Gamma-aminobutyric acid sustained release microsphere as well as preparation method and application thereof

Abstract

The invention relates to a food nutrition technology, and discloses gamma-aminobutyric acid sustained-release microspheres as well as a preparation method and application thereof. According to the sustained-release microspheres, covalent cross-linked hyaluronic acid or salt thereof-beta-glucan-konjac glucomannan-malic acid polymer and alginate form a composite wall material, and the defects that single sodium alginate microspheres are loose, porous and poor in compactness are overcome, so that the encapsulation efficiency of GABA is improved, and sudden release is not likely to happen. The sustained-release microspheres with different particle sizes and proportions thereof are selected, so that the GABA is continuously and stably released in the small intestine within 1-4 hours. The gamma-aminobutyric acid sustained-release microspheres can be widely applied to preparation of functional foods, health foods and special medical foods for soothing nerves, helping sleep, relieving anxiety and the like, and have good market application prospects and social benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of food nutrition, and in particular to gamma-aminobutyric acid sustained-release microspheres and a preparation method and application thereof. Background Art

[0002] Gamma-aminobutyric acid (GABA) is an important non-protein amino acid and a classic inhibitory neurotransmitter. GABA is a crystalline powder at room temperature, white or nearly white, with a relative molecular weight of 103.12. It is highly soluble in water, slightly soluble in hot ethanol, insoluble in common organic solvents, and has a melting point of 202-204°C. GABA is widely distributed in animals, plants, and microorganisms. In 1950, studies found that GABA exists in the central nervous system of mammals. Subsequent studies have shown that GABA is the main inhibitory neurotransmitter of the central nervous system and a multifunctional molecule with different functions. It has multiple physiological functions such as lowering blood pressure, lowering blood sugar, anti-tumor, anti-depression, sedation and analgesia, improving brain activity, and improving lipid metabolism. It has been used in food, health products, feed and other fields, and a series of products such as GABA capsules, GABA tea, and GABA soft candies have been developed.

[0003] As an inhibitory neurotransmitter, GABA plays a key role in regulating the balance of excitation and inhibition of neurons in the central nervous system. The allosteric sites on its three receptors (GABAA, GABAB and GABAC) can regulate the inhibition level of neurons in the relevant brain areas with high precision. These sites are also the molecular targets of hypnotic drugs and are related to the sleep-promoting effect. Studies have shown that exogenously ingested GABA can enter the blood. The GABA level in the blood reaches the highest level 30 minutes after oral administration of GABA. GABA can affect the GABA content level and receptor expression in the central nervous system through the vagus nerve pathway in the gut-brain axis. After oral administration of GABA, the mRNA and protein expression levels of GABAergic neurotransmitters in mice were significantly upregulated, especially in the intestine and brain, where the GABAA level increased by 7 times and 5 times, respectively. GABA has a relatively short half-life in the body, and oral GABA is metabolized quickly in the body. Therefore, if GABA is supplemented directly, it is difficult to maintain an effective concentration for a long time. However, making it into a sustained-release preparation and slowly releasing GABA can not only avoid the stimulation of high-concentration drugs on the gastrointestinal tract, but also help to maintain a relatively stable concentration of GABA in the blood for a certain period of time, thereby prolonging the action time of GABA. However, it is difficult for existing GABA sustained-release preparations to achieve an ideal release rate. If the release rate is too fast, the action time will be short, making it difficult to ensure the quality of sleep at night, while if the release rate is too slow, more GABA will still exist in the body after sleep, affecting the work and life status during the day.

[0004] Sodium alginate is a common base material for nanoparticles. However, the sodium alginate microspheres with a single wall material are loose and porous, with poor density, resulting in low encapsulation efficiency of effective substances and easy sudden release, and unable to achieve a sustained and stable release of effective ingredients at an appropriate release rate. Therefore, a sustained-release microsphere with a sustained and stable release rate of γ-aminobutyric acid (GABA) has a very broad application prospect. Summary of the invention

[0005] In view of the shortcomings and deficiencies of the prior art, the present invention provides a method for preparing gamma-aminobutyric acid (GABA) sustained-release microspheres, so that GABA can be continuously and stably released in the small intestine at an appropriate release rate. At the same time, the present invention also provides the use of the above-mentioned sustained-release GABA sustained-release microspheres in the preparation of functional foods, health foods, and special medical foods for calming the nerves, helping sleep, and relieving anxiety.

[0006] The present invention uses sodium alginate as a substrate, and achieves GABA encapsulation through an endogenous emulsification method to prepare microspheres that can be released in a controlled manner. Sodium alginate microspheres have excellent pH response characteristics, but sodium alginate microspheres with a single wall material are loose and porous, and have poor density, resulting in a low encapsulation rate of effective substances and easy sudden release, and cannot achieve continuous and stable release of effective ingredients at a suitable release rate.

[0007] The invention introduces covalently cross-linked polysaccharides as composite wall materials to solve the problems of low encapsulation efficiency and poor density. At the same time, the invention prepares GABA microspheres of different particle sizes through different emulsification methods. The preferred microsphere particle size ratio achieves the sustained and stable release of GABA in the small intestine within 1-4 hours. The preparation process of the controlled-release microspheres of the invention uses food-grade raw materials, which is green and safe and has good market application prospects. The specific scheme is as follows

[0008] In a first aspect, the present invention provides a γ-aminobutyric acid sustained-release microsphere, wherein the sustained-release microsphere comprises γ-aminobutyric acid or a salt thereof and a wall material.

