CN113999403B

CN113999403B - Quaternized soybean protein, quaternized soybean protein-dopamine, and preparation method and application thereof

Info

Publication number: CN113999403B
Application number: CN202111376464.9A
Authority: CN
Inventors: 陈云; 董齐; 陈飞翔; 柯梅芳; 梁萧; 杨心迪
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-08-30
Anticipated expiration: 2041-11-19
Also published as: CN113999403A

Abstract

The invention discloses a quaternized soybean protein, a quaternized soybean protein-dopamine, and a preparation method and application thereof, wherein the method for quaternizing the soybean protein comprises the following steps: dissolving soy protein in an alkaline solution to obtain a soy protein solution; uniformly mixing the soybean protein solution and epoxypropyltrimethylammonium chloride for ring-opening reaction to obtain a ring-opening reaction product; and carrying out post-treatment on the ring-opening reaction product to obtain the quaternized soybean protein. The method for quaternizing the soy protein-dopamine comprises the following steps: dissolving the quaternized soybean protein in deionized water to obtain a quaternized soybean protein solution; uniformly mixing the quaternized soybean protein solution with dopamine and EDC/NHS for crosslinking reaction to obtain a crosslinking reaction product; and carrying out post-treatment on the cross-linking reaction product to obtain the quaternized soybean protein-dopamine. The quaternized soybean protein has broad-spectrum antibacterial property; the quaternized soybean protein-dopamine has an antioxidant function.

Description

Quaternized soybean protein, quaternized soybean protein-dopamine, and preparation method and application thereof

Technical Field

The invention relates to the crossing field of polymer chemistry and tissue engineering, in particular to quaternized soybean protein, quaternized soybean protein-dopamine, and a preparation method and application thereof.

Background

The soybean protein (Soy protein isolate, SPI) is derived from soybean meal, the protein content of the soybean protein is higher than 90 percent, and the soybean protein has excellent biocompatibility and degradability, so the soybean protein has wide application potential in the field of tissue engineering. Under the action of strong hydrogen bonds, disulfide bonds and van der waals force, the molecular chains of the soy protein are folded and tightly combined to form a spherical structure. The soybean protein is insoluble in water and conventional organic solvents, which brings certain difficulties for the application of the soybean protein in the field of biological materials. Meanwhile, the active polypeptide contained in the soybean protein is difficult to exert biological functions under the influence of steric hindrance. Therefore, the modification of the soybean protein to enhance the water solubility is of great significance. Meanwhile, the water solubility of the antibacterial agent is improved, and the chemical modification can endow the synthesized product with broad-spectrum antibacterial property, so that the antibacterial agent has important significance. The removal of excessive active oxygen generated in a damaged microenvironment is necessary for promoting tissue regeneration, so that the oxidation resistance of the protein is obviously improved through modification, and the water-soluble soybean protein derivative is obtained, and has important significance.

However, in the prior art, the soybean protein is rich in source and is renewable, and a new way for researching and developing high-value utilization of the soybean protein conforms to the development concept of green chemistry. Soy protein is insoluble in water and conventional organic solvents, which greatly limits its use. And no protein modification method for endowing the synthesized product with broad-spectrum antibacterial property or improving the antioxidant capacity is available;

therefore, it is necessary to develop chemical synthesis, biological function and medical application research of water-soluble soy protein derivatives, which is not only helpful to fill up the international gap, but also expected to produce great economic benefits.

Disclosure of Invention

The invention aims to provide quaternized soybean protein, quaternized soybean protein-dopamine, and preparation methods and applications thereof. On the basis, the invention synthesizes the quaternized soybean protein-dopamine with the antioxidant function, and further enhances the efficacy and the applicability of the quaternized soybean protein-dopamine in the field of tissue engineering.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a process for the preparation of a quaternized soy protein, said process comprising:

dissolving soy protein in an alkaline solution to obtain a soy protein solution;

uniformly mixing the soybean protein solution and epoxypropyl trimethyl ammonium chloride for ring-opening reaction to obtain a ring-opening reaction product;

and carrying out post-treatment on the ring-opening reaction product to obtain the quaternized soybean protein.

Further, the alkaline solution is 0.5-5 (m/m)% of NaOH by mass, and the final concentration of the soybean protein solution is 2-20 (m/m)%.

Further, the percentage of the added mass of the epoxypropyltrimethylammonium chloride to the dry weight of the soybean protein in the soybean protein solution is more than or equal to 50%.

