Non-denatured type II collagen microcapsule as well as preparation method and application thereof

Abstract

The invention provides a non-denatured type II collagen microcapsule as well as a preparation method and application thereof, and relates to the technical field of medicine processing. The non-denatured type II collagen microcapsule provided by the invention is prepared from the following raw materials in parts by weight: 10-40 parts of a core material, 5-15 parts of a capsule material and 500-1200 parts of distilled water, the core material is composed of non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose according to the weight ratio of 20: 5: 1, and the capsule material is composed of chitosan and Chinese yam extract according to the weight ratio of 5: 1. The non-denatured type II collagen microcapsule provided by the invention has relatively good acid resistance and stability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug processing, and specifically relates to a non-denatured type II collagen microcapsule and a preparation method and application thereof. Background Art

[0002] Type II collagen is composed of three identical α1(II) peptide chains and has a triple helix structure. Non-denatured type II collagen (NC II) is a type II collagen product with a complete triple helix structure. Studies have shown that NC II has high physiological activity and can also relieve and improve the pain and inflammatory symptoms of osteoarthritis through "oral immune tolerance". Oral immune tolerance is an immune mechanism, and the immune tolerance effect of NC II is related to its triple helix structure. However, the triple helix structure of NC II is very fragile and is easily denatured by environmental conditions, enzymes, mechanical effects, etc., resulting in the loss of its physiological activity, which limits its application in complex food and health food systems.

[0003] In recent years, the research and application of NC II have gradually increased, but they are mostly concentrated in the research of extraction technology. Tablets and capsules are the main dosage forms in application. At present, some NC-II health products for preventing and treating joint diseases are generally made into capsules or tablets for swallowing due to their bad taste, and other types of food are rarely reported; therefore, broadening the application scope of NC-II in the food field is also an important development direction.

[0004] Microencapsulation is a technology that uses film-forming materials as shell materials to encapsulate liquids, solids, and even gases into tiny particles, which can effectively reduce the reaction of active substances to external environmental factors or control the release of substances. NC-II microencapsulation can greatly reduce its environmental sensitivity and expand its application scope and form. Therefore, it is urgent to study NC-II microencapsulation. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a non-denatured type II collagen microcapsule and a preparation method and application thereof.

[0006] In one aspect, the present invention provides a non-denatured type II collagen microcapsule.

[0007] Specifically, the non-denatured type II collagen microcapsules include the following raw materials in parts by weight: 10-40 parts of core material and 5-15 parts of capsule material.

[0008] Specifically, the core material includes non-denatured type II collagen, mannitol and sodium carboxymethylcellulose.

[0009] Specifically, the capsule material comprises chitosan and yam extract.

[0010] More specifically, the core material is composed of non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in a weight ratio of 20:1-10:0.2-4.

[0011] Preferably, the weight ratio of collagen, mannitol and sodium carboxymethylcellulose is 20:2-8:0.4-2.

[0012] More preferably, the weight ratio of collagen, mannitol and sodium carboxymethylcellulose is 20:4-6:0.4-1.

[0013] More specifically, the capsule material consists of chitosan and yam extract in a weight ratio of 5:0.2-2.

[0014] Preferably, the mass ratio of chitosan to yam extract is 5:0.6-1.2.

[0015] More preferably, the mass ratio of chitosan to yam extract is 5:0.8-1.2.

[0016] Specifically, the preparation steps of the yam extract are:

[0017] (1) crushing the Chinese yam, adding water and grinding to obtain a Chinese yam slurry;

[0018] (2) adding composite protease to the yam homogenate for enzymatic hydrolysis, and centrifuging to obtain a supernatant;

[0019] (3) The supernatant is heated to 90°C to inactivate the enzyme and obtain an enzymatic hydrolyzate;

[0020] (4) The enzymatic hydrolyzate is filtered, desalted, concentrated, and freeze-dried to obtain the yam extract.

[0021] More specifically, in the step (1) of preparing the yam extract, the ratio of yam to water is 1 kg:2.8 L.

[0022] More specifically, in the step (2) of preparing the yam extract, the composite protease comprises alkaline protease and ginger protease.

[0023] More specifically, in the step (2) of preparing the yam extract, the mass ratio of the alkaline protease to the ginger protease is 6:1.

