Type II collagen and preparation method thereof

Abstract

The preparation method comprises the following steps: degreasing: taking clean chicken sternal cartilage, mincing and crushing, transferring into a supercritical extraction device, carrying out degreasing treatment by using CO2 fluid, adding purified water of which the weight is 3-5 times of that of the chicken sternal cartilage, adding pancreatin of which the weight is 0.5-1.5% of that of the chicken sternal cartilage, carrying out enzymolysis, and filtering to obtain filtrate; defatted chicken breast cartilage is prepared; enzymolysis: after degreasing is completed, adjusting the pH value of the solution to 2.5-3.5 by using hydrochloric acid, continuing to add pepsase accounting for 0.5-1.5% of the weight of the chicken sternal cartilage, and carrying out enzymolysis treatment to obtain an enzymatic hydrolysate; concentrating: filtering the enzymatic hydrolysate until the enzymatic hydrolysate is clear, and concentrating the filtrate by 10 times of volume through a 500Da nanofiltration membrane; and spray-drying: feeding the filtrate into a spray dryer to prepare the type II collagen. According to the method, the degreasing process is carried out by adopting supercritical CO2 and adding pancreatin to carry out enzymolysis, so that the extraction rate of the collagen can be greatly improved, the extraction purity of the non-denatured type II collagen is high, the production process is simple, and the method can be suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen preparation, and in particular to type II collagen and a preparation method thereof. Background Art

[0002] Collagen is a white, odorless fibrous protein widely distributed in mammals. It is the most abundant protein in animals, accounting for about 30% of the total protein. There are more than 20 known types of collagen, among which type II collagen is mainly distributed in the articular cartilage of connective tissues such as skin, cartilage, tendons, blood vessels, and cornea of ​​animals, and is the main component of cartilage matrix.

[0003] Chicken sternum cartilage is a waste product in chicken processing, which not only causes great waste but also pollutes the environment. Studies have found that the content of type II collagen in chicken sternum cartilage is as high as more than 50%, so it can be used as a high-quality raw material for extracting type II collagen.

[0004] Type II collagen is composed of three entangled polypeptide chains. Due to its triple helical structure, it mainly plays the role of supporting organs and protecting the body in the body. However, its triple helical structure is easily destroyed and may be accompanied by the loss of biological activity. The maintenance of the triple helical structure of type II collagen depends on the synergistic effect of various intermolecular and intramolecular effects, such as hydrogen bonds, van der Waals forces, hydration and disulfide bonds, which play a role in maintaining its molecular structure. Hydrophobic and ionic interactions provide intermolecular forces. Therefore, the thermal stability of the triple helical structure of type II collagen is affected by many factors.

[0005] Most of the type II collagen products currently on the market are completely enzymatically hydrolyzed collagen products. Due to the limitations of the extraction and preparation processes, under the action of enzymes, high temperatures, pH values ​​and other factors, type II collagen loses its original triple helix structure and is decomposed into polypeptides and amino acids below 20,000 Daltons, losing its original prevention and adjuvant treatment functions for rheumatoid arthritis and arthritis.

[0006] At present, a variety of preparation processes of non-denatured type II collagen have been disclosed at home and abroad. CN116253791A discloses a method for preparing non-denatured type II collagen, including sterilization and impurity removal, solid-liquid separation, cartilage crushing, homogenization, enzymolysis, solid-liquid separation, pH adjustment, solid-liquid separation, drying, crushing and other steps. CN114045322A discloses a method for preparing non-denatured type II collagen, using chicken breast cartilage as raw material through defatting, grinding, acid hydrolysis, enzymolysis, electrophoresis dialysis, secondary grinding, sterilization, and obtaining type II collagen. CN113563458A discloses a method for preparing non-denatured type II collagen, using chicken breast cartilage as raw material, through material selection, cleaning, crushing, enzymolysis, drying, crushing, sieving and other steps. At present, the existing technology generally has the problems of complex procedures, long time consumption, and low extraction rate or low purity of non-denatured type II collagen. Therefore, it is very important to provide a method for extracting non-denatured type II collagen with a simple process, high collagen extraction rate and high purity.

