Collagen extraction process

Abstract

The invention relates to the technical field of protein extraction, and discloses a collagen extraction process which comprises the following steps: S1, pretreatment; s2, degreasing is conducted; s3, performing enzymolysis; and S4, purifying. According to the method, different degreasing fluids are used for degreasing, the degreasing efficiency is improved, and through repeated degreasing of ethyl alcohol and sodium hydroxide, better degreasing can be achieved, and endotoxin can be removed; glucose and diatomite are added, so that solid impurities and part of soluble impurities can be better cleaned; a proper amount of ammonium chloride is added to promote collagen precipitation and increase the recovery rate; the filter element is added for filtering, impurities and bacteria can be better removed, the final product meets the medical requirement, the protein purity is high, and the endotoxin is smaller than 0.5.

Description

Technical Field

[0001] The present invention relates to the technical field of protein extraction, and specifically to a collagen extraction process. Background Art

[0002] At present, the collagen extraction process is relatively mature. However, due to the similar physical and chemical properties of fat and protein, both being high-nutrient substances, and the instability of protein, the currently extracted finished products have problems such as a relatively high fat content and serious endotoxin over-standard, and thus need to be improved urgently. Summary of the Invention

[0003] The technical problem to be solved by the present invention is that the currently extracted collagen finished products by the existing collagen extraction process have a relatively high fat content and serious endotoxin over-standard.

[0004] To solve the above problems, the technical solution of the present invention is a collagen extraction process, including the following steps:

[0005] S1, Pretreatment: Thaw the pigskin and remove fat and impurities at low temperature;

[0006] S2, Degreasing:

[0007] S21, Put the pretreated pigskin into the first degreasing solution for degreasing, soak for 3h, and then wash three times with physiological saline;

[0008] S22, Freeze and cut into pieces at low temperature;

[0009] S23, Put the cut pigskin into the second degreasing solution for degreasing, soak for 5h, and then wash with physiological saline;

[0010] S24, Remove miscellaneous proteins;

[0011] S25, Put the pigskin after removing miscellaneous proteins into the third degreasing solution and soak for 3h, then wash with pure water, dry, and freeze at low temperature;

[0012] S3, Enzymatic hydrolysis:

[0013] S31, Take 1 Kg of degreased pigskin, add 1 L of 0.5 moL / L glacial acetic acid, and crush at low temperature;

[0014] S32, Take the pigskin treated in S31 and add glacial acetic acid again to 10%, soak for 3h, add 5% pepsin, and stir for enzymatic hydrolysis for 72h;

[0015] S33, Add 5 L of 0.5 mol / L glacial acetic acid for dilution, filter, and rinse the filter residue twice with 5 L of glacial acetic acid;

[0016] S34, Add 0.5% diatomaceous earth and 0.1% glucose, mix and filter to obtain a filtrate;

[0017] S4, Purification:

[0018] S41. Add the filtrate obtained in S34 to 2 L of 20% ammonium chloride solution, add sodium chloride to 0.5 - 1 moL / L, and perform salting - out for 12 h;

[0019] S42. Discard the supernatant, add 20 L of 0.5 mol / L low - temperature glacial acetic acid, and dissolve;

[0020] S43. Filter the dissolved solution through filter cartridges with pore sizes of 5 microns, 1 micron, and 0.45 microns respectively;

[0021] S44. Add sodium chloride to the filtrate to 0.5 - 1 moL / L, and perform salting - out for 12 h;

[0022] S45. Dissolve the solid with 0.5 mol / L glacial acetic acid and perform dialysis;

[0023] S46. After dissolution and dialysis, add sodium hydroxide solution, adjust the pH to 7, and ultra - filter and concentrate to obtain collagen.

[0024] Furthermore, in step S1:

[0025] The first degreasing solution is a mixture of 75% ethanol and chloroform, and the ratio of 75% ethanol to chloroform is 3:1;

[0026] The second degreasing solution is an aqueous solution containing 0.9% sodium chloride and 8% sodium carbonate;

[0027] The third degreasing solution is 75% ethanol.