[0009] Furthermore, the added amount of γ-aminobutyric acid is 3% to 15% of the total mass of the wall material, for example, it can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and any value in between.

[0010] Furthermore, the added amount of γ-aminobutyric acid is 5% to 10% of the total mass of the wall material.

[0011] Furthermore, the wall material comprises hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer (HA-DT-KGM-MA) and alginate.

[0012] Furthermore, the mass ratio of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer to alginate is (1-5):(5-20), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 and any value in between.

[0013] Furthermore, the microspheres also contain calcium carbonate, wherein the amount of calcium carbonate added is 0.5%-1% of the total mass of the wall material, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and any value therebetween.

[0014] Furthermore, the hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer is formed by mixing hyaluronic acid or its salt, β-glucan, konjac glucomannan and then mixing with malic acid.

[0015] 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. Exemplarily, the mass ratio can be 1:10:5, 2:15:10, 3:30:14, 2:11:6, 3:12:7:, 4:13:8, 5:4:9, 2:15:10, 3:16:11, 4:17:9, 5:18:10, 2:19:11, 3:20:12, 4:21:13, 5:22:14, 2:22:15, 3:22:13, 4:23:10, 4:24:15 and any value in between.

[0016] Furthermore, the mass ratio is (1-3):(10-30):(5-15).

[0017] Furthermore, the added amount of malic acid is 0.1-0.2% of the total mass of hyaluronic acid or its salt, β-glucan, and konjac glucomannan, for example, it can be 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2% or any value in between.

[0018] Furthermore, the hyaluronic acid or its salt comprises sodium hyaluronate.

[0019] In a second aspect, the present invention provides a γ-aminobutyric acid sustained-release preparation, comprising a first sustained-release microsphere and a second sustained-release microsphere.

[0020] The mass ratio of the first sustained-release microspheres to the second sustained-release microspheres is (2.5-3.5):1. For example, the mass ratio of the first sustained-release microspheres to the second sustained-release microspheres can be 2.5:1, 2.55:1, 2.6:1, 2.65:1, 2.7:1, 2.75:1, 2.8:1, 2.85:1, 2.9:1, 2.95:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1 or any ratio therebetween.

[0021] The first sustained-release microspheres comprise 80-200 mesh γ-aminobutyric acid sustained-release microspheres; for example, the sustained-release microspheres can be 80 mesh, 85 mesh, 90 mesh, 95 mesh, 100 mesh, 105 mesh, 110 mesh, 115 mesh, 120 mesh, 125 mesh, 130 mesh, 135 mesh, 140 mesh, 145 mesh, 150 mesh, 155 mesh, 160 mesh, 165 mesh, 170 mesh, 175 mesh, 180 mesh, 190 mesh, 200 mesh and any value therebetween.

[0022] The second sustained-release microspheres comprise γ-aminobutyric acid sustained-release microspheres having a size greater than 200 mesh. For example, the sustained-release microspheres may be 200 mesh, 205 mesh, 210 mesh, 215 mesh, 220 mesh, 225 mesh, 230 mesh, 235 mesh, 240 mesh, 245 mesh, 250 mesh, 255 mesh, 260 mesh, 265 mesh, 270 mesh, 275 mesh, 280 mesh, 285 mesh, 290 mesh, 295 mesh, 300 mesh, 350 mesh, 400 mesh, and any value thereof.

[0023] In a third aspect, the present invention provides a method for preparing gamma-aminobutyric acid (GABA) sustained-release microspheres, the method comprising the following steps:

[0024] S1. preparing a covalently cross-linked hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer, preferably, the cation of the hyaluronic acid or its salt is selected from at least one of H, K, Na, Ca or Zn, more preferably, the hyaluronate is sodium hyaluronate;

[0025] S2. The hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer, alginate, γ-aminobutyric acid and calcium carbonate prepared in step S1 are dispersed in water and mixed to obtain a wall material;

[0026] Preferably, the alginate includes but is not limited to one of sodium alginate, potassium alginate or ammonium alginate;

[0027] S3. Add the oil phase to the aqueous solution obtained in step S2 and emulsify to form an oil-in-water emulsion;

[0028] Preferably, the emulsification is at least a high-pressure homogenization emulsification method and / or a high-speed shear emulsification method;

[0029] More preferably, the emulsifier includes, but is not limited to, at least one of sucrose fatty acid esters, span, polylactic acid, polyglycolic acid, or polycaprolactone;

[0030] More preferably, the emulsifier includes but is not limited to at least one of sucrose fatty acid ester and Span;

[0031] S4. The pH of the emulsion prepared in step S3 is adjusted to be acidic, stirred at low speed, and then allowed to stand to collect the microspheres;

[0032] Preferably, the pH value is <7;

