Preparation method of collagen tripeptide and obtained product

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of collagen tripeptide and an obtained product. Compared with the prior art, the method has the advantages that by adding alkali to soak the cowhide particles and combining treatment means such as denaturation and enzymolysis, the destroy denaturation of a triple helix structure of collagen in the cowhide can be promoted, the collagen can be thoroughly subjected to enzymolysis, so that the yield of collagen tripeptide is higher, the molecular weight is small and concentrated, and meanwhile, the foreign flavor in the cowhide can be effectively removed; subsequently, mixed enzymes jointly act on collagen peptide bonds, so that generation of collagen tripeptide is facilitated; in the operation process of the method, acid or alkali does not need to be added for multiple times to adjust the pH, and the generated collagen tripeptide is low in ash content; after suction filtration and impurity removal, impurities in the collagen tripeptide are reduced, and the yield of the obtained collagen tripeptide freeze-dried product is high; under the synergistic effect of all the treatment processes, the prepared product is fine and smooth in component, free of fishy smell and foreign flavor and good in water solubility.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a preparation method of collagen tripeptide and the obtained product. Background Art

[0002] Collagen is an extracellular protein, a fibrous protein made of three peptide chains twisted into a spiral. Collagen is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% in some organisms. Collagen has been widely used in food, medicine, tissue engineering, cosmetics and other fields due to its good biocompatibility, biodegradability and biological activity.

[0003] Collagen is rich in amino acids such as glycine, proline, and hydroxyproline. Collagen molecules contain a highly repetitive glycine-XY sequence. X and Y are arbitrary, but are usually occupied by proline, hydroxyproline, and alanine. This composition makes collagen more resistant to degradation by general proteases.

[0004] Collagen tripeptide (CTP) is a highly purified, non-antigenic, hypoallergenic collagen fraction containing a high concentration of tripeptides containing the glycine-XY sequence. Some studies have shown that small collagen peptides, including CTP, have biological effects, such as activating the body's ability to produce new collagen and hyaluronic acid, preventing atherosclerosis, and strengthening bones and tendons. Collagen is rich in cowhide and is inexpensive, so it is often used as a raw material for extracting collagen. However, the existing methods for extracting collagen tripeptides are relatively complicated and cannot remove odors. The purity is not high, and the multi-level application of collagen tripeptides cannot be achieved. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for preparing collagen tripeptide. The present invention uses secondary enzymolysis, and the pH value does not need to be adjusted multiple times during the process. The molecular weight of the enzymolysis product is small and concentrated, the free amino acid content of the enzymolysis product is low, and the prepared collagen tripeptide powder has no odor, high yield, and good water solubility.

[0006] The present invention also provides collagen tripeptide prepared by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above collagen tripeptide.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing collagen tripeptide, comprising the following steps: (1) Pretreatment of cowhide: Take fresh cowhide, remove hair and fat, cut into small pieces, and wash; add the washed cowhide particles into an alkaline solution for alkaline treatment, and wash with water to obtain pretreated cowhide; (2) Modified cowhide: Mix the pre-treated cowhide and the treatment solution, keep them warm for a period of time and then boil them. After cooling, filter out the residue to obtain modified cowhide. (3) Homogenization: Add the denatured cowhide into purified water and homogenize under high pressure to obtain cowhide homogenate; (4) First enzymatic hydrolysis: adding mixed proteases to the cowhide homogenate for enzymatic hydrolysis; inactivating the enzymes to obtain a primary enzymatic hydrolyzate; (5) Second enzymatic hydrolysis: using the first enzymatic hydrolyzate as substrate, adding animal protein hydrolyzing enzyme to hydrolyze; after inactivating the enzyme, the second enzymatic hydrolyzate is obtained; (6) Filtration, adsorption and impurity removal: The secondary enzymatic hydrolyzate is cooled to room temperature and then filtered. Activated carbon is added to the filtrate, and the filtrate is centrifuged after being left for a period of time to obtain a supernatant. The supernatant is filtered to remove impurities and obtain a collagen tripeptide solution. The obtained collagen tripeptide solution is then centrifuged at 6000-9000 rpm for 10-15 min to obtain a supernatant. The supernatant is filtered to remove impurities and obtain.

