Separation preparation method and application of cattle hide collagen

Abstract

The invention discloses a separation preparation method and application of cattle hide collagen, and belongs to the technical field of collagen preparation. According to the method, the cow leather is subjected to freezing and two-step pre-fermentation treatment, so that the cow leather is fully swelled, dissolution of collagen is promoted, and the extraction rate is increased; an organic weak acid medium and a compound enzyme are used for co-extraction subsequently, so that the collagen is fully dissolved out, the content of impurity protein is reduced, and the purity and the extraction rate of the collagen are improved; the process is high in cowhide protein extraction rate, adopts biotechnology extraction in the whole process, avoids negative effects of strong acid and strong alkali on the collagen, is safe, environment-friendly and pollution-free, is also suitable for extraction of collagen in other raw materials, and is worthy of large-scale popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen preparation, and specifically relates to a method for separating and preparing cowhide collagen and application thereof. Background Art

[0002] Collagen is a biological macromolecule synthesized by animal cells. It is widely found in animal bones, tendons, cartilage, skin and other connective tissues. It has the function of supporting organs and protecting the body. In recent years, with the development of biotechnology, the research on the properties, structure and biological functions of collagen and collagen polypeptides has been continuously deepened. Collagen products have been valued by more and more people, and its research and application in food and cosmetics has become a hot spot.

[0003] As a major meat producer and consumer, a large number of by-products are produced during the production of beef. How to develop beef by-products and improve the economic benefits of the enterprise has become a problem faced by various meat-processing companies and research institutes today.

[0004] Cowhide is rich in collagen. Extracting collagen from cowhide is a high-yield scientific and technological project. The comprehensive utilization of these by-products can also reduce environmental pollution and has high economic and social benefits.

[0005] Traditionally, collagen is extracted by high-temperature boiling in water, acid hydrolysis, and alkaline hydrolysis, but each of these methods has its own fatal shortcomings. Although the high-temperature boiling method in water has low production requirements, simple operation, low equipment requirements, no pollution, and is beneficial to environmental protection, the collagen molecules are heated and denatured, and the triple helix is ​​unwound to form free polypeptide chains, which destroys the covalent cross-linking between molecules and causes random hydrolysis of the bonds in collagen, which easily turns into large molecular gelatin, which is not conducive to human absorption and reduces its utilization value. The acid hydrolysis method is rapid and thorough, but all tryptophan is destroyed, serine and tyrosine are partially destroyed, and the product yield is low, the equipment is severely corroded, and secondary pollution is generated. The alkaline hydrolysis method is rapid and thorough, but all amino acids containing hydroxyl groups and sulfhydryl groups are destroyed and racemization (structural variation) occurs.

[0006] Enzyme hydrolysis has incomparable advantages over other methods. It is environmentally friendly, has high purity of extracted hydrolyzed collagen, good water solubility, and stable physical and chemical properties. Moreover, with the improvement of biotechnology, the sources of enzyme preparations are extensive, the activity is constantly improving, and the cost-effectiveness ratio is greatly reduced, which provides great space for its application in industrial production. In particular, the application of protease preparations is not uncommon and has achieved significant results.

[0007] However, enzymatic hydrolysis is a mild extraction method. Although it can avoid problems such as collagen denaturation to a certain extent, the current enzymatic methods for preparing bovine skin collagen have many problems such as long extraction time and low extraction rate. Therefore, how to improve the extraction efficiency and quality of bovine skin collagen prepared by enzymatic hydrolysis and reduce costs is the key technical barrier currently hindering the industrialization of bovine skin collagen. Summary of the invention

[0008] The present invention aims at the many problems existing in the current enzymatic extraction of cowhide collagen, combines low-temperature freezing and biological fermentation technology, and greatly improves the production efficiency of preparing collagen by enzymatic hydrolysis of cowhide.

