Study On Protein-based Complexes And Assembled Particles For Protection Of Bioactives And The Interaction Between Proteins And Bioactives

Folate,an important B-group vitamin,participates in many metabolic pathways in the human body.However,both folate and folic acid are sensitive to environmental factors including light,oxygen,heat,and low p H,and degrade to form inactive products.The stability of folic acid can be improved by supplementation of low-molecular-weight antioxidants,complexation with proteins,and encapsulation.At present,the structure-activity relationship and mechanism of antioxidants for protection of folic acid are still in the stage of exploration.Meanwhile,few studies have paid attention to the protective role of supplemental bioactives and changes in their structure and function.As an important class of biological macromolecules,proteins have the properties of ligand binding,self-assembly and complexation with polysaccharides,and are considered as good carrier materials for encapsulation and protection of bioactive components.Under adverse environment conditions,the oxidative degradation of bioactive components will induce changes in the composition,structure and biological activity of the protein carriers,though its mechanism is not clear.In this study,soluble protein-ligand complexes and alcohol-soluble protein hollow/solid particles were prepared based on the study of the interaction among the bioactive components.Furthermore,protein-bioactive interation,and the instabilization/stabilization mechanisms were explored.These results may provide a suggestion for the design of appropriate protein carriers.Firstly,ultraviolet light was used as an environmental factor to induce the decomposition of folic acid.Antioxidants with different molecular structures were mixed with folic acid,and the structure-activity relationship of antioxidants for protection of folic acid was studied using fluorescence spectroscopy and high performance liquid chromatography(HPLC).Moreover,the DPPH and ABTS assays were used to study the antioxidant capacities under irradiation,revealing the impact of degradation on biological activity.On this basis,the protection mechanism was discussed in depth.It was found that the photo-stability of folic acid increased by the supplementation with antioxidants.The protective effect and antioxidant activity of phenolic compounds were higher than those of the compounds with olefin double bonds.The protective effect of various antioxidants against photodecomposition of folic acid ranked in order of effectiveness caffeic acid>catechol>quercetin>ferulic acid≈p-coumaric acid>EGCG>resveratrol>curcumin>p-benzoquinone>guaiacol>kojic acid>5-hydroxy-pnaphthoquinone>salicylic acid>β-ionone>retinol>linoleic acid>trans-stilbene.ABTS×+ scavenging capacity of caffeic aicd,p-benzoquinone,acetylsalicylic acid and β-ionone increased significantly during UVA irradiation,while there was a decrease in ABTS×+ scavenging capacity for ferulic acid,kojic acid and salicylic acid.ABTS×+ scavenging ability of the others remained constant during irradiation.The protective effect of antioxidants on folic acid was more dependent on their activity after irradiation than the initial activity.The increase in absorbance in the UVA region of antioxidants under UVA irradiation also contributed to the effective protection.Next,resveratrol was selected to study the inhibition of folic acid photodegradation and its induced β-lactoglobulin structural change by fluorescence spectroscopy,HPLC,absorbance spectroscopy and circular dichroism spectroscopy(CD).Meanwhile,the absorbance spectroscopy,HPLC and ABTS assay were used to study the transformation and degradation of resveratrol induced by folic acid photodegradation as well as the impact on its antioxidant activity.It was found that resveratrol could inhibit the folic acid decomposition and control the decomposition process,depending on the polyphenol concentration and addition time.Resveratrol at 1 μM added before irradiation could inhibit the decomposition of folic acid at 10 μM.Resveratrol at 10 μM added during irradiation could inhibit the decomposition process of folic acid at 10 μM.Resveratrol inhibited the decomposition of folic acid to form 6-formylpterin and p-aminobenzoylglutamate and the transformation from p-aminobenzoylglutamate to its decomposition products.Moreover,resveratrol could also inhibit the unfolding of betalactoglobulin caused by the folic acid decomposition.Transformation of resveratrol was accelerated by photodecomposition of folic acid,while resveratrol still retained its antioxidant capacities.Antioxidant activity of resveratrol during irradiation was important for the protective effect.Then,caffeic acid,the most effective protector was selected as another bioactive component to evaluate the protein damage induced by folic acid photodegradation and the ability of caffeic acid to inhibit protein damage.Folic acid photodecomposition-induced protein oxidation mechanism was also explored.Quantitative and qualitative analysis of bioactive components as well as the degradation products were performed using HPLC and LC-MS.The stabilization mechanism was also revealed.