Construction Of Soybean Protein Isolate And Ellagic Acid System With High Antioxidant Activity And Their Stabilization Effect On β-carotene Nanoemulsion

Plant protein is widely used in delivery systems instead of synthetic emulsifiers due to its advantages.However,its weak antioxidant capacity limits its application.The addition of extra synthetic antioxidants to the emulsion system will affect the safety of the emulsion.Therefore,the development of protein products with antioxidant properties is urgent.Currently,common methods to improve antioxidant capacity mainly include chemical grafting of polyphenols.However,the grafting rate is low and does not achieve good antioxidant results.Because ultrasound has sonochemical and mechanical effects,it provides the possibility of improving grafting rate and antioxidant capacity.Soy protein isolate（SPI）is often used as a carrier material to construct delivery systems to protect and improve food functional factors due to its availability,high nutritional value,low cost,and rich functional properties（solubility,emulsification,foaming,and gelling）.Ellagic acid has been shown to enhance protein stability and antioxidant capacity.Therefore,this study used SPI and pomegranate peel ellagic acid（EA）as research objects,prepared non-covalent/covalent composites using different methods,characterized their structure and physicochemical properties,and used them as carrier materials to construct a nanoemulsion delivery system to evaluate their stability toβ-carotene.The main research content and conclusions are as follows:（1）Research on the preparation of pomegranate peel ellagic acid assisted by ultrasound:Using pomegranate peel as raw material,ethanol crude extract was obtained.The optimal single-factor conditions for ultrasound-assisted preparation of ellagic acid from crude extract were ultrasound time of 40 min,ultrasound temperature of 60℃,and ultrasound power of 285 W.The conditions were optimized by response surface methodology to obtain the conditions of 42 min,58℃,and 275.5 W,and the yield of ellagic acid was 0.064 g/g dw.After the crude extract obtained by ultrasound treatment was further purified step by step by macroporous resin and semi-preparative liquid chromatography,it was found that the antioxidant activity of the extract increased significantly with the increase of ellagic acid purity,and showed purity dependence,and the purity could reach 95.3%.By analyzing the stability of punicalin and punicalagin under ultrasound treatment,it was found that the pathway for the former to generate ellagic acid was the chemical bond（C-C）of punicalin was broken,and the latter may contain two pathways:1)the chemical bond（C-C）of punicalagin was broken to generate ellagic acid;2)the degradation product first generated hexahydroxydiphenic acid and then underwent intramolecular esterification to generate ellagic acid.（2）The interaction between SPI and EA was studied by multispectral analysis and molecular docking simulation:This paragraph describes the study of the interaction between SPI and EA through multispectral analysis and molecular docking simulation.Using the quenching model,it was shown that the fluorescence quenching rate(5.8×10¹¹L/（mol·s）-1.18×10¹² L/（mol·s）)was higher than the maximum value controlled by diffusion collision(2×10¹⁰ L/（mol·s）),proving that EA has a static quenching effect on the fluorescence of SPI,and hydrophobic interaction dominates this interaction process,in which the binding of tyrosine（Tyr）residues plays a dominant role in the interaction,rather than tryptophan（Trp）residues.Secondary structure analysis showed that EA caused the SPI structure to become looser,with an increase inα-helix content from 46.85%to 50.01%,a decrease inβ-sheet content from 12.33%to 9.68%,a decrease inβ-turn content from 15.62%to 10.33%,and an increase in free coil content from 25.20%to 29.98%.Molecular docking simulation showed that there were ten active amino acid sites involved in the interaction between SPI and EA,including hydrophobic interaction and hydrogen bond interaction.（3）SPI-EA cojugate was prepared by the free radical method:This paragraph describes the use of free radical method to enhance the interaction between SPI and EA,and the preparation of SPI-EA cojugate.The study found that the optimal conditions for preparing cojugate by free radical method were:SPI 1 g,EA 60 mg,grafting temperature 50℃,and grafting time 24 h.The generation of new peaks in the infrared spectrum of SPI（3560 nm,1710 nm,1620 nm,and 1340 nm）and the decrease in the content of free amino and thiol groups both indicate the successful grafting of EA,and the covalent connection of EA will reduce theα-helix content and increase the random coil content in the secondary structure of SPI,making the protein structure more loose.Compared with the mixture of SPI and SPI-EA,the cojugate prepared by covalent grafting of SPI-EA for 24 h has better surface hydrophobicity（increased by 41.7%and 15.1%,respectively）,emulsifying stability（increased by 146.1%and 1.8%,respectively）,and antioxidant activity（increased by 121.4%and 29%m M/L Trolox Equivalent,respectively）.At the same time,the covalent grafting of EA improved the thermal stability of SPI.（4）U-SPI-EA conjugate was prepared by ultrasonic-assisted radical method:Using ultrasound to further enhance the covalent interaction between SPI and EA,ultrasound-assisted SPI-EA cojugate（U-SPI-EA cojugate）were prepared.The optimal grafting conditions were found to be ultrasound power of 380 W,ultrasound temperature of 50℃,and ultrasound time of 75 min.Compared with the cojugate prepared by traditional free radical method（21.3%）,the grafting rate of the cojugate prepared by ultrasound-assisted method（23.5%）was significantly improved.Theα-helix content in the secondary structure was lower and the random coil content was higher,the surface hydrophobicity was higher,and the spatial structure was looser.Although both grafting methods significantly improved the emulsifying properties and thermal stability of SPI,U-SPI-EA cojugate had the best antioxidant activity（72.9μg/mL TE）.（5）This study investigated the preparation ofβ-carotene nanoemulsions stabilized by different free radical grafting methods of cojugate,including SPI,SPI-EA mixture,SPI-EA cojugate,and U-SPI-EA cojugate:The study found that compared with SPI,SPI-EA mixture,and SPI-EA cojugate,theβ-carotene emulsion prepared by U-SPI-EA cojugate had better salt ion stability and thermal stability.In addition,the UV and thermal stability ofβ-carotene in the emulsion prepared by U-SPI-EA covalent cojugate were significantly enhanced,and the bioavailability was significantly improved.