Method for separating bovine serum albumin

Abstract

The invention provides a method for separating bovine serum albumin, and relates to the technical field of bovine serum albumin preparation. The method for separating bovine serum albumin mainly comprises the following steps: centrifuging a mixed anticoagulant, preparing a stabilizer from normal saline and a small amount of nicotinic acid, hirudin, sodium myristate and sodium alkyl benzene sulfonate, diluting the serum mixed stabilizer, repeatedly freezing and thawing with liquid nitrogen, adjusting the pH value with hydrochloric acid, adding salt for precipitation, dialyzing and the like. According to the method, the defects in the prior art are overcome, the extraction yield of the bovine serum albumin can be effectively improved through a simple treatment process, the purity of the bovine serum albumin is ensured, and the economic benefit of production is comprehensively improved.

Description

Technical Field

[0001] The invention relates to the technical field of bovine serum albumin preparation, and in particular to a method for separating bovine serum albumin. Background Art

[0002] Bovine serum albumin (BSA) is a globulin in bovine serum with many important biological functions and wide applications. Bovine serum albumin has a highly conservative structure, high stability, and is not easily affected by external factors.

[0003] The extraction of bovine serum albumin usually starts from bovine serum and is purified through multiple steps such as salting out, chromatography (such as hydrophobic chromatography, ion exchange chromatography, molecular sieve chromatography, etc.). During the extraction process, it is necessary to pay attention to controlling conditions such as temperature and pH value to ensure the stability and activity of bovine serum albumin; the overall processing cost is high, and the overall yield is also low, making it difficult to achieve large-scale production and preparation of bovine serum albumin. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for separating bovine serum albumin, which effectively improves the extraction rate of bovine serum albumin, ensures the purity of bovine serum albumin, and comprehensively improves the economic benefits of production.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0006] A method for separating bovine serum albumin, the method comprising the following steps:

[0007] S1. Take fresh bovine blood, mix with 3%-5% anticoagulant by weight of the bovine blood, place in an ice-water bath, stir evenly, let stand to degas, centrifuge, take the supernatant and place in an ice-water bath for standby use;

[0008] S2. Preparation of stabilizer: using physiological saline as solvent, adding 6-8 g / L nicotinic acid, 10-12 g / L hirudin, 30-60 g / L sodium myristic acid, and 10-20 g / L sodium alkylbenzene sulfonate as stabilizer for later use;

[0009] S3, mixing the above supernatant with a stabilizer, repeatedly freezing and thawing for 3-5 times, and then standing for 50-100 minutes to obtain a pre-treated solution for use;

[0010] S4, adjusting the pH of the pretreatment solution to 5.1-5.2 with hydrochloric acid, treating it in a water bath at 65-75°C for 60-100 minutes, filtering it, and collecting the filtrate for later use;

[0011] S5, adding 3%-5% of the total mass of ammonium sulfate to the filtrate, letting it stand for 40-50 minutes, and then taking the precipitate for later use;

[0012] S6. The precipitate is mixed with sterile deionized water for dialyzing, and then the dialysate is freeze-dried to obtain freeze-dried bovine serum albumin.

[0013] Preferably, the anticoagulant in step S1 is any one or more combinations of potassium oxalate, sodium fluoride, heparin, diethylaminetetraacetic acid sodium salt, sodium citrate, and the added concentration is 8%-10%.

[0014] Preferably, the centrifugal speed in step S1 is 8000-10000 r / min, and the centrifugal time is 10-15 min.

[0015] Preferably, in step S3, the mass ratio of the supernatant to the stabilizer is 1-2:1.

[0016] Preferably, the freeze-thaw method in step S3 is to freeze with liquid nitrogen and then thaw in a 30° C. water bath.

[0017] Preferably, the concentration of hydrochloric acid used in step S4 is 1 mol / L.

[0018] Preferably, the step S5 is performed by standing in an ice water bath.

[0019] Preferably, the method of mixing the precipitate with sterile deionized water in step S6 is to add 5-10 times the volume of sterile deionized water to the precipitate in an ice water bath, adjust the pH to 8.0-9.0, and fully dissolve and mix.

