Construction Of Delivery Systems With Whey Protein Isolate And Their Stabilization Effect On Bioactive Compounds

Whey protein isolate(WPI)was often used to construct delivery systems to improve the physichemical stability and bioavailability of bioactive ingredients.However,protein-based delivery systems are sensitive to environmental conditions(p H,high temperature,ion concentration,etc.),which limit their applicaiton in diverse food systems.The use of modified proteins as carrier materials is an effective way to overcome this drawback.The covalent bonding of polysaccharides and polyphenols can impart new functional properties to proteins and is of great significance for the construction of protein-based delivery systems.In this study,WPI,gum acacia(GA)and(-)-epigallocatechin-3-gallate(EGCG)were used to prepare binary and ternary conjugates by different methods.The structure and physicochemical properties of the complexes were studied,the protection and delivery of bioactive compounds by the delivery systems based on these covalent complexes were also evaluated.The main conclusions are as follows:(1)The WPI-GA conjugates were prepared by dry-heating Maillard reaction.It was found that the graft degree of the conjugates incubated for 1,3,5,and 7 days were28.14 %,44.98 %,49.50 %,and 51.20 %,respectively.And the higher the degree of grafting,the more severe the browning.The glycation reaction could decrease the α-helix content,fluorescence intensity and surface hydrophobicity of WPI.Functional properties of the conjugates,such as solubility,thermal stability and emulsion properties were all superior to the control WPI.However,the highest solubility and emulsion properties were obtained by the conjugates of reaction for 1 and 5 days,respectively,indicating that the reaction time suitable for producing conjugates with superior functional properties was therefore not necessarily the highest glycation degree that could be reached.(2)The WPI-GA conjugates were prepared by ultrasound assisited Maillard reaction.It was found that sonication could accelerate the glycation reaction between WPI and GA.The degree of glycation for the conjugate prepared by dry heating for 1day could be obtained by ultrasound treatment for 80 min under the optimized conditions(ultrasonic temperature: 70 °C,ultrasonic power: 500 W,initial p H: 11.0,protein concentration: 10.0 mg/m L,protein-polysaccharide mass ratio: 1 : 2).Structural analyses suggested that the conjugates obtained by ultrasound treatment showed smaller molecular weight,lower α-helix content,higher fluorescence intensity and surface hydrophobicity than those obtained by dry heating.The conjugates obtained by ultrasound treatment showed better solubility and emulsion activity,while the conjugates prepared by dry heating had better thermal stability and emulsion stability.(3)The WPI-GA conjugates were used to construct EGCG-loaded nanocomplexes.It was found that ultrasound-assisted glycosylation enhanced the binding ability of WPI to EGCG,while dry heating glycosylation reduced the binding ability of WPI to EGCG.Therefore,the conjugates prepared by ultrasound-assisted had higher EGCG loading efficiency.Based on the steric hindrance of polysaccharide chains,glycosylation could inhibit the EGCG-induced protein aggregation.The greatest protection of EGCG and its antioxidant activity were obtained by complexing it with the conjugate prepared by untrasound.Glycosylation could enhance the bioavailability of EGCG in nanocomplexes,but there were no significant difference(p > 0.05)between different glycosylation methods.In general,the conjugate prepared by untrasound is more suitable for constructing protein-polyphenol nanocomplexes delivery system.(4)The β-carotene nanoemulsion was prepared by WPI,WPI-GA mixture and WPI-GA conjugates.Compared with WPI and WPI-GA mixture stabilied nanoemulsions,the nanoemulsions stabilized by WPI-GA conjugates showed stronger stability against freeze-thaw,high ion concentration,acid-base and heat treatment,and improved chemical stability.The covalent linkage of GA could improve the bioavailability of β-carotene in WPI-stabilized nanoemulsions,but the bioavailability of β-carotene nanoemulsions stabilized with the WPI-GA conjugate prepared by ultrasound was significantly lower than that stabilized with WPI-GA mixture.Compared with the nanoemulsion stabilized by the conjugate prepared by ultrasound,the nanoemulsion stabilized by the conjugate prepared by dry heating showed better thermal stability and higher bioavailability of β-carotene,so it is more suitable for constructing nanoemulsion delivery systems.(5)The WPI-EGCG conjugates were prepared by alkali method,free radical method and enzymatic method,and used to stabilize β-carotene nanoemulsion.The highest grafting efficiency was obtained by enzymatic method,followed by alkali method.The conjugates showed less α-helix content,higher random coil content and fluorescence intensity than those of WPI.Covalent bonding of EGCG induced by alkali and enzymatic methods decreased the surface hydrophobicity and increased thermal stability of WPI,while the free radical method led to opposite results.The antioxidant activity of the conjugates was higher than that of WPI,and was proportional to the amount of bonded EGCG.Compared with the WPI-stabilized nanoemulsion,the WPIEGCG conjugates-stabilized nanoemulsions have smaller particle size,and the degradation of β-carotene under UV light and during storage were markedly inhibited.This inhibition effect was laso positively correlated to the amount of grafted EGCG.(6)Based on the WPI-EGCG binary conjugates,WPI-EGCG-GA ternary conjugates were prepared by Maillard reaction and used to stabilize β-carotene nanoemulsion.The covalent linkage of GA increased the molecular weight,and enhanced the thermal stability and antioxidant activity of binary conjugates.Compared with the nanoemulsions stabilized by WPI,binary conjugates and ternary mixture,the ternary conjugates stabilized nanoemulsion showed better freezing-thaw stability,salt ion stability,acid-base stability and thermal stability,the chemical stability of β-carotene against UV light and heat treatment were also improved.Moreover,the ternary conjugate as an emulsifier could enhance the stability of the nanoemulsion during digestion and thus increased the bioavailability of β-carotene.