Mineral-loaded ovalbumin-polyphenol nanoparticles and preparation method and application thereof
阅读说明：本技术 一种负载矿物质的卵清蛋白-多酚纳米颗粒及其制备方法与应用 (Mineral-loaded ovalbumin-polyphenol nanoparticles and preparation method and application thereof ) 是由 苏东晓 黄贞贞 杨欣禧 梁思月 陈乐祺 于 2021-08-06 设计创作，主要内容包括：本发明公开了一种负载矿物质的卵清蛋白-多酚纳米颗粒及其制备方法与应用。该方法包括如下步骤：(1)将卵清蛋白加入到水中,并调节pH值至7.0±0.1,然后静置使蛋白质充分水合,得到卵清蛋白分散液；(2)将多酚加入到水中,并调节pH值至7.0±0.1,得到多酚溶液；(3)将多酚溶液加入到卵清蛋白分散液中,并调节pH值至1.0～10.0,得到卵清蛋白-多酚复合溶液；(4)将矿物质加入到卵清蛋白-多酚复合溶液中,搅拌均匀后离心,取上清,得到负载矿物质的卵清蛋白-多酚纳米颗粒。本发明利用蛋白质的结构特征以及与多酚的相互作用,形成高荷载的蛋白纳米制品,可提高纳米颗粒的稳定性,可用于负载不同的矿物质。(The invention discloses an ovalbumin-polyphenol nanoparticle loaded with mineral substances, and a preparation method and application thereof. The method comprises the following steps: (1) adding ovalbumin into water, adjusting the pH value to 7.0 +/-0.1, and standing to fully hydrate the protein to obtain an ovalbumin dispersion liquid; (2) adding polyphenol into water, and adjusting the pH value to 7.0 +/-0.1 to obtain polyphenol solution; (3) adding the polyphenol solution into the ovalbumin dispersion liquid, and adjusting the pH value to 1.0-10.0 to obtain an ovalbumin-polyphenol composite solution; (4) adding the mineral substance into the ovalbumin-polyphenol composite solution, stirring uniformly, centrifuging, and taking the supernatant to obtain the ovalbumin-polyphenol nanoparticle loaded with the mineral substance. The invention utilizes the structural characteristics of protein and the interaction with polyphenol to form a high-load protein nano product, can improve the stability of nano particles, and can be used for loading different mineral substances.)
1. A preparation method of mineral-loaded ovalbumin-polyphenol nanoparticles is characterized by comprising the following preparation steps:
(1) adding ovalbumin into water, stirring uniformly, adjusting the pH value to 7.0 +/-0.1, and then standing to fully hydrate the protein to obtain an ovalbumin dispersion liquid;
(2) adding polyphenol into water, stirring uniformly, and adjusting the pH value to 7.0 +/-0.1 to obtain a polyphenol solution; wherein the polyphenol is at least one of ferulic acid, gallic acid, epigallocatechin gallate and catechin;
(3) adding the polyphenol solution obtained in the step (2) into the ovalbumin dispersion liquid obtained in the step (1), stirring and mixing uniformly, and adjusting the pH value to 1.0-10.0 to obtain an ovalbumin-polyphenol composite solution;
(4) and (4) adding mineral substances into the ovalbumin-polyphenol composite solution obtained in the step (3), stirring uniformly, centrifuging, and taking supernate to obtain the ovalbumin-polyphenol nanoparticles loaded with the mineral substances.
2. The method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 1, wherein:
the concentration of the egg white protein dispersion liquid in the step (1) is 5 mg/mL-50 mg/mL;
the concentration of the polyphenol solution in the step (2) is 1 mg/m/L-10 mg/mL;
the volume ratio of the egg white protein dispersion liquid to the polyphenol solution in the step (3) is 1-48: 1-4;
the pH value in the step (3) is 4.0-10.0;
the mineral in the step (4) is a mineral containing at least one of calcium, potassium, magnesium, sulfur, iron, copper, iodine, manganese and zinc.
3. The method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 2, wherein:
the concentration of the egg white protein dispersion liquid in the step (1) is 10 mg/mL;
the concentration of the polyphenol solution in the step (2) is 5 mg/L;
the volume ratio of the egg white protein dispersion liquid to the polyphenol solution in the step (3) is 2-6: 1;
the pH value in the step (3) is 6.0-10.0;
the mineral in the step (4) is a mineral containing at least one of calcium, potassium, magnesium and sulfur elements.
