Green preparation method of genipin cross-linked black soybean peptidyl tea polyphenol sustained-release gel balls

Abstract

The invention belongs to the technical field of biological material preparation, and provides genipin cross-linked black bean peptidyl tea polyphenol sustained-release gel balls and a preparation method thereof. The preparation method comprises the following steps: dissolving black bean protein in water, carrying out microwave treatment, heating and incubating, adding protease for enzymolysis modification, and carrying out enzyme deactivation and precipitate removal to obtain a black bean peptide solution; the preparation method comprises the following steps: uniformly mixing black soybean peptide with a sodium alginate solution, adding tea polyphenol, uniformly stirring and mixing, adding a genipin aqueous solution, carrying out constant-temperature water bath, and dropwise adding the obtained solution into a calcium chloride solution to form gel balls; the prepared genipin cross-linked black bean peptidyl tea polyphenol sustained-release gel ball has a compact and stable wrinkled three-dimensional network structure and is high in biocompatibility and dispersity, and the stability of tea polyphenol is remarkably improved; the pH-responsive tea polyphenol slow release performance is achieved, accurate slow release of the tea polyphenol in intestinal juice is achieved, and the bioavailability of the tea polyphenol is improved.

Description

Technical Field

[0001] The present invention relates to a green preparation method of genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads, belonging to the technical field of biomaterial preparation. Background Art

[0002] Tea polyphenols have good antioxidant properties, play an important role in inhibiting cardiovascular and cerebrovascular diseases, can accelerate the decomposition and metabolism of excess fat in the human body, and reduce the deposition of cholesterol and triglycerides on the blood vessel wall. Tea polyphenols exhibit various biological functions, such as antibacterial, antiviral, tumor inhibition, prevention of cardiovascular diseases, etc., and thus have important applications in the fields of anti-aging, cosmetics, food, etc. The main absorption site of tea polyphenols is the small intestine. However, before reaching the small intestine, tea polyphenols are often destroyed by gastric acid, microorganisms, and various enzymes in the body, which destroys their phenolic hydroxyl structure, thereby losing their antioxidant and other physiological activities, and reducing the bioavailability. Currently, the most effective method for protecting and transporting bioactive components of tea polyphenols is to embed them in a biodegradable hydrogel network structure.

[0003] Common gel networks include sodium alginate-based and protein-based ones, both of which have certain application limitations. Proteins have high nutritional value, good gelation properties, and biodegradability, but protein-based gels generally have the problem of thermal denaturation, resulting in the loss of physiological activities of some thermosensitive drug-loading molecules. "Preparation and Performance Evaluation of Avermectin Gel Microspheres" prepared sustained-release microspheres loaded with avermectin using a crosslinking material of sodium alginate and calcium chloride, and coated PVA and chitosan on the surface of the microspheres. The drug dissolution rate of the prepared avermectin gel microspheres increases with the increase of pH. There is no reported sodium alginate-based gel that inhibits drug sustained release in simulated gastric juice and simulated saliva.

[0004] Genipin is the product of the hydrolysis of geniposide by β-glucosidase. Its use as a crosslinking agent has advantages such as natural low toxicity, good biocompatibility, mild crosslinking conditions, controllable crosslinking kinetics, and excellent gel properties in gel formation. Genipin can crosslink with various biomolecules (such as proteins, polysaccharides, etc.) to form a stable gel structure; its crosslinking kinetics is controllable, and the crosslinking degree and properties of the gel can be precisely controlled by adjusting parameters such as the concentration of genipin, crosslinking time, and temperature, such as gels with high strength, high toughness, or high elasticity, to meet the needs of different application fields. CN102362853A discloses a genipin-crosslinked soy protein-based theophylline controlled-release gel preparation, which uses a polymer of chitosan, soy protein, and genipin as the gel layer to coat theophylline to form a pH-sensitive sustained-release gel. The prepared gel releases too fast in simulated gastric juice and simulated saliva, and releases slowly in simulated intestinal juice, which is likely to cause waste of the sustained-release drug. Summary of the Invention

[0005]

Technical Problem

[0006] The main absorption site of tea polyphenols is the small intestine, but their phenolic hydroxyl structures are easily destroyed by enzymes in gastric juice and saliva, reducing their bioavailability. Alginate-based and protein-based drug sustained-release gels have a slow enough release rate in simulated gastric juice and simulated saliva, but too slow a release rate in simulated intestinal fluid, which easily causes waste of the sustained-release drug. There is a need for a tea polyphenol sustained-release gel with high release accuracy, low release rates in gastric juice and saliva, and high release rates in intestinal fluid, and to improve the stability of tea polyphenols.