[0033] Preferably, the pH value is 6.95, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.95, 3.9, 3.85, 3.8, 3.75, 3.7, 3.65, 3.6, 3.55, 3.5, 3.45, 3.4, 3.35, 3.3, 3.25, 3.2, 3.15, 3.1, 3.05, 3.0, 2.95, 2.9, 2. 85, 2.8, 2.75, 2.7, 2.65, 2.6, 2.55, 2.5, 2.45, 2.4, 2.35, 2.3, 2.25, 2.2, 2.15, 2.1, 2.05, 2.0, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.5, 1.45, 1.4, 1.35, 1.3, 1.25, 1.2, 1.15, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.001, 0.0001, 0.00001, 0 or any value in between;

[0034] S5. solidifying the microspheres prepared in step S4 to obtain sustained-release microspheres of γ-aminobutyric acid;

[0035] Preferably, the curing agent is at least one of calcium chloride and calcium lactate;

[0036] Furthermore, the mass concentration of the curing agent is 0.25% to 1.5%, for example, it can be 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% and any value in between.

[0037] In a specific embodiment of the present invention, the mass concentration of the curing agent is 0.25%, 0.30%, or 0.40%.

[0038] Furthermore, the preparation of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer comprises the following steps:

[0039] S1-1. The hyaluronic acid or its salt, β-glucan, konjac glucomannan are mixed and placed in a granulator;

[0040] Preferably, the hyaluronic acid or its salt cation is selected from at least one of H, K, Na, Ca or Zn, and more preferably, the hyaluronate salt is sodium hyaluronate;

[0041] Preferably, the mass ratio of sodium hyaluronate, β-glucan and konjac glucomannan is (1-5):(10-50):(5-30), for example, the mass ratio of hyaluronic acid or its salt can be 1, 2, 3, 4, 5, the mass ratio of β-glucan can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and the mass ratio of konjac glucomannan can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, illustratively, the mass ratio can be 1:10:5, 2:15:10, 3:30:14, 2:11:6, 3:12:7:, 4:13:8, 5:14:9, 2:15:10, 3:16:11, 4:17:9, 5:18:10, 2:19:11, 3:20:12, 4:21:13, 5:22:14, 2:22:15, 3:22:13, 4:23:10, 4:24:15 and any value therebetween;

[0042] More preferably, the mass ratio is (1-3):(10-30):(5-15);

[0043] S1-2. Malic acid (MA) solution was added to the granulator in step S1-1 to obtain sodium hyaluronate-β-glucan-konjac glucomannan-malic acid polymer;

[0044] Preferably, the amount of malic acid solution added is 0.1%-0.2% of the total mass of sodium hyaluronate, β-glucan, and konjac glucomannan in step S1-1; for example, it can be 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2% or any value therebetween.

[0045] Further, the initial inlet air temperature is 50-60°C, for example, the initial inlet air temperature is 50°C, 50.5°C, 51°C, 51.5°C, 52°C, 52.5°C, 53°C, 53.5°C, 54°C, 54.5°C, 55°C, 55.5°C, 56°C, 56.5°C, 57°C, 57.5°C, 58°C, 58.5°C, 59°C, 59.5°C, 60°C or any temperature therebetween.

[0046] Further, the increased inlet air temperature is 90-110°C, for example, the increased inlet air temperature is 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C or any temperature therebetween.

[0047] More preferably, the inlet air temperature is 90-100°C.

[0048] Furthermore, the inlet and outlet air temperatures of the general conventional granulation of the granulator can all meet the requirements of the present technical solution, and the embodiments are only exemplary solutions.

[0049] Furthermore, the mass concentration of the wall material described in step S2 is 0.5% to 2%;

[0050] Preferably, the mass ratio of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer to sodium alginate is (1-5):(5-20), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 and any value in between.

[0051] More preferably, the added amount of GABA is 3% to 15% of the total mass of the wall material, for example, it can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and any value in between.

[0052] Most preferably, the addition amount of calcium carbonate is 0.5%-1% of the total mass of the composite wall material, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and any value therebetween.

[0053] Furthermore, the mass ratio of pure water in the aqueous phase to oil in the oil phase in step S3 is (1-3):(1-6), for example, the mass ratio of pure water in the aqueous phase to oil in the oil phase can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 2:1, 2:1.5, 2:2.5, 2:3.5, 2:4.5, 2:5.5, 3:1, 3:1.5, 3:2, 3:2.5, 3:3, 3:3.5, 3:4, 3:5, 3:5.5 or any ratio therebetween.

[0054] Furthermore, the amount of the emulsifier used is 0.25% to 1.5% of the mass of the oil in the oil phase. For example, the amount of the emulsifier used can be 0.25%, 0.3%, 0.35%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.57%, 0.59%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.80%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5% or any number in between.

[0055] Further, the emulsifier includes but is not limited to at least one of sucrose fatty acid ester and Span;

[0056] Furthermore, the oil phase comprises at least one vegetable oil or animal oil.

[0057] Preferably, the vegetable oil comprises oil extracted from fruits, seeds or germs of plants.

[0058] More preferably, the vegetable oil includes but is not limited to any one of soybean oil, linseed oil, sunflower seed oil, cottonseed oil, sesame oil, olive oil, coconut oil, castor oil and the like.

[0059] Preferably, the animal oil comprises animal fat.

[0060] More preferably, the animal oil includes at least one of lard and fish oil.