[0020] Preferably, the dialysis method in step S6 is to dialyze the solution obtained by mixing the precipitate with sterile deionized water in sterile deionized water for 10-16 hours.

[0021] The present invention provides a method for separating bovine serum albumin, which has the following advantages compared with the prior art:

[0022] The overall process flow of the invention is short and easy to operate. Nicotinic acid, hirudin, sodium myristate and sodium alkylbenzene sulfonate are used as a composite stabilizer. After multiple freeze-thaw treatments and combined with a subsequent salting-out method, the yield of bovine serum albumin is effectively improved, and the purity of the bovine serum albumin is guaranteed. At the same time, the extraction time is reduced, the production efficiency is comprehensively improved, and large-scale factory production can be achieved. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Embodiment 1:

[0025] Method for separation of bovine serum albumin:

[0026] S1. Take fresh bovine blood, mix it with 10% potassium oxalate solution (3% by mass of bovine blood), stir it evenly in an ice-water bath, let it stand to degas, centrifuge it at 8000r / min for 15min, take the supernatant and let it stand in an ice-water bath for later use;

[0027] S2. Preparation of stabilizer: using physiological saline as solvent, adding 6 g / L nicotinic acid, 10 g / L hirudin, 30 g / L sodium myristic acid, and 10 g / L sodium alkylbenzene sulfonate as stabilizer for later use;

[0028] S3, the supernatant and the stabilizer are mixed in a mass ratio of 1:1, frozen in liquid nitrogen and then thawed in a 30°C water bath, the cold-melting operation is repeated 3 times, and then allowed to stand for 100 minutes to obtain a pre-treated solution for use;

[0029] S4, using 1 mol / L hydrochloric acid solution to adjust the pH of the pre-treated solution to a range of 5.1-5.2, then treating it in a water bath at 65°C for 100 min, filtering it, and collecting the filtrate for later use;

[0030] S5, adding 3% ammonium sulfate of the total mass of the filtrate to the filtrate, letting it stand for 40 minutes, and then taking the precipitate for later use;

[0031] S6. Add 5 times the volume of sterile deionized water to the precipitate in an ice-water bath, adjust the pH to 8.0-9.0 with 1 mol / L hydrochloric acid solution, fully dissolve and mix, and dialyze in sterile deionized water for 10 hours. Then, freeze-dry the dialysate to obtain freeze-dried bovine serum albumin.

[0032] Embodiment 2:

[0033] Method for separation of bovine serum albumin:

[0034] S1. Take fresh bovine blood, mix it with 8% sodium citrate solution (5% by mass of bovine blood), stir it evenly in an ice-water bath, let it stand to degas, centrifuge it at 10000r / min for 10min, take the supernatant and let it stand in an ice-water bath for later use;

[0035] S2. Preparation of stabilizer: using physiological saline as solvent, adding nicotinic acid 8g / L, hirudin 12g / L, sodium myristate 60g / L, sodium alkylbenzene sulfonate 20g / L as stabilizer;

[0036] S3, the supernatant and the stabilizer are mixed in a mass ratio of 2:1, frozen in liquid nitrogen and then thawed in a 30°C water bath, the cold-melt-thaw operation is repeated 5 times, and then allowed to stand for 50 minutes to obtain a pre-treated solution for use;

[0037] S4, using 1 mol / L hydrochloric acid solution to adjust the pH of the pre-treated solution to a range of 5.1-5.2, then treating it in a water bath at 75°C for 60 minutes, filtering it, and collecting the filtrate for later use;

[0038] S5, adding 5% ammonium sulfate of the total mass of the filtrate to the filtrate, letting it stand for 50 minutes, and then taking the precipitate for later use;

[0039] S6. Add 10 times the volume of sterile deionized water to the precipitate in an ice-water bath, adjust the pH to 8.0-9.0 with 1 mol / L hydrochloric acid solution, fully dissolve and mix, and dialyze in sterile deionized water for 16 hours. Then, freeze-dry the dialysate to obtain freeze-dried bovine serum albumin.