[0007]

Technical Solution

[0008] To solve the above technical problems, the present invention provides a genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel bead, which belongs to a pH-sensitive hydrogel and has a wrinkled network structure, which is beneficial to increasing the stability of tea polyphenols. Both genipin and black bean peptides have good biocompatibility and low toxicity. Tea polyphenols are evenly distributed in the gel layer, and the gel layer swells when it encounters water, which can control the release rate of tea polyphenols and achieve the purpose of directional slow release in the small intestine.

[0009] The first object of the present invention is to provide a preparation method of the above genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel bead, including the steps of:

[0010] S1. Dissolve black bean protein in water, subject it to microwave treatment and heating incubation, add protease for enzymatic modification, inactivate the enzyme and remove the precipitate to obtain a black bean peptide solution;

[0011] S2. Mix the black bean peptide solution and the sodium alginate solution evenly to obtain solution A;

[0012] S3. Add tea polyphenols to solution A and stir evenly to obtain solution B;

[0013] S4. Add an aqueous solution of genipin to solution B and perform a constant temperature water bath to obtain solution C;

[0014] S5. Drop solution C into calcium chloride solution to form gel beads.

[0015] Black bean protein is often used as a nutritional supplement. Black bean peptide is a small molecule peptide obtained by enzymatic hydrolysis of black bean protein. It has a small molecular weight and has various physiological functions, such as antioxidant, lipid-lowering, and immune-enhancing effects. The presence of these bioactive peptide segments makes black bean peptide more diverse in function. Black bean peptide is relatively stable in acidic and hot environments. Compared with black bean protein, it has the advantages of higher absorption utilization rate and stronger biological activity.

[0016] Genipin molecules contain many active functional groups and can undergo chemical reactions with the active groups on the amino acid residues in black bean peptides. Specifically, the epoxy group of genipin can undergo a ring-opening addition reaction with the amino group on the amino acid residue to form a stable covalent bond; at the same time, the carboxyl group in the genipin molecule may also undergo an amidation reaction with the amino group in the black bean peptide, further strengthening the cross-linking effect. In addition, the introduction of sodium alginate is of great significance. It can significantly enhance the density and stability of the peptide-based hydrogel bead network through strong intermolecular and intramolecular hydrogen bond interactions with calcium chloride, playing a positive promoting role in improving the mechanical strength and sustained-release performance of the gel beads. The flow chart of the preparation method is as shown in Figure 4 shown.

[0017] In one embodiment of the present invention, the preparation method of black bean protein is as follows: disperse black bean powder in water, adjust the pH to be alkaline and stir, take the supernatant after centrifugation and adjust the pH to be acidic, let it stand for sedimentation, take the precipitate after centrifugation and freeze-dry to obtain black bean protein.

[0018] In one embodiment of the present invention, in the preparation method of black bean protein, the mass ratio of black bean powder to water is 1:5 to 1:10; adjusting the pH to be alkaline means adjusting the pH to 10.0 to 10.5; the stirring time is 2 to 3 h; adjusting the pH to be acidic means adjusting the pH to 4.0 to 4.5; the standing sedimentation time is 30 to 60 min; the centrifugation speed is 6000 to 10000 rpm, and the centrifugation time is 10 to 15 min.

[0019] In one embodiment of the present invention, in step S1, the mass ratio of black bean protein to water is 1:5 to 1:10.

[0020] In one embodiment of the present invention, in step S1, the power of microwave treatment is 100 to 600 W, and the treatment time is 5 to 20 min.

[0021] In one embodiment of the present invention, in step S1, the temperature of heating incubation is 45 to 55 °C, and the heating incubation time is 15 to 30 min.

[0022] In one embodiment of the present invention, in step S1, the protease is one or more of papain, bromelain, and ficin.

[0023] In one embodiment of the present invention, in step S1, the addition amount of the protease is 1 to 3% of the mass of black bean protein; the enzyme activity of the protease is 100 to 300 units / mg.

[0024] In one embodiment of the present invention, in step S1, the enzymatic modification is to stir at a temperature of 50 to 55 °C for 0.5 to 2.5 h.