[0009] (7) Vacuum freeze drying: The supernatant is vacuum freeze dried to obtain collagen tripeptide.

[0010] Furthermore, in step (1), the mass volume concentration of the alkaline solution is 3-6%; the mass ratio of the cowhide particles to the alkaline solution is 1:2-10; and the alkaline treatment is stirred at 3-5°C for 12-24h.

[0011] Furthermore, in step (2), the mass ratio of the pretreated cowhide to the treatment liquid is 1:3-8; the treatment liquid is prepared by the following raw materials: 10 parts of water, 0.3-0.5 parts of glucose; 5 mL of lactic acid streptococcus liquid is added to every 100 mL of water; the specific treatment is: heat preservation treatment at 30-32°C for 50-60 minutes; boiling for 20-40 minutes; then cooling to 50-60°C and filtering.

[0012] Furthermore, in step (3), the mass ratio of the denatured cowhide to purified water is 1:3-5; and the high-pressure homogenization is carried out at a pressure of 20-50 MPa for 8-15 min.

[0013] Furthermore, in step (4), the amount of the mixed protease added is 0.1-0.25% of the mass of the denatured cowhide; the mixed protease includes 20-30% of pancreatic enzyme and 80%-70% of alkaline protease; and the enzymatic hydrolysis is performed at 55° C. for 4 hours.

[0014] Furthermore, in step (5), the amount of the animal protein hydrolase added is 0.2-0.5% of the primary enzymatic hydrolysis solution; and the hydrolysis is carried out at 55° C. for 12-24 hours.

[0015] Furthermore, in step (6), the amount of activated carbon added is 0.2%-0.6% of the mass of the filtrate; the standing time is 12-18 hours; and the filtration is performed using filter paper with a pore size of 20-40 μm.

[0016] Furthermore, in step (7), the vacuum freeze-drying procedure is divided into three parts: pre-freezing, low-temperature drying and heating drying; the pre-freezing temperature is between -20 and 80°C, and the pre-freezing time is 5-12 hours; after pre-freezing, the freeze dryer is evacuated, and then the temperature is increased to -10-0°C at 5-10°C / h and then kept constant at that temperature, and low-temperature drying is performed for a constant temperature time of 12-24 hours; then the temperature is increased to 0-30°C at 5-10°C / h and then kept constant at that temperature, and the temperature is increased and dried for a constant temperature time of 16-48 hours.

[0017] The present invention discloses a collagen tripeptide prepared by the preparation method described in any one of claims 1 to 8.

[0018] The beneficial effects of the present invention are: (1) The present invention pre-treats the cowhide particles by soaking them in alkali, thereby causing the cowhide particles to swell and remove excess fat and non-collagen components; (2) The present invention can promote the destruction and denaturation of the triple helix structure of collagen in cowhide through denaturation and enzymatic hydrolysis, which can not only completely enzymatically hydrolyze collagen, so that the amount of collagen tripeptide is higher, the molecular weight is small and concentrated, but also can effectively remove the foreign smell in the cowhide; the subsequent mixed enzyme acts on the collagen peptide bond, which is conducive to the generation of collagen tripeptide; during the operation of this method, there is no need to add acid or alkali multiple times to adjust the pH, and the generated collagen tripeptide has a low ash content; after filtration and impurity removal, the impurities in the collagen tripeptide are reduced, and the yield of the obtained collagen tripeptide freeze-dried product is high; (3) Under the synergistic effect of various treatment processes, the product prepared by the present invention has fine ingredients, no fishy smell, no foreign taste, and good water solubility.