4. The method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 3, wherein:
the volume ratio of the egg white protein dispersion liquid to the polyphenol solution in the step (3) is 4: 1;
the pH value in the step (3) is 6.0 +/-0.1;
the mineral in the step (4) is a multi-vitamin tablet 21.
5. The method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 1, wherein:
the mass ratio of the mineral substances to the ovalbumin in the step (4) is 0.15-0.2: 1.2.
6. the method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 5, wherein:
the mass ratio of the mineral substances to the ovalbumin in the step (4) is 0.2: 1.2.
7. the method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 1, wherein:
in the step (4), the step (c),
when the loaded mineral is a mineral containing calcium element, the polyphenol in the step (2) is ferulic acid;
when the loaded mineral is a mineral containing potassium element, the polyphenol in the step (2) is epigallocatechin gallate;
when the loaded mineral is a mineral containing magnesium element, the polyphenol in the step (2) is catechin;
when the loaded mineral is a mineral containing elemental sulfur, the polyphenol in step (2) is gallic acid.
8. The method of preparing mineral-loaded ovalbumin-polyphenol nanoparticles of claim 1, wherein:
the conditions for fully hydrating the protein by standing in the step (1) are as follows: standing at the low temperature of 4 ℃ for 12-24 hours;
the water in the steps (1), (2) and (4) is deionized water;
the stirring conditions in the steps (1), (2) and (4) are as follows: continuously stirring for more than 2h at room temperature;
the regulators used for regulating the pH value in the steps (1), (2) and (3) are 0.5-1 mol/L HCl solution and 0.5-1 mol/L NaOH solution;
the stirring conditions in the step (3) are as follows: stirring at 400-1200 rpm for 5-30 min;
the stirring conditions in the step (4) are as follows: stirring at 700-1500 rpm for 10-30 min;
the centrifugation conditions in the step (4) are as follows: centrifuging at 2000-8000 rpm for 5-20 min.
9. A mineral-loaded ovalbumin-polyphenol nanoparticle, characterized by: prepared by the method of any one of claims 1 to 8.
10. Use of the mineral-loaded ovalbumin-polyphenol nanoparticles of claim 9 in a food, health product, cosmetic or pharmaceutical product.
Technical Field
The invention belongs to the technical field of food embedding, and particularly relates to an ovalbumin-polyphenol nanoparticle loaded with mineral substances, and a preparation method and application thereof.
Background
Minerals are one of six nutrients required by animals, including macroelements and trace elements. The human body needs minerals to maintain normal life activities, and most people suffer from various diseases due to mineral deficiency. In modern society, people often take dietary supplements such as various vitamins or minerals to improve health conditions, but the vitamins and minerals are affected by other nutrients in the absorption process of the human body, so that the using effect is reduced, which is also an important problem facing at present. Therefore, there is a need to develop an effective preparation which is natural and healthy, can be loaded with various minerals, and can be used for health products or functional foods.
Ovalbumin (OVA) is the most abundant protein in egg white, and has the functional characteristics of foamability, gelling property, water retention, self-assembly and the like. It has been shown that protein complexes with other natural products can improve the stability of the system. It has been found that the production of ovalbumin-coupled ferulic acid reagents, which are formed by chemical cross-linking, has been established and that many studies have been made on protein-polyphenol systems, e.g.different pH, CaCl2Zein-ferulic acid interaction at concentrationThe research on the action, the structural characterization and the physicochemical property, but the load rate of the carrier with high calcium ion load rate and other mineral substances is not necessarily high. The deep processing rate of the existing eggs is relatively low, egg white protein in the eggs is not reasonably applied, polyphenol and mineral substances have multiple functions, but the utilization rate is low, and the mineral substances (such as calcium, potassium, magnesium, sulfur and the like) are seriously lost due to improper carriers, so that the mineral substance resource waste is caused, and the like. However, no reports have been made so far about loading different minerals (calcium, potassium, magnesium and sulfur) by using ovalbumin-ferulic acid, ovalbumin-gallic acid, ovalbumin-EGCG and ovalbumin-catechin nanoparticle carriers.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method of mineral-loaded ovalbumin-polyphenol nanoparticles.