Further, post-treating the reaction product to obtain the quaternized soy protein, comprising:

adjusting the pH value of the reaction product to 6.5-7.5 by adopting glacial acetic acid, then precipitating the product by adopting an organic solvent, carrying out solid-liquid separation, collecting the solid, washing and drying to obtain the water-soluble quaternized soybean protein.

In a second aspect of the invention, there is provided a quaternized soy protein prepared by the method.

In a third aspect of the invention, there is provided a method of preparing quaternized soy protein-dopamine, the method comprising:

dissolving the quaternized soybean protein in deionized water to obtain a quaternized soybean protein solution;

uniformly mixing the quaternized soybean protein solution with dopamine and EDC/NHS for crosslinking reaction to obtain a crosslinking reaction product;

and carrying out post-treatment on the cross-linking reaction product to obtain the quaternized soybean protein-dopamine.

Further, the final concentration of the quaternized soybean protein solution is 2-20 (m/m)%.

Further, the percentage of the added mass of the dopamine to the dry weight of the quaternized soybean protein is more than or equal to 10%, and the ratio of the mass of the EDC and the mass of the NHS to the mass of the dopamine is (1: 1: 0.5) - (1: 1: 10).

In a fourth aspect of the invention, there is provided quaternized soy protein-dopamine prepared by the method.

In a fifth aspect of the invention, there is provided an application comprising: the quaternary ammonium soybean protein is applied to being used as an antibacterial biological material, and/or the quaternary ammonium soybean protein-dopamine is applied to being used as an antioxidant biological material.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides a quaternized soybean protein, a quaternized soybean protein-dopamine, and a preparation method and application thereof, (1) the quaternized soybean protein and the quaternized soybean protein-dopamine are respectively synthesized by a two-step method by using the soybean protein as a raw material, the reaction conditions are mild, and the environmental pollution is small; (2) after chemical modification, the water solubility of the obtained soybean protein derivative is remarkably improved, and the quaternized soybean protein-dopamine respectively show excellent antibacterial property and antioxidant activity; (3) the obtained quaternized soybean protein and quaternized soybean protein-dopamine belong to brand new bioactive polymers, have good efficacy and applicability, and can be widely and rapidly applied to the field of tissue engineering after being compatible with the existing polymer materials.

Specifically, the method comprises the following steps:

1. the quaternized soybean protein and the preparation method thereof provided by the embodiment of the invention take soybean protein and epoxypropyltrimethylammonium chloride as raw materials, and generate ring-opening reaction under alkaline conditions to generate the quaternized soybean protein. The molecular chain of the soy protein is broken under alkaline conditions, exposing reactive functional groups. At the moment, the amino group on the soybean protein and the epoxy group on the epoxypropyltrimethylammonium chloride carry out ring-opening reaction, and the introduction of the quaternary ammonium group destroys the acting force in the soybean protein molecule, thereby stabilizing the stretched molecular structure of the soybean protein. The synthesis method of the quaternized soybean protein provided by the invention obviously enhances the water solubility of the soybean protein and expands the application of the quaternized soybean protein in the field of biological materials. On the other hand, quaternized soy proteins also exhibit excellent antibacterial properties. The antibacterial mechanism of the quaternized soybean protein is independent of a biological enzyme system of bacteria, so that the risk of drug resistance does not exist, and the quaternized soybean protein is very suitable for patients infected by drug-resistant bacteria and patients with high infection risk. The results of the antibacterial experiments show that: the quaternized soybean protein has obvious antibacterial effect on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus.

2. The quaternized soybean protein-dopamine and the preparation method thereof provided by the embodiment of the invention have the advantages that the quaternized soybean protein and dopamine are used as raw materials for the first time, and EDC/NHS is used as a cross-linking agent to react to obtain the quaternized soybean protein-dopamine with excellent antioxidant activity. The quaternized soybean protein-dopamine has strong antioxidant capacity and is used as a bulk antioxidant polymer, and the quaternized soybean protein-dopamine can be added into various existing material systems, such as chitosan composite hydrogel, gelatin composite sponge, polycaprolactone composite nanofiber and the like, so that the existing materials are endowed with the commonly-lacking antioxidant function. The DPPH clearance test results show that: the clearance rate of 0.1mg/mL quaternized soybean protein-dopamine to DPPH reaches 59.4%, and the in vitro antioxidant effect is good.

