Co-production process for extracting collagen peptide, chondroitin sulfate, cartilage extract and hyaluronic acid

Abstract

The invention belongs to the technical field of biological product preparation, and particularly relates to a co-production process for extracting collagen peptide, chondroitin sulfate, cartilage extract and hyaluronic acid. The technology comprises the steps of cartilage pretreatment, enzymolysis pretreatment, homogenization, enzymolysis and collagen separation, cartilage is treated with an alkaline salt solution before enzymolysis, ammonia gas is introduced at the same time, and the protein extraction rate can be remarkably increased. In addition, when the cartilage homogenate is subjected to enzymolysis, a quaternary ammonium salt cationic compound is also added, so that the extraction rate of the non-denatured type II collagen is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological product preparation, and specifically relates to a co-production process for extracting collagen peptides, chondroitin sulfate, cartilage extracts and hyaluronic acid. Background Art

[0002] Collagen is a functional protein with a high content in the human body, accounting for one-third of the total protein in the human body. It contains 29 subtypes and is distributed in different connective tissues according to different biological functions. Collagen has characteristic GXY repeating sequences and triple helical structure characteristics, among which glycine (Gly) sequence has the highest content, accounting for 33% of the total content of all amino acids. Positions X and Y are usually proline and hydroxyproline, which can form complex high-level structures through multi-level self-assembly to provide structural integrity for connective tissue; collagen is the main component of the extracellular matrix, and regulates cell proliferation, migration and differentiation through interactions with multiple receptors on the cell surface; type II collagen is the extracellular matrix of articular cartilage Type II collagen is the main component of cartilage protein, accounting for 90-95%. Type II collagen can form specific collagen fibers with shock-absorbing function with other tissue-specific collagen and glycoproteins. Type II collagen maintains its triple helix structure through hydrogen bonding, dipole-dipole bonding, ionic bonds and van der Waals forces. However, the triple helix structure of type II collagen is easily affected by the environment. For example, when the temperature rises, the hydrogen bonding force between the peptide chains will be weakened, thereby dehelicalizing the peptide chains and causing the overall disorder of the protein. Such changes can easily lead to the denaturation of type II collagen, causing changes in structure and activity, and weakening the interaction between related cells and II collagen molecules, thereby failing to maintain normal joint function. Chicken cartilage is rich in collagen, accounting for more than 50% of the wet weight of chicken cartilage. The collagen in chicken cartilage is mainly type II collagen, which forms a grid structure through the interweaving of fiber bundles to maintain the stability of the cartilage. Chicken cartilage also includes highly hydrophilic glycoprotein molecules, which are composed of a core glycoprotein connected to aminopolysaccharide side chains to form chondroitin sulfate and keratan sulfate, which can increase the elasticity of chicken cartilage. Chondroitin sulfate and type II collagen can be extracted from chicken cartilage, but during the extraction process, type II collagen is easily affected by the environment and denatured, thereby losing the activity of type II collagen. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a method for preparing non-denatured type II collagen based on cartilage, comprising the following steps:

[0004] S1. Initial treatment of cartilage:

[0005] Obtain animal cartilage, soak it in drinking water for 1 hour; steam the cartilage for 3 to 4 hours; remove the cartilage and rinse it with clean water, drain it and crush it into pieces less than 2 mm to obtain cartilage particles;

[0006] S2. Alkaline hydrolysis pretreatment:

[0007] Dispersing cartilage particles in a mixed solution of NaOH and NaCl, wherein the concentration of NaOH is 0.2wt% to 0.8wt% and the concentration of NaCl is 0.5mol / L to 2.0mol / L; optionally introducing ammonia gas; the treatment time is 3 to 4 hours to obtain alkaline hydrolyzed cartilage particles;

[0008] S3. Homogenization:

[0009] The alkaline hydrolyzed cartilage particles are mixed with water for homogenization to obtain a homogenate; the mass ratio of the cartilage particles to water is 1:2-5;

[0010] S4, Enzymatic hydrolysis:

[0011] A quaternary ammonium salt cationic compound is added to the homogenate in an amount of 2-8% of the mass of the homogenate, and the pH, temperature and time are adjusted according to the selected enzyme species for enzymolysis; after the enzymolysis is completed, solid-liquid separation is performed to obtain a supernatant and an enzymolysis residue: the supernatant contains soluble collagen peptides, chondroitin sulfate and hyaluronic acid; and the enzymolysis residue retains a portion of cartilage extract that has not been completely hydrolyzed.