The invention also aims to provide the mineral-loaded ovalbumin-polyphenol nanoparticles prepared by the method.
It is a further object of the present invention to provide the use of said mineral-loaded ovalbumin-polyphenol nanoparticles.
The purpose of the invention is realized by the following technical scheme:
a preparation method of mineral-loaded ovalbumin-polyphenol nanoparticles comprises the following preparation steps:
(1) adding ovalbumin into water, stirring uniformly, adjusting the pH value to 7.0 +/-0.1, and then standing to fully hydrate the protein to obtain an ovalbumin dispersion liquid;
(2) adding polyphenol into water, stirring uniformly, and adjusting the pH value to 7.0 +/-0.1 to obtain a polyphenol solution; wherein the polyphenol is at least one of Ferulic Acid (FA), Gallic Acid (GA), epigallocatechin gallate (EGCG), and Catechin (CT);
(3) adding the polyphenol solution obtained in the step (2) into the ovalbumin dispersion liquid obtained in the step (1), stirring and mixing uniformly, and adjusting the pH value to 1.0-10.0 to obtain an ovalbumin-polyphenol composite solution;
(4) and (4) adding mineral substances into the ovalbumin-polyphenol composite solution obtained in the step (3), stirring uniformly, centrifuging, and taking supernate to obtain the ovalbumin-polyphenol nanoparticles loaded with the mineral substances.
The conditions for fully hydrating the protein by standing in the step (1) are as follows: standing at the low temperature of 4 ℃ for 12-24 hours; preferably: standing at 4 deg.C for 24 hr.
The water used in steps (1), (2) and (4) is preferably deionized water.
The stirring conditions in the steps (1), (2) and (4) are as follows: stirring at room temperature for more than 2 h.
The concentration of the egg white protein dispersion liquid in the step (1) is 5 mg/mL-50 mg/mL; preferably 10 mg/mL.
The regulator for regulating the pH value in the steps (1), (2) and (3) is preferably 0.5-1 mol/L HCl solution and 0.5-1 mol/L NaOH solution.
The concentration of the polyphenol solution in the step (2) is 1 mg/m/L-10 mg/mL; preferably 5 mg/L.
The volume ratio of the egg white protein dispersion liquid to the polyphenol solution in the step (3) is (1-48): (1-4); more preferably (2-6) 1; still more preferably 4: 1.
The pH value in the step (3) is preferably 4.0-10.0; more preferably 6.0 to 10.0; still more preferably 6.0. + -. 0.1.
The stirring conditions in the step (3) are as follows: stirring at 400-1200 rpm for 5-30 min.
The mineral in the step (4) is a mineral which can be loaded by the ovalbumin-polyphenol composite solution, can be a mineral containing a single element, such as potassium chloride, calcium chloride and the like, and can also be a mineral containing more than two elements (the loaded minerals are required not to have chemical reaction, have no influence on a loading system, and have no influence on the loading rate of each mineral); preferably a mineral containing at least one of the elements calcium, potassium, magnesium, sulfur, iron, copper, iodine, manganese and zinc; further preferably a mineral containing at least one of calcium, potassium, magnesium and sulfur; still more preferably minerals containing calcium, potassium, magnesium, sulfur and other elements; wherein, the other elements are at least one of iron, copper, iodine, manganese and zinc elements; most preferably a multi-dimensional element sheet (21).
The mass ratio of the mineral substances to the ovalbumin in the step (4) is 0.15-0.2: 1.2; preferably 0.2: 1.2.
in the step (4), the step (c),
when the loaded mineral is a mineral containing calcium element, the polyphenol in the step (2) is preferably Ferulic Acid (FA);
when the loaded mineral is a mineral containing potassium element, the polyphenol in the step (2) is preferably epigallocatechin gallate (EGCG);
when the loaded mineral is a mineral containing magnesium element, the polyphenol in the step (2) is preferably Catechin (CT);
when the mineral to be loaded is a mineral containing elemental sulfur, the polyphenol in step (2) is preferably Gallic Acid (GA).
The stirring conditions in the step (4) are as follows: stirring at 700-1500 rpm for 10-30 min.