[0007] In view of this, this application is hereby filed. Summary of the invention

[0008] The problem with the prior art is that the preparation of type II collagen is complicated and time-consuming, and the extraction rate or purity of the non-denatured type II collagen is low. The present invention provides a type II collagen and a preparation method thereof. The degreasing process is carried out by combining supercritical CO2 with the addition of pancreatic enzymes for enzymatic hydrolysis, which can greatly improve the extraction rate of collagen, and the non-denatured type II collagen has a high extraction purity, a simple production process, and is suitable for large-scale production.

[0009] The present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing type II collagen, wherein the raw material is subjected to defatting, enzymatic hydrolysis, concentration and spray drying steps in sequence;

[0011] The specific method of degreasing process is as follows:

[0012] After pretreatment, the raw materials are transferred to a supercritical extraction device and subjected to extraction and degreasing treatment using CO2 fluid;

[0013] After the extraction is completed, 0.5-1.5% pancreatin is added to the raw material for enzymatic hydrolysis to complete the defatting process.

[0014] In a specific embodiment, the CO2 fluid extraction conditions in the degreasing process are: extraction flow rate 25-30 L / h, 20-25 MPa, 30-35°C treatment for 2h.

[0015] In a specific embodiment, the enzymatic hydrolysis treatment conditions in the defatting process are: 40-45°C, pH 8.0-8.2, and enzymatic hydrolysis for 2 hours.

[0016] In a specific embodiment, in the degreasing process, the specific steps of raw material pretreatment are as follows: the clean raw materials are minced and crushed so that they can pass through a 20-40 mesh sieve.

[0017] In a specific embodiment, the specific method of the enzymatic hydrolysis step is as follows: the defatted raw material is treated with hydrochloric acid to adjust the pH value of the solution to 2.5-3.5, and 0.5-1.5% of the weight of the raw material pepsin is added for enzymatic hydrolysis to obtain an enzymatic solution.

[0018] In a specific embodiment, in the enzymatic hydrolysis step, the enzymatic hydrolysis treatment conditions are: ultrasonic power 310W, enzymatic hydrolysis treatment at 36-38°C for 1 to 2 hours.

[0019] In a specific embodiment, the specific method of the concentration step is as follows: the enzymatic hydrolyzate is filtered until it is clarified, and the filtrate is concentrated to 10 times the volume through a 500Da nanofiltration membrane.

[0020] In a specific embodiment, the specific method of the spray drying process is as follows: the filtrate enters the spray dryer, the inlet air temperature is 190-200°C, the outlet air temperature is 80°C, and the feed frequency is 45-50HZ to obtain type II collagen.

[0021] In a specific embodiment, the raw material is chicken breast cartilage.

[0022] In a second aspect, the present invention provides a type II collagen prepared by the preparation method.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] The embodiment of the present invention provides a type II collagen and a preparation method thereof. By combining supercritical CO2 with the addition of pancreatic enzymes for enzymatic hydrolysis to carry out the degreasing process, the extraction rate of collagen can be greatly improved. The non-denatured type II collagen has high extraction purity, a simple production process, and is suitable for large-scale production. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0026] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to one of ordinary skill in the art that these specific details need not be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, well-known materials or methods are not specifically described.

[0027] Throughout the specification, references to "one embodiment," "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment of the present invention. Therefore, the phrases "one embodiment," "an embodiment," "an example," or "an example" appearing in various places throughout the specification do not necessarily all refer to the same embodiment or example. In addition, particular features, structures, or characteristics may be combined in one or more embodiments or examples in any suitable combination and / or subcombination. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.

[0029] In the description of the present invention, unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0030] In the description of the present invention, unless otherwise specified, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0031] In the description of the present invention, unless otherwise specified, the words "include" and "comprising" mentioned in the present application may be open-ended or closed-ended. For example, the words "include" and "comprising" may mean that other components not listed may also be included or comprised, or may only include or comprise the listed components.