[0009] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: A method for separating and preparing bovine skin collagen comprises the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in a 5-8% sodium chloride solution, slowly stir at room temperature for 1-2 hours to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1-1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 1-2 hours; (4) Primary fermentation: inoculate the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and ferment at a constant temperature of 28-30° C. for 5-10 h; (5) Second fermentation: Add 0.05-0.1% pepsin by weight of the cowhide to the primary fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30°C for 5-10 hours to complete the fermentation process; (6) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.1-0.3% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 4000-5000 r / min for 5-6 minutes to obtain the supernatant; (7) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h, and then dialyze with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0010] Furthermore, the mass concentration of the sodium carbonate solution in step (1) is 6-10%.

[0011] Furthermore, in step (2), the solid-to-liquid ratio of the cowhide to the sodium chloride solution is 1 g:20 ml.

[0012] Furthermore, in step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 1-3% of the weight of the cowhide.

[0013] Furthermore, the preparation method of the Lactobacillus acidophilus fermentation liquid in step (4) is as follows: after activating the Lactobacillus acidophilus strain numbered CGMCC.NO.1.3342, inoculate it on a seed culture medium, and culture it at 28-30°C and a rotation speed of 50-100rpm for 10-12h to obtain a seed liquid; then inoculate the seed liquid into a fermentation medium at an inoculum amount of 2-5%, and the fermentation conditions are: 28-30°C shaking culture, a rotation speed of 50-150r / min, and fermentation for 24h to obtain a Lactobacillus acidophilus fermentation liquid.

[0014] The Lactobacillus acidophilus used in the present invention was purchased from the General Microbiological Center of China Microbiological Culture Collection Administration, with the strain number of CGMCC.NO. 1.3342 and the original preservation time being November 26, 2002.

[0015] The composition of the seed culture medium is as follows: 15-25 g / L glucose, 8-10 g / L trypsin, 3-5 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L magnesium sulfate, and the balance is water.

[0016] The composition of the fermentation medium is: 15-25 g / L glucose, 10-12 g / L tryptone, 5-8 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, and the balance is water.

[0017] The present invention firstly freezes the cowhide, so that the secondary structure composition of the cowhide collagen is changed, while the spatial network structure, subunit composition and triple helix structure have no obvious changes, thereby reducing the denaturation of the protein and being more conducive to subsequent fermentation and enzymolysis.

[0018] The subsequent two-step fermentation is used to pre-treat the cowhide. In the initial fermentation, the active weak acid produced by specific Lactobacillus acidophilus makes the structure of the cowhide loose, which increases the repulsive force between the original collagen molecules and destroys the ionic bonds and Schiff bonds between the collagen molecules. The large amount of H +Help water molecules enter collagen fibers, so that the protein molecules in the raw materials swell sufficiently, and promote the dissolution of collagen while ensuring the integrity of the collagen structure; the subsequent second step is combined with pepsin for fermentation, so that the collagen is fully dissolved, and the purity and extraction rate of collagen are improved. At the same time, the two-step fermentation treatment of Lactobacillus acidophilus can effectively reduce the odor of cowhide collagen and improve product quality.

[0019] Furthermore, in step (6), the composite protease is a mixture of pepsin and papain in a mass ratio of 1:1.

[0020] Furthermore, the composition of the weak acid extraction medium in step (6) is: 30% acetic acid, 10% citric acid, 10% ferulic acid, 1% chitosan, 0.5% sodium dodecyl sulfate, and the balance is water, calculated by mass percentage.

[0021] Furthermore, in step (6), the solid-liquid ratio of the cowhide to the weak acid extraction medium is 1 g:20 ml.