[0019] (4) In the freeze-drying process of the method provided by the present invention, temperature control is crucial. The pre-freezing stage requires rapid cooling to avoid the formation of large ice crystals; the sublimation drying and analytical drying stages require gradual heating to promote the evaporation and escape of water. Temperature control not only affects the morphology and quality of the product, but also directly affects the structure and activity of collagen. Therefore, accurate temperature control is an important factor in ensuring product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Collagen tripeptide powder prepared by the present invention; Figure 2 It is a comparison chart of the dissolved solutions of the samples prepared in the embodiment and the comparative example. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0022] The lactic acid streptococcus used in the present invention is commercially available with a product number of BMZ068611.

[0023] Example 1 (1) Take fresh cowhide, remove the hair and fat, cut into small pieces of 4cm×4cm, add purified water to wash and set aside; (2) Pretreatment of cowhide: Take 100g of cowhide particles and place them in a container, wash them repeatedly with purified water, then add them into a sodium hydroxide solution with a concentration of 3% by mass volume twice, stir them evenly at 3-5°C, treat them with alkali for 12h, and then wash them with water 3-5 times to obtain pretreated cowhide; (3) Modified cowhide: Add 5 times the weight of the treatment solution (10 parts of water, 0.5 parts of glucose; add 5 mL of lactic acid streptococcus liquid to every 100 mL of water) to the pretreated cowhide, and then keep it at 30-32°C for 60 minutes; boil it for 30 minutes; then cool it to about 50°C, filter out the residue, and obtain modified cowhide; (4) Homogenization: Add purified water to denatured cowhide at a solid-liquid ratio of 1:3, and homogenize under high pressure at 20 MPa for 10 min; (5) First enzymatic hydrolysis: Add 0.2% of the weight of the hide into a mixed protease (20% pancreatic enzyme and 80% alkaline protease) and perform enzymatic hydrolysis at 55°C for 4 h. (6) Inactivation of enzyme: After the first enzymatic hydrolysis is completed, the solution is inactivated in boiling water at 100°C for 30 min; (7) Second enzymatic hydrolysis: using the first enzymatic hydrolysis solution as substrate, add 0.5% of the liquid mass of animal protein hydrolase, and hydrolyze at 55°C for 12 h; (8) Inactivation of enzyme: After the second enzymatic hydrolysis is completed, the solution is inactivated in boiling water at 100°C for 15 min; (9) Filtration and adsorption: After the enzymatic hydrolysate is cooled to room temperature, it is filtered through a sieve, 0.5% activated carbon is added, and the solution is left for 12 hours; (10) Impurity removal: The obtained collagen tripeptide solution was centrifuged at 8000 rpm for 10 min to obtain a supernatant. The supernatant was filtered through a 30 μm pore size filter paper to obtain a collagen tripeptide solution; (11) Vacuum freeze drying: The collagen tripeptide solution was transferred into a vacuum freeze dryer for vacuum freeze drying: pre-freeze at -50°C for 8 h; after pre-freezing, the freeze dryer was evacuated, and then the temperature was increased to 0°C at 5-10°C / h and then kept constant for 12 h; then the temperature was increased to 20°C at 5-10°C / h and then kept constant, heated and dried, and the constant temperature time was 48 h to obtain a collagen tripeptide freeze-dried product; the freeze-dried product was ground to obtain a collagen tripeptide powder sample.

[0024] Example 2 Steps (1) to (3) are the same as in Example 1; (4) Homogenization: Add purified water to denatured cowhide at a solid-liquid ratio of 1:5, and homogenize under high pressure at 30 MPa for 10 min; (5) First enzymatic hydrolysis: Add 0.1% of the weight of the hide into a mixed protease (30% pancreatic enzyme and 70% alkaline protease) and perform enzymatic hydrolysis at 55°C for 6 h. (6) Inactivation of enzyme: After the first enzymatic hydrolysis is completed, the solution is inactivated in boiling water at 100°C for 20 min; (7) Second enzymatic hydrolysis: using the first enzymatic hydrolysis solution as substrate, add 0.5% of the liquid mass of animal protein hydrolase, and hydrolyze at 55°C for 12 h; (8) Inactivation of enzyme: After the second enzymatic hydrolysis is completed, the solution is inactivated in boiling water at 100°C for 20 min; (9) Filtration and adsorption: After the enzymatic hydrolysate is cooled to room temperature, it is filtered through a sieve, 0.6% activated carbon is added, and the solution is left for 15 hours; (10) Impurity removal: The obtained collagen tripeptide solution was centrifuged at 6000 rpm for 15 min to obtain a supernatant. The supernatant was filtered through a 30 μm pore size filter paper to obtain a collagen tripeptide solution; (11) Vacuum freeze drying: The collagen tripeptide solution was transferred to a vacuum freeze dryer for vacuum freeze drying: pre-freeze at -30°C for 12 h; after pre-freezing, the freeze dryer was evacuated, and then the temperature was increased to 10°C at a rate of 5-10°C / h and then kept constant for 6 h; then the temperature was increased to 25°C at a rate of 5-10°C / h and then kept constant, heated and dried, and the constant temperature time was 30 h to obtain a collagen tripeptide freeze-dried product. The freeze-dried product was ground to obtain a collagen tripeptide powder sample.