[0032] The problem with the prior art is that the preparation of type II collagen is complicated and time-consuming, and the extraction rate or purity of the non-denatured type II collagen is low.

[0033] The present invention is achieved through the following technical solutions:

[0034] In a first aspect, the present invention provides a method for preparing type II collagen, wherein the raw material is subjected to defatting, enzymatic hydrolysis, concentration and spray drying steps in sequence;

[0035] The specific method of degreasing process is as follows:

[0036] After pretreatment, the raw materials are transferred to a supercritical extraction device and subjected to extraction and degreasing treatment using CO2 fluid;

[0037] After the extraction is completed, 0.5-1.5% pancreatin is added to the raw material for enzymatic hydrolysis to complete the defatting process.

[0038] In a specific embodiment, the CO2 fluid extraction conditions in the degreasing process are: extraction flow rate 25-30 L / h, 20-25 MPa, 30-35°C treatment for 2h.

[0039] In a specific embodiment, the enzymatic hydrolysis treatment conditions in the defatting process are: 40-45°C, pH 8.0-8.2, and enzymatic hydrolysis for 2 hours.

[0040] In a specific embodiment, in the degreasing process, the specific steps of raw material pretreatment are as follows: the clean raw materials are minced and crushed so that they can pass through a 20-40 mesh sieve.

[0041] In a specific embodiment, the specific method of the enzymatic hydrolysis step is as follows: the defatted raw material is treated with hydrochloric acid to adjust the pH value of the solution to 2.5-3.5, and 0.5-1.5% of the weight of the raw material pepsin is added for enzymatic hydrolysis to obtain an enzymatic solution.

[0042] In a specific embodiment, in the enzymatic hydrolysis step, the enzymatic hydrolysis treatment conditions are: ultrasonic power 310W, enzymatic hydrolysis treatment at 36-38°C for 1 to 2 hours.

[0043] In a specific embodiment, the specific method of the concentration step is as follows: the enzymatic hydrolyzate is filtered until it is clarified, and the filtrate is concentrated to 10 times the volume through a 500Da nanofiltration membrane.

[0044] In a specific embodiment, the specific method of the spray drying process is as follows: the filtrate enters the spray dryer, the inlet air temperature is 190-200°C, the outlet air temperature is 80°C, and the feed frequency is 45-50HZ to obtain type II collagen.

[0045] In a specific embodiment, the raw material is chicken breast cartilage.

[0046] In a second aspect, the present invention provides a type II collagen prepared by the preparation method.

[0047] The present invention provides a type II collagen and a preparation method thereof. The degreasing process is carried out by combining supercritical CO2 with the addition of pancreatic enzymes for enzymatic hydrolysis, which can greatly improve the extraction rate of collagen. The non-denatured type II collagen has high extraction purity, a simple production process, and is suitable for large-scale production.

[0048] Example 1

[0049] The present invention provides a method for preparing type II collagen, comprising the following steps:

[0050] (1) Degreasing:

[0051] Take 1kg of cleaned chicken breast cartilage, mince and crush it so that it can pass through a 20-mesh sieve, transfer the crushed and sieved chicken breast cartilage to a supercritical extraction device, use CO2 fluid for degreasing, set the extraction flow rate to 25-30L / hour, 20-25Mpa, 30-35℃ for 2 hours. The extract enters the separation kettle for separation, collects oil and other substances at the bottom, and collects CO2 gas for recycling.

[0052] Add purified water 3 to 5 times the weight of the chicken sternal cartilage, add pancreatin 1.0% of the weight of the chicken sternal cartilage, and perform enzymolysis for 2 hours at 40 to 45° C. and pH 8.0 to 8.2 to obtain defatted chicken sternal cartilage.