[0022] Beneficial Effects (1) The present invention freezes the cowhide and performs two-step pre-fermentation treatment to fully swell the cowhide, promote the dissolution of collagen, and improve the extraction rate; (2) Subsequently, an organic weak acid medium and a complex enzyme are used for extraction to fully dissolve the collagen, reduce the content of impurity proteins, and improve the purity and extraction rate of the collagen; (3) The process has a high extraction rate for cowhide protein, and the whole process is extracted by biotechnology, which completely avoids the negative impact of strong acids and alkalis on collagen. The process is safe, environmentally friendly and pollution-free. It is also applicable to the extraction of collagen from other raw materials and is worthy of large-scale promotion and application; (4) According to pharmacological experiments, the obtained cowhide collagen helps to enhance immunity and improve bone density, and can be used in the preparation of health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The UV spectra of collagen of Example 3 of the present invention and the standard product are shown; Figure 2 This is a scanning electron microscope image of the sample of Example 3 of the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0025] Example 1, a method for separating and preparing bovine skin collagen, comprising the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in a 5% by mass sodium chloride solution, slowly stir at room temperature for 1 hour to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add sterile water 1 times the weight of the cowhide, stir, and place in a refrigerator at 0℃ for 1 hour; (4) Primary fermentation: inoculate the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and ferment at a constant temperature of 28-30° C. for 5 h; (5) Second fermentation: add pepsin at 0.05% of the weight of the cowhide to the primary fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30°C for 5 hours to complete the fermentation process; (6) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.1% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 4000 r / min for 5-6 minutes to obtain the supernatant; (7) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0026] The mass concentration of the sodium carbonate solution in step (1) is 6%.

[0027] In step (2), the solid-to-liquid ratio of the cowhide to the sodium chloride solution is 1 g:20 ml.

[0028] In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 1% of the weight of the cowhide.

[0029] The preparation method of the Lactobacillus acidophilus fermentation liquid in step (4) is as follows: after activating the Lactobacillus acidophilus strain numbered CGMCC.NO.1.3342, inoculate it on a seed culture medium, and culture it at 28-30°C and a rotation speed of 50-100 rpm for 10 hours to obtain a seed liquid; then inoculate the seed liquid into a fermentation medium at an inoculum amount of 2%, and ferment under the following fermentation conditions: culture on a shaking table at 28-30°C, a rotation speed of 50-150 r / min, and ferment for 24 hours to obtain a Lactobacillus acidophilus fermentation liquid.

[0030] The Lactobacillus acidophilus used in this example was purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration. The strain number is CGMCC.NO. 1.3342 and the preservation date is November 26, 2002.

[0031] The composition of the seed culture medium is: glucose 15-25 g / L, trypsin 8-10 g / L, yeast extract 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.1-0.3 g / L, and the balance is water.

[0032] The composition of the fermentation medium is: 15-25 g / L glucose, 10-12 g / L tryptone, 5-8 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, and the balance is water.

[0033] In step (6), the composite protease is a mixture of pepsin and papain in a mass ratio of 1:1.

[0034] The composition of the weak acid extraction medium in step (6) is: 30% acetic acid, 10% citric acid, 10% ferulic acid, 1% chitosan, 0.5% sodium dodecyl sulfate, and the balance is water.

[0035] In step (6), the solid-liquid ratio of the cowhide to the weak acid extraction medium is 1 g:20 ml.

[0036] Example 2, a method for separating and preparing bovine skin collagen, comprising the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 2 hours; (4) Primary fermentation: inoculate the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and ferment at a constant temperature of 28-30° C. for 8 h; (5) Second fermentation: add pepsin at 0.05% of the weight of the cowhide to the primary fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30°C for 8 hours to complete the fermentation process; (6) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.2% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 5000 r / min for 5-6 minutes to obtain the supernatant; (7) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h, and then dialyze with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0037] The mass concentration of the sodium carbonate solution in step (1) is 8%.

[0038] In step (2), the solid-to-liquid ratio of the cowhide to the sodium chloride solution is 1 g:20 ml.

[0039] In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 2% of the weight of the cowhide.

[0040] The preparation method of the Lactobacillus acidophilus fermentation liquid in step (4) is as follows: after activating the Lactobacillus acidophilus strain numbered CGMCC.NO.1.3342, inoculate it on a seed culture medium, and culture it at 28-30°C and a rotation speed of 50-100 rpm for 12 hours to obtain a seed liquid; then inoculate the seed liquid into a fermentation medium at an inoculum amount of 3%, and ferment under the following conditions: culture on a shaking table at 28-30°C, a rotation speed of 50-150 r / min, and ferment for 24 hours to obtain a Lactobacillus acidophilus fermentation liquid.

[0041] The Lactobacillus acidophilus used in this example was purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration. The strain number is CGMCC.NO. 1.3342 and the preservation date is November 26, 2002.

