Novel simplified method for extracting and preparing bullfrog skin collagen

Abstract

The invention provides a novel method for efficiently extracting and preparing collagen by taking bullfrog skin as a raw material. The process comprises the following steps. The preparation method comprises the following steps: 1) removing impurities from bullfrog skin, cleaning, chopping and homogenizing, stirring and soaking the bullfrog skin with an alkaline solution to remove grease and non-collagen in the bullfrog skin, and washing the bullfrog skin with pure water until the bullfrog skin is neutral; the preparation method comprises 1) preparing collagen powder, 2) hydrolyzing with pepsin and an acetic acid solution, and filtering to obtain a collagen hydrolysate, 3) precipitating to remove macromolecules and non-collagen containing pigment in the collagen hydrolysate by an alkali precipitation method, and centrifuging or filtering to obtain a collagen extracting solution, and 4) concentrating the collagen extracting solution by a membrane, and freeze-drying to obtain the high-purity collagen powder.

Description

Technical Field

[0001] The present invention belongs to the technical field of amphibian bioengineering, and particularly relates to a simplified new method for extracting and preparing high-purity collagen from bullfrog skin as a raw material. Background Art

[0002] Collagen is the main component of the extracellular matrix, widely distributed in organisms, with the highest proportion in animals, and is the macromolecular protein with the highest content and three-dimensional high-level structure. At present, 29 types of collagen are known, and different types of collagen are distributed in different tissues. Type I collagen mainly exists in tissues such as the bones, skin, ligaments, tendons, and corneas of animals, and is the most common, highest-content, and most abundantly distributed type among all types of collagen.

[0003] As a functional structural protein with a triple helix structure, collagen has biological characteristics such as non-toxicity, low allergenicity, good biocompatibility, and biodegradability. It can form collagen fibers with a certain tensile strength and stability through self-assembly and cross-linking, and is widely used in the fields of medical and bioengineering materials as a biomaterial with various biological functions.

[0004] At present, the collagen products used clinically are mainly from terrestrial animals. However, with the frequent occurrence of zoonotic diseases such as foot-and-mouth disease and mad cow disease, the safety issues of collagen from terrestrial animals have been continuously raised and questioned. In addition, due to the troubles of factors such as animal protection, the application scope of collagen from terrestrial animals has been greatly restricted. Compared with terrestrial animals, the living environment of aquatic organisms is relatively clean, and they are safer than terrestrial animals in terms of risk control such as virus carriage and cross-infection, with obvious advantages; in addition, the application scope is wider. Therefore, it is very feasible to choose aquatic animals as the source for extracting natural collagen.

[0005] The bullfrog belongs to Amphibia, Anura, and Ranidae. Because of its tender meat, delicious taste, and rich nutrition, it is deeply loved by consumers and has become one of the important aquatic products in aquaculture in China. Consuming bullfrogs will produce a large amount of by-products of frog skin, with low added value. Therefore, establishing a production process for frog skin collagen and preparing high-quality frog skin collagen can not only protect the environment and reduce waste, but also increase the added value of bullfrogs. Summary of the Invention

[0006] The present invention aims to provide a simplified method for extracting collagen from bullfrog skin as a raw material. This method has a high extraction rate and good quality of collagen, can be applied to the fields of medical and bioengineering materials, and has good industrialization prospects. Technical Solution

[0007] A new method for efficiently extracting and preparing bullfrog skin collagen.

[0008] 1) Clean the fresh bullfrog skin, then crush and homogenize it.

[0009] 2) Remove grease and miscellaneous proteins during alkali treatment: Stir and soak the bullfrog skin homogenate in step 1) with an alkali solution for 24 - 48 hours, and change the alkali solution every 24 hours; wash it with pure water until neutral, and then filter.

[0010] 3) Extract collagen by acid - enzyme method. Extract collagen from the bullfrog skin after alkali treatment in step 2) with pepsin and acetic acid solution, and the ratio of material to liquid is 1:10 - 1:50 (w / v); the amount of pepsin is 5 - 50 U / g, the treatment temperature is 20 - 25 °C, and the treatment time is 24 - 48 h; centrifuge or filter to obtain the collagen extract.

[0011] 4) Purify collagen by alkali precipitation method. Adjust the pH of the collagen extracted by acid - enzyme method in step 3) to 8 - 11 with NaOH solution, and let it stand to precipitate macromolecular soluble and pigment - containing non - collagen proteins. Remove the precipitate by centrifugation or filtration to obtain the purified collagen extract.