[0025] In one embodiment of the present invention, in step S1, the enzyme inactivation treatment is to stir at a temperature of 95 - 100°C for 10 - 20 min.

[0026] In one embodiment of the present invention, in step S1, the precipitate is removed by centrifugation; the parameters of the centrifugation treatment are a temperature of 4 - 15°C, a rotation speed of 8000 - 10000 rpm, and a time of 10 - 30 min.

[0027] In one embodiment of the present invention, in step S2, the sodium alginate concentration of the sodium alginate solution is 0.5 - 2.0 wt%.

[0028] In one embodiment of the present invention, in step S2, the mixing volume ratio of the black bean peptide solution to the sodium alginate solution is 1 - 1:3 - 1.

[0029] In one embodiment of the present invention, in step S3, the tea polyphenol concentration of solution B is 0.3 - 1.0 wt%.

[0030] In one embodiment of the present invention, in step S4, the genipin concentration of the genipin aqueous solution is 20 - 40 mmol / L.

[0031] In one embodiment of the present invention, in step S4, the temperature of the constant temperature water bath is 35 - 45°C, and the time of the constant temperature water bath is 20 - 60 min.

[0032] In one embodiment of the present invention, in step S5, the calcium chloride concentration of the calcium chloride solution is 0.5 - 2.5 wt%.

[0033] The second object of the present invention is the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads prepared by the above method.

[0034] The third object of the present invention is the application of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads in the food field.

[0035] Beneficial effects:

[0036] The beneficial effects of the present invention compared with the prior art are:

[0037] The prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads have natural and safe raw materials.

[0038] The prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads improve the stability of tea polyphenols and significantly extend the sustained-release time of tea polyphenols. Specifically, the gel network structure formed by genipin-crosslinked black bean peptides provides a physical barrier for tea polyphenols, encapsulating the tea polyphenols within it, reducing the direct contact of tea polyphenols with factors such as oxygen, moisture, and light in the external environment, and decreasing the oxidation, hydrolysis, etc. of tea polyphenols by these factors, thereby improving the stability of tea polyphenols. The three-dimensional network structure of the gel restricts the movement of tea polyphenol molecules, slowing down their diffusion rate within the gel. At the same time, there may be interactions such as van der Waals forces and hydrogen bonds between black bean peptide molecules and tea polyphenol molecules, further hindering the movement of tea polyphenol molecules, thus extending their release time.

[0039] The prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads have a certain degree of swelling and can absorb a certain amount of water and swell in an aqueous solution. When the gel contacts a solution containing tea polyphenols, the tea polyphenols will be adsorbed into the gel. During the release process, water molecules gradually enter the gel network, causing the gel to swell and simultaneously pushing the tea polyphenol molecules to diffuse outwards. However, the swelling degree and diffusion path of the gel are relatively complex, which results in a slow release process of tea polyphenols. The pore size and distribution inside the gel play a key role in controlling the diffusion of tea polyphenols. The pore structure of the genipin-crosslinked black bean peptide gel is relatively uniform and the pore size is moderate, and the diffusion of tea polyphenol molecules in the gel pores is restricted. The smaller pore size makes it necessary for tea polyphenol molecules to release from the gel through a tortuous path, increasing the diffusion distance and thus extending the sustained-release time. At the same time, the pore structure of the gel can also be adjusted as needed, and the release rate of tea polyphenols can be controlled by changing parameters such as the crosslinking degree, further improving its stability and sustained-release effect.

[0040] The prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads are easily absorbed. In the in vivo environment, gels with good biocompatibility will not be quickly cleared by the immune system as foreign substances, thus having more time and opportunities to be absorbed. The gel formed by genipin-crosslinked black bean peptides has a unique microstructure, usually a three-dimensional network structure. This structure has a large specific surface area, which can increase the contact area with the organism and is beneficial to the exchange and absorption of nutrients or drugs. At the same time, the pore size of the gel is moderate, allowing small molecule substances such as tea polyphenols and some absorption media in the organism (such as water, ions, etc.) to freely diffuse therein, thus promoting the overall absorption process of the gel. Black bean peptides themselves have a certain hydrophilicity, and genipin may also retain some hydrophilic groups after crosslinking, making the gel have good hydrophilicity. The hydrophilic gel can quickly absorb the water in the surrounding environment. When contacting the organism, it is easy to form a hydrated layer on the surface. This hydrated layer can not only reduce the interfacial tension between the gel and biological tissues, making the gel easier to fit with the tissues, but also promote the diffusion and absorption of nutrients or drugs with water as the medium.