[0061] Furthermore, the processing condition of the high-pressure homogenization emulsification method is a homogenization pressure of 35 to 50 MPa; for example, the homogenization pressure can be 35 MPa, 36 MPa, 37 MPa, 38 MPa, 39 MPa, 40 MPa, 41 MPa, 42 MPa, 43 MPa, 44 MPa, 45 MPa, 46 MPa, 47 MPa, 48 MPa, 49 MPa, 50 MPa or any pressure value therebetween.

[0062] The processing condition of the high-speed shear emulsification method is a shear speed of 1000 to 2500 rpm. For example, the shear speed can be 1000 rpm, 1050 rpm, 1100 rpm, 1150 rpm, 1200 rpm, 1250 rpm, 1300 rpm, 1350 rpm, 1400 rpm, 1450 rpm, 1500 rpm, 1550 rpm, 1600 rpm, 1650 rpm, 1700 rpm, 1750 rpm, 1800 rpm, 1850 rpm, 1900 rpm, 1950 rpm, 2000 rpm, 2050 rpm, 2100 rpm, 2150 rpm, 2200 rpm, 2250 rpm, 2300 rpm, 2350 rpm, 2400 rpm, 2450 rpm, 2500 rpm or any speed value therebetween.

[0063] Conventional high-pressure homogenization emulsification methods can satisfy the technical solution of this invention. The embodiments are only exemplary solutions for preparing microspheres with a mesh size of 200 or more.

[0064] Conventional high-speed shear emulsification methods can satisfy the present technical solution. The embodiments are only exemplary solutions for preparing microspheres with a mesh size of 80-200.

[0065] You can also choose any one of the high-pressure homogenization emulsification method or the high-speed shearing emulsification method, or choose both the high-pressure homogenization emulsification method and the high-speed shearing emulsification method to prepare microspheres of different mesh sizes.

[0066] Furthermore, the method further comprises the step of washing and drying the γ-aminobutyric acid microspheres prepared in step S5.

[0067] Preferably, the cleaning step comprises cleaning with an acidic aqueous solution having a pH of 3 to 4.5, for example, the pH value of the acidic aqueous solution may be 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.95, 3.9, 3.85, 3.8, 3.75, 3.7, 3.65, 3.6, 3.55, 3.5, 3.45, 3.4, 3.35, 3.3, 3.25, 3.2, 3.15, 3.1, 3.05, 3.0 or any pH value therebetween.

[0068] Preferably, the cleaning step may be repeated at least once, for example, once, twice, three times, four times, five times or more.

[0069] Furthermore, the drying method is selected from at least one of vacuum drying and freeze drying.

[0070] Furthermore, the washed and dried microspheres need to be mixed in different particle size ranges according to a certain ratio.

[0071] Preferably, the collected microspheres are in the range of 80-200 mesh and microspheres above 200 mesh.

[0072] Preferably, the mixing ratio of the microspheres of 80-200 mesh and those larger than 200 mesh is (2.5-3.5):1.

[0073] Preferably, the mixing ratio of the microspheres of 80-200 mesh and larger than 200 mesh is 2.5:1, 2.55:1, 2.6:1, 2.65:1, 2.7:1, 2.75:1, 2.8:1, 2.85:1, 2.9:1, 2.95:1, 3.0:1, 3.05:1, 3.1:1, 3.2:1, 3.25:1, 3.3:1, 3.35:1, 3.4:1, 3.45:1, 3.5:1 or any ratio therebetween.

[0074] The fourth invention of the present invention provides a covalently cross-linked polymer of hyaluronic acid or a salt thereof-β-glucan-konjac glucomannan-malic acid.

[0075] Preferably, the cation of the hyaluronic acid or its salt is selected from at least one of H, K, Na, Ca or Zn, and more preferably, the hyaluronate salt is sodium hyaluronate.

[0076] Preferably, the mass ratio of hyaluronic acid or its salt, β-glucan and konjac glucomannan in the polymer is (1-5):(10-50):(5-30), and more preferably, the mass ratio is (1-3):(10-30):(5-15).

[0077] The fifth aspect of the present invention provides the use of the covalently cross-linked polymer of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid in the preparation of a γ-aminobutyric acid sustained-release preparation.

[0078] Preferably, the cation of the hyaluronic acid or its salt is selected from at least one of H, K, Na, Ca or Zn, and more preferably, the hyaluronate salt is sodium hyaluronate.

[0079] Preferably, the sustained-release preparation is selected from at least one of sustained-release microspheres, sustained-release suspensions, sustained-release composite solutions, film-coated preparations, sustained-release capsules, and sustained-release compressed tablets.

[0080] The sixth aspect of the present invention provides the use of the sustained-release microspheres or the sustained-release preparations or the sustained-release microspheres prepared by the method or the hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymers in the preparation of functional foods, health foods, and special medical foods.

[0081] Preferably, the functional food, health food, special medical food is a functional food, health food, special medical food that can calm the nerves, help sleep, relieve anxiety, etc.

[0082] The beneficial effects of the present invention include but are not limited to:

[0083] 1. The hyaluronic acid or its salt, β-glucan, konjac glucomannan and malic acid are covalently cross-linked to obtain sodium hyaluronate-β-glucan-konjac glucomannan-malic acid polymer, which overcomes the defects of single sodium alginate microspheres that are loose and porous and have poor density, thereby improving the encapsulation rate of γ-aminobutyric acid and preventing sudden release.