[0042] The composition of the seed culture medium is: glucose 15-25 g / L, trypsin 8-10 g / L, yeast extract 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.1-0.3 g / L, and the balance is water.

[0043] The composition of the fermentation medium is: 15-25 g / L glucose, 10-12 g / L tryptone, 5-8 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, and the balance is water.

[0044] In step (6), the composite protease is a mixture of pepsin and papain in a mass ratio of 1:1.

[0045] The composition of the weak acid extraction medium in step (6) is: 30% acetic acid, 10% citric acid, 10% ferulic acid, 1% chitosan, 0.5% sodium dodecyl sulfate, and the balance is water.

[0046] In step (6), the solid-liquid ratio of the cowhide to the weak acid extraction medium is 1 g:20 ml.

[0047] Example 3, a method for separating and preparing bovine skin collagen, comprising the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 2 hours; (4) Primary fermentation: inoculate the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and ferment at a constant temperature of 28-30° C. for 10 h; (5) Second fermentation: add 0.1% pepsin by weight of the cowhide to the primary fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30°C for 10 hours to complete the fermentation process; (6) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.3% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 5000 r / min for 5-6 minutes to obtain the supernatant; (7) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0048] The mass concentration of the sodium carbonate solution in step (1) is 10%.

[0049] In step (2), the solid-to-liquid ratio of the cowhide to the sodium chloride solution is 1 g:20 ml.

[0050] In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 3% of the weight of the cowhide.

[0051] The preparation method of the Lactobacillus acidophilus fermentation liquid in step (4) is as follows: after activating the Lactobacillus acidophilus strain numbered CGMCC.NO.1.3342, inoculating it on a seed culture medium, and culturing it at 28-30°C and a rotation speed of 50-100 rpm for 12 hours to obtain a seed liquid; then inoculating the seed liquid into a fermentation medium at an inoculum amount of 5%, and fermenting under the following conditions: culturing on a shaking table at 30-42°C, a rotation speed of 50-150 r / min, and fermenting for 24 hours to obtain a Lactobacillus acidophilus fermentation liquid.

[0052] The Lactobacillus acidophilus used in this example was purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration. The strain number is CGMCC.NO. 1.3342 and the preservation date is November 26, 2002.

[0053] The composition of the seed culture medium is: glucose 15-25 g / L, trypsin 8-10 g / L, yeast extract 3-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.1-0.3 g / L, and the balance is water.

[0054] The composition of the fermentation medium is: 15-25 g / L glucose, 10-12 g / L tryptone, 5-8 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, and the balance is water.

[0055] In step (6), the composite protease is a mixture of pepsin and papain in a mass ratio of 1:1.

[0056] The composition of the weak acid extraction medium in step (6) is: 30% acetic acid, 10% citric acid, 10% ferulic acid, 1% chitosan, 0.5% sodium dodecyl sulfate, and the balance is water.

[0057] In step (6), the solid-liquid ratio of the cowhide to the weak acid extraction medium is 1 g:20 ml.

[0058] Comparative Example 1: In this comparative example, except that the freezing treatment is not performed in step (3), the remaining preparation methods and raw material selections are the same as those in Example 3. That is: A method for separating and preparing bovine skin collagen comprises the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization: Sterilize the cowhide after impurities are removed and add sterile water 1.3 times the weight of the cowhide and stir; (4) Primary fermentation: inoculate the sterilized material in step (3) with Lactobacillus acidophilus fermentation liquid and ferment at a constant temperature of 28-30° C. for 10 h; (5) Second fermentation: add 0.1% pepsin by weight of the cowhide to the primary fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30°C for 10 hours to complete the fermentation process; (6) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.3% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 5000 r / min for 5-6 minutes to obtain the supernatant; (7) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0059] Comparative Example 2: In this comparative example, except that only the first fermentation step is performed, the remaining preparation methods and raw materials are the same as those of Example 3.

[0060] A method for separating and preparing bovine skin collagen comprises the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 2 hours; (4) Fermentation: inoculating the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and fermenting at a constant temperature of 28-30° C. for 20 h to complete the fermentation process; (5) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.3% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 5000 r / min for 5-6 minutes to obtain the supernatant; (6) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0061] In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 3% of the weight of the cowhide.