[0041] The prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads have a high accuracy of active ingredient release. There are specific interactions between tea polyphenols and black bean peptides, such as hydrogen bonds, van der Waals forces, and electrostatic interactions. These interactions enable tea polyphenols to be stably bound in the gel network formed by black bean peptides. During the release process, these interactions will gradually weaken with the change of environmental conditions (such as pH value, temperature, etc.), so that tea polyphenols can be released from the gel at a specific rate. For example, under the pH value conditions simulating the human intestinal environment, the electrostatic interaction between tea polyphenols and black bean peptides will change, resulting in the gradual dissociation and release of tea polyphenols from the gel. This release mechanism based on specific interactions improves the accuracy of release. Description of the Drawings

[0042] Figure 1 are the appearance image and scanning electron microscope image of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads.

[0043] Figure 2 are the appearance images of the gel beads prepared in Comparative Examples 1 to 3.

[0044] Figure 3 is the cumulative release rate curve of tea polyphenols of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads prepared in Example 1.

[0045] Figure 4 is the preparation flow chart of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads of the present invention. Detailed Embodiments

[0046] The enzyme preparations used in the examples and comparative examples are as follows:

[0047] Papain is derived from McLean, with a Cas number of 9001-73-4 and an enzyme activity of 200U / mg. The enzyme activity is defined as: using ultraviolet spectrophotometry, under the measurement conditions (37±0.2℃; pH 7.0), when the trichloroacetic acid soluble matter released by hydrolyzing casein per minute has an absorbance at a wavelength of 275nm equivalent to that of 1 microgram of tyrosine, the amount of enzyme required is one activity unit, expressed as u / g.

[0048] Bromelain (BROMELAIN) comes from McLean, with Cas number 9001-00-7 and enzyme activity of 300U / mg. The enzyme activity is defined as: using ultraviolet spectrophotometry, under the measuring conditions (37±0.2℃; pH 7.0), when the trichloroacetic acid soluble matter released by casein hydrolysis per minute has an absorbance at a wavelength of 275nm equivalent to that of 1 microgram of tyrosine, the amount of enzyme required is one activity unit, expressed in u / g.

[0049] Ficin comes from Aladdin, with Cas number 9001-33-6 and enzyme activity of 100U / mg. The enzyme activity is defined as: using ultraviolet spectrophotometry, under the measuring conditions (37±0.2℃; pH 7.0), when the trichloroacetic acid soluble matter released by casein hydrolysis per minute has an absorbance at 275nm wavelength equivalent to that of 1 microgram of tyrosine, the amount of enzyme required is one activity unit, expressed as u / g.

[0050] Test method:

[0051] (1) Scanning electron microscopy test: After the samples were freeze-fractured in liquid nitrogen, surface samples and cross-section samples were taken and gold was sprayed. Then, the microstructure of the gel spheres at 1 mm, 200 μm, and 100 μm was analyzed by scanning electron microscopy (SEM, S-4800, Hitachi Co., Tokyo, Japan) at an accelerating voltage of 5 kV.

[0052] (2) Simulated digestion experiment:

[0053] The chemical composition of the simulated digestive fluid at each stage in the in vitro digestion model is shown in Table 1 , and the experimental methods at each stage in the in vitro digestion model are shown in Table 2 .

[0054] Table 1 Chemical composition of simulated digestive fluid at each stage in the in vitro digestion model

[0055]

[0056]

[0057] Table 2 Experimental methods at each stage in the in vitro digestion model

[0058]

[0059] (3) Test for the release performance of tea polyphenols:

[0060] Take 1 mL of the supernatant collected at the oral, gastric, and intestinal digestion stages respectively, add 4 mL of absolute ethanol, shake and mix well to precipitate impurities such as proteins. Centrifuge the mixture at a speed of 10000 rpm for 15 min, take the supernatant and filter it through a 0.45 μm microporous filter membrane, and the filtrate is used for HPLC analysis.