The centrifugation conditions in the step (4) are as follows: centrifuging at 2000-8000 rpm for 5-20 min; preferably: centrifuging at 4000rpm for 5-15 min.
Mineral-loaded ovalbumin-polyphenol nanoparticles prepared by any one of the methods described above.
The mineral-loaded ovalbumin-polyphenol nanoparticles are applied to the fields of food, health products, cosmetics or medicines (medicines).
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention mixes the egg white protein aqueous solution and the polyphenol aqueous solution, obtains the egg white protein-polyphenol nano particles with smaller particle size and larger potential absolute value by regulating and controlling the adding proportion of the egg white protein aqueous solution and the polyphenol aqueous solution and the pH value of the composite solution, and then utilizes the egg white protein-polyphenol nano particles to load different mineral substances, thereby having good dispersibility and biocompatibility.
(2) The invention expands the application of the eggs to a certain extent, expands the cognition of people on egg protein, solves the problem of excessive yield, increases the social and economic values, and also solves the problems of difficult utilization of polyphenol and mineral substances and mineral substance resource waste to a certain extent.
(3) The invention utilizes the structural characteristics of protein and the interaction with polyphenol to form a high-load protein nano product, can greatly improve the stability of nano particles, can successfully obtain high-stability and high-load protein-polyphenol nano particle composite liquid on the premise of not adding any organic solvent, and has good protection effect on calcium, potassium, magnesium and sulfur ions.
(4) The ovalbumin-polyphenol nanoparticles selectively loaded with specific minerals have the advantages of high embedding rate, good stability, simple and safe preparation, low cost, low energy consumption, controllable operation, avoidance of mineral waste and the like, are suitable for large-scale industrial production and processing, and have wide application space in the industries of foods, health care products, daily chemical products and medicines.
Drawings
FIG. 1 is an appearance diagram and a particle size diagram of an ovalbumin-polyphenol nanoparticle solution obtained when the concentration of Ovalbumin (OVA) is 10mg/mL and the concentration of polyphenol is 5mg/mL, both are at pH 7.0 + -0.1, and the mixing ratio is (48:1) to (1:4) (in the diagram, FA: ferulic acid; GA: gallic acid; CT: catechin; EGCG: epigallocatechin gallate); wherein: (a) the (b), (c) and (d) are respectively the appearance diagrams of the ovalbumin-ferulic acid (OVA-FA), the ovalbumin-epigallocatechin gallate (OVA-EGCG), the ovalbumin-gallic acid (OVA-GA) and the ovalbumin-catechin (OVA-CT) nanoparticle solution; (e) and (f), (g) and (h) are particle size diagrams of ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solution nanoparticle solutions, respectively.
FIG. 2 is a potential diagram of an ovalbumin-polyphenol nanoparticle solution obtained when the concentration of Ovalbumin (OVA) is 10mg/mL and the concentration of polyphenol is 5mg/mL, both of which are at pH 7.0. + -. 0.1 at the mixing ratio of (48:1) to (1: 4); wherein: (a) and (b), (c) and (d) are potential maps of ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solutions respectively.
FIG. 3 is an appearance diagram and a particle size diagram of an ovalbumin-polyphenol nanoparticle solution obtained when the concentration of Ovalbumin (OVA) is 10mg/mL, the concentration of polyphenol is 5mg/mL, the mixing ratio is 4:1, and the pH of the composite solution is 1-10; wherein, (a), (b), (c) and (d) are respectively the appearance diagram of the ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solution; (e) and (f), (g) and (h) are particle size diagrams of ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solution nanoparticle solutions, respectively.
FIG. 4 is a potential diagram of an ovalbumin-polyphenol nanoparticle solution obtained when the Ovalbumin (OVA) concentration is 10mg/mL, the polyphenol concentration is 5mg/mL, the mixing ratio is 4:1, and the pH of the composite solution is 1-10; wherein: (a) and (b), (c) and (d) are potential maps of ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solutions respectively.