[0012] 5) Concentrate and freeze - dry collagen by membrane. Concentrate the purified collagen extract obtained in step 4) with a membrane concentrator and vacuum freeze - dry it to obtain high - purity collagen powder.

[0013] Furthermore, for the crushing and homogenizing of the frog skin in step 1), a cutting machine, a wall - breaker, a colloid mill, etc. are selected.

[0014] Furthermore, the alkali solution in step 2) is 0.1 mol / L NaOH solution, and the ratio of the volume of the alkali solution to the mass of the material is 6:1.

[0015] Furthermore, in step 3), the ratio of material to liquid is 1:20, the acetic acid concentration is 0.6 mol / L, the amount of pepsin is 5 U / g, the extraction temperature is 25 °C, and the extraction time is 24 h.

[0016] Furthermore, the concentration of the NaOH solution in step 4) is 10 mol / L.

[0017] Furthermore, the ultrafiltration membrane concentration in step 5) is membrane concentration with a molecular weight cut - off of 50 - 100 kDa.

[0018] Furthermore, the freeze - drying in step 5) is freeze - drying at - 50 °C for 48 h.

[0019] Beneficial effects 1) The present invention provides a highly simplified new method for extracting and preparing collagen from bullfrog skin. This method can greatly remove non-collagen proteins and pigments. The product is white in color and of high quality. The prepared collagen retains the unique triple helix structure of collagen, ensuring the integrity and activity of the collagen structure.

[0020] 2) In the present invention, alkali treatment is used to remove both grease and miscellaneous proteins simultaneously. The traditional method is to use organic solvents to remove grease and then use an alkali solution to remove miscellaneous proteins. The extraction and preparation method of the present invention completes the removal of grease and miscellaneous proteins in one step. That is, the grease is saponified with an alkali solution, and the saponified grease is dissolved in the alkali solution and removed, and the alkali solution also removes the miscellaneous proteins at the same time.

[0021] 3) In the present invention, the alkali precipitation method is used to remove macromolecular non-collagen proteins, pigment-containing proteins and other miscellaneous proteins in the enzymatic hydrolysate, and high-purity collagen can be obtained. Compared with the traditional salting-out method, it is simple and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Process flow chart for the extraction of bullfrog skin collagen. Figure 2 : SDS-PAGE electrophoresis analysis of the extracted and prepared bullfrog skin collagen. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments usually follow conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0024] Example 1 (1) Take fresh bullfrog skin, remove the remaining frog meat, wash it with pure water, and then store it at -20°C after cutting, homogenizing and draining.

[0025] (2) Mix the frog skin with 0.15 M NaOH solution at a material-liquid ratio of 1:6 (w / v), stir and soak for 48 hours, and change the alkali solution every 24 hours.

[0026] (3) Wash the alkali-soaked frog skin with pure water until it is neutral, filter and weigh it.

[0027] (4) Mix the alkali-treated frog skin with 0.6 M acetic acid solution at a material-liquid ratio of 1:10 (w / v), homogenize and then add 10 U / g of pepsin, and enzymatically hydrolyze for 24 h. After enzymatic hydrolysis, filter out the insoluble matter.

[0028] (5) Use 10 M NaOH solution to adjust the pH of the enzymatic hydrolysate to 10, and let it stand for 2 - 4 h.

[0029] (6) The alkaline precipitation solution is first passed through a 0.22 μm filter membrane and then concentrated by a membrane concentrator to obtain a clarified concentrated solution.

[0030] (7) The concentrated solution is pre-frozen at -20 °C in a refrigerator for 24 h and then freeze-dried at -50 °C for 72 h to obtain the finished product of collagen. Example

[0031] (1) Take fresh bullfrog skins, remove the remaining frog meat, wash them with pure water, and then store them at -20 °C after cutting, homogenizing, and draining.

[0032] (2) Mix the frog skins with 0.1 M NaOH solution at a material-liquid ratio of 1:6 (w / v), stir and soak for 48 hours, and change the alkaline solution every 24 hours.

[0033] (3) Wash the alkali-soaked frog skins with pure water until neutral, filter and dry them, and weigh them.

