Preparation method of EGCG (epigallocatechin gallate)-loaded soybean enzymatic emulsion

Abstract

The invention discloses a preparation method of EGCG (epigallocatechin gallate)-loaded soybean enzymatic emulsion. The method comprises the following steps: (1) preparing soybean emulsion by adopting an enzymatic method; (2) preparing a soybean enzymatic emulsion dispersion liquid; and (3) preparing the EGCG-loaded soybean enzymatic emulsion. The particle size of the prepared EGCG-loaded soybean enzymatic emulsion is increased, the zeta-potential absolute value is increased, and the EGCG-loaded soybean enzymatic emulsion has good emulsion stability.

Description

Technical Field

[0001] The invention mainly relates to a method for preparing an EGCG-loaded soybean enzymatic emulsion. Background Art

[0002] Epigallocatechin gallate (EGCG) is the most abundant polyphenol in green tea and is known for its antioxidant, anti-inflammatory and anti-cancer properties. In food systems, EGCG can interact covalently and non-covalently with proteins to change the structure and function of proteins. Studies have shown that EGCG affects interfacial proteins in oil-in-water (O / W) emulsions, thereby effectively resisting protein-lipid co-oxidation during storage. However, as a natural polyphenol, the phenolic hydroxyl group of EGCG is easily oxidized and usually degrades, oxidizes and isomerizes under light and high temperature conditions. These shortcomings limit the application of EGCG in food. Therefore, it is necessary to design an appropriate delivery system to improve the stability of EGCG in order to expand its application in the food field.

[0003] Oil-in-water (O / W) emulsions have become a topic of increasing concern in the food industry, as they can change the appearance, texture, flavor, and functionality of food. Conventional methods for preparing emulsions include homogenization, homogenization-solvent removal, emulsion cooling, and interfacial polycondensation. Homogenization is the most commonly used method for preparing emulsions, but the emulsion system prepared by this method is unstable and prone to aggregation, precipitation, flocculation, or phase separation.

[0004] The formation mechanism of soybean enzymatic emulsion is: based on physical mechanical crushing, enzymes are added to act on soybean tissue cells to degrade macromolecular complexes such as lipoproteins, lipopolysaccharides and cell walls in cells, and the process is improved according to the optimal temperature and pH value of the enzyme. The enzyme can fully destroy the oil tissue in soybeans, destroy its cell structure, enhance the flow properties of oil and release oil into the system. With the destruction of soybean oil tissue, the enzymatic process divides the system into different parts by utilizing the different affinities of different components in soybeans to oil and water, and then uses the different densities to achieve the purpose of separation and extraction. Under the action of enzymes, a system consisting of free oil, emulsion, hydrolyzate and residue is formed. Compared with conventional emulsions, during the formation of soybean enzymatic emulsions, the naturally existing proteins and a small amount of phospholipids in soybeans can be adsorbed on the oil-water interface as amphiphilic emulsifiers, reduce interfacial tension, and spontaneously form a stable, core-shell structured oil-in-water (O / W) emulsion system. During the formation of enzymatic emulsions, proteins are hydrolyzed into polypeptides by protease treatment. The emulsion contains a large amount of polypeptides, which can greatly increase the stability of the emulsion. In addition, the preparation process of soybean enzymatic emulsion using conventional enzyme-assisted stirring hydrolysis consumes less energy and has the advantages of mild reaction conditions, simple and safe operation, and no solvent residue.

[0005] The invention selects puffed soybean powder as raw material, obtains soybean enzymatic emulsion by alkaline protease enzymolysis, loads EGCG with the soybean enzymatic emulsion, optimizes the ratio, studies the particle size and potential of the emulsion after adding EGCG, and expands the application of EGCG and soybean enzymatic emulsion in food processing. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing an EGCG-loaded soybean enzymatic emulsion, thereby improving the stability of EGCG and expanding its application in food.

[0007] To achieve the above object, the present invention comprises the following steps:

[0008] 1. A method for preparing an EGCG-loaded soybean enzymatic emulsion, the method comprising the following steps:

[0009] (1) Preparation of soybean enzymatic emulsion: Grind the puffed soybean powder into 60 mesh fine powder, mix 1200 mL of distilled water with 200 g of soybean powder, and stir them evenly in a water bath at 55°C with high-speed stirring. Use NaOH to adjust the pH value of the solution to 9.0. Add alkaline protease Protex 6 L for enzymatic hydrolysis. After the enzymatic hydrolysis, adjust the pH of the enzymatic solution to 7.0, inactivate the enzyme in a boiling water bath, cool to room temperature, and centrifuge at 4°C and 9000×g for 20 min to obtain soybean enzymatic emulsion.

[0010] (2) Preparation of soybean enzymatic emulsion dispersion: The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01 M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion.

[0011] (3) Preparation of soybean enzymatic emulsion loaded with EGCG: EGCG was dispersed in the emulsion dispersion at different solid-liquid ratios (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL), the solution pH was maintained at 7.0 and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the emulsion protein in the solution.

[0012] Preferably, the amount of alkaline protease Protex 6 L added in step (1) is 1.5%.

[0013] Preferably, the enzymatic hydrolysis time in step (1) is 90 min.

[0014] Preferably, the concentration of the soybean enzymatic emulsion in step (2) is 40% w / v.

[0015] Preferably, the optimal concentration of EGCG in step (3) is 0.8 mg / mL.