[0025] Example 3 Steps (1) to (3) are the same as those in Example 1; (4) homogenization: adding purified water to the denatured cowhide at a solid-liquid ratio of 1:5, and high-pressure homogenization at a pressure of 30 MPa for 10 minutes; (5) First enzymatic hydrolysis: Add 0.25% of the weight of the hide into a mixed protease (25% pancreatic enzyme and 75% alkaline protease) and perform enzymatic hydrolysis at 55°C for 8 h. (6) Inactivation of enzyme: After the first enzymatic hydrolysis is completed, the solution is inactivated in boiling water for 20 min; (7) Second enzymatic hydrolysis: using the first enzymatic hydrolysis solution as substrate, add 0.4% of the liquid mass of animal protein hydrolase, and hydrolyze at 55°C for 18 h; (8) Inactivation of enzyme: After the second enzymatic hydrolysis is completed, the solution is inactivated in boiling water for 15 min; (9) Filtration and adsorption: After the enzymatic hydrolysate is cooled to room temperature, it is filtered through a sieve, 0.6% activated carbon is added, and the solution is left for 18 hours.

[0026] (10) Impurity removal: The obtained collagen tripeptide solution was centrifuged at 9000 rpm for 10 min to obtain a supernatant. The supernatant was filtered through a 30 μm pore size filter paper to obtain a collagen tripeptide solution; The above-mentioned filtration (11) Vacuum freeze drying: The collagen tripeptide solution was transferred into a vacuum freeze dryer for vacuum freeze drying: pre-freeze at -40°C for 8 h; after pre-freezing, the freeze dryer was evacuated, and then the temperature was increased at 5-10°C / h to 0°C and then kept constant for 10 h; then the temperature was increased at 5-10°C / h to 25°C and then kept constant, heated and dried, and the constant temperature time was 48 h to obtain a collagen tripeptide freeze-dried product. The freeze-dried product was ground to obtain a collagen tripeptide powder sample.

[0027] Comparative Example 1 (1) Take fresh cowhide, remove the hair and fat, cut into small pieces of 4cm×4cm, add purified water to wash and set aside; (2) Pretreatment of cowhide: Take 100g of cowhide particles and place them in a container, wash them repeatedly with purified water, then add them into a sodium hydroxide solution with a concentration of 3% by mass volume twice, stir them evenly at 3-5°C, treat them with alkali for 12h, and then wash them with water 3-5 times to obtain pretreated cowhide; (3) Modified cowhide: Add 5 times the weight of water to the pretreated cowhide; boil for 30 minutes; then cool to about 50°C, filter out the residue, and obtain modified cowhide; The other steps are the same as in Example 1.