FIG. 5 is a graph showing the effect of polyphenols on ovalbumin fluorescence intensity at an Ovalbumin (OVA) concentration of 10mg/mL, a polyphenol concentration of 5mg/mL, a mixing ratio of 4:1, and a pH of the complex solution of 6 (the concentration in the graph is the concentration of polyphenols in the OVA-polyphenol complex solution in mg/mL); wherein: (a) and (b), (c) and (d) are fluorescence intensity maps of ovalbumin-ferulic acid (OVA-FA), ovalbumin-epigallocatechin gallate (OVA-EGCG), ovalbumin-gallic acid (OVA-GA) and ovalbumin-catechin (OVA-CT) nanoparticle solutions respectively.
FIG. 6 is a graph showing the effect of polyphenols on the infrared spectra of ovalbumin at an Ovalbumin (OVA) concentration of 10mg/mL, a polyphenol concentration of 5mg/mL, a mixing ratio of 4:1, and a complex solution pH of 6; wherein A is infrared spectrogram of ovalbumin and ovalbumin-polyphenol (OVA-FA, OVA-EGCG, OVA-GA and OVA-CT) nanoparticle solution; b is the infrared amide I-band Gaussian fitting result of the ovalbumin and ovalbumin-polyphenol (OVA-FA, OVA-EGCG, OVA-GA and OVA-CT) nanoparticle solution.
FIG. 7 is a microscopic image of ovalbumin-polyphenol nanoparticles loaded with calcium, potassium, magnesium and sulfide ions when the concentration of Ovalbumin (OVA) is 10mg/mL, the concentration of polyphenol is 5mg/mL, the mixing ratio is 4:1 and the pH of the composite solution is 6.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The acid-base solution for adjusting the pH value is a pH value adjusting agent which is conventional in the field, such as hydrochloric acid, sodium hydroxide and the like; preferably 0.5-1 mol/L HCl solution and 0.5-1 mol/L NaOH solution.
The present invention relates to ovalbumin (biotech grade) purchased from Shanghai Michelin Biotechnology, Inc. (China, Shanghai).
Example 1
(1) Accurately weighing 1g of Ovalbumin (OVA) powder, dispersing the powder in 100g of deionized water (the default density of the deionized water is 1.00mg/mL), continuously stirring for 3h at room temperature, adjusting the pH to 7.0 +/-0.1 by using an acid-base solution, and then placing at 4 ℃ for 12h to fully hydrate the protein so as to obtain an ovalbumin stock solution with the concentration of 10 mg/mL.
(2) 0.5g of Ferulic Acid (FA), epigallocatechin gallate (EGCG), Gallic Acid (GA) and Catechin (CT) powder are respectively weighed and added into 100g of deionized water, and continuously stirred for 2h at room temperature, pH is adjusted to 7.0 +/-0.1 by alkali liquor, and polyphenol is fully and uniformly mixed to obtain polyphenol solution with the concentration of 5 mg/mL.
(3) And (2) mixing the ovalbumin solution obtained in the step (1) with the polyphenol solution obtained in the step (2) according to the volume ratio of 48:1, 24:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:2 and 1:4 (with no polyphenol solution added as a contrast), dropwise adding the polyphenol solution into the ovalbumin solution under magnetic stirring (at room temperature), wherein the magnetic stirring speed is 400-1200 rpm, and the stirring time is 5-30 min, so as to obtain the ovalbumin-polyphenol composite solution.
(4) And finally, respectively adjusting the pH of the ovalbumin-polyphenol composite solution obtained in the step (3) to 1-10 by using acid and alkali liquor (respectively named as pH1, pH2, pH3, pH4, pH5, pH6, pH7, pH8, pH9 and pH 10).
In this example, the change of particle size potential was examined when the ovalbumin concentration was 10mg/mL and the polyphenol concentration was 5mg/mL, both of which were mixed at different volume ratios. The appearance and the particle size of the ovalbumin-polyphenol nanoparticle composite liquid with different volume ratios are shown in figure 1, and the potential is shown in figure 2. The mixed volume ratio of the ovalbumin and the polyphenol is different, and the color of the composite solution is different. With the increase of the proportion of the polyphenol, the particle size and the absolute potential value of the complexing solution are gradually increased, because the interaction between the ovalbumin and the polyphenol occurs, a compound with the size of nanometer is formed, and when the ovalbumin and the polyphenol are mixed according to the volume ratio of 4:1, the particle size and the absolute potential value of the compound are the largest, so the optimal proportion of the mixture of the ovalbumin and the polyphenol is 4:1, which indicates that the nanoparticles are formed most under the condition of quantifying the ovalbumin and the polyphenol.