[0016] The present invention prepares soybean emulsion by enzyme method, does not involve chemical reagents and is environmentally friendly. Then, the soybean enzymatic emulsion is treated with EGCG of different concentrations to obtain a stable EGCG enzymatic emulsion, and at the same time, the efficient delivery of EGCG is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 Particle size of different EGCG enzymatic emulsion samples

[0018] Attached Figure 2 Zeta potential values ​​of different EGCG enzymatic emulsion samples DETAILED DESCRIPTION

[0019] In order to introduce the effects achieved by the technical solution of the present invention in more detail, a detailed description is given below in conjunction with specific implementation cases.

[0020] A method for preparing EGCG-loaded soybean enzymatic emulsion:

[0021] (1) Preparation of soybean enzymatic emulsion: Grind the puffed soybean powder into 60 mesh fine powder, mix 1200 mL of distilled water with 200 g of soybean powder, and stir them evenly in a water bath at 55°C with high-speed stirring. Use NaOH to adjust the pH value of the solution to 9.0. Add alkaline protease Protex 6 L for enzymatic hydrolysis. After the enzymatic hydrolysis, adjust the pH of the enzymatic hydrolyzate to 7.0, inactivate the enzyme in a boiling water bath, cool to room temperature, and centrifuge at 4°C and 9000×g for 20 min to obtain soybean enzymatic emulsion. (2) Preparation of soybean enzymatic emulsion dispersion: Disperse the soybean enzymatic emulsion in freshly prepared PBS (0.01 M, pH 7.0) to prepare soybean enzymatic emulsion dispersion. (3) Preparation of soybean enzymatic emulsion loaded with EGCG: EGCG was dispersed in the emulsion dispersion at different solid-liquid ratios (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL), the solution pH was maintained at 7.0 and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the emulsion protein in the solution. Example 1

[0022] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios at 0.2 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 983.61±8.40 nm and the potential was -31.26±0.27 mV. Example 2

[0023] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios of 0.4 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 993.23±4.59 nm and the potential was -31.78±0.14 mV. Example 3

[0024] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios of 0.6 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 1123.09±5.97 nm and the potential was -33.57±0.31 mV. Example 4

[0025] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios of 0.8 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 1167.17±2.49 nm and the potential was -36.60±0.26 mV. Example 5

[0026] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios at 1.0 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 1208.63±7.59 nm and the potential was -36.28±0.43 mV. Example 6

[0027] The puffed soybean powder was ground into 60 mesh fine powder, and 1200 mL of distilled water was mixed with 200 g of soybean powder and stirred evenly in a water bath at 55°C with high-speed stirring. The pH value of the solution was adjusted to 9.0 with NaOH. Alkaline protease Protex 6 L was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the pH of the enzymatic solution was adjusted to 7.0, the enzyme was inactivated in a boiling water bath, cooled to room temperature, and centrifuged at 4°C and 9000×g for 20 min to obtain a soybean enzymatic emulsion. The soybean enzymatic emulsion was dispersed in freshly prepared PBS (0.01M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion. EGCG was dispersed in the emulsion dispersion at different material-liquid ratios of 1.2 mg / mL, the solution pH was maintained at 7.0, and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the latex protein in the solution. The sample particle size obtained at this time was 1427.45±8.45 nm and the potential was -36.58±0.13 mV.

Claims

1. A method for preparing an EGCG-loaded soybean enzymatic emulsion, the method comprising the following steps: (1) Preparation of soybean enzymatic emulsion: Grind the puffed soybean powder into 60 mesh fine powder, mix 1200 mL of distilled water with 200 g of soybean powder, and stir them evenly in a water bath at 55°C with high-speed stirring. Use NaOH to adjust the pH value of the solution to 9.

0. Add alkaline protease Protex 6 L for enzymatic hydrolysis. After the enzymatic hydrolysis, adjust the pH of the enzymatic solution to 7.0, inactivate the enzyme in a boiling water bath, cool to room temperature, and centrifuge at 4°C and 9000×g for 20 min to obtain soybean enzymatic emulsion.

2. (2) Preparation of soybean enzymatic emulsion dispersion: Disperse the soybean enzymatic emulsion in freshly prepared PBS (0.01 M, pH 7.0) to prepare a soybean enzymatic emulsion dispersion.

3. (3) Preparation of EGCG-loaded soybean enzymatic emulsion: EGCG was dispersed in the emulsion dispersion at different solid-liquid ratios (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL), the solution pH was maintained at 7.0 and stirred in an anaerobic environment (25°C, 500 rpm, 2 h) to non-covalently bind to the emulsion protein. The emulsion dispersion without EGCG was used as the control group. All sample solutions were transferred to a dialysis bag (3500 Da) and dialyzed for 24 h to remove EGCG that was not bound to the emulsion protein in the solution.

4. The method for preparing a soybean enzymatic emulsion loaded with EGCG according to claim 1, characterized in that: The amount of alkaline protease Protex 6 L used in step (1) is 1.5%.

5. The method for preparing a soybean enzymatic emulsion loaded with EGCG according to claim 1, characterized in that: The enzymatic hydrolysis time selected in step (1) was 90 min.

6. The method for preparing a soybean enzymatic emulsion loaded with EGCG according to claim 2, characterized in that: The mass concentration of soybean enzyme emulsion was 40% w / v.

7. The method for preparing a soybean enzymatic emulsion loaded with EGCG according to claim 3, characterized in that: The optimal EGCG concentration was 0.8 mg / mL.

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