[0028] Furthermore, in step S21, the weight ratio of pigskin to the first degreasing solution is 1:2.

[0029] Furthermore, the method for removing miscellaneous proteins is to soak the pigskin obtained in step S23 in an aqueous solution containing 2% sodium hydroxide and 2% sodium carbonate for 5 h, and then wash it with physiological saline to obtain pigskin with miscellaneous proteins removed.

[0030] The advantages of the present invention compared with the existing technology are as follows: By using different degreasing solutions for degreasing, the degreasing efficiency is increased. Degreasing and endotoxin removal can be better achieved through multiple degreasings with ethanol and sodium hydroxide; adding glucose and diatomaceous earth can better clean solid impurities and some soluble impurities; adding an appropriate amount of ammonium chloride can promote the precipitation of collagen and increase the recovery rate; increasing filter cartridge filtration can better remove impurities and bacteria. The final product meets medical requirements, with a high protein purity and an endotoxin content less than 0.5. Brief Description of the Drawings

[0031] Figure 1 is the flow chart of the present invention. Detailed Embodiments

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] A collagen extraction process includes the following steps:

[0034] S1. Pretreatment: Thaw the pigskin and remove fat and impurities at low temperature;

[0035] S2. Degreasing:

[0036] S21. Put the pretreated pigskin into the first degreasing solution for degreasing, soak for 3 h, and then wash three times with physiological saline. The first degreasing solution is a mixed solution of 75% ethanol and chloroform, and the ratio of 75% ethanol to chloroform is 3:1. The weight ratio of pigskin to the first degreasing solution is 1:2;

[0037] S22. Freeze and cut into small pieces at low temperature;

[0038] S23. Put the cut pigskin into the second degreasing solution for degreasing, soak for 5 h, and then wash with physiological saline. The second degreasing solution is an aqueous solution containing 0.9% sodium chloride and 8% sodium carbonate;

[0039] S24. Soak the pigskin obtained in step S23 in an aqueous solution containing 2% sodium hydroxide and 2% sodium carbonate for 5 h, and then wash with physiological saline to obtain pigskin with removed miscellaneous proteins;

[0040] S25. Put the pigskin with removed miscellaneous proteins into the third degreasing solution and soak for 3 h, then wash with pure water, dry, and freeze at low temperature. The third degreasing solution is 75% ethanol;

[0041] S3. Enzymatic hydrolysis:

[0042] S31. Take 1 kg of degreased pigskin, add 1 L of 0.5 moL / L glacial acetic acid, and break at low temperature;

[0043] S32. Take the pigskin treated in S31 and add glacial acetic acid again to 10%, soak for 3 h, add 5% pepsin, and stir for enzymatic hydrolysis for 72 h;

[0044] S33. Add 5 L of 0.5 mol / L glacial acetic acid for dilution, filter, and rinse the filter residue twice with 5 L of glacial acetic acid;

[0045] S34. Add 0.5% diatomaceous earth and 0.1% glucose, mix and filter to obtain a filtrate;

[0046] S4. Purification:

[0047] S41. Add the filtrate obtained in S34 to 2 L of 20% ammonium chloride solution, add sodium chloride to 0.5 - 1 moL / L, and perform salting - out for 12 h;

[0048] S42. Discard the supernatant, add 20 L of 0.5 mol / L low - temperature glacial acetic acid, and dissolve;

[0049] S43. Filter the dissolved solution through filter cartridges with pore sizes of 5 microns, 1 micron, and 0.45 microns respectively;

[0050] S44. Add sodium chloride to the filtrate to 0.5 - 1 moL / L, and perform salting - out for 12 h;

[0051] S45. Dissolve the solid with 0.5 mol / L glacial acetic acid and perform dialysis with a molecular weight cut - off of 10 Kda;

[0052] S46. After dissolution and dialysis, add sodium hydroxide solution and adjust the pH to 7, then ultra - filter and concentrate to obtain collagen.