ROS scavenging experimental results show that: with the increase of the concentration of the quaternized soybean protein-dopamine, the intracellular ROS content is obviously reduced, and the quaternized soybean protein-dopamine has the in vivo antioxidant potential.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a total reflection Fourier transform infrared spectrum and nuclear magnetic resonance hydrogen spectrum of quaternized soy proteins and quaternized soy protein-dopamine obtained in examples 1 and 4; FIG. 1a is a total reflection Fourier transform infrared spectrum of the quaternized soy protein obtained in example 1; FIG. 1b is a total reflectance Fourier transform infrared spectrum of quaternized soy protein-dopamine obtained in example 4; FIG. 1c is a NMR hydrogen spectrum of the quaternized soy protein obtained in example 1; FIG. 1d is a NMR spectrum of quaternized soy protein-dopamine obtained in example 4;

FIG. 2 is a graph showing the degree of substitution of different masses of glycidyltrimethylammonium chloride for quaternized soy protein as reacted in examples 1-5 and comparative example 1;

FIG. 3 is the result of DPPH scavenging experiments corresponding to quaternized soy protein-dopamine when different qualities of dopamine of examples 6-10 and comparative example 2 are involved in the reaction;

FIG. 4 is the results of an antimicrobial experiment with quaternized soy protein obtained in example 2;

FIG. 5 is the results of the DPPH scavenging assay for the quaternized soy protein-dopamine at different concentrations obtained in example 9;

FIG. 6 is the results of the quaternized soy protein-dopamine scavenging intracellular ROS assay obtained in example 9;

FIG. 7 is a flow chart of a method of making a quaternized soy protein provided by embodiments of the present invention;

fig. 8 is a flowchart of a method for preparing quaternized soy protein-dopamine according to an embodiment of the present invention.

Detailed Description

The present invention will be specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

It will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or be indirectly on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.

It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "first," "second," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, refer to an orientation or positional relationship indicated in the drawings for convenience in describing the present application and to simplify description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be operated in a particular manner, and is not to be construed as limiting the present application.

In addition, in the description of the present application, "a plurality" or "a plurality" means two or more unless specifically defined otherwise.

The technical scheme of the application has the following general idea:

according to an exemplary embodiment of the present invention, there is provided a method of preparing quaternized soy protein, as shown in fig. 7, the method comprising:

step S101, dissolving soy protein in an alkaline solution to obtain a soy protein solution;

in the step S101, the alkaline solution is NaOH with the mass fraction of 0.5-5%; if the concentration of the solution less than 2 percent is very low, the reaction is not facilitated, and if the concentration of the solution more than 20 percent is relatively high, the dissolution is incomplete. 0.5-20% NaOH can be replaced by strong alkaline solution, such as urea, urea/alkali and the like;

the final concentration of the soybean protein solution is 2-20 (m/m)%. The soy protein solution is preferably at a concentration of 6 (m/m)%.

Stirring at room temperature for 10-60min to accelerate dissolution;

step S102, uniformly mixing the soybean protein solution and epoxypropyltrimethylammonium chloride for ring-opening reaction to obtain a ring-opening reaction product;

in the step S102, the percentage of the added mass of the epoxypropyltrimethylammonium chloride to the dry weight of the soybean protein in the soybean protein solution is more than or equal to 50%. Preferably, the added mass of the epoxypropyltrimethylammonium chloride is 50-300% of the dry weight of the soybean protein in the soybean protein solution. The mass ratio is for full reaction, if the added mass of the epoxypropyltrimethylammonium chloride is less than 50% of the dry weight of the soybean protein, the substitution is lower, and grafted quaternary ammonium salt groups are less; if the added mass of the epoxypropyltrimethylammonium chloride is more than 300% of the dry weight of the soybean protein, the reaction is close to saturation, and raw material waste exists. Experimental results figure 2 shows that the degree of substitution increases with increasing mass of the added glycidyltrimethylammonium chloride, 72% when the glycidyltrimethylammonium chloride is increased to 200% of the dry weight of the soy protein, and that the degree of substitution changes very little as the amount of reaction of the glycidyltrimethylammonium chloride continues to increase, indicating that the reaction is substantially saturated.

In order to fully react, stirring at room temperature for 3-48 h in the reaction process.

And step S103, carrying out post-treatment on the ring-opening reaction product to obtain the quaternized soybean protein.