[0062] Comparative Example 3: In this comparative example, except that only one-step fermentation of Lactobacillus acidophilus and pepsin is performed, that is, no step-by-step pre-fermentation treatment is performed, the rest of the preparation method and raw materials are the same as those of Example 3.

[0063] A method for separating and preparing bovine skin collagen comprises the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 2 hours; (4) Fermentation: Inoculate the material with Lactobacillus acidophilus fermentation liquid, add pepsin at 0.1% of the weight of the cowhide, stir evenly, and ferment at a constant temperature of 28-30°C for 20 hours to complete the fermentation process; (5) Collagen extraction: the fermented cowhide is crushed and placed in a weak acid extraction medium, 0.3% composite protease by weight of the cowhide is added, the pH is adjusted to 6-6.5, and the extraction is carried out for 3 hours; after the extraction is completed, the filtrate is filtered and collected, and the filtrate is centrifuged in a high-speed refrigerated centrifuge at a speed of 5000 r / min for 5-6 minutes to obtain the supernatant; (6) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0064] In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 3% of the weight of the cowhide.

[0065] Comparative Example 4: This comparative example does not undergo any fermentation treatment, and the remaining preparation methods and raw materials are the same as those of Example 3.

[0066] A method for separating and preparing bovine skin collagen comprises the following preparation steps: (1) Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3 mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; (2) Impurity removal: Soak the defatted cowhide in an 8% sodium chloride solution, slowly stir at room temperature for 2 h to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; (3) Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0°C for 2 hours; (4) Collagen extraction: Crush the cowhide and place it in a weak acid extraction medium, add 0.3% composite protease by weight of the cowhide, adjust the pH to 6-6.5, and extract for 3 hours; after the extraction, filter and collect the filtrate, centrifuge the filtrate at 5000 r / min for 5-6 minutes in a high-speed refrigerated centrifuge, and take the supernatant; (5) Add NaCl to the supernatant to a concentration of 0.8 mol / L and perform salting out for 12 h. Centrifuge at 5000 r / min for 3 min. Add 0.5 mol / L acetic acid solution to the precipitate and stir to fully dissolve. After complete dissolution, dialyze with 0.1 mol / L acetic acid for 24 h and then with distilled water for 24 h. Freeze-dry to obtain bovine skin collagen.

[0067] Performance Testing 1. Determination of collagen content Hydroxyproline is a characteristic amino acid of collagen, so the extraction rate of collagen can be investigated by measuring the content of hydroxyproline in collagen.

[0068] The determination of hydroxyproline is in accordance with GB / T9695.23-2008 method.

[0069] The mass fraction of collagen in cowhide = the mass fraction of hydroxyproline × 100%; 2. Calculation of collagen extraction rate Collagen extraction rate (%) = mass fraction of hydroxyproline in solution × volume × dilution factor / mass fraction of hydroxyproline in cowhide × 100 3. Identification of Collagen The sample was placed in a 0.5 mol / L acetic acid solution to prepare a 0.5 mg / mL solution. After diluting 10 times, the sample was tested on a UV-visible spectrophotometer. The collagen solution was scanned in the near-ultraviolet region of 200-400 nm. The results were compared with those of the standard. Figure 1 .

[0070] 4. Scanning Electron Microscope Observation The samples were pressed and fixed on a slide, and then their morphological characteristics were observed using a scanning electron microscope. An accelerating voltage of 10 kV was used and images were collected at a magnification of 200.

[0071] Table 1 Extraction experimental results

[0072] It can be seen from the data in the table that the method of the embodiment of the present invention achieves efficient extraction of cowhide collagen, with an extraction rate of more than 85% and a purity of more than 95%. However, in Comparative Examples 1-4, in which the process steps were changed, the extraction rate showed a significant downward trend because the cowhide was not fully pretreated for fermentation. It can be seen that freezing pretreatment + two-step efficient fermentation are the key to achieving the extraction of cowhide collagen in the present invention. If one of them is missing, the effect will be weak.