[0034] (4) Mix the alkali-treated frog skins with 0.6 M acetic acid solution at a material-liquid ratio of 1:20 (w / v), homogenize and then add 5 U / g of pepsin, and enzymolyze for 24 h. After enzymolysis, filter out the insoluble substances.

[0035] (5) Use 10 M NaOH solution to adjust the pH of the enzymolysis solution to 10, and let it stand for 2 - 4 h.

[0036] (6) The alkaline precipitation solution is first passed through a 0.22 μm filter membrane and then concentrated by a membrane concentrator to obtain a clarified concentrated solution.

[0037] (7) The concentrated solution is pre-frozen at -20 °C in a refrigerator for 24 h and then freeze-dried at -50 °C for 72 h to obtain the finished product of collagen. Example

[0038] (1) Take fresh bullfrog skins, remove the remaining frog meat, wash them with pure water, and then store them at -20 °C after cutting, homogenizing, and draining.

[0039] (2) Mix the frog skins with 0.1 M NaOH solution at a material-liquid ratio of 1:6 (w / v), Stir and soak for 24 hours.

[0040] (3) Wash the alkali-soaked frog skins with pure water until neutral, filter and dry them, and weigh them.

[0041] (4) Mix the alkali-treated frog skins with 0.6 M acetic acid solution at a material-liquid ratio of 1:20 (w / v), homogenize and then add 10 U / g of pepsin, and enzymolyze for 24 h. After enzymolysis, filter out the insoluble substances.

[0042] (5) Use 10 M NaOH solution to adjust the pH of the enzymolysis solution to 10, fully react and then let it stand for 2 h.

[0043] (6) The alkaline precipitation solution is first passed through a 0.22 μm filter membrane and then concentrated by a membrane concentrator to obtain a clarified concentrated solution.

[0044] (7) The concentrated solution is pre-frozen in a -20 °C refrigerator for 24 h and freeze-dried at -50 °C for 72 h to obtain the finished product of collagen.

Claims

1. A simplified new method for extracting and preparing bullfrog skin collagen, characterized in that: 1) Clean the fresh bullfrog skin, crush and homogenize; 2) Alkali treatment to remove oil and foreign proteins at the same time: stir and soak the bullfrog skin homogenate in the above 1) in an alkaline solution for 24-48 hours, and replace the alkaline solution every 24 hours; wash with pure water until neutral, and filter; 3) Extract collagen by acid-enzyme method. Extract collagen from bullfrog skin treated with alkali in 2) above with pepsin and acetic acid solution, with a solid-liquid ratio of 1:10-1:50 (w / v); pepsin amount of 5-50U / g, treatment temperature of 20-25℃, and treatment time of 24-48h; centrifuge or filter to obtain collagen extract; 4) Alkaline precipitation method to purify collagen. Adjust the pH of the collagen extracted by the acid enzymatic method in 3) to 8-11 with NaOH solution, and let it stand to precipitate macromolecular soluble and pigmented non-collagen proteins. Remove the precipitate by centrifugation or filtration to obtain a purified collagen extract; 5) Membrane concentration and freeze-drying of collagen: The purified collagen extract obtained in 4) above is concentrated using a membrane concentrator and vacuum freeze-dried to obtain high-purity collagen powder.

2. A simplified new method for extracting and preparing bullfrog skin collagen according to claim 1, characterized in that: The alkaline solution in step 2) is a 0.05-0.2 mol / L NaOH solution, and the volume ratio of the alkaline solution to the material mass is 5-10:1 (v / w).

3. A simplified new method for extracting and preparing bullfrog skin collagen according to claim 1, characterized in that: Step 3) The solid-liquid ratio is 1:10-50 (w / v), the amount of pepsin is 5-50 U / g, the extraction temperature is 25°C, the extraction time is 24-48h, and the acetic acid concentration is 0.2-1.0 mol / L.

4. A simplified new method for extracting and preparing bullfrog skin collagen according to claim 1, characterized in that: The alkaline solution in step 4) is a 10 mol / L NaOH solution.

5. A simplified new method for extracting and preparing bullfrog skin collagen according to claim 1, characterized in that: Step 5) The ultrafiltration membrane is a membrane with a molecular weight cutoff of 50-100 kDa.

6. A simplified new method for extracting and preparing bullfrog skin collagen according to claim 1, characterized in that: The freeze drying in step 5) is freeze drying at -50°C for 48 hours.

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