The step S103 specifically includes:

Other calculations can also be used to adjust the pH in the above technical scheme, such as dilute hydrochloric acid;

in the technical scheme, the purpose of adjusting the pH value to 6.5-7.5 is to neutralize the alkaline solution; below 6.5, the product contains acidic solvent, needs to be washed to neutrality after subsequent filtration, and is wasted; above 7.5, the product contains alkaline solvent, needs to be washed to neutrality after subsequent filtration, and is wasted; the preferred pH is 7.0.

The organic solvent is one or more of methanol, ethanol, acetone, diethyl ether, petroleum ether and cyclohexane.

Compared with the prior art, the technical scheme of the invention has the advantages that the soybean protein and the epoxypropyl trimethyl ammonium chloride are used as raw materials, and the ring-opening reaction is carried out under the alkaline condition to generate the quaternized soybean protein. The molecular chain of the soy protein is broken under alkaline conditions, exposing reactive functional groups. At the moment, the amino group on the soybean protein and the epoxy group on the epoxypropyltrimethylammonium chloride generate ring-opening reaction, and the introduction of the quaternary ammonium group destroys the acting force in the soybean protein molecule and stabilizes the stretched molecular structure of the soybean protein. The synthesis method of the quaternized soybean protein provided by the invention obviously enhances the water solubility of the soybean protein and expands the application of the quaternized soybean protein in the field of biological materials. On the other hand, quaternized soy proteins also exhibit excellent antibacterial properties. The antibacterial mechanism of the quaternized soybean protein is independent of a biological enzyme system of bacteria, so that the risk of drug resistance does not exist, and the quaternized soybean protein is very suitable for patients infected by drug-resistant bacteria and patients with high infection risk.

Meanwhile, the synthesis conditions of the steps S101 to S103 are optimized, such as pH value and reaction time adjustment, so that the reaction rate and efficiency are improved;

according to another exemplary embodiment of the present invention, there is provided a quaternized soy protein produced by the method. The results of the antibacterial experiments show that: the quaternized soybean protein has obvious antibacterial effect on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus.

According to another exemplary embodiment of the present invention, there is provided a method for preparing quaternized soy protein-dopamine, as shown in fig. 6, the method comprising:

step S201, dissolving the quaternized soybean protein into deionized water to obtain a quaternized soybean protein solution;

in the step S201, the final concentration of the quaternized soybean protein solution is 2-20 (m/m)%. The quaternized soy protein solution preferably has a concentration of 6 (m/m)%; if the concentration of the solution less than 2 percent is very low, the reaction is not facilitated, and if the concentration of the solution more than 20 percent is relatively high, the dissolution is incomplete.

Stirring for 5-15 min at room temperature for accelerating dissolution;

step S202, uniformly mixing the quaternized soybean protein solution with dopamine and EDC/NHS for cross-linking reaction to obtain a cross-linking reaction product;

in the step S202, the first step,

the percentage of the added mass of the dopamine to the dry weight of the quaternized soybean protein is more than or equal to 10%, and preferably, the added mass of the dopamine accounts for 10-50% of the dry weight of the quaternized soybean protein; if the adding mass of the dopamine is less than 10% of the dry weight of the quaternized soybean protein, the substitution is low, and if the adding mass of the dopamine is more than 50%, the reaction is saturated; as shown in fig. 3, the DPPH removal efficiency gradually increased with the increase of the reaction mass, and when the addition amount of dopamine reached 40% of the dry weight of the quaternized soybean protein, the DPPH removal efficiency of the 0.1mg/mL quaternized soybean protein-dopamine solution reached saturation of 56%, and the DPPH removal efficiency changed little with the increase of the addition amount of dopamine.

The ratio of the substance quantities of EDC and NHS to the substance quantity of dopamine is (1: 1: 0.5) - (1: 1: 10); this ratio favors crosslinking; wherein the preferred ratio is 1: 1: 1, the reaction efficiency is highest, EDC/NHS is a reaction catalyst, and the reaction efficiency is improved in the following steps that 1: the catalytic efficiency is highest at 1.

And step S203, carrying out post-treatment on the crosslinking reaction product to obtain the quaternized soybean protein-dopamine.

The step S203 specifically includes:

and precipitating the crosslinking reaction product by adopting an organic solvent, carrying out solid-liquid separation, collecting solids, washing and drying to obtain the water-soluble quaternized soybean protein-dopamine.