[0040] Comparative Example 1:

[0041] Method for separation of bovine serum albumin:

[0042] S1. Take fresh bovine blood, mix it with 8% sodium citrate solution (5% by mass of bovine blood), stir it evenly in an ice-water bath, let it stand to degas, centrifuge it at 10000r / min for 10min, take the supernatant and let it stand in an ice-water bath for later use;

[0043] S2, the supernatant and physiological saline were mixed in a mass ratio of 2:1, and then 0.7 g of sodium palmitate was added per 100 mL of supernatant, the mixture was mixed and stirred evenly, and then frozen in liquid nitrogen and thawed in a 30°C water bath, and the cold-melting operation was repeated 5 times, and then allowed to stand for 50 minutes to obtain a pre-treated solution for use;

[0044] S3, using 1 mol / L hydrochloric acid solution to adjust the pH of the pre-treated solution to a range of 5.1-5.2, then treating it in a water bath at 75°C for 60 minutes, filtering it, and collecting the filtrate for later use;

[0045] S4, adding 5% ammonium sulfate of the total mass of the filtrate to the filtrate, letting it stand for 50 minutes, and then taking the precipitate for later use;

[0046] S5. Add 10 times the volume of sterile deionized water to the precipitate in an ice-water bath, adjust the pH to 8.0-9.0 with 1 mol / L hydrochloric acid solution, fully dissolve and mix, and dialyze in sterile deionized water for 16 hours. Then, freeze-dry the dialysate to obtain freeze-dried bovine serum albumin.

[0047] Comparative Example 2:

[0048] Method for separation of bovine serum albumin:

[0049] S1. Take fresh bovine blood, mix it with 8% sodium citrate solution (5% by mass of bovine blood), stir it evenly in an ice-water bath, let it stand to degas, centrifuge it at 10000r / min for 10min, take the supernatant and let it stand in an ice-water bath for later use;

[0050] S2. Preparation of stabilizer: using physiological saline as solvent, adding nicotinic acid 8g / L, hirudin 12g / L, sodium myristate 60g / L, sodium alkylbenzene sulfonate 20g / L as stabilizer;

[0051] S3, mixing the above supernatant with the stabilizer in a mass ratio of 2:1, and letting it stand for 50 minutes to obtain a pre-treated solution for use;

[0052] S4, using 1 mol / L hydrochloric acid solution to adjust the pH of the pre-treated solution to a range of 5.1-5.2, then treating it in a water bath at 75°C for 60 minutes, filtering it, and collecting the filtrate for later use;

[0053] S5, adding 5% ammonium sulfate of the total mass of the filtrate to the filtrate, letting it stand for 50 minutes, and then taking the precipitate for later use;

[0054] S6. Add 10 times the volume of sterile deionized water to the precipitate in an ice-water bath, adjust the pH to 8.0-9.0 with 1 mol / L hydrochloric acid solution, fully dissolve and mix, and dialyze in sterile deionized water for 16 hours. Then, freeze-dry the dialysate to obtain freeze-dried bovine serum albumin.

[0055] Comparative Example 3:

[0056] Method for separation of bovine serum albumin:

[0057] S1. Take fresh bovine blood, mix it with 8% sodium citrate solution (5% by mass of bovine blood), stir it evenly in an ice-water bath, let it stand to degas, centrifuge it at 10000r / min for 10min, take the supernatant and let it stand in an ice-water bath for later use;

[0058] S2, the supernatant and physiological saline are mixed in a mass ratio of 2:1, and then 0.7 g of sodium palmitate is added per 100 mL of the supernatant, the mixture is mixed and stirred evenly, and then allowed to stand for 50 minutes to obtain a pre-treated solution for use;

[0059] S3, using 1 mol / L hydrochloric acid solution to adjust the pH of the pre-treated solution to a range of 5.1-5.2, then treating it in a water bath at 75°C for 60 minutes, filtering it, and collecting the filtrate for later use;

[0060] S4, adding 5% ammonium sulfate of the total mass of the filtrate to the filtrate, letting it stand for 50 minutes, and then taking the precipitate for later use;

[0061] S5. Add 10 times the volume of sterile deionized water to the precipitate in an ice-water bath, adjust the pH to 8.0-9.0 with 1 mol / L hydrochloric acid solution, fully dissolve and mix, and dialyze in sterile deionized water for 16 hours. Then, freeze-dry the dialysate to obtain freeze-dried bovine serum albumin.