[0053] (2) Enzymatic hydrolysis:

[0054] The pH value of the solution was adjusted to 2.5-3.5 with 1 mol / L hydrochloric acid, and 0.5% pepsin by weight of chicken sternal cartilage was added. The ultrasonic power was 310 W and the enzymolysis was performed at 36-38° C. for 1 hour to obtain an enzymolysis solution.

[0055] (3) Concentration:

[0056] The enzymatic hydrolysate was filtered until clear, and the filtrate was concentrated 10 times by volume through a 500Da nanofiltration membrane.

[0057] (4) Spray drying:

[0058] The filtrate enters the spray dryer with an inlet air temperature of 190-200°C, an outlet air temperature of 80°C, and a feed frequency of 45-50HZ to produce type II collagen.

[0059] Example 2

[0060] The present invention provides a method for preparing type II collagen, comprising the following steps:

[0061] (1) Degreasing:

[0062] Take 1kg of cleaned chicken breast cartilage, mince and crush it so that it can pass through a 20-mesh sieve, transfer the crushed and sieved chicken breast cartilage to a supercritical extraction device, use CO2 fluid for degreasing, set the extraction flow rate to 25-30L / hour, 20-25Mpa, 30-35℃ for 2 hours. The extract enters the separation kettle for separation, collects oil and other substances at the bottom, and collects CO2 gas for recycling;

[0063] Add purified water 3 to 5 times the weight of the chicken sternal cartilage, add pancreatin 1.0% of the weight of the chicken sternal cartilage, and perform enzymolysis at 40 to 45° C. and pH 8.0 to 8.2 for 2 hours to obtain defatted chicken sternal cartilage.

[0064] (2) Enzymatic hydrolysis:

[0065] Purified water 3 to 6 times the weight of chicken sternal cartilage was added, and the pH value of the solution was adjusted to 2.5-3.5 with 1 mol / L hydrochloric acid. Pepsin 1.5% of the weight of chicken sternal cartilage was added, and the enzymatic hydrolysis was performed at 36-38° C. with an ultrasonic power of 310 W for 2 hours to obtain an enzymatic solution.

[0066] (3) Concentration:

[0067] The enzymatic hydrolysate was filtered until clear, and the filtrate was concentrated 10 times by volume through a 500Da nanofiltration membrane.

[0068] (4) Spray drying:

[0069] The filtrate enters the spray dryer with an inlet air temperature of 190-200°C, an outlet air temperature of 80°C, and a feed frequency of 45-50HZ to produce type II collagen.

[0070] Comparative Example 1

[0071] This comparison provides a method for preparing type II collagen, comprising the following steps:

[0072] (1) Degreasing:

[0073] The chicken breast cartilage is thawed and drained, and coarsely chopped to obtain chicken cartilage pieces with a particle size of 0.5 to 10 mm; 3 kg of sodium hydroxide solution (concentration 0.2%) is added to 1.0 kg of chicken cartilage pieces to sterilize and remove impurities; after sterilization and removal of impurities for 8 hours, the mixture is filtered with a filter cloth, the precipitate is taken, and it is washed twice with distilled water, and then filtered to obtain sterilized and impurity-free cartilage pieces.

[0074] Add purified water 3 to 5 times the weight of the chicken sternal cartilage, add pancreatin 1.0% of the weight of the chicken sternal cartilage, and perform enzymolysis for 2 hours at 40 to 45° C. and pH 8.0 to 8.2 to obtain defatted chicken sternal cartilage.

[0075] (2) Enzymatic hydrolysis:

[0076] Add purified water 10-12 times the weight of chicken sternal cartilage, and use 1 mol / L hydrochloric acid to adjust the solution pH to 2.5-3.5, continue to add pepsin 0.5-1.5% of the weight of chicken sternal cartilage, ultrasonic power 310W, 36-38°C condition enzymolysis treatment for 2 hours to obtain enzymolysis solution.

[0077] (3) Concentration:

[0078] The enzymatic hydrolysate was filtered until clear, and the filtrate was concentrated 10 times by volume through a 500Da nanofiltration membrane.