In recent years, tissue engineering has put higher demands on the performance of biomedical materials, and on the basis of materials, the method has great challenge on organically intervening specific biological processes of organisms and optimizing microenvironment to promote tissue regeneration through technical means of materials and chemistry. Traditional bio-inert materials clearly do not meet the requirements. The tissue damage microenvironment is very complex, the technical problem of clearing excessive active oxygen generated in an organism is preferably selected as an entry point, the quaternized soybean protein and dopamine are used as raw materials for the first time, and EDC/NHS is used as a cross-linking agent to react to obtain the quaternized soybean protein-dopamine with excellent antioxidant activity. As a bulk antioxidant macromolecule, quaternized soybean protein-dopamine is added into various existing material systems, such as chitosan composite hydrogel, gelatin composite sponge, polycaprolactone composite nanofiber and the like, so that the oxidation resistance which is generally lacked in the existing materials is endowed. It is not difficult to assume that the range of applications for quaternized soy protein-dopamine is extremely wide. If the material can be organically integrated with the existing commercial tissue engineering material, the material is expected to pass the examination and approval process of national medical instruments in a short time and realize the maximum economic benefit in the clinical application process.

According to another exemplary embodiment of the present invention, there is provided quaternized soy protein-dopamine prepared by the method. The DPPH clearance test results show that: the clearance rate of 0.1mg/mL quaternized soybean protein-dopamine to DPPH reaches 59.4%, and the in vitro antioxidant effect is good.

The ROS scavenging experiment result shows that: with the increase of the concentration of the quaternized soybean protein-dopamine, the ROS content in cells is remarkably reduced, and the quaternized soybean protein-dopamine has the potential of resisting oxidation in vivo.

A quaternized soy protein, a quaternized soy protein-dopamine, and a preparation method and application thereof of the present application will be described in detail below with reference to experimental data.

Example 1 Quaternary Soy protein and Process for its preparation

Step S101, adding 6g of soy protein isolate and 0.5g of NaOH into 93.5g of water, and stirring for 1 hour to completely dissolve the soy protein isolate and the NaOH to obtain a soy protein solution with the concentration of 6%;

step S102, adding 3g of 2, 3-epoxypropyltrimethylammonium chloride (which is 50% of the dry weight of the soybean protein) into the soybean protein solution with the concentration of 6%, and stirring for 24 hours at room temperature;

and S103, adjusting the pH value to 7 by using glacial acetic acid after the reaction is finished, then precipitating by using ethanol, filtering, washing by using the ethanol, and drying for 24 hours at the temperature of 60 ℃ to obtain the quaternized soybean protein isolate.

Example 2 Quaternary Soy protein and Process for its preparation

step S102, adding 6g of 2, 3-epoxypropyltrimethylammonium chloride (accounting for 100% of the dry weight of the soybean protein) into the soybean protein solution with the concentration of 6%, and stirring for 24 hours at room temperature;

and step S103, after the reaction is finished, adjusting the pH value to 7 by using glacial acetic acid, then precipitating by using ethanol, filtering, washing by using ethanol, and drying for 24 hours at the temperature of 60 ℃ to obtain the quaternized soybean protein isolate.

Example 3 Quaternary Soy protein and Process for its preparation

step S102, adding 9g of 2, 3-epoxypropyltrimethylammonium chloride (which is 150% of the dry weight of the soybean protein) into the soybean protein solution with the concentration of 6%, and stirring for 24 hours at room temperature;

Example 4 quaternized Soybean proteins and methods of making same

step S102, adding 12g of 2, 3-epoxypropyltrimethylammonium chloride (which is 200% of the dry weight of the soybean protein) into the soybean protein solution with the concentration of 6%, and stirring for 24 hours at room temperature;

Example 5 quaternized Soybean proteins and methods of making same

Step S101, adding 6g of soy protein isolate and 0.5g of NaOH into 93.5g of water, and stirring for 1 hour to completely dissolve the soy protein isolate and the NaOH to obtain a soy protein solution with the concentration of 20%;

step S102, adding 18g of 2, 3-epoxypropyltrimethylammonium chloride (which is 300% of the dry weight of the soybean protein) into the soybean protein solution with the concentration of 6%, and stirring for 24 hours at room temperature;

Comparative example 1

This comparative example 1.5g of 2, 3-epoxypropyltrimethylammonium chloride in step S102 was 25% of the dry weight of soy protein; the other steps were the same as in example 1.