[0061] HPLC analysis conditions: The chromatographic column is a C18 column (250 mm × 4.6 mm, 5 μm); mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, and a gradient elution program is adopted: 0 - 5 min, 5% B; 5 - 20 min, 5% - 30% B; 20 - 30 min, 30% - 50% B; 30 - 40 min, 50% - 95% B; 40 - 45 min, 95% B; 45 - 50 min, 95% - 5% B; flow rate 1.0 mL / min; detection wavelength 280 nm; column temperature 30 °C.

[0062] Calculate the content of tea polyphenols in the supernatant at each digestion stage according to the standard curve, and thus calculate the cumulative release rate of tea polyphenols at different digestion stages.

[0063] Example 1

[0064] A genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel bead, and its preparation method includes the steps:

[0065] S1. Grind the commercially available black bean raw materials into powder and pass through a 100-mesh sieve to obtain black bean powder; disperse the black bean powder in water, the mass ratio of black bean powder to water is 1:10, adjust the pH = 10.5, stir for 2 h, centrifuge at 8000 rpm for 10 min, take the supernatant and place it in a 1 L beaker, adjust the pH = 4.5, let it stand and sediment for 30 min, centrifuge at 8000 rpm for 10 min, take the precipitate and freeze-dry it to obtain black bean protein;

[0066] S2. Dissolve the black bean protein in water, the mass ratio of black bean protein to water is 1:5, treat it with 600 W microwave for 5 min to obtain a black bean protein solution; heat and incubate the black bean protein solution at 55 °C for 20 min, then add papain accounting for 2% of the mass of the black bean protein, enzymatically modify it at 55 °C for 2 h, carry out enzyme inactivation treatment at 95 °C for 10 min and centrifugation treatment at 4 °C and 10000 rpm for 10 min to remove the precipitate, and obtain a black bean peptide solution;

[0067] S3. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 2.0 wt%, mix the black bean peptide solution and the sodium alginate solution evenly according to a volume ratio of 1:1 to obtain solution A;

[0068] S4. Add tea polyphenols to Solution A, stir and mix evenly to obtain Solution B, where the tea polyphenol concentration of Solution B is 0.5 wt%.

[0069] S5. Add an aqueous genipin solution with a genipin concentration of 40 mmol / L to Solution B, and keep it in a constant temperature water bath at 45 °C for 30 min to obtain a highly viscous solution C of genipin-crosslinked black bean peptide-based tea polyphenols.

[0070] S6. Prepare a 2.0 wt% calcium chloride solution, and slowly drip Solution C into the calcium chloride solution using a syringe to form gel beads.

[0071] Example 2

[0072] A preparation method of a genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel bead includes the steps:

[0073] S1. The method of extracting black bean protein is the same as that in Step S1 of Example 1.

[0074] S2. Dissolve black bean protein in water with a mass ratio of black bean protein to water of 1:5, and treat it with 100 W microwave for 15 min to obtain a black bean protein solution; heat and incubate the black bean protein solution at 55 °C for 20 min, then add 2% of the mass of black bean protein of bromelain, enzymatically modify it at 50 °C for 1 h, perform enzyme inactivation treatment at 95 °C for 10 min, and centrifuge at 4 °C and 10,000 rpm for 10 min to remove the precipitate to obtain a black bean peptide solution.

[0075] S3. Dissolve sodium alginate in an aqueous solution to obtain sodium alginate with a concentration of 2.0 wt%, and mix the black bean peptide solution and the sodium alginate solution evenly at a volume ratio of 1:1 to obtain Solution A.

[0076] S4. Add tea polyphenols to Solution A, stir and mix evenly to obtain Solution B, where the tea polyphenol concentration of Solution B is 0.8 wt%.

[0077] S5. Add an aqueous genipin solution with a genipin concentration of 20 mmol / L to Solution B, and keep it in a constant temperature water bath at 35 °C for 30 min to obtain a highly viscous solution C of genipin-crosslinked black bean peptide-based tea polyphenols.

[0078] S6. Prepare a 1.0 wt% calcium chloride solution, and slowly drip Solution C into the calcium chloride solution using a syringe to form gel beads.

