proteins had merged into each other,and the oil phase played a stronger role as an "active filler",but the formation of the emulsion gel was adversely affected.The GC-OVA emulsion gel with 15%oil phase ratio achieved a Cur embedding rate of 96%and a loading rate of 28%.In the simulated in vitro digestion process,the dissociation rate of OVA emulsion gel(O-EG)in the stomach and the release rate of Cur increased rapidly,while the digestion and absorption site of GC-OVA emulsion gel(GO-EG)system was mainly in the intestine,and the slow-release effect of Cur was obvious.Compared with free Cur and Cur/O-EG,the bioavailability of Cur in Cur/GOEG system increased by 43%and 31%,respectively.These results indicate that glycosylation modification effectively enhances the anti-gastrointestinal digestion ability,slow-release potential,and bioavailability of bioactive ingredients in the emulsion gel carrier system. |
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