[0012] S5, Filtration and ion exchange:

[0013] The supernatant was filtered through a filter membrane with a pore size of 5 μm, purified by an ion exchange column, and treated by a nano-scale filter membrane to obtain a preliminarily clarified mixed solution;

[0014] S6, segmentation separation and decolorization:

[0015] The solution obtained in step S5 is adjusted to pH 8-9 for decolorization, and filtered again with a pore size of 5 μm;

[0016] Separating and extracting chondroitin sulfate and hyaluronic acid from the solution by graded ultrafiltration, osmotic pressure regulation or organic solvent precipitation;

[0017] The filtrate containing low molecular weight proteins or peptides formed during the separation process can be further concentrated and dried to obtain collagen peptide powder.

[0018] S7, cartilage extract and terminal treatment:

[0019] The enzymatic residue obtained in step S4 is subjected to a short-term enzymatic hydrolysis again as needed or directly precipitated and separated under pH 4.0 conditions to obtain a cartilage extract;

[0020] The cartilage extract or collagen peptide solution is concentrated at 70-80°C and then sterilized at a temperature greater than 95°C for 1 hour;

[0021] A drying method with an inlet air temperature of 200 to 260°C is adopted to obtain a corresponding powder product;

[0022] Finally, the obtained powder is screened, metal separated and packaged to obtain four products: collagen peptide, chondroitin sulfate, cartilage extract and hyaluronic acid.

[0023] Furthermore, in step S2, the concentration of NaOH is 0.2wt% to 0.6wt%, and the concentration of NaCl is 0.5mol / L to 1.5mol / L.

[0024] Furthermore, in step S2, the mass ratio of the cartilage particles to the mixed solution is 1:3-5.

[0025] Furthermore, in step S3, the mass ratio of cartilage particles to water is 1:3-4.

[0026] Furthermore, the quaternary ammonium salt cationic compound used in step S4 is selected from benzyltributylammonium bromide.

[0027] Furthermore, the enzyme in step S4 is pepsin, pancreatin or alkaline protease; when pepsin is used, the enzymolysis time is 12h to 24h; when pancreatin or alkaline protease is used, the enzymolysis time is 3h to 4h.

[0028] Furthermore, the ion exchange flow rate in steps S5 and S6 is 3 to 6 t / h, and the pH is adjusted to 8 to 9 for decolorization after the nano-filtration membrane treatment.

[0029] Furthermore, in step S7, the drying method is spray drying or hot air drying, and the air inlet temperature is 200-260°C.

[0030] The present invention also provides a type II collagen prepared by the method described herein.

[0031] Furthermore, the protein content of the non-denatured type II collagen in the type II collagen prepared by the method described herein is greater than 75%.

[0032] Beneficial effects of the present invention

[0033] In order to improve the protein extraction rate, the present invention uses an alkaline salt solution to treat the cartilage before enzymatic hydrolysis, which can not only sterilize the material and remove oil and alkali-soluble impurities in the raw material, but also swell and soften the cartilage. On this basis, the introduction of ammonia gas for treatment can accelerate the removal of impurities, swelling and softening of the cartilage, which is crucial to improving the protein extraction rate.

[0034] In addition, the present invention also adds a quaternary ammonium salt cationic compound when enzymatically hydrolyzing the cartilage homogenate. The long-chain alkyl and benzyl groups of the quaternary ammonium salt cationic compound can effectively promote the phase separation effect during extraction, improve the separation efficiency, and at the same time maintain the stability of the collagen structure, thereby improving the extraction rate of non-denatured type II collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a step diagram of the process of extracting type II collagen based on cartilage according to the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Example 1

[0038] A co-production process for extracting collagen peptides, chondroitin sulfate, cartilage extract, and hyaluronic acid, such as Figure 1 As shown, the following steps are included:

[0039] S1. Initial treatment of cartilage:

[0040] Obtain frozen chicken cartilage, thaw it at room temperature or low temperature, and soak it in drinking water for 1 hour to remove blood stains and soluble impurities.

[0041] Place the soaked cartilage in a cooking pot (or a double-layer pot), heat to 80-100°C, and cook for 3-4 hours to further remove grease, blood and other impurities.