[0079] Example 3

[0080] A preparation method of a genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel bead includes the steps:

[0081] S1. Grind the commercially available black bean raw material into powder and pass through an 80-mesh sieve to obtain black bean powder. Disperse the black bean powder in water with a mass ratio of black bean powder to water of 1:5, adjust the pH to 10.5, stir for 2 h, centrifuge at 10,000 rpm for 15 min, take the supernatant and place it in a 1-L beaker, adjust the pH to 4.0, let it stand and sediment for 30 min, centrifuge at 10,000 rpm for 15 min, and freeze-dry the precipitate to obtain black bean protein;

[0082] S2. Dissolve the black bean protein in water with a mass ratio of black bean protein to water of 1:5, treat it with 300-W microwave for 15 min to obtain a black bean protein solution. Heat and incubate the black bean protein solution at 55 °C for 20 min, then add ficin accounting for 3% of the mass of the black bean protein, enzymatically modify it at 50 °C for 2.5 h, perform heat inactivation treatment at 95 °C for 10 min and centrifugation treatment at 4 °C and 10,000 rpm for 10 min to remove the precipitate and obtain a black bean peptide solution;

[0083] S3. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 2.0 wt%, and mix the black bean peptide solution and the sodium alginate solution evenly at a volume ratio of 2:1 to obtain solution A;

[0084] S4. Add tea polyphenols to solution A, stir and mix evenly to obtain solution B with a tea polyphenol concentration of 1.0 wt% in solution B;

[0085] S5. Add an aqueous solution of genipin with a genipin concentration of 30 mmol / L to solution B, and keep it in a constant temperature water bath at 45 °C for 30 min to obtain a genipin-crosslinked black bean peptide-based tea polyphenol highly viscous solution C;

[0086] S6. Prepare a 2.0 wt% calcium chloride solution, and slowly drip solution C into the calcium chloride solution using a syringe to form gel beads.

[0087] Comparative Example 1

[0088] A tea polyphenol gel, and its preparation method includes the steps:

[0089] S1. Grind the commercially available black bean raw material into powder and pass through an 80-mesh sieve to obtain black bean powder. Disperse the black bean powder in water with a mass ratio of black bean powder to water of 1:5, adjust the pH to 10.5, stir for 2 h, centrifuge at 10,000 rpm for 15 min, take the supernatant and place it in a 1-L beaker, adjust the pH to 4.0, let it stand and sediment for 30 min, centrifuge at 10,000 rpm for 15 min, and freeze-dry the precipitate to obtain black bean protein;

[0090] S2. Dissolve black bean protein in water at a mass ratio of black bean protein to water of 1:5 without microwave treatment to obtain a black bean protein solution; incubate the black bean protein solution at 55 °C for 20 min, then add papain at 3% of the mass of the black bean protein, enzymatically modify it at 50 °C for 2 h, perform enzyme inactivation treatment at 95 °C for 10 min, and centrifuge at 4 °C and 10,000 rpm for 10 min to remove the precipitate, obtaining a black bean peptide solution;

[0091] S3. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 2.0 wt%, and mix the black bean peptide solution and the sodium alginate solution evenly at a volume ratio of 2:1 to obtain solution A;

[0092] S4. Add tea polyphenols to solution A and stir evenly to obtain solution B with a tea polyphenol concentration of 1.0 wt%;

[0093] S5. Add an aqueous solution of genipin with a concentration of 10 mmol / L of genipin to solution B and perform a constant temperature water bath at 45 °C for 30 min to obtain solution C. At this time, genipin and black bean peptides cannot form a highly viscous solution;

[0094] S6. Prepare a 2.0 wt% calcium chloride solution, and slowly drip solution C into the calcium chloride solution using a syringe to form a gel structure that is relatively loose.

[0095] Comparative Example 2

[0096] A tea polyphenol gel, the preparation method of which includes the steps:

[0097] S1. The extraction method of black bean protein is the same as that in step S1 of Comparative Example 1;

[0098] S2. The method for obtaining the black bean protein solution is the same as that in step S2 of Comparative Example 1; incubate the black bean protein solution at 55 °C for 20 min, then add papain at 3% of the mass of the black bean protein, enzymatically modify it at 50 °C for 0.5 h, perform enzyme inactivation treatment at 95 °C for 10 min, and centrifuge at 4 °C and 10,000 rpm for 10 min to remove the precipitate, obtaining a black bean peptide solution;

[0099] S3. Dissolve sodium alginate in water to prepare a sodium alginate solution with a concentration of 1.0 wt%, and mix the black bean peptide solution and the sodium alginate solution evenly at a volume ratio of 2:1 to obtain solution A;

[0100] S4. Add tea polyphenols to solution A and stir evenly to obtain solution B with a tea polyphenol concentration of 1.0 wt%;

[0101] S5. Prepare a 2.0 wt% calcium chloride solution, and slowly drip solution B into the calcium chloride solution using a syringe to form a gel structure that is relatively loose.