[0053] Degreasing stage: Use different degreasing agents for degreasing to increase the degreasing efficiency; Pre - clean with 75% ethanol and chloroform, which can reduce the risk of introducing impurities during subsequent crushing; Add sodium carbonate to the second degreasing agent, and sodium carbonate can be used as a swelling agent to promote the penetration ability of the reagent and increase the degreasing effect; Degrease multiple times with ethanol and sodium hydroxide to better degrease and remove endotoxins.

[0054] Enzymatic hydrolysis and purification stage: Add glucose and diatomaceous earth. Glucose is used as a flocculant and diatomaceous earth is used as a filter aid to better remove solid impurities and some soluble impurities; Add an appropriate amount of ammonium chloride to promote the precipitation of collagen and increase the recovery rate; Increase filter cartridge filtration to better remove impurities and bacteria.

[0055] The parts not disclosed in the present invention are all prior arts, and their specific structures and working principles will not be elaborated herein.

[0056] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non - exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0057] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0058] The above description of the present invention and its embodiments is not restrictive. If those of ordinary skill in the art are inspired by it and, without departing from the purpose of the present invention, design similar structural forms and embodiments to this technical solution without creative efforts, they shall fall within the protection scope of the present invention.

Claims

1. A collagen extraction process, characterized in that: The steps include: S1. Pretreatment: thawing the pig skin and removing fat and impurities at low temperature; S2, degreasing: S21, taking the pretreated pigskin and putting it into the first degreasing solution for degreasing, soaking for 3 hours, and then washing it three times with physiological saline; S22, frozen, cut into pieces at low temperature; S23, putting the shredded pigskin into the second degreasing solution for degreasing, soaking for 5 hours, and then washing with physiological saline; S24, remove foreign proteins; S25, soaking the pigskin after removing foreign proteins in the third degreasing liquid for 3 hours, then washing with pure water, drying, and freezing at low temperature; S3, Enzymatic hydrolysis: S31, take 1 kg of defatted pig skin, add 1 L of 0.5 mol / L glacial acetic acid, and crush at low temperature; S32, take the pigskin treated in S31, add glacial acetic acid to 10% again, soak for 3 hours, add pepsin 5%, stir and enzymolyze for 72 hours; S33, add 5L of 0.5mol / L glacial acetic acid to dilute, filter, and rinse the filter residue twice with 5L of glacial acetic acid; S34, adding 0.5% diatomaceous earth and 0.1% glucose, mixing and filtering to obtain a filtrate; S4, purification: S41, add 2L of 20% ammonium chloride solution to the filtrate obtained in S34, add sodium chloride to 0.5-1 mol / L, and salt out for 12 hours; S42, discard the supernatant, add 20L of 0.5mol / L low-temperature glacial acetic acid to dissolve; S43, the dissolved liquid was filtered using 5 micron, 1 micron, and 0.45 micron filter elements respectively; S44, add sodium chloride to the filtrate to 0.5-1 mol / L, and salt out for 12 hours; S45, the solid was dissolved and dialyzed with 0.5 mol / L glacial acetic acid; S46. After dissolution and dialysis, sodium hydroxide solution is added to adjust the pH to 7, and the collagen is concentrated by ultrafiltration.

2. A collagen extraction process according to claim 1, characterized in that: In step S1: The first degreasing solution is a mixture of 75% ethanol and chloroform, and the ratio of 75% ethanol to chloroform is 3:1; The second degreasing liquid is an aqueous solution containing 0.9% sodium chloride and 8% sodium carbonate; The third degreasing liquid is 75% ethanol.

3. A collagen extraction process according to claim 2, characterized in that: In step S21, the weight ratio of the pigskin to the first degreasing liquid is 1:

2.

4. A collagen extraction process according to claim 1, characterized in that: The method for removing impurities and proteins is to soak the pigskin obtained in step S23 in an aqueous solution containing 2% sodium hydroxide and 2% sodium carbonate for 5 hours, and then wash it with physiological saline to obtain the pigskin with impurities and proteins removed.

5. A collagen extraction process according to claim 1, characterized in that: In step S45, when the dialysis is performed by dissolving with glacial acetic acid, the molecular weight cut-off is 10Kda.

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