[0084] 2. The present invention prepares γ-aminobutyric acid microspheres of different particle size ranges, wherein the first sustained-release microspheres contain 80-200 mesh γ-aminobutyric acid sustained-release microspheres, and the second sustained-release microspheres contain γ-aminobutyric acid sustained-release microspheres larger than 200 mesh. By adjusting the mass ratio of the first sustained-release microspheres to the second sustained-release microspheres to (2.5-3.5):1, a sustained and stable release of γ-aminobutyric acid within 1-4 hours in the small intestine is achieved.

[0085] 3. The preparation process of the controlled-release microspheres of the present invention uses food-grade raw materials, has no toxic reagent residues, is green and safe, has a simple process, and can be industrially produced. It can be widely used in the preparation of functional foods, health foods, and special medical foods that can calm the nerves, help sleep, and relieve anxiety, and has good market application prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0087] Figure 1 1 is a schematic diagram of the effect of the polymer component ratio on the encapsulation rate and dissolution rate of GABA in the embodiment of the present invention, wherein FIG (A) is a schematic diagram of the encapsulation rate; FIG (B) is a schematic diagram of the dissolution rate;

[0088] Figure 2 1 is a schematic diagram of the effect of the amount of malic acid added on the encapsulation rate and dissolution rate of GABA in the embodiments of the present invention, wherein FIG (A) is a schematic diagram of the encapsulation rate; FIG (B) is a schematic diagram of the dissolution rate;

[0089] Figure 3 1 is a schematic diagram showing the effect of the ratio of the composite wall material components on the encapsulation rate and dissolution rate of GABA in an embodiment of the present invention, wherein FIG (A) is a schematic diagram showing the results of the encapsulation rate; FIG (B) is a schematic diagram showing the results of the dissolution rate;

[0090] Figure 4 1 is a schematic diagram of the effect of the amount of calcium carbonate added on the encapsulation rate and dissolution rate of GABA in the embodiments of the present invention, wherein FIG (A) is a schematic diagram of the results of the encapsulation rate; FIG (B) is a schematic diagram of the results of the dissolution rate;

[0091] Figure 5 1 is a schematic diagram of the effect of the number and ratio of microspheres on the encapsulation rate and dissolution rate of GABA in the embodiments of the present invention, wherein Figure (A) is a schematic diagram of the encapsulation rate; Figure (B) is a schematic diagram of the dissolution rate. DETAILED DESCRIPTION

[0092] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0093] Sodium hyaluronate: Bloomage Biotechnology, product number 22060152.

[0094] Example 1

[0095] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0096] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0097] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0098] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0099] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0100] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0101] Example 2

[0102] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0103] (1) 200 g of sodium hyaluronate, 1500 g of β-glucan, and 1000 g of konjac glucomannan were mixed and placed in a one-step granulator. 2.7 g of malic acid was dissolved in 267.3 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0104] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0105] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0106] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0107] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0108] Example 3

[0109] (1) 300 g of sodium hyaluronate, 3000 g of β-glucan, and 1400 g of konjac glucomannan were mixed and placed in a one-step granulator. 4.7 g of malic acid was dissolved in 465.3 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0110] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0111] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0112] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0113] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0114] Example 4

[0115] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0116] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 2.4 g of malic acid was dissolved in 237.6 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the malic acid solution was added by spray gun atomization. After the malic acid was added, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0117] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0118] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0119] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0120] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0121] Example 5

[0122] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0123] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 3.2 g of malic acid was dissolved in 316.8 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0124] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0125] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0126] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0127] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0128] Example 6

[0129] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0130] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0131] (2) 2 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 40 g of sodium alginate were dispersed in 4158 g of pure water, and then 2.52 g of GABA and 0.21 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0132] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, and each part was added to 4158 g of soybean oil, and 20.79 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form a water-in-oil emulsion;

[0133] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0134] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0135] Example 7

[0136] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0137] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0138] (2) 30 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 30 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0139] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0140] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0141] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0142] Example 8

[0143] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0144] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0145] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.45 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0146] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0147] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0148] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0149] Example 9

[0150] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0151] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0152] (2) Dispersing 10 g of the covalently cross-linked HA-DT-KGM-MA of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid prepared by the polymer in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.6 g of calcium carbonate, dispersing and mixing to obtain an aqueous solution;

[0153] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0154] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0155] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0156] Example 10

[0157] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0158] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0159] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0160] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0161] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0162] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, the precipitate was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres with 80-200 mesh and those with more than 200 mesh were collected and mixed at a mass ratio of 3.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0163] Embodiment 11

[0164] A method for preparing GABA microspheres for sustained release in the small intestine, the specific steps are as follows:

[0165] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain a covalently cross-linked polymer of sodium hyaluronate-β-glucan-konjac glucomannan-malic acid, HA-DT-KGM-MA;

[0166] (2) 10 g of the sodium hyaluronate-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate were dispersed in 5940 g of pure water, and then 3.6 g of GABA and 0.3 g of calcium carbonate were added, and the mixture was dispersed and mixed to obtain an aqueous solution;

