A soy protein-based composite nanogel material and a preparation method and application thereof

Abstract

The application belongs to the technical field of gel materials, and particularly relates to a composite nanogel material based on soybean protein and a preparation method and application thereof. The composite nanogel material is based on thymol and folic acid to induce self-assembly of soybean protein into a gel, realizes steady-state delivery of active substances and creates a water gel with antibacterial activity. The method is based on the discovery of the structure-activity relationship between soybean protein and thymol, and the interaction between the two is directionally regulated by pH shift treatment. Then, deprotonation of water-soluble functional factors such as folic acid is used to assist in promoting the formation of a soybean protein-thymol complex into a gel. Finally, a soybean protein cold-induced gel with antibacterial activity is constructed, and the embedding and delivery of thymol and folic acid are realized. The prepared composite nanogel material has superior antioxidant activity and antibacterial activity.

Description

Technical Field

[0001] This invention belongs to the technical field of gel materials, specifically relating to a composite nanogel material based on soybean protein, its preparation method, and its application. Background Technology

[0002] Plant essential oils possess antioxidant, antibacterial, and antiviral activities, as well as being green and safe, making them promising candidates for use as biological preservatives. However, plant essential oils often exhibit high volatility, low water solubility, and strong odors, hindering their industrial application. Research on the solubilization, stabilization, and sustained-release properties of plant essential oils will facilitate their industrialization as biological preservatives. Among plant essential oils, thymol (also known as thymol), as one of the main active ingredients in thyme and oregano essential oils, has been listed as a GRAS (Generally Recognized As Safe) food additive by the European Union (EU) and the US Food and Drug Administration (FDA), and is also a food additive permitted for use under Chinese national standards.

[0003] Proteins are natural macromolecules, and constructing bioactive substance delivery carriers using proteins as a base is a current research hotspot. The main forms include emulsions, capsules, molecular complexes, gels, core-shell particles, and micelles. Among these, gel-based delivery carriers, which are three-dimensional network structures formed by physical or chemical cross-linking of protein molecules, are excellent delivery carriers for the encapsulation and sustained release of bioactive substances. Soy protein, a byproduct of soybean oil processing, has cholesterol-lowering effects. Structurally, it contains both polar and non-polar groups and has a reasonable amino acid composition, giving it a superior ability to bind to small molecule bioactive substances, making it a research hotspot for bioactive substance delivery carriers in recent years. The formation of soybean protein gels often requires thermal denaturation and the assistance of cross-linking agents or coagulants. Research on directly inducing soybean protein gels based on small molecule bioactive substance complexes is still rare. Our team's previous research found that under pH shift treatment conditions, thymol complexes can promote the formation of soybean protein gel structures under non-thermal conditions (CN201910468544.3), but this process involves alkalization and acidification neutralization, making the process relatively complex.

[0004] pH shift treatment has proven to be an effective method for protein functional modification, improving the gelation properties of proteins and their ability to bind to polyphenols. Furthermore, under pH shift conditions, polyphenol complexation can induce protein gelation. For example, patent CN202010423972.7 reports the preparation of soy protein cryogenic gels via tannic acid crosslinking. However, this method requires high protein concentrations (12-13%), a long reaction time (9-10 hours) between soy protein and tannic acid, and the need to maintain contact between the mixture and oxygen during the reaction. Overall, the process is time-consuming and difficult to scale up for mass production. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a composite nanogel material based on soybean protein, its preparation method, and its application. The prepared gel material exhibits superior antioxidant and antibacterial activities and has broad application prospects in fields such as biological preservatives and nutrient fortifiers.

[0006] The technical content of this invention is as follows:

[0007] This invention provides a method for preparing a composite nanogel material based on soybean protein, comprising the following steps:

[0008] 1) Protein denaturation: Soy protein is fully dispersed in water to obtain a soy protein solution. Insoluble matter is removed, and the pH is adjusted to alkaline to allow the protein to denature appropriately and its structure to unfold.

[0009] The soy protein includes commercially available soy protein isolate (SPI) or SPI prepared from defatted soy flour;

[0010] The concentration (mass fraction) of the soybean protein solution is 5-10%, preferably 7.5%;

[0011] The pH condition is alkaline, ranging from 10 to 12, preferably 11.

[0012] 2) Nanocomposite: Thymol crystals are added to soybean protein solution and stirred thoroughly. The deprotonation of thymol under alkaline conditions is used to improve its solubility and promote the non-covalent binding of thymol and soybean protein to form a nanocomposite.

[0013] The amount of thymol crystals added is 4-15 wt% of the soybean protein solution;

[0014] 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 30–50°C;

[0015] The amount of folic acid added is 10-25 wt% of the soybean protein solution;

[0016] Furthermore, after adding folic acid powder, the mixture is thoroughly stirred and mixed at 40°C;

[0017] The deprotonation of folic acid gradually lowers the pH of the system, which helps promote the self-assembly of the soybean protein-thymol complex into a gel.