[0073] From the UV spectrum ( Figure 1 ) It can also be seen that the maximum ultraviolet absorption wavelength of the collagen extracted in Example 3 of the present invention is at 232nm, which is consistent with the standard collagen, and no absorption peak appears at 280nm, indicating that the content of aromatic amino acids is low and the extracted collagen has a higher purity.

[0074] From the electron microscopy image of Example 3 ( Figure 2 ) It can be seen that under the scanning electron microscope, a large number of porous and loose cross-sections are more clearly displayed, showing an intertwined curly structure, which shows that it has the fiber characteristics of collagen and the prepared collagen is of good quality.

[0075] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A method for separating and preparing cowhide collagen, characterized in that: The method comprises the following preparation steps: Degreasing: Take fresh cowhide, scrape off the subcutaneous fat, cut into small pieces of 3×3mm, soak in sodium carbonate solution to remove residual fat, and wash thoroughly with water; Impurity removal: Soak the defatted cowhide in a 5-8% sodium chloride solution, slowly stir at room temperature for 1-2 hours to remove salt-soluble non-collagen components, and then rinse repeatedly with clean water; Sterilization and rapid cooling treatment: sterilize the cowhide after impurities are removed, add 1-1.3 times the weight of the cowhide with sterile water, stir, and place in a refrigerator at 0℃ for 1-2 hours; Primary fermentation: inoculate the sterilized and cooled material in step (3) with Lactobacillus acidophilus fermentation liquid, and ferment at a constant temperature of 28-30°C for 5-10 hours; Second fermentation: add 0.05-0.1% pepsin of the cowhide weight to the first fermentation liquid, stir evenly, and ferment at a constant temperature of 28-30℃ for 5-10h to complete the fermentation process; Collagen extraction: crush the fermented cowhide and place it in a weak acid extraction medium, add 0.1-0.3% compound protease by weight of the cowhide, adjust the pH to 6-6.5, and extract for 3 hours; after the extraction, filter and collect the filtrate, centrifuge the filtrate in a high-speed refrigerated centrifuge at a speed of 4000-5000r / min for 5-6 minutes, and take the supernatant; NaCl was added to the supernatant to a concentration of 0.8 mol / L, and then salted out for 12 h, centrifuged at 5000 r / min for 3 min, 0.5 mol / L acetic acid solution was added to the precipitate, and stirred to fully dissolve; after complete dissolution, dialyzed with 0.1 mol / L acetic acid for 24 h, and then dialyzed with distilled water for 24 h, and freeze-dried to obtain bovine skin collagen.

2. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: The mass concentration of the sodium carbonate solution in step (1) is 6-10%.

3. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: In step (2), the solid-to-liquid ratio of the cowhide to the sodium chloride solution is 1 g:20 ml.

4. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: In step (4), the amount of Lactobacillus acidophilus fermentation liquid added is 1-3% of the weight of the cowhide.

5. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: The preparation method of the Lactobacillus acidophilus fermentation liquid in step (4) is as follows: after activating the Lactobacillus acidophilus strain numbered CGMCC.NO.1.3342, inoculate it on a seed culture medium, and culture it at 28-30°C and a rotation speed of 50-100 rpm for 10-12 hours to obtain a seed liquid; then inoculate the seed liquid into a fermentation medium at an inoculum amount of 2-5%, and ferment under the following conditions: culture on a shaking table at 28-30°C, a rotation speed of 50-150 r / min, and ferment for 24 hours to obtain a Lactobacillus acidophilus fermentation liquid.

6. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: In step (6), the composite protease is a mixture of pepsin and papain in a mass ratio of 1:

1.

7. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: The composition of the weak acid extraction medium in step (6) is: 30% acetic acid, 10% citric acid, 10% ferulic acid, 1% chitosan, 0.5% sodium dodecyl sulfate, and the balance is water, calculated by mass percentage.

8. The method for separating and preparing cowhide collagen according to claim 1, characterized in that: In step (6), the solid-liquid ratio of the cowhide to the weak acid extraction medium is 1 g:20 ml.

9. Application of cowhide collagen in the preparation of products for enhancing immunity and improving bone density, characterized in that: The bovine skin collagen is prepared by the method described in any one of claims 1-8.

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