Study On The Interaction Of Dietary Polyphenols With Soybean Protein And The Functional Properties Of Their Complexes

Polyphenols are highly effective antioxidants widely found in plant foods.It can be added to processed foods as a dietary supplement or functional ingredient.Proteins and polyphenols often coexist in foods,and the interaction between them has a certain influence on protein conformation,processing characteristics,nutritional properties and physiological activities of polyphenols.The interaction can further affect the structure and quality of food.The reported studies of protein-polyphenol interactions are mostly based on protein models such as milk proteins or enzymes.Soybean protein is an important dietary protein with good physiological function and processing characteristics.At present,there are few studies on the interaction between soy protein and polyphenol.The interaction mechanism between soy protein and polyphenols,as well as the structure,nutrient properties and functional properties of the complexes need to be further studied.Through the research of covalent and non-covalent interaction between soybean protein and polyphenols,by analyzing the interaction mechanism and the effects on the activity and functional properties of proteins and polyphenols,this study aims to provide better theoretical support for the application of soybean protein-polyphenol complex as a functional component of high value-added food and the development of soybean protein products containing polyphenol.At the same time,the effect of soybean protein-polyphenol complex as bioactive material embedding material is discussed,in order to expand its application in the field of functional materials.The research mainly includes the following four parts:1.Research on the non-covalent interaction between soybean 7S globulin and three polyphenols.Firstly,the mechanism and difference of action of non-co valent interaction between soybean protein and different polyphenols were studied.The experimental subjects were soybean 7S globulin and three different kinds of polyphenols,gallic acid(GA),chlorogenic acid(CA)and epigallocatechin gallate(EGCG).The quenching effect of polyphenols on the fluorescence of 7S globulin was studied by fluorescence spectra,and the effects of polyphenol binding on amino acid residues and peptide chains in primary structures of protein were discussed.Changes in the secondary structure of 7S globulin by polyphenol binding were determined by circular dichroism spectra.With the combined application of fluorescence thermodynamics and isothermal titration calorimetry technology,various thermodynamic parameters in the binding process of polyphenols to 7S globulin were measured to determine the type of interaction force.Finally,molecular simulation docking was used to intuitively observe the binding state of polyphenols on 7S globulin,and the previous experimental results were simulated and verified.The research findings showed:(1)GA,CA and EGCG had strong fluorescence quenching effects on the fluorescence of 7S globulin.The binding of polyphenols has a great effect on the tryptophan residues of the protein.The interaction could reduce the hydrophobicity of protein surface.The more polyphenol hydroxyl groups were introduced,the weaker the hydrophobicity of protein surface would be.(2)Polyphenol binding caused the decrease of ?-helix and increase of irregular crimp in the secondary structure of 7S globulin,and the protein structure was loose and unfolded.(3)Thermodynamic analysis showed that the interaction between the three polyphenols and 7S globulin belonged to exothermic reaction with spontaneous binding.The fluorescence quenching of GA and EGCG on 7S globulin are both static quenching.The fluorescence quenching of CA on 7S globulin belongs to the coexistence of static quenching and dynamic quenching,or static quenching with scope of action.CA had the strongest binding affinity on 7S globulin.Hydrogen bond and van der Waals force play a major role in the binding process of CA and EGCG with 7S globulin,while the main binding force between GA and protein is hydrophobic.(4)The simulated calculation results of the force by molecular simulation docking were basically consistent with the thermodynamic experimental results.Polyphenols mainly entered the hydrophobic region of proteins and bond to it.2.Study on EGCG covalently modified soybean protein isolate(SPI)based on proteomics technology.In this part,covalent interaction between soybean protein and polyphenols was studied mainly based on molecular level.SPI-EGCG covalent complex was successfully prepared under alkaline conditions.Firstly,changes of protein side chain and EGCG binding amount were analyzed-to discuss the effect of polyphenol binding on SPI side chain.Then,the covalent complex was identified and analyzed by proteomics technology based on Nano-LC-Q-Orbitrap-MS/MS system,the types and number of amino acid modification sites of EGCG in soybean protein isolate were analyzed qualitatively and quantitatively,and the covalent binding rule of EGCG and SPI was expounded.The results showed that:(1)with the increase of EGCG concentration,the content of sulfhydryl group and free amino group of soybean protein isolate decreased,the amount of polyphenol binding increased,and the protein structure had a great influence on the modified rate of side chain.(2)Through mass spectrometry,a total of 51 peptides with EGCG addition reaction were identified.These peptides belonged to soybean storage proteins,functional enzymes,and some unidentified proteins.(3)Among 51 EGCG-modified peptides,the number of peptides containing arginine,histidine,cysteine,and lysine were 40,11,8,and 47,respectively.There were 59 modification sites in total for modified peptides,including 3 cysteine modification sites,6 histidine modification sites,17 arginine modification sites and 33 lysine modification sites.