[0018] 4) Refrigerated gel: Centrifuge the mixture obtained in step 3) to remove unbound thymol and folic acid, and place the supernatant at low temperature to form a gel material.

[0019] The present invention also provides an application of a soybean protein-based composite nanogel material in the preparation of biopreservatives, nutrient fortifiers, etc.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention relates to a soybean protein-based composite nanogel material and its preparation. The material is based on the induction of soybean protein self-assembly into a gel by thymol and folic acid, achieving a dual function of steady-state delivery of active substances and creating a hydrogel with antibacterial activity. The method first utilizes the discovery of the structure-activity relationship between soybean protein and thymol, and then uses pH shifting to directionally regulate their interaction. Secondly, it utilizes the deprotonation of water-soluble functional factors such as folic acid to promote the formation of a soybean protein-thymol nanocomposite gel, ultimately constructing a soybean protein cryogenic gel with antibacterial activity and achieving the encapsulation and delivery of thymol and folic acid.

[0022] This invention fully utilizes the nanocomposite properties of soybean protein. By adjusting the pH to allow the interaction between soybean protein and thymol and folic acid, and by using thymol to induce protein fibrillation and aggregation and folic acid to deprotonate and acidify and promote coagulation, a soybean protein composite cryogenic gel can be constructed in one step. No additional heating or cross-linking agent is required throughout the process. The process is simple, low-cost, and suitable for industrial production.

[0023] The prepared composite nanogel material has superior antioxidant and antibacterial activities, and also has the characteristic of targeted delivery of folic acid in the small intestine, showing broad application prospects in the fields of biological preservatives and nutrient fortifiers. It breaks through the technical bottleneck of passive encapsulation of functional factors in the preparation of functional factor nanodelivery carriers, and purposefully selects active substances. Through a specific reaction system, the active substances are controlled to co-assemble with soybean protein into a gel. The active substances play multiple functions such as coagulant, cross-linking agent, antioxidant, and antibacterial. The preparation of the gel of this invention is applicable to other poorly soluble active substances, and even, as needed, gel products with multiple active substances can be prepared. Attached Figure Description

[0024] Figure 1 This is a scanning electron microscope image of the composite nanogel. Detailed Implementation

[0025] The present invention will be further described in detail below through specific implementation examples. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0026] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0027] Example 1

[0028] A method for preparing a composite nanogel material based on soybean protein

[0029] 1) Protein denaturation: Soy protein isolate (SPI) was fully dispersed in water to obtain a 10% (w / w) soy protein solution. Insoluble matter was removed, and the pH was adjusted to 12 using 4M NaOH to achieve appropriate protein denaturation and structural unfolding.

[0030] 2) Nanocomposite: 4 wt% thymol crystals were added to the soybean protein solution and stirred thoroughly. The deprotonation of thymol under alkaline conditions was used to improve its solubility and promote the non-covalent binding of thymol and soybean protein to form a nanocomposite.

[0031] 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 50°C;

[0032] The amount of folic acid added is 10 wt% of the soybean protein solution;

[0033] 4) Refrigerated gel: Centrifuge the mixture from step 3) at 5000 rpm for 5 minutes to remove unbound thymol and folic acid, and place the supernatant at 4°C for 12 hours to form a gel material.

[0034] Example 2

[0035] A method for preparing a composite nanogel material based on soybean protein

[0036] 1) Protein denaturation: Commercial soy protein isolate (SPI) was fully dispersed in water to obtain a soy protein solution with a mass fraction of 7.5%. Insoluble matter was removed, and the pH was adjusted to 11 using 4M NaOH to achieve appropriate protein denaturation and structural unfolding.

[0037] 2) Nanocomposite: 13.33 wt% thymol crystals were added to the soybean protein solution and stirred thoroughly. The deprotonation of thymol under alkaline conditions was used to improve its solubility and promote the non-covalent binding of thymol and soybean protein to form a nanocomposite.

[0038] 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 40°C;

[0039] The amount of folic acid added is 16.67 wt% of the soybean protein solution;

[0040] 4) Refrigerated gel: Centrifuge the mixture from step 3) at 5000 rpm for 5 minutes to remove unbound thymol and folic acid, and place the supernatant at 4°C for 12 hours to form a gel material.

[0041] Example 3

[0042] A method for preparing a composite nanogel material based on soybean protein

[0043] 1) Protein denaturation: Soy protein isolate (SPI) was fully dispersed in water to obtain a 7.5% (w / w) soy protein solution. Insoluble matter was removed, and the pH was adjusted to 11 using 4M NaOH to achieve appropriate protein denaturation and structural unfolding.

[0044] 2) Nanocomposite: 13.33 wt% thymol crystals were added to the soybean protein solution and stirred thoroughly. The deprotonation of thymol under alkaline conditions was used to improve its solubility and promote the non-covalent binding of thymol and soybean protein to form a nanocomposite.