[0024] More specifically, in the preparation step (2) of the yam extract, the ratio of the added composite protease to the yam in step (1) is 5 g:1 kg.

[0025] More specifically, in the preparation step (4) of the yam extract, the freeze-drying conditions are -40°C and 36h.

[0026] In another aspect, the present invention provides a method for preparing non-denatured type II collagen microcapsules.

[0027] Specifically, the steps include:

[0028] S1: taking various raw materials according to weight, suspending non-denatured type II collagen in a mixed solution of mannitol and sodium carboxymethyl cellulose, and stirring evenly to obtain a core material;

[0029] S2: dissolving chitosan and yam extract in acetic acid solution, mixing evenly and adjusting the pH value of the solution to obtain capsule material;

[0030] S3: slowly drip the core material obtained in S1 into the capsule material obtained in S2;

[0031] S4: Collect the wet capsules obtained in S3 and freeze-dry them to obtain non-denatured type II collagen microcapsules.

[0032] More specifically, in S1, the concentration ratio of the non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in the core material solution is 10wt%: 0.5-5wt%: 0.1-2wt%.

[0033] Preferably, the concentration ratio of the components of non-denatured type II collagen, mannitol and sodium carboxymethylcellulose is 10wt%: 1-4wt%: 0.2-1wt%.

[0034] More preferably, the concentration ratio of the components of non-denatured type II collagen, mannitol and sodium carboxymethylcellulose is 10wt%: 1-3wt%: 0.2-0.5wt%.

[0035] More specifically, in S2, the concentration ratio of chitosan to yam extract in the capsule material is 1.7wt%:0.07-0.7wt%

[0036] Preferably, in the capsule material, the concentration ratio of chitosan to yam extract is 1.7wt%:0.2-0.4wt%. More preferably, in the capsule material, the concentration ratio of chitosan to yam extract is 1.7wt%:0.27-0.4wt%.

[0037] More specifically, in S2, the pH range is 2-7.

[0038] Preferably, in S2, the pH range is 4-6.

[0039] More preferably, in S2, the pH range is 5-6.

[0040] In certain specific embodiments of the present invention, the preparation steps are:

[0041] S1: Weigh each raw material by weight, and evenly disperse 20 parts of non-denatured type II collagen in a solution obtained by mixing 5 parts of mannitol, 1 part of sodium carboxymethyl cellulose and 200 parts of distilled water;

[0042] S2: dissolving 5 parts of chitosan and 1 part of yam extract in 300 parts of acetic acid solution, mixing well, and adjusting the pH value of the solution to 5.5 to obtain capsule material;

[0043] S3: slowly drip the core material obtained in S2 into the capsule material obtained in S3 and let it stand;

[0044] S4: freeze-dry the wet capsules obtained in S3 at -50°C for 40 h to obtain non-denatured type II collagen microcapsules.

[0045] In another aspect, the present invention provides use of non-denatured type II collagen microcapsules in the preparation of a medicament for alleviating and / or improving pain and inflammatory symptoms of osteoarthritis.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The present invention uses chitosan and yam extract to make composite capsule materials, which can protect the core material mainly composed of non-denatured type II collagen. The chitosan capsule shell forms a structure under a soft acid environment, and the network structure is revealed under strong acid or alkaline conditions, which has a good sustained release effect. Chitosan is the only natural polysaccharide with a positive charge, has good water solubility, and forms an electrostatic interaction with the negatively charged non-denatured type II collagen under a neutral environment, so as to better embed the non-denatured type II collagen particles; yam is rich in active proteins and polysaccharides. The present invention finds that the yam extract modified by protease has better acid resistance and stability, and its functionality is also enhanced. It forms a mixed network structure with chitosan, improves the expansion properties of chitosan microcapsules, further protects non-denatured type II collagen, reduces the environmental sensitivity of non-denatured type II collagen, reduces the loss of non-denatured type II collagen during production, processing, transportation, and storage, and improves the heat resistance and stability of non-denatured type II collagen microcapsules.

[0048] 2. The present invention also found that mannitol can protect type II collagen and significantly reduce its denaturation rate in the solution, and an appropriate amount of sodium carboxymethyl cellulose can enhance the dispersion and stability of type II collagen and improve the embedding rate of the wall material to the core material.