Example 6 Quaternary Soy protein-dopamine and Process for its preparation

Step S201, adding 6g of the quaternized soybean protein isolate obtained in the embodiment 1 into 94g of deionized water, and stirring for 1 hour to completely dissolve the quaternized soybean protein isolate to obtain a quaternized soybean protein solution with the concentration of 6%;

step S202, adding 0.304g of EDC and 0.225g of NHS into the quaternized soybean protein solution with the concentration of 6%, stirring for 30 minutes, then adding 0.6g of dopamine with the dry weight of 10% of quaternized soybean protein, wherein the mass ratio of EDC, NHS and dopamine is 1: 1: 1. stirring for 6 hours at room temperature to react;

and S203, after the reaction is finished, precipitating with ethanol, filtering, washing with ethanol, and drying at 60 ℃ for 24 hours to obtain the quaternized soybean protein-dopamine.

Example 7 Quaternary Soy protein-dopamine and Process for its preparation

step S202, adding 0.304g of EDC and 0.225g of NHS into the quaternized soybean protein solution with the concentration of 6%, stirring for 30 minutes, then adding 1.2g of dopamine with the dry weight of 10% of quaternized soybean protein, wherein the mass ratio of EDC, NHS and dopamine is 1: 1: 1. stirring for 6 hours at room temperature to react;

Example 8 Quaternary Soy protein-dopamine and Process for its preparation

step S202, adding 0.304g of EDC and 0.225g of NHS into the quaternized soybean protein solution with the concentration of 6%, stirring for 30 minutes, then adding 1.8g of dopamine with the dry weight of 10% of quaternized soybean protein, wherein the mass ratio of EDC, NHS and dopamine is 1: 1: 1. stirring for 6 hours at room temperature to react;

Example 9 Quaternary Soy protein-dopamine and Process for its preparation

step S202, adding 2.430g of EDC and 1.803g of NHS into the quaternized soybean protein solution with the concentration of 6%, stirring for 30 minutes, then adding 2.4g of dopamine with the dry weight of 40% of quaternized soybean protein, wherein the mass ratio of EDC, NHS and dopamine is 1: 1: 1. stirring for 6 hours at room temperature to react;

Example 10 Quaternary Soy protein-dopamine and Process for its preparation

Step S202, adding 6g of the quaternized soybean protein isolate obtained in the example 1 into 94g of deionized water, and stirring for 1 hour to completely dissolve the quaternized soybean protein isolate to obtain a quaternized soybean protein solution with the concentration of 6%;

step S202, adding 3.04g of EDC and 2.254g of NHS into the quaternized soybean protein solution with the concentration of 6%, stirring for 30 minutes, then adding 3g of dopamine with 50% of the dry weight of the quaternized soybean protein, wherein the mass ratio of EDC, NHS and dopamine is 1: 1: 1. stirring for 6 hours at room temperature to react;

Comparative example 2

This comparative example 0.3g dopamine addition in step S202 is 10% of the dry weight of quaternized soy protein; the other steps were the same as in example 1. The degree of substitution of the reaction product is low, and the efficiency of removing DPPH is low.

Different masses of glycidyltrimethylammonium chloride according to degree of substitution in the reactions of Experimental example 1, examples 1-5 and comparative example 1

The degree of substitution corresponding to the incorporation of epoxypropyltrimethylammonium chloride of different masses in examples 1 to 5 and comparative example 1 was determined by comparing the amino group contents of the soybean protein before and after the reaction by the o-phthalaldehyde method (OPA). The determination method comprises the following steps: firstly, preparing an o-phthalaldehyde solution: 40mg of o-phthalaldehyde, 1mL of methanol, 100. mu.l of β -mercaptoethanol and 2.5mL of 20 m/m% SDS were added to 25mL of 0.1m sodium borate buffer (pH 9.75), and the mixture was diluted to 50mL with deionized water. Subsequently, 4mL of OPA reagent was reacted with 200. mu.L of 0.4% sample solution at 37 ℃ for 2 min. The absorption at a wavelength of 336nm was measured with an ultraviolet spectrophotometer.

Degree of substitution (%) - (A) ₀ -A _s )/A ₀ ]×100％，

Wherein: a. the ₀ I.e. the absorbance of the soy protein, A _s Absorbance of the quaternized soy protein.

The results are shown in FIG. 2 and Table 1.

TABLE 1

From the data in table 1, it can be seen that:

the substitution degree gradually increases with the increase of the mass of the epoxypropyltrimethylammonium chloride participating in the reaction, reaches 72 percent when the mass of the epoxypropyltrimethylammonium chloride is 12g which is 200 percent of the dry weight of the soybean protein, and basically reaches saturation, and the substitution degree changes little when the mass of the epoxypropyltrimethylammonium chloride continuously increases to 18g which is 300 percent of the dry weight of the soybean protein. FIG. 2 is a graph showing the degree of substitution of quaternized soy proteins corresponding to different masses of glycidyltrimethylammonium chloride incorporation reactions.