[0079] (4) Spray drying:

[0080] The filtrate enters the spray dryer with an inlet air temperature of 190-200°C, an outlet air temperature of 80°C, and a feed frequency of 45-50HZ to produce type II collagen.

[0081] The difference between this comparative example and the embodiment is that the supercritical CO2 degreasing technology in the degreasing process is replaced by the saponification degreasing technology, and the pancreatic enzyme hydrolysis treatment in the degreasing process is retained.

[0082] Comparative Example 2

[0083] This comparative example provides a method for preparing type II collagen, comprising the following steps:

[0084] (1) Degreasing:

[0085] Take 1kg of cleaned chicken breast cartilage, mince and crush it so that it can pass through a 20-mesh sieve, transfer the crushed and sieved chicken breast cartilage to a supercritical extraction device, use CO2 fluid for degreasing, set the extraction flow rate to 25-30L / hour, 20-25Mpa, 30-35℃ for 2 hours. The extract enters the separation kettle for separation, collects oil and other substances at the bottom, and collects CO2 gas for recycling.

[0086] Add purified water 3 to 5 times the weight of the chicken sternal cartilage, add pancreatin 1.0% of the weight of the chicken sternal cartilage, and perform enzymolysis for 2 hours at 40 to 45° C. and pH 8.0 to 8.2 to obtain defatted chicken sternal cartilage.

[0087] (2) Enzymatic hydrolysis:

[0088] Add purified water 10-12 times the weight of chicken sternal cartilage, and use 1 mol / L hydrochloric acid to adjust the solution pH to 2.5-3.5, continue to add pepsin 0.5-1.5% of the weight of chicken sternal cartilage, ultrasonic power 310W, 36-38°C condition enzymolysis treatment for 2 hours to obtain enzymolysis solution.

[0089] (3) Concentration:

[0090] The enzymatic hydrolysate was filtered until clear, and the filtrate was concentrated 10 times by volume through a 500Da nanofiltration membrane.

[0091] (4) Spray drying:

[0092] The filtrate enters the spray dryer with an inlet air temperature of 190-200°C, an outlet air temperature of 80°C, and a feed frequency of 45-50HZ to produce type II collagen.

[0093] The difference between this comparative example and the embodiment is that the supercritical CO2 degreasing technology in the degreasing process is retained, the pancreatic enzyme hydrolysis treatment in the degreasing process is omitted, and other operations remain unchanged.

[0094] Comparative Example 3

[0095] This comparative example provides a method for preparing type II collagen, comprising the following steps:

[0096] (1) Degreasing:

[0097] Take 1kg of cleaned chicken breast cartilage, mince and crush it so that it can pass through a 20-mesh sieve, transfer the crushed and sieved chicken breast cartilage to a supercritical extraction device, use CO2 fluid for degreasing, set the extraction flow rate to 25-30L / hour, 20-25Mpa, 30-35℃ for 2 hours. The extract enters the separation kettle for separation, collects oil and other substances at the bottom, and collects CO2 gas for recycling.

[0098] Add purified water 3 to 5 times the weight of the chicken sternal cartilage, add pancreatin 0.1% of the weight of the chicken sternal cartilage, and perform enzymolysis at 40 to 45° C. and pH 8.0 to 8.2 for 2 hours to obtain defatted chicken sternal cartilage.

[0099] (2) Enzymatic hydrolysis:

[0100] Add purified water 10-12 times the weight of chicken sternal cartilage, and use 1 mol / L hydrochloric acid to adjust the solution pH to 2.5-3.5, continue to add pepsin 0.5-1.5% of the weight of chicken sternal cartilage, ultrasonic power 310W, 36-38°C condition enzymolysis treatment for 2 hours to obtain enzymolysis solution.

[0101] (3) Concentration:

[0102] The enzymatic hydrolysate was filtered until clear, and the filtrate was concentrated 10 times by volume through a 500Da nanofiltration membrane.

[0103] (4) Spray drying:

[0104] The filtrate enters the spray dryer with an inlet air temperature of 190-200°C, an outlet air temperature of 80°C, and a feed frequency of 45-50HZ to produce type II collagen.