[0045] 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 40°C;

[0046] The amount of folic acid added is 20 wt% of the soybean protein solution;

[0047] 4) Refrigerated gel: Centrifuge the mixture from step 3) at 5000 rpm for 5 minutes to remove unbound thymol and folic acid, and place the supernatant at 4°C for 12 hours to form a gel material.

[0048] Example 4

[0049] A method for preparing a composite nanogel material based on soybean protein

[0050] 1) Protein denaturation: Soy protein isolate (SPI) was fully dispersed in water to obtain a 5% (w / w) soy protein solution. Insoluble matter was removed, and the pH was adjusted to 11 using 4M NaOH to achieve appropriate protein denaturation and structural unfolding.

[0051] 2) Nanocomposite: 15wt% thymol crystals were added to the soybean protein solution and stirred thoroughly. The deprotonation of thymol under alkaline conditions was used to improve its solubility and promote the non-covalent binding of thymol and soybean protein to form a nanocomposite.

[0052] 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 30°C;

[0053] The amount of folic acid added is 25 wt% of the soybean protein solution;

[0054] 4) Refrigerated gel: Centrifuge the mixture from step 3) at 5000 rpm for 5 minutes to remove unbound thymol and folic acid, and place the supernatant at 4°C for 12 hours to form a gel material.

[0055] The loading of thymol and folic acid, functional activity, and textural properties of the soybean protein-based composite nanogel materials prepared in Examples 1-4 were tested, and the results are shown in Tables 1 and 2.

[0056] Table 1. Loading capacity and functional activity of composite nanogel materials for thymol and folic acid.

[0057]

[0058]

[0059] Note: The half-maximal inhibitory concentration (MIC) of the inhibitory activity of Escherichia coli and Staphylococcus aureus is calculated based on the concentration of thymol; the release in simulated gastrointestinal fluid is calculated based on the folic acid release rate.

[0060] Table 2. Textural properties of composite nanogel materials

[0061]

[0062] Note: The control is a soybean protein-thymol composite gel.

[0063] As can be seen from Tables 1 and 2, the gel prepared in Example 1 is a yellow semi-solid with good antibacterial and antioxidant activities, and has the characteristic of releasing folic acid in the small intestine. However, the gel has poor texture properties such as hardness and elasticity.

[0064] The gels prepared in Examples 2, 3 and 4 are yellow solids with good antibacterial and antioxidant activities, and have the characteristic of releasing folic acid in the small intestine. The gels also have good textural properties such as hardness and elasticity.

[0065] Figure 1 The images show the microstructure of the composite nanogel materials prepared in the examples. It can be seen that the composite gel structures of Examples 1 and 2 are relatively loose with large network pores, the composite gel structure of Example 3 is dense, and the composite gel structure of Example 4 has large pores. This indicates that composite gels with different microstructures can be constructed by controlling factors such as matrix concentration, reaction pH, and folic acid addition.

[0066] Therefore, as can be seen from the above, the soybean protein-thymol-folic acid composite gel prepared by this invention has superior antioxidant and antibacterial activities, and also has the characteristic of targeted delivery of folic acid in the small intestine, and has broad application prospects in the fields of biological preservatives and nutrient fortifiers.

Claims

1. A method for preparing a composite nanogel material based on soybean protein, characterized in that, Includes the following steps: 1) Protein denaturation: Soy protein is fully dispersed in water to obtain a soy protein solution, insoluble matter is removed, and the pH is adjusted to alkaline; The soy protein includes commercially available soy protein isolate (SPI) or SPI prepared from defatted soy flour; 2) Nanocomposite: Thymol crystals are added to soybean protein solution and stirred thoroughly to form a nanocomposite. 3) Acidification and coagulation: Add folic acid powder to the nanocomposite from step 2) and mix thoroughly at 30~50℃; 4) Refrigerated gel: Centrifuge the mixture obtained in step 3) to remove unbound thymol and folic acid, and place the supernatant at low temperature to form a gel material.

2. The method for preparing the soybean protein-based composite nanogel material according to claim 1, characterized in that, Step 1) The soybean protein solution has a mass fraction of 5-10%.

3. The method for preparing the soybean protein-based composite nanogel material according to claim 1, characterized in that, Step 1) involves adjusting the pH to an alkaline level of 10-12.

4. The method for preparing the soybean protein-based composite nanogel material according to claim 1, characterized in that, Step 2) The amount of thymol crystals added is 4-15 wt% of the soybean protein solution.

5. The method for preparing the soybean protein-based composite nanogel material according to claim 1, characterized in that, Step 3) The amount of folic acid added is 10-25 wt% of the soybean protein solution.

6. A soybean protein-based composite nanogel material prepared by the method of claim 1.

7. The application of the soybean protein-based composite nanogel material as described in claim 6 in the preparation of biological preservatives and nutrient fortifiers.

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