[0028] Comparative Example 2 (1) Take fresh cowhide, remove the hair and fat, cut into small pieces of 4cm×4cm, add purified water to wash and set aside; (2) Pretreatment of cowhide: Take 100g of cowhide particles and place them in a container, wash them repeatedly with purified water, then add twice the mass volume concentration of 3% sodium hydroxide solution, stir evenly at 3-5°C, treat with alkali for 12h, and then wash with water 3-5 times to obtain pretreated cowhide; (3) Modified cowhide: Add 5 times the weight of the treatment solution (10 parts of water, 0.5 parts of glucose; add 5 mL of lactic acid streptococcus liquid to every 100 mL of water) to the pretreated cowhide, and then keep it at 30-32°C for 60 minutes; boil it for 30 minutes; then cool it to about 50°C, filter out the residue, and obtain modified cowhide; (4) Homogenization: Add purified water to denatured cowhide at a solid-liquid ratio of 1:3, and homogenize under high pressure at 20 MPa for 10 min; (5) First enzymatic hydrolysis: Add 0.2% of the weight of the cowhide mixed protease (20% pancreatic enzyme and 80% alkaline protease), and perform enzymatic hydrolysis at 55°C for 4 h to obtain the enzymatic hydrolyzate; (6) Inactivation of enzyme: After the first enzymatic hydrolysis is completed, the solution is inactivated in boiling water at 100°C for 30 min; (7) Filtration and adsorption: After the enzymatic hydrolysate is cooled to room temperature, it is filtered through a sieve, 0.5% activated carbon is added, and the solution is left for 12 hours; (8) Impurity removal: The obtained collagen tripeptide solution was centrifuged at 8000 rpm for 10 min to obtain a supernatant. The supernatant was filtered through a 30 μm pore size filter paper to obtain a collagen tripeptide solution; (9) Vacuum freeze drying: The collagen tripeptide solution was transferred into a vacuum freeze dryer for vacuum freeze drying: pre-freeze at -50°C for 8 h; after pre-freezing, the freeze dryer was evacuated, and then the temperature was increased to 0°C at 5-10°C / h and then kept constant for 12 h; then the temperature was increased to 20°C at 5-10°C / h and then kept constant, heated and dried, and the constant temperature time was 48 h to obtain a collagen tripeptide freeze-dried product; the freeze-dried product was ground to obtain a collagen tripeptide powder sample.

[0029] Effect Example (I) Samples of collagen tripeptide obtained in Example 1-3 and Comparative Example 1-2 were tested for indexes. The molecular weight distribution was measured according to the method in Appendix A of "National Standard of the People's Republic of China GB / T22729-2008"; the hydroxyproline detection method was measured according to "National Standard of the People's Republic of China GB / T9695.23-2008", on a dry basis; the total nitrogen detection method was measured according to "National Standard of the People's Republic of China GB / T5009.5-2016", on a dry basis; and the ash detection method was measured according to "National Standard of the People's Republic of China GB / T5009.4-2016". The results are shown in Table 1 below.

[0030] Table 1 (II) Solubility and transmittance testing 10 g of the collagen tripeptide powder prepared in Example 1-3 and Comparative Example 1-2 were respectively poured into different cups and dissolved with 500 mL of warm water at about 60° C. to obtain a collagen tripeptide solution. The solution was observed with the naked eye after 5 seconds, and the transmittance of the collagen tripeptide solution was detected by an ultraviolet spectrophotometer at 620 nm. The results are shown in Table 2.

[0031] Table 2 The collagen tripeptide products obtained according to the experimental procedures in Examples 1-3 have a high yield and a relatively concentrated molecular weight, with >89% of the molecular weight concentrated in 200-500Da, and high hydroxyproline and total nitrogen contents. The preparation method provided by the present invention does not require pH adjustment and does not introduce a large amount of salt components, so the ash content is extremely low, the quality is good, the performance is excellent, it can be quickly dissolved in water, and the solution is clear and transparent without impurities; and the collagen tripeptide obtained by the preparation method of the present invention has no fishy smell or other foreign smells. In the comparative examples, due to the lack of key steps, the yield of collagen tripeptide is low, the quality is poor, and it has a certain fishy smell. The molecular weight distribution is relatively dispersed, with a portion exceeding >1000Da. The hydroxyproline and total nitrogen contents are slightly reduced, and the ash value is increased. It is possible that the obtained sample contains more impurities, so it is dissolved in water, the light transmittance is reduced, and there is even a trace amount of precipitation. Specifically, Figure 2 shown.