[0042] After steaming, remove the cartilage, rinse with clean water not higher than 37°C and drain, and use a grinder to crush the cartilage into cartilage particles less than 2 mm.

[0043] S2. Alkaline hydrolysis pre-treatment:

[0044] The crushed cartilage particles are mixed with a mixed solution of NaOH and NaCl at a mass ratio of 1:4, wherein the mass percentage of NaOH in the mixed solution is 0.2% and the concentration of NaCl is 1.5 mol / L; ammonia gas is introduced under stirring conditions, and the treatment time is appropriately extended from 2 hours to 3 to 4 hours to further defat and remove impurities such as glycosaminoglycans.

[0045] After processing, rinse the cartilage particles with clean water until they are nearly neutral, drain and set aside.

[0046] S3. Homogenization:

[0047] The alkaline hydrolyzed cartilage particles were mixed with water at a mass ratio of 1:3, and homogenized at 5-25° C. to obtain a homogenate.

[0048] S4, Enzymatic Hydrolysis:

[0049] Add benzyltributylammonium bromide to the homogenate to make its mass concentration in the homogenate reach 6%, and stir thoroughly for 10 to 30 minutes.

[0050] The pH was adjusted to 2.0 using hydrochloric acid; 0.5 wt % pepsin was added based on the dry weight of the chicken cartilage, and enzymolysis was performed at 25±5° C. for 18 hours.

[0051] After the enzymatic hydrolysis is completed, centrifuge (10000r / min, 10min) or filter through a 5μm microfiltration membrane, and take the supernatant for later use.

[0052] The supernatant contains soluble collagen peptides, chondroitin sulfate and hyaluronic acid; the enzymatic hydrolysis residue retains some cartilage extracts that have not been completely hydrolyzed.

[0053] S5, Filtration and Ion Exchange:

[0054] The supernatant was pre-filtered through a filter membrane with a pore size of 5 μm;

[0055] The filtrate is sent to an ion exchange column for purification (the ion exchange flow rate is controlled at 3-6 t / h) to remove more inorganic salts and impurities;

[0056] The liquid after ion exchange is then processed through a nano-scale filtration membrane (with an optional molecular weight cut-off range of 10 to 100 kDa) to further remove small molecule impurities.

[0057] S6, decolorization:

[0058] The solution obtained in step S5 is adjusted to pH 8-9 for decolorization, and filtered again with a pore size of 5 μm;

[0059] Separating and extracting chondroitin sulfate and hyaluronic acid from the solution by means of graded ultrafiltration, osmotic pressure regulation or organic solvent precipitation;

[0060] The filtrate containing low molecular weight proteins or peptides formed during the separation process can be further concentrated and dried to obtain collagen peptide powder.

[0061] S7, Concentration, Sterilization and Drying:

[0062] Under stirring conditions, adjust the decolorized solution back to pH 4.0. After the pH stabilizes, centrifuge at 10,000 r / min for 10 min to obtain a precipitate (if separation by precipitation is required);

[0063] Or directly concentrate the decolorized solution under vacuum at 70-80°C to reduce the volume to 1 / 3-1 / 5 of the original volume;

[0064] The concentrated solution is sterilized at a temperature greater than 95° C. for 1 hour, and after sterilization, the collagen powder is obtained by spray drying (or hot air drying) at an air inlet temperature of 200 to 260° C.;

[0065] The powder is screened (80-200 mesh) and the metal is separated, and then the inner and outer packaging and storage are completed.

[0066] Product indicators: The protein extraction rate of the non-denatured type II collagen peptide produced by this process can reach 72.3%; the content of non-denatured type II collagen protein in the finished product can reach 78.9%, and the triple helix structure is well maintained, which is suitable for application in food, health products or other related fields.

[0067] Comparative Example 1

[0068] A co-production process for extracting collagen peptides, chondroitin sulfate, cartilage extract, and hyaluronic acid, which differs from Example 1 in that step S2 is not included. The results show that the protein extraction rate is 52.7% and the non-denatured type II collagen protein content is 78.1%.

[0069] Comparative Example 2

[0070] A process for extracting type II collagen based on cartilage, which differs from Example 1 in that ammonia ventilation treatment is not performed in step S2. The results show that the protein extraction rate is 63.6% and the non-denatured type II collagen protein content is 79.2%.