[0102] Comparative Example 3

[0103] A tea polyphenol gel, the preparation method thereof comprising the steps of:

[0104] S1. The extraction method of black bean protein is the same as that of step S1 in Comparative Example 1;

[0105] S2. The method for obtaining the black bean protein solution is the same as that of step S2 in Comparative Example 1; the black bean protein solution is centrifuged at 10000 rpm for 10 min at 4°C to remove the precipitate, and a black bean peptide solution is obtained;

[0106] S3. Sodium alginate is dissolved in water to prepare a sodium alginate solution with the concentration of sodium alginate being 1.0 wt%, and the black bean peptide solution and the sodium alginate solution are mixed evenly at a volume ratio of 1:1 to obtain solution A;

[0107] S4. Tea polyphenols are added into solution A and stirred evenly to obtain solution B, and the tea polyphenol concentration of solution B is 1.0 wt%;

[0108] S5. An aqueous solution of genipin with the concentration of genipin being 40 mmol / L is added into solution B, and a constant temperature water bath is carried out at 45°C for 30 min to obtain solution C. At this time, genipin and black bean peptides cannot form a relatively viscous solution;

[0109] S6. A 2.0 wt% calcium chloride solution is prepared, and the obtained solution C is slowly dropped into the calcium chloride solution by using a needle syringe, and the formed gel structure is relatively loose.

[0110] Genipin and black bean peptides form a stable three-dimensional network structure through cross-linking reaction. This structure has good mechanical properties and stability, can maintain its integrity under different environmental conditions, and provides a stable carrier for the active ingredient tea polyphenols. For example, in the experiment of simulating the human gastrointestinal environment, the gel structure can resist the erosion of digestive juices, reduce the phenomenon of sudden release of tea polyphenols caused by structural damage, and thus ensure the accuracy of release. The genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel has certain environmental responsiveness. It can respond to some factors in the human physiological environment, such as pH value, temperature, enzymes, etc., so as to achieve the accurate release of active ingredients. For example, there are differences in the pH values in different parts of the gastrointestinal tract. The swelling degree and molecular structure of the gel change in acidic environment (such as in the stomach) and alkaline environment, thereby affecting the release rate of tea polyphenols. In the stomach, the swelling degree of the gel is small and the release of tea polyphenols is slow; while in the small intestine, the swelling degree of the gel increases and the release rate of tea polyphenols accelerates. This precise release according to the pH values in different parts of the gastrointestinal tract improves the accuracy of release.

[0111] Figure 1These are the appearance image, apparent scanning electron micrograph, and cross-sectional scanning electron micrograph of genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads. The genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads prepared in Examples 1 to 3 have an appearance of opaque near-spherical particles with a smooth surface, and the cross-section has a wrinkled network structure. Tea polyphenols can be evenly distributed in the wrinkled network structure, avoiding the destruction of their phenolic hydroxyl group structure by gastric acid, microorganisms, and various enzymes in the body, reducing the direct contact of tea polyphenols with factors such as oxygen, moisture, and light in the external environment, and reducing the oxidation, hydrolysis, etc. of these factors on tea polyphenols, thereby improving their stability.