This example also examined the effect of pH on the particle size potential of ovalbumin-polyphenol nanoparticle composite solutions. The appearance and particle size of the ovalbumin-polyphenol composite solutions with different pH values are shown in figure 3, the potential is shown in figure 4, and the ovalbumin-polyphenol composite solutions with different pH values have different colors. The aggregation of the complex around pH4 is caused by the fact that the isoelectric point of ovalbumin is about 4.5, and mainly because the charge of the ovalbumin around the isoelectric point is almost 0, the intermolecular attraction is strong, and the aggregation phenomenon exists. As can be seen from fig. 4, the particle size of the ovalbumin-polyphenol complex solution is the smallest at pH6, and the absolute value of the potential is the largest, indicating that the complex solution is the most stable at pH6, so the pH optimum condition for complexing ovalbumin and polyphenol is pH6, and the most stable ovalbumin-polyphenol nanoparticles can be formed under this pH condition.
This example further investigated the effect of polyphenols on ovalbumin fluorescence intensity. As shown in fig. 5, the fluorescence intensity of the complexes decreased with increasing concentration, except ovalbumin-catechin, indicating that interaction with polyphenols quenches the fluorescence intensity of OVA, probably due to the development of OVA. There are reports of increased fluorescence intensity of OVA-CT complexes that are under investigation. In addition, the red-shift indicates that the interaction of ovalbumin with polyphenols results in a change in the microenvironment of tryptophan (Trp) and tyrosine (Tyr), exposing tryptophan to a polar environment. In particular, the amplitude of the red shift of OVA-EGCG reached 14.8nm, which was more pronounced than the red shift of the other complexes. It can therefore be concluded that EGCG significantly alters the molecular conformation of OVA. In addition, OVA-GA undergoes a slight blue shift, probably due to pi stacking of the benzene ring of the aromatic amino acid residue in OVA with GA.
This example also investigated the effect of polyphenols on the infrared spectra of ovalbumin:
amide I (1650 cm)-1) And III band (1312 cm)-1) Fig. 6A shows the results of OVA characteristic peaks caused by coupling of C ═ O stretching vibration and N — H bending vibration with C — N stretching vibration, respectively. As can be seen from FIG. 6A, after binding to ferulic acid, gallic acid, catechin, no amide III band of OVA was found, probably because the N-H bending of OVA combined with C-N stretching vibration participated in the interaction with polyphenol. Furthermore, 3298.3cm-1The strong broad peak is generated by coupling N-H stretching vibration and hydrogen bonds, and several protein polyphenol compounds are displaced to different degrees, which shows that the N-H of the ovalbumin and the phenolic hydroxyl interact to form the hydrogen bonds. Furthermore, the peak red-shifted degree of OVA binding to EGCG was greater, probably due to stronger hydrogen bonding between OVA and EGCG.
The secondary structure of the protein can be reflected by the amide I band, and thus Gaussian fitting was performed on the amide I band, and the α -helix (α -helix), β -sheet (β -sheet), β -turn (β -turn), and random coil (random coil) contents of the amide I band were calculated, as shown in FIG. 6B. As shown in FIG. 6B, compared with pure ovalbumin, the addition of ferulic acid and EGCG increases the alpha-helix and beta-sheet content of the complex, and reduces the random coil content, and studies show that beta-sheet is favorable for the formation of hydrogen bonds, and alpha-helix is favorable for the stability of the protein structure, which is probably because part of the random coil in the protein is converted into alpha-helix and beta-sheet, so that the protein structure is more stable. On the contrary, the addition of gallic acid and catechin reduces alpha-helix content, increases random coil content, and reduces stability of nanoparticle system.
Example 2
(1) Accurately weighing 1g of Ovalbumin (OVA) powder, dispersing the powder in 100g of deionized water (the default density of the deionized water is 1.00mg/mL), continuously stirring for 3h at room temperature, adjusting the pH to 7.0 +/-0.1 by using an acid-base solution, and then placing at 4 ℃ for 12h to fully hydrate the protein so as to obtain an ovalbumin stock solution with the concentration of 10 mg/mL.