[0167] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0168] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0169] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 3:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0170] Comparative Example 1

[0171] The preparation method is the same as that of Example 1, except that sodium hyaluronate, β-glucan, and konjac glucomannan are not covalently cross-linked. The specific steps are as follows:

[0172] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, 500 g of konjac glucomannan, and 1.6 g of malic acid were mixed uniformly to obtain a composite polysaccharide;

[0173] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0174] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts and added to 5940 g of soybean oil and 29.7 g of sucrose fatty acid ester respectively, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form a water-in-oil emulsion;

[0175] (4) adjusting the pH of the emulsion obtained in step (3) to 3, stirring at a low speed for 30 minutes and then letting it stand, collecting the wet microspheres obtained by different emulsification methods respectively. Then adding them to 0.5% calcium chloride solution respectively, stirring and solidifying at room temperature, and then separating and collecting the bottom quicksand precipitate respectively;

[0176] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0177] Comparative Example 2

[0178] (1) 50 g of sodium hyaluronate, 500 g of β-glucan, and 100 g of konjac glucomannan were mixed and placed in a one-step granulator. 0.65 g of malic acid was dissolved in 64.35 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0179] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0180] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0181] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0182] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0183] Comparative Example 3

[0184] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 0.8 g of malic acid was dissolved in 79.2 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0185] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0186] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0187] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0188] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0189] Comparative Example 4

[0190] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 4 g of malic acid was dissolved in 396 g of pure water, and the air inlet temperature of the one-step granulator was set to 55° C. All of the above malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95° C. and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0191] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0192] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0193] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0194] (5) The precipitate obtained in step (4) is washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, is sieved through an 80-mesh sieve and a 200-mesh sieve, respectively, and microspheres of 80-200 mesh and larger than 200 mesh are collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres that can be sustained-released in the small intestine.

[0195] Comparative Example 5

[0196] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0197] (2) Dispersing 6 g of the polymer HA-DT-KGM-MA prepared in step (1) and 24 g of sodium alginate in 2970 g of pure water, then adding 1.8 g of GABA and 0.15 g of calcium carbonate, dispersing and mixing to obtain an aqueous solution;

[0198] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, and each part was added to 2970 g of soybean oil, and 14.85 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min, to form a water-in-oil emulsion;

[0199] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0200] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0201] Comparative Example 6

[0202] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0203] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.06 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0204] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0205] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0206] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0207] Comparative Example 7

[0208] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0209] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.9 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0210] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0211] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes, and then let it stand to collect the two emulsified wet microspheres. Then add them to 0.5% calcium chloride solution, stir and solidify at room temperature, and then separate and collect the bottom quicksand precipitate;

[0212] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2.5:1 to obtain GABA microspheres.

[0213] Comparative Example 8

[0214] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0215] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0216] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0217] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification respectively, then add them to 0.5% calcium chloride solution respectively, stir and solidify at room temperature, then separate and collect the bottom quicksand precipitate respectively;

[0218] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres above the 80-mesh sieve were collected to obtain GABA microspheres.

[0219] Comparative Example 9

[0220] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0221] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0222] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0223] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0224] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 2:1 to obtain GABA microspheres.

[0225] Comparative Example 10

[0226] (1) 100 g of sodium hyaluronate, 1000 g of β-glucan, and 500 g of konjac glucomannan were mixed and placed in a one-step granulator. 1.6 g of malic acid was dissolved in 158.4 g of pure water, and the air inlet temperature of the one-step granulator was set to 55°C. All of the malic acid solution was added by spray gun atomization. After the addition of malic acid, the air inlet temperature of the one-step granulator was raised to 95°C, and cross-linked for 4 hours to obtain the polymer HA-DT-KGM-MA;

[0227] (2) Dispersing 10 g of the polymer HA-DT-KGM-MA prepared in step (1) and 50 g of sodium alginate in 5940 g of pure water, then adding 3.6 g of GABA and 0.3 g of calcium carbonate, and dispersing and mixing to obtain an aqueous solution;

[0228] (3) the aqueous phase solution obtained in step (2) was divided into two equal parts, each of which was added to 5940 g of soybean oil, and 29.7 g of sucrose fatty acid ester was added to each part, one part was homogenized at 40 MPa for 3 times, and the other part was sheared at 1500 rpm for 15 min to form an oil-in-water emulsion;

[0229] (4) Adjust the pH of the emulsion obtained in step (3) to 3, stir at a low speed for 30 minutes and then let it stand, collect the two wet microspheres obtained by emulsification, add them into 0.5% calcium chloride solution, stir and solidify at room temperature, separate and collect the bottom quicksand precipitate;

[0230] (5) The precipitate obtained in step (4) was washed three times with a pH 3.8 aqueous solution to remove free GABA, and after freeze-drying, it was sieved through an 80-mesh sieve and a 200-mesh sieve, and microspheres of 80-200 mesh and larger than 200 mesh were collected and mixed at a mass ratio of 4:1 to obtain GABA microspheres.