[0071] Comparative Example 3

[0072] A co-production process for extracting collagen peptides, chondroitin sulfate, cartilage extract, and hyaluronic acid, which differs from Example 1 in that benzyltributylammonium bromide is not added in step S4. The results show that the protein extraction rate is 59.8% and the non-denatured type II collagen protein content is 72.4%.

[0073] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A co-production process for extracting collagen peptides, chondroitin sulfate, cartilage extract, and hyaluronic acid, characterized in that: The following steps are involved: S1. Initial treatment of cartilage: Obtain animal cartilage, soak it in drinking water for 1 hour; steam the cartilage for 3 to 4 hours; remove the cartilage and rinse it with clean water, drain it and crush it into pieces less than 2 mm to obtain cartilage particles; S2. Alkaline hydrolysis pretreatment: Dispersing cartilage particles in a mixed solution of NaOH and NaCl, wherein the concentration of NaOH is 0.2wt% to 0.8wt% and the concentration of NaCl is 0.5mol / L to 2.0mol / L; optionally introducing ammonia gas; the treatment time is 3 to 4 hours to obtain alkaline hydrolyzed cartilage particles; S3. Homogenization: The alkaline hydrolyzed cartilage particles are mixed with water for homogenization to obtain a homogenate; the mass ratio of the cartilage particles to water is 1:2-5; S4, Enzymatic hydrolysis: A quaternary ammonium salt cationic compound is added to the homogenate in an amount of 2-8% of the mass of the homogenate, and the pH, temperature and time are adjusted according to the selected enzyme species for enzymolysis; after the enzymolysis is completed, solid-liquid separation is performed to obtain a supernatant and an enzymolysis residue: the supernatant contains soluble collagen peptides, chondroitin sulfate and hyaluronic acid; and the enzymolysis residue retains a portion of cartilage extract that has not been completely hydrolyzed. S5, Filtration and ion exchange: The supernatant was filtered through a filter membrane with a pore size of 5 μm, purified by an ion exchange column, and treated by a nano-scale filter membrane to obtain a preliminarily clarified mixed solution; S6, segmentation separation and decolorization: The solution obtained in step S5 is adjusted to pH 8-9 for decolorization, and filtered again with a pore size of 5 μm; Separating and extracting chondroitin sulfate and hyaluronic acid from the solution by graded ultrafiltration, osmotic pressure regulation or organic solvent precipitation; The filtrate containing low molecular weight proteins or peptides formed during the separation process can be further concentrated and dried to obtain collagen peptide powder. S7, cartilage extract and terminal treatment: The enzymatic residue obtained in step S4 is subjected to a short-term enzymatic hydrolysis again as needed or directly precipitated and separated under pH 4.0 conditions to obtain a cartilage extract; The cartilage extract or collagen peptide solution is concentrated at 70-80°C and then sterilized at a temperature greater than 95°C for 1 hour; A drying method with an inlet air temperature of 200 to 260°C is adopted to obtain a corresponding powder product; Finally, the obtained powder is screened, metal separated and packaged to obtain four products: collagen peptide, chondroitin sulfate, cartilage extract and hyaluronic acid.

2. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: In step S2, the concentration of NaOH is 0.2wt% to 0.6wt%, and the concentration of NaCl is 0.5mol / L to 1.5mol / L.

3. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: In step S2, the mass ratio of the cartilage particles to the mixed solution is 1:3-5.

4. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: In step S3, the mass ratio of cartilage particles to water is 1:3-4.

5. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: The quaternary ammonium salt cationic compound used in step S4 is selected from benzyltributylammonium bromide.

6. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: The enzyme in step S4 is pepsin, pancreatin or alkaline protease; when pepsin is used, the enzymolysis time is 12h to 24h; when pancreatin or alkaline protease is used, the enzymolysis time is 3h to 4h.

7. The method for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: The ion exchange flow rate in steps S5 and S6 is 3 to 6 t / h, and the pH is adjusted to 8 to 9 for decolorization after the nano-scale filtration membrane treatment.

8. A process for extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to claim 1, characterized in that: In step S7, the drying method is spray drying or hot air drying, and the air inlet temperature is 200-260°C.

9. Non-denatured type II collagen prepared by the co-production process of extracting collagen peptides, chondroitin sulfate, cartilage extract and hyaluronic acid according to any one of claims 1 to 8.

Images