β-Lactoglobulin,α-lactalbumin,bovine serum albumin and β-casein were studied as model proteins.Results showed that the increase in protein carbonyls caused by folic acid photodegradation ranked as follows: β-LG>BSA>α-LA>β-CS,and the tryptophan loss ranked in the order BSA>β-LG>α-LA>β-CS.Folic aicd photodecomposition resulted in the reduction of surface and total free sulfhydryls content of β-LG and BSA,while the total sulfhydryls content remained invariable,indicating a conversion between free thiol groups and disulfide bonds instead of formation of other oxidized species.In the presence of folic acid,unfolding of β-LG and formation of β-LG dimers,the loss of α-LA as a monomeric state,and the formation of BSA trimers and tetramers and reduction in the total secondary structure content of BSA were observed,while the secondary structure of β-CS didn’t change after irradiation.Results indicated that triplet excited state of folic acid directly oxidized the amino acids in proteins via electron transfer pathway,giving corresponding amino acid radicals cation and folic acid radical anion,further leading to degradation,aggregation and unfolding of proteins.Co-stabilization of proteins,folic acid and caffeic acid under UVA irradiation was observed.The protective effectiveness of the four protein complexes on folic acid and caffeic acid was exactly the same,ranking in the order β-CS≈α-LA>β-LG>BSA.Caffeic acid and its product,esculetin,inhibited the folic acid photodecomposition and its induced protein composition and structure change via electron transfer pathway.The antioxidant capacities of the proteins also play a role in improving the stability of folic acid.Folic acid acts as a UVA absorber and hence reduces the absorption of UVA light by caffeic acid.Besides,the remarkable antioxidant capacity and absorbance in the UVA region of esculetin contributed to improved stability of caffeic acid.Finally,hydrophilic folic acid and caffeic acid were co-encapsulated into chitosan-coated hollow zein nanoparticles prepared by using calcium phosphate carbonate as a sacrificing template,in comparison with solid nanoparticles without addition of calcium ions.The interactions among the chemicals were characterized by infrared and fluorescence spectroscopy.The partitioning and distribution of the bioactive components in nanoparticles were analyzed through ultrafiltration centrifuge tube and ultracentrifugation methods.The photostability and antioxidant activity of the bioactive components were also studied.And the advantages and disadvantages of two carriers were revealed.The optimized hollow and solid nanoparticles exhibited a size of 169.4 nm and 98.1 nm with a PDI of only 0.13 and 0.19 and a ζ-potential of +41.1 and +40.8 m V,respectively,indicating their small and uniform dimension with great colloidal stability.The encapsulation of 0.15 m M folic acid and 0.03 m M caffeic acid did not induce expansion of particle size of hollow particles,while it dramatically increased particle size of solid particles to 137 nm.Folic acid complexed to chitosan first and then was coated on the surface of zein nanoparticles,while caffeic acid was encapsulated in the hollow core,driven by coordination with calcium ions.The co-encapsulation of folic acid and caffeic acid within nanoparticles masked the antioxidant activity of caffeic acid but significantly improved their photostability against UVA light.To sum up,the supplementation with antioxidants can improve the photostability of folic acid.The activity and UVA absorbance of antioxidants after irradiation play a more decisive role in protecting folic acid than the antioxidants themselves.Antioxidants inhibited the photodegradation of folic acid and thus resulting protein structure and composition changes.The transformation and degradation of resveratrol were accelerated,while the photostability of caffeic acid was significantly improved.The zein hollow nanoparticles coated with chitosan using calcium phosphate as a sacrificing template could be used for encapsulation and protection of hydrophilic bioactive components.