[0112] Figure 2 The gels prepared in Comparative Example 1 (without microwave treatment), Comparative Example 2 (without genipin crosslinking treatment), and Comparative Example 3 (without black bean protein hydrolysis treatment) have insufficient crosslinking density and do not form gel beads. Among them, microwave treatment is beneficial to promoting the hydrolysis of black bean protein to form small-molecule black bean peptides, thereby increasing the crosslinking network density of the gel. Without microwave treatment, the globulin with a high content in black bean protein cannot be completely exposed, and the black bean peptides prepared by hydrolysis are mostly albumin with a low content in black bean protein. The small molecular weight peptide segments formed by peptide bond cleavage have insufficient crosslinking effect, so gel beads cannot be formed in Comparative Example 1. Genipin can undergo a crosslinking reaction with black bean peptides and can interact with tea polyphenols through covalent bonds or other chemical bonds, making tea polyphenols more firmly bound in the gel network. At the same time, functional groups such as hydroxyl groups in tea polyphenols may undergo chemical reactions with certain groups on genipin or black bean peptides to form new chemical bonds or complexes, thereby improving the stability of tea polyphenols and enabling them to be slowly released in the gel. The lack of genipin crosslinking treatment in Comparative Example 2 results in a relatively loose gel structure. Without black bean protein hydrolysis treatment, in step S2 of Comparative Example 3, the black bean peptides are basically the supernatant of black bean protein dissolved in water, with a low concentration and a high content of hydrophilic amino acids, and the crosslinking reaction effect with genipin is not good, so a stable gel structure cannot be formed in Comparative Example 3. Microwave treatment, black bean protein hydrolysis treatment, and genipin crosslinking treatment increase the crosslinking network density of the gel, promote the formation of gel beads, protect tea polyphenols and improve the stability of tea polyphenols, and improve the directional sustained-release performance of gel beads.

[0113] Figure 3The tea polyphenol release performance curves of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads prepared in Example 1 in simulated oral fluid (pH 6.8), simulated gastric fluid (pH 1.2), and simulated intestinal fluid (pH 6.8). During the simulated oral digestion stage for 10 minutes, the cumulative sustained-release rate of tea polyphenols was only 44.17 ± 1.46%. During the simulated gastric digestion stage for 120 min, the cumulative sustained-release rate of tea polyphenols was only 74.73 ± 2.50%. During the simulated intestinal digestion stage for 120 min, the cumulative sustained-release rate of tea polyphenols was as high as 92.67 ± 2.08%. It shows that the prepared genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads can protect tea polyphenols in simulated oral fluid and simulated gastric fluid, enabling the directional release of tea polyphenols in the intestinal digestion stage and improving the bioavailability of tea polyphenols.

[0114] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A preparation method of genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads, characterized in that, Including the steps: S1. Dissolve black bean protein in water, subject it to microwave treatment and heating incubation, add protease for enzymatic modification, inactivate the enzyme and remove the precipitate to obtain a black bean peptide solution; S2. Mix the black bean peptide solution and sodium alginate solution evenly to obtain solution A; S3. Add tea polyphenols to solution A and stir evenly to obtain solution B; S4. Add genipin aqueous solution to solution B and carry out a constant temperature water bath to obtain solution C; S5. Drop solution C into calcium chloride solution to form gel beads.

2. The preparation method according to claim 1, wherein In step S1, the mass ratio of black bean protein to water is 1:5 - 1:10; the parameters of microwave treatment are 100 - 600W, 5 - 20min; the temperature of heating incubation is 45 - 55°C, and the time of heating incubation is 15 - 30min.

3. The preparation method according to claim 1, characterized in that, In step S1, the protease is one of papain, bromelain, ficin; the addition amount of protease is 1 - 3% of the mass of black bean protein; the enzyme activity of protease is 100 - 300 units / mg.

4. The preparation method according to claim 1, characterized in that, In step S1, the enzymatic modification is to stir at a temperature of 50 - 55°C for 0.5 - 2.5h; the enzyme inactivation treatment is to stir at a temperature of 95 - 100°C for 10 - 20min; remove the precipitate by centrifugation; the parameters of centrifugation treatment are a temperature of 4 - 15°C, a rotation speed of 8000 - 10000rpm, and a time of 10 - 30min.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of sodium alginate in the sodium alginate solution is 0.5 - 2.0wt%; the mixing volume ratio of the black bean peptide solution to the sodium alginate solution is 1 - 1:3 - 1.

6. The preparation method according to claim 1, characterized in that, In step S3, the concentration of tea polyphenols in solution B is 0.3 - 1.0wt%.

7. The preparation method according to claim 1, wherein In step S4, the concentration of genipin in the genipin aqueous solution is 20 - 40mmol / L; the temperature of the constant temperature water bath is 35 - 40°C, and the time of the constant temperature water bath is 20 - 60min.

8. The preparation method according to claim 1, characterized in that, In step S5, the concentration of calcium chloride in the calcium chloride solution is 0.5 - 2.5wt%.

9. Genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads prepared by any of the preparation methods of claims 1 - 8.

10. Application of the genipin-crosslinked black bean peptide-based tea polyphenol sustained-release gel beads described in claim 9 in the food field.

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