[0231] In order to evaluate the sustained release effect of the GABA microspheres prepared by the present invention in the small intestine, the encapsulation efficiency and dissolution curve of the GABA microspheres prepared in the above examples and comparative examples were measured, as follows:

[0232] (1) Determination of encapsulation efficiency:

[0233] 2 g of GABA microspheres prepared in the examples and comparative examples were placed in a mixed disintegration solution containing 0.2 mol / L NaHCO3 and 0.06 mol / L Na3C6H5O7·2H2O (pH>8.0), stirred for 6 h until the microspheres were completely disintegrated, and the GABA content in the disintegration solution was determined by high performance liquid chromatography, recorded as W 总 The cleaning solutions of all the examples and comparative examples were collected, and the GABA content in the cleaning solutions was determined by high performance liquid chromatography to determine the free GABA content, which was recorded as W 游离 The encapsulation efficiency of GABA in different embodiments and comparative examples was calculated. The formula is as follows:

[0234]

[0235] The GABA encapsulation efficiency results of Examples 1-11 and Comparative Examples 1-10 are shown in Table 1.

[0236] Table 1 Encapsulation efficiency results

[0237]

[0238]

[0239] (2) Determination of dissolution curve:

[0240] Take 10g of the finished GABA microspheres prepared by the examples and comparative examples, add simulated gastric juice (SGF, pH 1.2) to simulate digestion for 2h, centrifuge and remove the microspheres and transfer them to simulated intestinal fluid (SIF, pH 7.4) for 4h of simulated digestion, and continue stirring until the microspheres are completely disintegrated. The content of GABA in the reaction solution after 1h of simulated gastric juice digestion, 2h of simulated gastric juice digestion, 1h of simulated intestinal juice digestion, 2h of simulated intestinal juice digestion, 3h of simulated intestinal juice digestion, 4h of simulated intestinal juice digestion and complete disintegration is determined by high performance liquid chromatography, and the dissolution rate of GABA at different time points during the simulated gastrointestinal digestion process is calculated, and the dissolution curve is drawn.

[0241] Test results such as Figure 1-Figure 5 :

[0242] from Figure 1It can be found that the encapsulation rate and controlled release effect of the microspheres prepared in the embodiment on GABA are significantly better than those in the comparative example. The encapsulation rate of Example 1 is increased by more than 70% compared with that of Comparative Example 1. After 1h of simulated intestinal fluid digestion, the GABA dissolution rate of the microspheres prepared in Examples 1-3 is about 50%, while the dissolution rate of Comparative Example 1 is more than 71%, and the dissolution rate of Comparative Example 2 has reached more than 89%, indicating that the GABA microspheres prepared in Comparative Examples 1 and 2 are released suddenly in the intestinal fluid within 1h, which is not conducive to the continuous effect of GABA. The polymer formed by covalent cross-linking of sodium hyaluronate, β-glucan, and konjac glucomannan with a suitable ratio can form a good interpenetrating network structure with sodium alginate, overcoming the defects of loose porous and poor compactness of single sodium alginate microspheres, so that the microspheres prepared by the technology of the present invention have a higher encapsulation rate and better controlled release effect on GABA.

[0243] from Figure 2 It can be found that the encapsulation rate of GABA in the microspheres prepared in Example 3 and Comparative Example 4 is significantly better than that in Comparative Example 3, wherein insufficient malic acid is added in Comparative Example 3, resulting in the inability to achieve covalent crosslinking of sodium hyaluronate, β-glucan, and konjac glucomannan, and the prepared microspheres have poor compactness, resulting in an encapsulation rate of only 52%. Due to poor compactness, a sudden release occurs in the simulated intestinal fluid digestion process in Comparative Example 3, and excessive addition of malic acid in polymer crosslinking in Comparative Example 4 results in a slow release of GABA in the simulated intestinal fluid digestion process in the prepared microspheres, and the GABA dissolution rate after 4 hours of simulated intestinal fluid digestion is only 73%, which is not conducive to the sleep-inducing effect of GABA, and the long release time affects the mental state during the day.

[0244] from Figure 3 It can be found that the encapsulation rate and controlled release effect of the microspheres prepared in the embodiment on GABA are significantly better than those in comparative example 5. The ratio of polymer to sodium alginate in comparative example 5 is not within the scope required by the present invention. Compared with comparative example 5, the encapsulation rate of embodiment 7 is increased by more than 130%. After 1h of simulated intestinal fluid digestion, the dissolution rate of comparative example 5 has reached more than 70%. The polymer formed by covalent cross-linking of sodium hyaluronate, β-glucan, and konjac glucomannan can form a good interpenetrating network structure with sodium alginate at a certain ratio, overcoming the defects of loose porous and poor density of single sodium alginate microspheres. The microspheres prepared by the technology of the present invention have a high encapsulation rate and good controlled release effect on GABA.

[0245] from Figure 4It can be found that the encapsulation rate of GABA in the microspheres prepared in Example 6 and Comparative Example 7 is significantly higher than that in Comparative Example 6. The content of calcium carbonate added in the GABA microspheres prepared in Comparative Example 6 is too low, and the content of calcium carbonate added in the GABA microspheres prepared in Comparative Example 7 is higher than the requirements of the present invention. Compared with Comparative Example 6, the encapsulation rate of Example 9 is increased by more than 220%. After 1 hour of simulated intestinal fluid digestion, the GABA dissolution rate of the microspheres prepared in Comparative Example 6 is about 79%, and the GABA dissolution rate of the microspheres prepared in Comparative Example 7 is 23%, indicating that the range of calcium carbonate addition is a key factor in the quality of GABA microspheres. Too low calcium carbonate addition leads to low microsphere encapsulation rate and easy sudden release in intestinal fluid. Too high calcium carbonate addition leads to too slow release of microspheres during simulated intestinal fluid digestion, and GABA cannot be exerted.