DPPH removal efficiency of quaternized Soy protein-dopamine corresponding to different mass dopamine incorporation reactions in Experimental example 2, example 6-example 10, and comparative example 2

DPPH removal efficiency of quaternized soy protein-dopamine in examples 6-10 and comparative example 2 was determined by: the dopamine with different masses of example 6-example 10 and comparative example 2 were added to the reacted products respectively, and reacted with DPPH respectively, and after 0.5h of reaction, samples were taken out and centrifuged to obtain DPPH solution after reaction. And measuring the absorbance of a characteristic peak of the free radical at 516nm by using an ultraviolet spectrophotometer, and obtaining the DPPH clearance rate of the quaternized soybean protein-dopamine through statistical analysis.

The results are shown in FIG. 3 and Table 2.

TABLE 2

From the data in table 2, it can be seen that:

the grafting rate of dopamine is gradually increased along with the increase of the quality of dopamine participating in the reaction, and the DPPH (active free radical) scavenging efficiency is gradually increased. When the amount of dopamine 2.4g is 40% of the dry weight of the soy protein, the DPPH (active free radical) scavenging efficiency reaches 56%, and is substantially saturated. When the dopamine mass continued to increase to 3g at 50% of the dry weight of soy protein, the DPPH removal efficiency varied very little, with 59% DPPH removal efficiency for a 0.1mg/mL quaternized soy protein-dopamine solution. Figure 3 is a graph of DPPH removal efficiency for quaternized soy protein-dopamine corresponding to different mass dopamine incorporation reactions.

Experimental example 3 chemical Structure detection of quaternized Soybean protein and quaternized Soybean protein dopamine

The quaternized soy proteins obtained in example 1 and example 4 and quaternized soy protein dopamine were ground to powders. And detecting the chemical structure of the sample by using a total reflection Fourier infrared spectrometer and a nuclear magnetic resonance hydrogen spectrometer.

FIG. 1a is a total reflection Fourier transform infrared spectrum of the quaternized soy protein obtained in example 1. Quaternary soy protein at wavenumber of 1051.56cm, in contrast to SPI ^-1 、969.68cm ^-1 、879.87cm ^-1 A change occurs. Wave number of 1051.56cm ^-1 Is secondary hydroxyl C-O stretching vibration with wave number of 969.68cm ^-1 Is a characteristic peak of C-N stretching vibration, is also a characteristic absorption peak of the quaternary ammonium salt compound, and has a wave number of 879.87cm ^-1 Is the out-of-plane vibration of N-H. These newly appearing characteristic absorption peaks can indicate successful grafting of the quaternary ammonium salt groups onto the soy protein polypeptide chain.

FIG. 1c is a NMR hydrogen spectrum of the quaternized soy protein obtained in example 1. Quaternary ammonium soy protein at 3.14 ppm-N ⁺ (CH ₃ ) ₃ Further confirms that the quaternized soybean protein is successfully prepared by the method.

FIG. 1b is a total reflectance Fourier transform infrared spectrum of quaternized soy protein-dopamine obtained in example 4. Quaternized soy protein-dopamine is 875cm ^-1 And 813cm ^-1 A new characteristic peak appears nearby, and the peak is a characteristic absorption that two hydroxyl groups are positioned at the ortho position of a benzene ringAnd (4) peak narrowing, which is just consistent with the characteristic absorption peak of dopamine.

FIG. 1d is a NMR spectrum of quaternized soy protein-dopamine obtained in example 4. As shown in the figure, a catechol aromatic proton peak appears at the position of delta-7 ppm, a benzenediol methylene proton peak appears at the positions of delta-3.1 ppm and delta-2.8, and the quaternized soybean protein-dopamine is proved to be successfully prepared by the method.