[0049] 3. The composite capsule material used in the present invention has a denser capsule wall structure and higher strength than a single capsule material; compared with the emulsification method, it does not require a large amount of oil continuous phase, and the raw material cost is low; at the same time, the preparation method is simple and easy to operate. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0051] Example 1

[0052] Components:

[0053] The invention is made of the following raw materials in parts by weight: 40 parts of core material, 12 parts of capsule material and 900 parts of distilled water; the core material is composed of non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in a weight ratio of 20:4:0.8, and the capsule material is composed of chitosan and yam extract in a weight ratio of 5:1.

[0054] Preparation method of yam extract:

[0055] 1 kg of washed white jade yam was crushed, added with 2.8 L of water and then put into a colloid mill for circulation for 10 min to obtain a homogenate, the pH value of the slurry was adjusted to 9, 5 g of composite protease (alkaline protease and ginger protease weight ratio of 6:1) was added and heated to 35°C, enzymolysis was performed for 2 h, centrifuged at 6000 rpm for 20 min, the supernatant was taken and heated to 90°C to inactivate the enzyme for 15 min to obtain an enzymolysis solution, the enzymolysis solution passed through a 500 mesh filter cloth, and then passed through an anion resin and a cationic resin in turn to obtain a solution with a conductivity of 230 μS / cm, and concentrated under reduced pressure to a solid content of 18%, the concentrate was frozen at -20°C overnight, and then freeze-dried at -40°C for 36 h.

[0056] Preparation method:

[0057] (1) weighing and uniformly dispersing non-denatured type II collagen in a mixed solution prepared by mannitol, sodium carboxymethyl cellulose and 400 parts of distilled water to obtain a core material;

[0058] (2) dissolving chitosan and yam extract in an appropriate amount of 1% acetic acid solution, adding distilled water to adjust the solution mass to 500 parts, mixing well, and then adjusting the solution pH to 5.5 to obtain a capsule material solution;

[0059] (3) Slowly drip the core material obtained in step (1) into the capsule material obtained in step (2) and mix well.

[0060] (4) freeze-drying the wet capsules obtained in step (3) at -50°C for 40 hours to obtain non-denatured type II collagen microcapsules.

[0061] Example 2

[0062] Components:

[0063] The invention is made of the following raw materials in parts by weight: 30 parts of core material, 10 parts of capsule material and 600 parts of distilled water; the core material is composed of non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in a weight ratio of 20:5:0.7, and the capsule material is composed of chitosan and yam extract in a weight ratio of 5:1.2.

[0064] The preparation steps of yam extract are the same as those in Example 1.

[0065] Preparation method:

[0066] (1) Weighing each raw material by weight, dispersing non-denatured type II collagen in a solution prepared by mannitol, sodium carboxymethyl cellulose and 300 parts of distilled water;

[0067] (2) dissolving chitosan and yam extract in an appropriate amount of 1% acetic acid solution, adding water to 300 parts, mixing well, and adjusting the pH value of the solution to 6 to obtain a capsule material solution;

[0068] (3) slowly dripping the core material obtained in step (1) into the capsule material obtained in step (2) and letting it stand;

[0069] (4) Drying and crushing the wet microcapsules obtained in step (3) at -50°C to obtain non-denatured type II collagen microcapsules.

[0070] Comparative Example 1

[0071] According to the weight proportions and preparation process of the components in Example 1, the core material is pure non-denatured type II collagen.

[0072] Comparative Example 2

[0073] The weight proportions of the components and the preparation process were the same as those in Example 1, except that the core material was replaced with equal proportions of non-denatured type II collagen, sucrose and pectin.

[0074] Comparative Example 3

[0075] The difference from Example 1 is that the wall materials are chitosan and yam extract in different proportions.

[0076] Comparative Example 3-1 The weight ratio of chitosan to yam extract is 5:0.

[0077] Comparative Example 3-2 The weight ratio of chitosan to yam extract is 5:1.6.

[0078] Comparative Example 4

[0079] The difference from Example 1 is that the wall material is replaced by chitosan and gum arabic, and the mass ratio of chitosan to gum arabic is 5:1.

[0080] Experimental Example 1 Test of the triple helix structure content of non-denatured type II collagen

[0081] Test method: Take 0.5 g of the microcapsules obtained in Examples 1-2 and Comparative Examples 1-4, and measure the content of non-denatured type II collagen in the dry basis of the microcapsules. The results are expressed in g / 100 g. The results are shown in the table.