[0062] Detection:

[0063] The same batch of fresh bovine blood was used to prepare freeze-dried bovine serum albumin according to the methods of Examples 1-2 and Comparative Examples 1-3, and the yield and purity of freeze-dried bovine serum albumin obtained in each group were detected;

[0064] Where: yield = freeze-dried bovine serum albumin mass / total mass of protein in original serum × 100%;

[0065] Purity = total mass of albumin in freeze-dried bovine serum albumin / total mass of freeze-dried bovine serum albumin × 100%.

[0066] The specific results are shown in the following table:

[0067] Group Yield (%) purity(%) Example 1 36.7％ 97.2％ Example 2 36.8％ 96.9％ Comparative Example 1 32.5％ 96.1％ Comparative Example 2 31.4％ 95.5％ Comparative Example 3 27.8％ 95.4％

[0068] It can be seen from the above tests that the methods of Example 1 and Example 2 can effectively improve the yield and purity of bovine serum albumin.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating bovine serum albumin, characterized in that: The method comprises the following steps: S1. Take fresh bovine blood, mix with 3%-5% anticoagulant by weight of the bovine blood, place in an ice-water bath, stir evenly, let stand to degas, centrifuge, take the supernatant and place in an ice-water bath for standby use; S2. Preparation of stabilizer: using physiological saline as solvent, adding 6-8 g / L nicotinic acid, 10-12 g / L hirudin, 30-60 g / L sodium myristic acid, and 10-20 g / L sodium alkylbenzene sulfonate as stabilizer for later use; S3, mixing the above supernatant with a stabilizer, repeatedly freezing and thawing for 3-5 times, and then standing for 50-100 minutes to obtain a pre-treated solution for use; S4, adjusting the pH of the pretreatment solution to 5.1-5.2 with hydrochloric acid, treating it in a water bath at 65-75°C for 60-100 minutes, filtering it, and collecting the filtrate for later use; S5, adding 3%-5% of the total mass of ammonium sulfate to the filtrate, letting it stand for 40-50 minutes, and then taking the precipitate for later use; S6. The precipitate is mixed with sterile deionized water for dialyzing, and then the dialysate is freeze-dried to obtain freeze-dried bovine serum albumin.

2. The method for separating bovine serum albumin according to claim 1, characterized in that: The anticoagulant in step S1 is any one or more combinations of potassium oxalate, sodium fluoride, heparin, diethylaminetetraacetic acid sodium salt, and sodium citrate, and the added concentration is 8%-10%.

3. The method for separating bovine serum albumin according to claim 1, characterized in that: The centrifugal speed in step S1 is 8000-10000 r / min, and the centrifugal time is 10-15 min.

4. The method for separating bovine serum albumin according to claim 1, characterized in that: In step S3, the mass ratio of the supernatant mixed with the stabilizer is 1-2:

1.

5. The method for separating bovine serum albumin according to claim 1, characterized in that: The freeze-thaw method in step S3 is to freeze with liquid nitrogen and then thaw in a 30° C. water bath.

6. The method for separating bovine serum albumin according to claim 1, characterized in that: The concentration of hydrochloric acid used in step S4 is 1 mol / L.

7. The method for separating bovine serum albumin according to claim 1, characterized in that: The step S5 is to allow the mixture to stand in an ice-water bath.

8. The method for separating bovine serum albumin according to claim 1, characterized in that: The method of mixing the precipitate with sterile deionized water in step S6 is to add 5-10 times the volume of sterile deionized water to the precipitate under ice water bath conditions, adjust the pH to 8.0-9.0, and fully dissolve and mix.

9. The method for separating bovine serum albumin according to claim 1, characterized in that: The dialysis method in step S6 is to dialyze the solution of the precipitate mixed with sterile deionized water in sterile deionized water for 10-16 hours.