Experimental example 4 detection of bacteriostatic Effect of quaternized Soybean protein

And detecting the bacteriostatic performance of the quaternized soybean protein on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus. According to the national standard GB15979-2002, the tested sample is prepared into aqueous solutions or dispersions with different concentrations, each tube is 5mL, and the control group is sterile water. The bacterial solution was diluted to a concentration of 1X 10 with PBS ⁶ CFU/mL of bacterial suspension. And (3) taking the suspension liquid, dropwise adding 100 mu L of the suspension liquid into each tube of sample liquid and the control sample liquid, and uniformly mixing. After 12h, sucking 0.5mL from each tube of sample liquid and the control sample liquid, diluting properly, coating 0.05mL into an LB solid culture medium plate, culturing at 37 ℃ for 24h, counting the number of colonies, and calculating the bacteriostasis rate according to the following formula:

X(％)＝(A-B)/A×100，

in the formula: x: bacteriostasis rate, a: average colony number of control samples, B: average colony number of the samples to be tested.

FIG. 4 is the results of the antimicrobial experiment for quaternized soy protein obtained in example 2. With the increase of the concentration of the quaternized soybean protein, the bacteriostasis rate of the quaternized soybean protein is obviously increased. The quaternized soybean protein has good antibacterial effect on staphylococcus aureus and methicillin-resistant staphylococcus aureus.

Experimental example 5 measurement of DPPH removal Rate by Quaternary Soybean protein-dopamine

4mg of DPPH was first dissolved in 100mL of methanol for use. And dispersing the sample in a methanol solution to prepare a sample dispersion to be detected. 3mL of DPPH solution was mixed with 3mL of sample dispersions of quaternized soy protein-dopamine (0.0001-1mg/mL) at various concentrations. The methanol solution was mixed with the DPPH solution as a control. After the reaction time of 0.5h, the sample was taken out and centrifuged to obtain a reacted DPPH solution. And measuring the absorbance of a characteristic peak of the free radical at 516nm by using an ultraviolet spectrophotometer, and obtaining the DPPH clearance rate of the quaternized soybean protein-dopamine through statistical analysis.

Fig. 5 is the results of the quaternized soy protein-dopamine DPPH clearance experiment obtained in example 9. When the concentration of the quaternary ammonium soybean protein-dopamine is 0.1mg/mL, the DPPH clearance rate reaches 59.4%. The quaternized soybean protein-dopamine provided by the invention has a good effect of removing active free radicals DPPH.

Experimental example 6 detection of ROS scavenging Effect of quaternized Soybean protein-dopamine

Schwann cells were seeded in 12-well plates and cultured in a cell incubator at 37 ℃ for 120 min. Adding quaternized soy protein-dopamine serum-free culture medium with different concentrations into each hole, and continuously culturing for 12 h. By means of H ₂ O ₂ Oxidizing and stimulating the cells for 6h by using the solution, then adding a DCFH-DA probe, and detecting the content of intracellular ROS by using a flow cytometer; wherein the control group is prepared by adding no quaternized soy protein-dopamine and H ₂ O ₂ The normal culture group of (4).

Fig. 6 is the results of the quaternized soy protein-dopamine ROS scavenging experiment obtained in example 9. With the increase of the concentration of the quaternized soybean protein-dopamine, the content of ROS in Schwann cells is obviously reduced, which indicates that the quaternized soybean protein-dopamine has the potential of in vivo antioxidation.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method of making a quaternized soy protein, the method comprising:

dissolving soy protein in an alkaline solution to obtain a soy protein solution; the alkaline solution is 0.5-5% of NaOH by mass, and the final concentration of the soybean protein solution is 2-20%;

uniformly mixing the soybean protein solution and epoxypropyltrimethylammonium chloride for ring-opening reaction to obtain a ring-opening reaction product; the percentage of the added mass of the epoxypropyltrimethylammonium chloride to the dry weight of the soybean protein in the soybean protein solution is more than or equal to 50 percent;

2. A quaternized soy protein made by the method of claim 1.

3. A method of preparing quaternized soy protein-dopamine, the method comprising:

dissolving the quaternized soy protein of claim 2 in deionized water to obtain a quaternized soy protein solution with a final concentration of 2-20%;

uniformly mixing the quaternized soybean protein solution with dopamine and EDC/NHS for crosslinking reaction to obtain a crosslinking reaction product; the percentage of the added mass of the dopamine to the dry weight of the quaternized soybean protein is more than or equal to 10%, and the ratio of the mass of the EDC and the mass of the NHS to the mass of the dopamine is (1: 1: 0.5) - (1: 1: 10);

4. A quaternized soy protein-dopamine made by the method of claim 3.

5. Use of the quaternized soy protein of claim 2 in the preparation of an antimicrobial biomaterial.

6. Use of the quaternized soy protein-dopamine of claim 4 in the preparation of an antioxidant biomaterial.