(2) 0.5g of Ferulic Acid (FA), epigallocatechin gallate (EGCG), Gallic Acid (GA) and Catechin (CT) are respectively weighed and added into 100g of deionized water, and continuously stirred for 2h at room temperature, pH is adjusted to 7.0 +/-0.1 by alkali liquor, and polyphenol is fully and uniformly mixed to obtain polyphenol solution with the concentration of 5 mg/mL.
(3) Mixing the ovalbumin solution obtained in the step (1) and the polyphenol solution obtained in the step (2) according to a volume ratio of 4:1, dropwise adding the polyphenol solution into the ovalbumin solution under magnetic stirring, wherein the magnetic stirring speed is 400-1200 rpm, and the stirring time is 5-30 min, so as to obtain the ovalbumin-polyphenol composite solution.
(4) Respectively adjusting the pH of the ovalbumin-polyphenol nanoparticle composite solution obtained in the step (3) to 6.0 +/-0.1 by using acid-alkali liquor.
(5) Respectively weighing 0.15-0.2 g of mineral (the mineral used in the experiment is a multi-dimensional element sheet (21) purchased from a pharmacy in Guangzhou with the product batch number of P20C 010; the multi-dimensional element sheet (21) contains calcium, potassium, magnesium, sulfur and other elements, and after load measurement, the load rates of the four elements of potassium, calcium, magnesium and sulfur are higher than those of other elements, and the four mineral substances all have polyphenol with the highest load rate corresponding to the four elements, so that the subsequent experiments are carried out on the four elements) and adding the four elements into the nanoparticle composite solution (150mL) obtained in the step (4) under magnetic stirring (the stirring speed is 700-1500 rpm, and the stirring time is 10-60 min), and then centrifuging the obtained nanoparticle composite solution for a certain time (about 10min) at 4000rpm, removing insoluble substances, and obtaining supernatant which is the ovalbumin-polyphenol nanoparticle composite solution loaded with calcium, potassium, magnesium and sulfur ions.
This example examines microscopic images of calcium, potassium, magnesium, and sulfide-loaded ovalbumin-ferulic acid (OVA-FA), ovalbumin-gallic acid (OVA-GA), ovalbumin-epigallocatechin gallate (OVA-EGCG), and ovalbumin-catechin (OVA-CT) nanoparticle complexes. The contents of calcium, potassium, magnesium and sulfur atoms in the sample are detected by SEM-EDS, and the result is shown in FIG. 7: different mineral (calcium, potassium, magnesium and sulfur) loads of the pure ovalbumin are lower than those of the ovalbumin-ferulic acid, the ovalbumin-gallic acid, the ovalbumin-epigallocatechin gallate and the ovalbumin-catechin nanoparticle carrier system. From the results, it is also found that compared with OVA-GA, OVA-EGCG and OVA-CT nanoparticle carriers, the loading rate of OVA-FA on calcium ions is higher (33.9%), the loading rate on other minerals (potassium, magnesium and sulfur) is lower than that of other nanoparticle loading systems, and the loading effect of the OVA-EGCG nanoparticle loading system on potassium ions is best (35.4%), and the loading effect is obviously higher than that of other nanoparticle loading systems; moreover, the OVA-GA nanoparticle loading system has the best loading effect on sulfur ions (32.60%), but is obviously inferior to other nanoparticle loading systems in terms of loading of other minerals, while the OVA-CT nanoparticle loading system has the best loading effect on magnesium ions (13.88%), is obviously higher than other nanoparticle loading systems, but is lower than the corresponding nanoparticle loading systems in terms of loading of other minerals. It can be seen that different minerals (calcium, potassium, magnesium, sulfur) have the corresponding optimal load of OVA-FA, OVA-GA, OVA-EGCG OVA-CT nanoparticle carriers. The research finds that different mineral substances (calcium, potassium, magnesium and sulfur), OVA-FA, OVA-GA and OVA-EGCG OVA-CT nanoparticle carriers have different loading rates, and the addition of polyphenol improves the loading rate of the mineral substances (calcium, potassium, magnesium and sulfur), so that the research result can provide theoretical basis for the mineral substances (calcium, potassium, magnesium and sulfur) to reach the maximum loading rate and utilization rate and avoid the waste of calcium, potassium, magnesium and sulfur ion resources.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.