[0082] Experimental Example 2 Embedding Rate Test

[0083] Test method: Take 0.5g of non-denatured type II collagen microcapsules (Examples 1-2, Comparative Examples 1-4), wash them three times with 5mL of water, test the collagen content (on a dry basis) in the microcapsules before and after washing, calculate the embedding rate, and the results are shown in the table.

[0084] Embedding rate = collagen content after washing / collagen content before washing × 100%.

[0085] Experimental Example 3: Stability Test

[0086] Test method: several non-denatured type II collagen microcapsules were placed in a constant temperature box at 60°C for 7 days, samples were taken, and the non-denatured type II collagen triple helix content in the dry basis of the microcapsules before and after insulation was determined by ELISA, and the triple helix structure retention rate was calculated. The test results are shown in the table.

[0087] Triple helix structure retention rate = ELISA test value after incubation / ELISA test value before incubation × 100%.

[0088] Table 1 Non-denatured type II collagen content, embedding rate and triple helix structure storage retention rate in microcapsules of Examples and Comparative Examples

[0089] Non-denatured type II collagen content / g / 100g Embedding rate / % Triple helix structure retention rate / % Example 1 52.2 85.4 82.2 Example 2 56.0 86.2 80.0 Comparative Example 1 57.3 89.3 47.7 Comparative Example 2 40.3 78.1 55.6 Comparative Example 3-1 50.3 60.3 62.3 Comparative Example 3-2 43.7 71.8 70.3 Comparative Example 4 46.4 79.4 50.3

[0090] The content of non-denatured type II collagen in the microcapsules of Examples 1-2 is substantially higher than that of the comparative example, and the wall material used in the present method has well embedded the non-denatured type II collagen, and also plays an effective protective role during the heated storage process, and the triple helix retention rate is higher. The microcapsules of the present invention significantly improve the stability of non-denatured type II collagen.

[0091] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A non-denatured type II collagen microcapsule, characterized in that: The invention comprises the following raw materials in parts by weight: 10-40 parts of core material and 5-15 parts of capsule material; the core material comprises non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose; and the capsule material comprises chitosan and yam extract.

2. The non-denatured type II collagen microcapsule according to claim 1, characterized in that: The core material is composed of non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in a weight ratio of 20:1-10:0.2-4. The weight ratio of the three is preferably 20:2-8:0.4-2, and more preferably 20:4-6:0.4-1.

3. The non-denatured type II collagen microcapsule according to claim 1, characterized in that: The capsule material is composed of chitosan and yam extract in a weight ratio of 5:0.2-2, and the ratio of the two is preferably 5:0.6-1.2, and more preferably 5:0.8-1.

2.

4. The method for preparing the non-denatured type II collagen microcapsules according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: taking various raw materials according to weight, dispersing non-denatured type II collagen in a mixed solution of mannitol and sodium carboxymethyl cellulose, and stirring evenly to obtain a core material; S2: dissolving chitosan and yam extract in acetic acid solution, mixing evenly and adjusting the pH value of the solution to obtain capsule material; S3: slowly drip the core material obtained in S1 into the capsule material obtained in S2 and let it stand; S4: Collect the non-denatured type II collagen microcapsules obtained in S3 and freeze-dry them.

5. The preparation method according to claim 4, characterized in that: In S1, the concentrations of the non-denatured type II collagen, mannitol and sodium carboxymethyl cellulose in the core material solution are 10wt%: 0.5-5wt%: 0.1-2wt%, preferably 10wt%: 1-4wt%: 0.2-1wt%, and more preferably 10wt%: 1-3wt%: 0.2-0.5wt%.

6. The preparation method according to claim 4, characterized in that: In S2, the concentration of each component of the capsule material is 1.7wt%: 0.07-0.7wt%, preferably 1.7wt%: 0.2-0.4wt%, and more preferably 1.7wt%: 0.27-0.4wt%.

7. The preparation method according to claim 4, characterized in that: In S2, the pH range of the capsule material solution is 2-7, preferably 4-6, and more preferably 5-6.

8. Use of the non-denatured type II collagen microcapsules according to any one of claims 1 to 7 in the preparation of a drug for alleviating and / or improving the pain and inflammatory symptoms of osteoarthritis.