Preparation And Modification Of Glycated Whey Protein Isolate Nanofibrils And Its Application In Pickering Emulsion System

With the development of dairy processing technology,whey protein isolate has gradually become an important raw material and added component of functional foods,nutritional products and other products with its good protein nutritional value,and is widely used in the food industry.However,whey protein isolate is prone to denaturation and aggregation under heat treatment conditions,which greatly affects its application in food emulsification systems.As a potential new functional food material,protein fibrils aggregates are expected to solve the problem of protein aggregation under heating conditions.In recent years,with the in-depth study of Pickering emulsion,the system is developing in the direction of high efficiency,safety and functionalization.The application of Pickering emulsion in the field of food has also become a hot topic for current scholars.Therefore,in view of the improvement of the thermal stability of whey protein isolates and the current lack of green and efficient Pickering emulsion stabilizers in the food industry.In this paper,acid-heated whey protein isolates were used to self-assemble to form nanofibrils aggregates.At the same time,protein fibrils were glycosylated to prepare acid-heat-stable amphiphilic anisotropic protein nanofibrils,and further used glycosylated protein nanofibrils in ethanol-induced modification and compounding with tannic acid to improved its functional properties,and explored the application of glycosylated whey protein isolate nanofibrils and its modified products to the stability of Pickering emulsion system and nutrient delivery.The main results are listed as follows:(1)In this study,nanofibrils were produced from whey protein isolate glycated with glucose,lactose,or maltodextrin by heating at p H 2.0 and 90 ~oC,and the properties of nanofibrils forming and stabilizing Pickering O/W emulsions were studied.Glycation reduced fibril length,among them,the length of whey protein isolate-glucose glycosylated protein nanofibrils corresponded to the average length measured by atomic force microscopy and transmission electron microscopy of 1.2and 1.6?m respectively,with the highest degree of inhibition,however,glycation had no effect on the width of the nanofibrils,all about 3-4 nm.Nanofibrils derived from glycated WPI had the increased hydrophobicity and formed a thick layer on oil droplets,and observed the interface structure of Pickering emulsion by laser confocal microscope and made a statistical analysis of the thickness of the emulsion interface layer(in percentage of droplet outer diameter)increased from 11.75±0.82%to 33.05±0.66%.Compared with whey protein isolate nanofibrils,glycosylated protein nanofibrils stable Pickering emulsion were more stable during prolonged storage at room temperature and at p H 2.0–8.0,0–250 m M Na Cl.The carbohydrate chain length positively impacted the emulsion viscosity while negatively impacted lipolysis and bioaccessibility of curcumin encapsulated in emulsions.With the growth of carbohydrate chains,the degree of lipolysis was reduced from 67.62%to 66.37%,and the bioaccessibility of curcumin was reduced from 72.00%to 65.57%.(2)The feasibility of whey protein isolate nanofibril fabrication on glycation and the different levels of ethanol-water mixtures at p H 2.0 and 85°C as well as the preparation of Pickering emulsions with ethanol-modified nanofibrils were investigated.The glycated WPI was reported for the first time to be fibrillated into semiflexible and highly flexible worm-like fibrils in different ethanol-water solutions.As the ethanol concentration increased from 0%to 50%,the height of whey protein isolate nanofibrils decreased from 3.3±0.2 nm to 2.0±0.4 nm,and the height of glycosylated protein nanofibrils decreased from 3.7±0.3 nm to 2.7±0.2 nm.At the same ethanol level,there was no significant difference between the height of them.Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to monitor the composition and the set of polypeptide changes during the reaction.Results support that worm-like nanofibrils derived from the glycated protein were built by hydrolyzed peptides,with the MW decreasing during the reaction.Based on the thioflavin T assay,the effect of different levels of ethanol on glycated WPI polypeptide fibrillation may contribute to steric restrictions and disrupt hydrophobic interactions.Through DPPH free radical scavenging ability,total antioxidant capacity analysis and ABTS free radical scavenging ability test on ethanol-induced modified protein nanofibrils,it was concluded that ethanol-induced modification enhanced the antioxidant capacity of the original protein nanofibrils.In addition,WPI and g WPI nanofibrils modified under different ethanol conditions can stabilize Pickering emulsions,and glycosylation can improve the adsorption of protein considerably at the oil–water interface.The g WPI nanofibrils prepared with 50%ethanol can significantly improve the thermal stability and oxidation stability of the Pickering emulsion under acidic conditions.(3)In this study,non-covalent and covalent complexes were prepared by using whey protein isolate/protein fibrils and glycosylated whey protein isolate/protein fibrils with tannic acid,the effects of different compounding methods on the structure and functional properties of protein/protein fibrils,and the properties of its forming and stabilizing Pickering emulsions were studied.Whey protein isolate/protein fibrils-lactose-tannic acid ternary complex were constructed successfully based on the simple dry heat Maillard reaction and the alkali method.Zeta potential data showed that the absolute value of Zeta potential of protein fibrils complex was increased by1-3m V compared to protein complex.The results of fluorescence spectrum and Th T fluorescence analysis showed that protein/protein fibrils and tannic acid covalent interaction were stronger than non-covalent interaction.The addition of tannic acid had a strong fluorescence quenching effect on protein/protein fibrils.Fourier infrared spectroscopy results showed that the interaction between protein/protein fibrils and tannic acid caused a change in the secondary structure of the protein.The introduction of tannic acid in the protein fibrils-tannic acid binary complex reduced the surface hydrophobicity of the protein/protein fibrils from 9324.0 to 1744.6,while the introduction of tannic acid in the glycosylated protein/protein fibrils-tannic acid ternary complex reduced the surface hydrophobicity of glycosylated protein/protein fibrils from 28916 to 388.2.The modification of whey protein isolates and the formation of complexes increased the thermal denaturation temperature of protein by9-15 ~oC.Besides,compared with the non-covalent complex of protein/protein fibrils and tannic acid,the covalent complex of protein/protein fibrils and tannic acid were more conducive to the adsorption of protein/protein fibrils at the oil-water interface of Pickering emulsion.The particle size of the Pickering emulsion was smaller,and the thermal stability,salt ion stability and storage stability of Pickering emulsion were improved significantly.Moreover,the glycosylated protein-tannic acid ternary complex was used to stabilize Pickering emulsion loaded curcumin,and after the emulsion was stored at room temperature in the dark for 5 weeks,the retention rate of curcumin in Pickering emulsion was as high as 95%or more,which played a good role in protecting the degradation of curcumin in the oil phase.