[0105] The difference between this comparative example and the embodiment is that the supercritical CO2 degreasing technology in the degreasing process is retained, the pancreatic enzyme hydrolysis treatment in the degreasing process is retained, the amount of pancreatic enzyme added is reduced to 0.1%, and other operations remain unchanged.

[0106] Relevant data tests were performed on the type II collagen prepared in Example 1-Example 2 and Comparative Example 1-Comparative Example 3, and the results are shown in Table 1 below.

[0107] Table 1

[0108]

[0109]

[0110] As can be seen from Table 1, compared with the traditional degreasing and washing operation, the process time of the embodiment of the present invention is shortened by about 50%, and the collagen purity is higher, and the content of hydroxyproline and chondroitin sulfate is also higher. The traditional preparation method needs to remove impurities and other operations after degreasing, which is cumbersome.

[0111] This application adopts supercritical cleaning technology with SCCO2 as the main body, combined with the effect of pancreatic enzymes to decompose fats and proteins. The oil removal rate of this method reaches 98.2% to 99.5%. Compared with traditional cleaning methods such as soaking in salt water, the cleaning time is greatly shortened, and the raw material cleaning process can be completed within 5 hours.

[0112] The invention creatively uses pancreatic enzyme in the degreasing step, which can not only decompose fat, but also separate impurity protein and polysaccharide, thus significantly improving product quality. After reducing pancreatic enzyme or not using pancreatic enzyme, the degreasing rate and product quality are significantly reduced.

[0113] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing type II collagen, characterized in that: The raw materials are sequentially subjected to defatting, enzymatic hydrolysis, concentration and spray drying processes; The specific method of degreasing process is as follows: After pretreatment, the raw materials are transferred to a supercritical extraction device and subjected to extraction and degreasing treatment using CO2 fluid; After the extraction is completed, pancreatic enzyme is added for enzymatic hydrolysis to complete the degreasing process.

2. The method for preparing type II collagen according to claim 1, characterized in that: The CO2 fluid extraction conditions in the degreasing process are: extraction flow rate 25-30 L / h, 20-25 MPa, 30-35° C. for 2 hours.

3. The method for preparing type II collagen according to claim 2, characterized in that: In the degreasing process, the amount of pancreatin added is 0.5-1.5%, and the enzymatic treatment conditions are: 40-45° C., pH 8.0-8.2, and enzymatic hydrolysis for 2 hours.

4. The method for preparing type II collagen according to claim 1, characterized in that: In the degreasing process, the specific steps of raw material pretreatment are as follows: the clean raw materials are minced and crushed so that they can pass through a 20-40 mesh sieve.

5. The method for preparing type II collagen according to claim 1, characterized in that: The specific method of the enzymatic hydrolysis process is as follows: the defatted raw material is treated with hydrochloric acid to adjust the pH value of the solution to 2.5-3.5, and 0.5-1.5% of the weight of the raw material pepsin is added for enzymatic hydrolysis to obtain an enzymatic solution.

6. The method for preparing type II collagen according to claim 5, characterized in that: In the enzymatic hydrolysis step, the enzymatic hydrolysis treatment conditions are: ultrasonic power 310W, enzymatic hydrolysis treatment at 36-38°C for 1-2h.

7. The method for preparing type II collagen according to claim 1, characterized in that: The specific method of the concentration process is as follows: the enzymatic hydrolysate is filtered until it is clear, and the filtrate is concentrated to 10 times the volume through a 500Da nanofiltration membrane.

8. The method for preparing type II collagen according to claim 1, characterized in that: The specific method of the spray drying process is as follows: the filtrate enters the spray dryer, the inlet air temperature is 190-200°C, the outlet air temperature is 80°C, and the feed frequency is 45-50HZ to obtain type II collagen.

9. The method for preparing type II collagen according to claim 1, characterized in that: The raw material is chicken breast cartilage.

10. A type II collagen, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 9.

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