[0246] from Figure 5 It can be found that the microspheres prepared in the embodiment and the comparative example have a good encapsulation rate for GABA, but the microspheres prepared in comparative examples 8 and 10 release GABA too slowly in the simulated intestinal digestion process, and the microspheres prepared in comparative example 9 release GABA suddenly in the simulated intestinal digestion process. The present invention optimizes the appropriate ratio of the two microspheres, and through the ratio regulation, GABA releases more than 40% of GABA in the intestine within 1 hour, which quickly achieves the sleep-aiding effect for the human body, and at the same time realizes the continuous and stable release of GABA in the small intestine within 1-4 hours, ensuring that GABA continues to play a role during sleep, and at the same time, GABA is basically released completely after 4 hours, which will not affect the mental state after getting up. The GABA microspheres prepared by the technology of the present invention have good market prospects and social benefits.

[0247] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Gamma-aminobutyric acid sustained-release microspheres, characterized in that: The sustained-release microspheres include gamma-aminobutyric acid or its salt and wall materials, and the added amount of the gamma-aminobutyric acid is at least 3% to 15% of the total weight of the wall materials.

2. The γ-aminobutyric acid sustained-release microspheres according to claim 1, characterized in that: The wall material comprises hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer and alginate.

3. A γ-aminobutyric acid sustained-release preparation, characterized in that: The γ-aminobutyric acid sustained-release preparation comprises a first sustained-release microsphere and a second sustained-release microsphere; The mass ratio of the first sustained-release microspheres to the second sustained-release microspheres is (2.5-3.5):1; The first sustained-release microspheres include 80-200 mesh γ-aminobutyric acid sustained-release microspheres; The second sustained-release microspheres contain γ-aminobutyric acid sustained-release microspheres larger than 200 meshes.

4. A method for preparing γ-aminobutyric acid sustained-release microspheres, characterized in that: The method comprises the following steps: S1. preparing a covalently cross-linked hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer; S2. dispersing the hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer prepared in step S1, alginate, γ-aminobutyric acid and calcium carbonate in water, and mixing to obtain an aqueous solution, wherein the alginate is at least one selected from sodium alginate, potassium alginate or ammonium alginate; S3. Add the oil phase to the aqueous solution obtained in step S2 and emulsify to form an oil-in-water emulsion; S4. The emulsion prepared in step S3 is adjusted to an acidic pH and stirred at a low speed, and then allowed to stand to collect the microspheres; S5. Curing the microspheres prepared in step S4 to obtain sustained-release microspheres of γ-aminobutyric acid. Preferably, the curing agent is at least one of calcium chloride and calcium lactate.

5. The method according to claim 4, characterized in that The preparation of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer comprises the following steps: S1-1. The hyaluronic acid or its salt, β-glucan and konjac glucomannan are mixed and placed in a granulator. Preferably, the mass ratio of the hyaluronic acid or its salt, β-glucan and konjac glucomannan is (1-5):(10-50):(5-30), more preferably, the mass ratio is (1-3):(10-30):(5-15); S1-2. Add malic acid (MA) solution to the granulator in step S1-1 to obtain hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer. Preferably, the amount of malic acid added is 0.1%-0.2% of the total mass of hyaluronic acid or its salt, β-glucan, and konjac glucomannan in step S1-1.

6. The method according to claim 4, characterized in that The mass concentration of the composite wall material described in step S2 is 0.5% to 2%. Preferably, the mass ratio of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid polymer and sodium alginate is (1-5): (5-20). More preferably, the addition amount of γ-aminobutyric acid is 5% to 10% of the total mass of the composite wall material. Most preferably, the addition amount of calcium carbonate is 0.5% to 1% of the total mass of the composite wall material.

7. The method according to claim 4, characterized in that The method further comprises the step of washing and drying the γ-aminobutyric acid microspheres prepared in step S5.

8. A covalently cross-linked polymer of hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid prepared according to any one of claims 4 to 7, wherein the mass ratio of hyaluronic acid or its salt, β-glucan and konjac glucomannan in the polymer is (1-5):(10-50):(5-30), and more preferably, the mass ratio is (1-3):(10-30):(5-15).

9. Use of the polymer according to claim 8 in the preparation of a sustained-release preparation of γ-aminobutyric acid, preferably, the sustained-release preparation is selected from at least one of sustained-release microspheres, sustained-release suspensions, sustained-release composite solutions, film-coated preparations, sustained-release capsules, and sustained-release compressed tablets.

10. Use of the sustained-release microspheres according to any one of claims 1 to 2, the sustained-release preparation according to claim 3, or the sustained-release microspheres prepared by the method according to any one of claims 4 to 7, or the hyaluronic acid or its salt-β-glucan-konjac glucomannan-malic acid covalently cross-linked polymer according to claim 8 in the preparation of functional foods, health foods, and special medical foods.

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