[0032] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing collagen tripeptide, characterized in that: The following steps are involved: (1) Pretreatment of cowhide: Take fresh cowhide, remove hair and fat, cut into small pieces, and wash; add the washed cowhide particles into an alkaline solution for alkaline treatment, and wash with water to obtain pretreated cowhide; (2) Modified cowhide: Mix the pre-treated cowhide and the treatment solution, keep them warm for a period of time and then boil them. After cooling, filter out the residue to obtain modified cowhide. (3) Homogenization: Add the denatured cowhide into purified water and homogenize under high pressure to obtain cowhide homogenate; (4) First enzymatic hydrolysis: adding mixed proteases to the cowhide homogenate for enzymatic hydrolysis; inactivating the enzymes to obtain a primary enzymatic hydrolyzate; (5) Second enzymatic hydrolysis: using the first enzymatic hydrolyzate as substrate, adding animal protein hydrolyzing enzyme to hydrolyze; after inactivating the enzyme, the second enzymatic hydrolyzate is obtained; (6) Filtration, adsorption and impurity removal: The secondary enzymatic hydrolyzate is cooled to room temperature and then filtered. Activated carbon is added to the filtrate, and the filtrate is centrifuged after being left for a period of time to obtain a supernatant. The supernatant is filtered to remove impurities and obtain a collagen tripeptide solution. The obtained collagen tripeptide solution is then centrifuged at 6000-9000 rpm for 10-15 min to obtain a supernatant. The supernatant is filtered to remove impurities and obtain. (7) Vacuum freeze drying: The supernatant is vacuum freeze dried to obtain collagen tripeptide.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass volume concentration of the alkaline solution is 3-6%; the mass ratio of the cowhide particles to the alkaline solution is 1:2-10; and the alkaline treatment is stirred at 3-5°C for 12-24h.

3. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the pretreated cowhide to the treatment liquid is 1:3-8; the treatment liquid is prepared from the following raw materials: 10 parts of water and 0.3-0.5 parts of glucose; 5 mL of lactic acid streptococcus liquid is added to every 100 mL of water; the specific treatment is: heat preservation at 30-32°C for 50-60 minutes; boiling for 20-40 minutes; then cooling to 50-60°C and filtering.

4. The preparation method according to claim 1, characterized in that: In step (3), the mass ratio of the modified cowhide to purified water is 1:3-5; and the high-pressure homogenization is carried out at a pressure of 20-50 MPa for 8-15 min.

5. The preparation method according to claim 1, characterized in that: In step (4), the amount of the mixed protease added is 0.1-0.25% of the mass of the denatured cowhide; the mixed protease includes 20-30% of pancreatic enzyme and 80%-70% of alkaline protease; and the enzymatic hydrolysis is performed at 55° C. for 4 hours.

6. The preparation method according to claim 1 or 5, characterized in that: In step (5), the amount of the animal protein hydrolase added is 0.2-0.5% of the primary enzymatic hydrolysis solution; and the hydrolysis is carried out at 55° C. for 12-24 hours.

7. The preparation method according to claim 1, characterized in that: In step (6), the amount of activated carbon added is 0.2%-0.6% of the mass of the filtrate; the standing time is 12-18 hours; and the filtration is performed using filter paper with a pore size of 20-40 μm.

8. The preparation method according to claim 1, characterized in that: In step (7), the vacuum freeze drying procedure is divided into three parts: pre-freezing, low-temperature drying and heating drying; the pre-freezing temperature is between -20--80°C, and the pre-freezing time is 5-12 hours; after pre-freezing, the freeze dryer is vacuumed, and then the temperature is increased to -10-0°C at 5-10°C / h and then kept constant at that temperature, low-temperature drying, and the constant temperature time is 12-24 hours; then the temperature is increased to 0-30°C at 5-10°C / h and then kept constant at that temperature, heating drying, and the constant temperature time is 16-48 hours.

9. A collagen tripeptide prepared by the preparation method according to any one of claims 1 to 8.

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