(4)There was one covalent binding sites which can bind to the imidazolyl of His,the amino group of lysine and arginine,and the mercapto group of cysteine of the protein on each of the two phenol rings of EGCG molecule.(5)Amino acid composition,amino acid residue content and reactivity have effects on the ratio of various EGCG covalently binding sites on SPI.Amino acid residues with high content,high reactivity and located on the protein surface are more likely to covalently bind to EGCG.The sequences of amino acid affect the EGCG binding sites,resulting in the phenomenon of one or more covalent binding of EGCG to the same or similar peptides.3.Study on functional properties of SPI-EGCG complexesIn this part,the functional properties of non-covalent and covalent complexes of SPI and EGCG were systematically compared.The physical and chemical properties of the complexes with different interactions were analyzed by spectroscopic,optical,and thermodynamic methods such as circular dichroism spectra,differential scanning calorimetry,particle size distribution,transmission electron microscopy,etc.Through in vitro digestion experiment,and the analysis of following protein digestibility,polyphenol retention rate,amino acid analysis,antioxidant capacity,the effects on functional activities of non-covalent and covalent complexes were compared and discussed.Meanwhile,the effect of covalent binding of EGCG on the allergenic of SPI was also discussed.The results showed that:(1)Both the covalent and non-covalent combination of SPI and EGCG caused changes in SPI secondary structure,in which the amount of ?-helix and random crimp increased.(2)The combination of SPI and EGCG can improve the thermal stability of the complexes.The covalent complex has better thermal stability,and the higher the amount of EGCG binding,the higher the thermal stability.(3)The particle size distribution of covalent complexes is less affected by electrostatic action.(4)SPI-EGCG covalent complexes and non-covalent complexes are prone to form network structures in case that the concentration of EGCG is low and high respectively,and high concentration of EGCG hinders the formation of the network structure of covalent complex.(5)The presence of EGCG all has a negative effect on SPI digestibility,but SPI-EGCG non-covalent complexes are more digestible than covalent complexes,while the covalent complexes have higher stability,polyphenol protection and antioxidant activity.(6)The effect of covalent modification of EGCG on the allergenic of soybean protein was determined,and it was found that polyphenol covalent modification had a reducing effect on the allergenic of SPI.4.Application of SPI-EGCG complexes in the preparation of ?-carotene nanoparticlesIn this part,the feasibility-of SPI-EGCG complexes as carrier material of active material was studied.Using SPI-EGCG non-covalent and covalent complexes and SPI as protein bases,the nanoparticles containing ?-carotene was successfully prepared in combination with soybean soluble polysaccharides.The effects of different protein bases on the functional properties of the nanoparticles were discussed.The results showed that:(1)the protein bases,?-carotene and soybean soluble polysaccharides combined with each other through electrostatic,hydrogen bond and hydrophobic force,and ?-carotene encapsulated nanoparticles were formed.(2)The embedding rate of ?-carotene in SPI-EGCG covalent combination was higher,and it could protect the particle better,increase the rigidity of the granule structure and make it more stable.In addition,it reduced the aggregation of nanoparticles and enhanced the stability of the solution system.(3)The digestibility and stability of protein bases of nanoparticles affect the release rate of?-carotene.The release rate of SPI-EGCG complex protein-based nanoparticles was lower than that of SPI protein-based nanoparticles.The SPI-EGCG covalent complex nanoparticles have the effect of controlled-releasing ?-carotene in the intestinal tract.(4)The dissolution rate of composite particles added with EGCG was higher than that of the unadded composite particles,among which the dissolution rate of SPI-EGCG covalent complex nanoparticles was the highest,indicating that the binding mode of EGCG and SPI affected the solubility of ?-carotene carrier,thus affecting the dissolution behavior of composite particles.Except the common polyphenols,there are diversified other antioxidative compositions in foods.In order to rapidly screen and identify the antioxidant in complex samples and evaluate the effect of processing method on antioxidative compositions in samples,the online HPLC-PDA-Triple-TOF-MS/MS-ABTS system was established in this study.According to online antioxidant screening system,the identification and evaluation of antioxidant ingredients of the Eurotiun cristatum fermenting Angelica dahurica was carried out.The research results show that:(1)The fermentation of Eurotium cristatum improves the oxidation resistance of Angelica dahurica.The antioxidant activity of organic solution extractive in fermentative Angelica dahurica was higher than that in the unfermented extractive.(2)The chemical components of fermentative Angelica dahurica make a great difference by comparing HPLC spectrogram.Various of new chemical compounds with low performance were generated.(3)According to the established HPLC-PDA-Triple-TOF-MS/MS-ABTS system,12 kinds of antioxidative active compound would be screened and identified from the fermentative Angelica dahurica.(4)The fermentation of Eurotium cristatum has small influence on the original antioxidative composition in Angelica dahurica.However,secondary metabolites generated by Eurotium cristatum have a higher antioxidant activity.(5)The online ABTS antioxidant detection system can screen and identify different kinds of antioxidant components in mixture conveniently and quickly.