Method for purifying bovine lactoferrin by using hydrophobic chromatography technology

Abstract

The invention discloses a method for purifying bovine lactoferrin by using a hydrophobic chromatography technology, and belongs to the technical field of protein purification. The method comprises the following steps: centrifuging fermentation liquor containing bovine lactoferrin, collecting thalli, resuspending, homogenizing at high pressure, centrifuging, collecting supernate, carrying out ultrafiltration and concentration, loading a sample into a hydrophobic chromatographic column subjected to column balance, and then washing and eluting the column to obtain purified bovine lactoferrin eluent. By controlling the pH values of the loading solution, the equilibrium buffer solution and the eluent, the high-purity bovine lactoferrin can be obtained from the fermentation liquor, and the cost of the whole purification process is reduced by about 30% compared with that of other chromatographic methods.

Description

Technical Field

[0001] The invention relates to the technical field of protein purification, and in particular to a method for purifying bovine lactoferrin by using a hydrophobic chromatography technique. Background Art

[0002] At present, bovine lactoferrin is mainly obtained from cow milk in China and abroad, which is difficult to separate and has high cost. With the development of gene editing technology, bovine lactoferrin can now be expressed from a variety of genetically engineered bacteria, and there are many studies on separation methods, mainly salting out, ultrafiltration, chromatography, etc.

[0003] The main advantage of the salting-out method and ultrafiltration method is that a large amount of dairy samples can be processed in a short period of time, but the purity of the bovine lactoferrin obtained is relatively low, and it is only suitable for industries such as health products and food additives that do not have high requirements for the purity of bovine lactoferrin.

[0004] Purification of bovine lactoferrin by heparin chromatography or ion chromatography is currently considered to be a relatively good method for purifying bovine lactoferrin, but the filler is expensive and the production cost is high. Based on this, the present invention provides an option for industrial production of bovine lactoferrin. Compared with traditional affinity chromatography, the present invention achieves efficient and highly selective recovery and purification of bovine lactoferrin from Pichia pastoris fermentation broth while reducing costs. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for purifying bovine lactoferrin using hydrophobic chromatography technology, so as to overcome the technical problems of the prior art in purifying bovine lactoferrin, such as high cost and low purity of the obtained bovine lactoferrin.

[0006] In a first aspect, the present invention provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0007] (1) centrifuging the fermentation broth containing bovine lactoferrin, collecting the bacterial cells, and resuspending the bacterial cells in an equilibrium buffer having a pH value of 7.6 to 7.8;

[0008] (2) high-pressure homogenization and centrifugation of the bacterial solution obtained after resuspension, and collecting the supernatant;

[0009] (3) ultrafiltration and concentration of the supernatant to 1 / 5 to 1 / 3 of the original volume to obtain a concentrated solution;

[0010] (4) Column equilibration: Use equilibration buffer with a pH value of 7.6 to 7.8 and 2 to 5 column volumes to equilibrate the hydrophobic chromatography column;

[0011] (5) Loading: The pH value of the concentrated solution obtained in step (3) is adjusted to 7.6 to 7.8 and then loaded onto a hydrophobic chromatography column as a loading solution, and the linear velocity of the loading solution is controlled to be 50 to 100 cm / h; the UV280 curve is observed, and after the peak appears, the effluent of the chromatography column is collected, and after the loading is completed, the collected effluent is repeated with step (5) and loaded again;

[0012] (6) Column flushing: flushing the hydrophobic chromatography column with 2 to 5 column volumes of a flushing solution, wherein the pH value of the flushing solution is 7.6 to 7.8;

[0013] (7) Elution: Elute the bovine lactoferrin on the hydrophobic chromatography column using an eluent at a linear speed of 50 to 100 cm / h; observe the UV280 curve, start collecting the eluent when the main peak of bovine lactoferrin appears, and stop collecting when the baseline drops steadily;

[0014] The eluent is a 10-50 mmol / L phosphate buffer or a 10-50 mmol / L Tris-HCl buffer; the pH value of the eluent is 6-6.4.

[0015] Furthermore, the bacterial cell is Pichia pastoris.

[0016] Furthermore, when the bacterial cells are resuspended in step (1), 5 to 20 ml of equilibration buffer is added for each gram of bacterial cells for resuspending.

[0017] Furthermore, in step (2), the pressure of high pressure homogenization is 1000-1500 bar, the temperature is 2-10° C., and the homogenization is performed 2-5 times.

[0018] Furthermore, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 20-30 min.

[0019] Furthermore, in step (3), the pore size of the ultrafiltration membrane is 20 to 50 kDa, and the ultrafiltration pressure is 0.05 to 0.15 MPa.

[0020] Furthermore, during the step (5) of loading the sample, the temperature of the loading liquid and the effluent liquid is controlled to be 4 to 20°C.

[0021] Furthermore, the balancing buffer and the flushing solution have the same composition, namely: 10-50 mmol / L phosphate buffer or 10-50 mmol / L Tris-HCl buffer, the phosphate buffer or the Tris-HCl buffer contains 1-2 mol / L of an additive, and the additive is any one or more of ammonium sulfate, ammonium chloride, sodium chloride, and imidazole.

[0022] Furthermore, the eluent in step (7) also contains 0 to 0.1 mmol / L of an additive, wherein the additive is any one or more of ammonium sulfate, ammonium chloride, sodium chloride, and imidazole.

[0023] Furthermore, the filler of the hydrophobic chromatography column is a matrix filler containing a hydrophobic group; the matrix filler includes an agarose-based filler, a polyacrylate filler or a silica-based filler; the hydrophobic group includes any one or more of phenyl, butyl, pentyl, ethyl, benzyl, and a long-chain alkyl.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] (1) Compared with the prior art, the present invention adjusts the pH value of the sample solution to 7.6-7.8 when loading, and the pH value of the equilibration buffer used during column equilibration is also 7.6-7.8. By controlling the pH values ​​of the two within the range of 7.6-7.8, the binding between the hydrophobic groups on the bovine lactoferrin in the sample solution and the hydrophobic groups on the hydrophobic chromatography column filler can be made stronger. At the same time, the present invention adjusts the pH value of the eluent to 6-6.4. Within this pH value range, the hydrophobic interaction between bovine lactoferrin and the chromatography medium can be further weakened, making bovine lactoferrin easier to elute. The present invention can obtain high-purity bovine lactoferrin from the fermentation broth by controlling the pH values ​​of the sample solution, the equilibration buffer and the eluent, and the cost of the entire purification process is reduced by about 30% compared with other chromatography methods.

[0026] (2) The present invention defines that the components of the balancing buffer and the flushing solution are the same, and optimizes the salt ion concentrations in the two. The balancing buffer and the flushing solution of the present invention both have relatively high concentrations of salt ions. The high concentration of salt ions in the balancing buffer can, on the one hand, promote the hydrophobic groups on bovine lactoferrin to combine with the hydrophobic groups on the chromatography column when loading, so that more bovine lactoferrin can be bound to the chromatography column; on the other hand, it can also maintain the natural conformation of bovine lactoferrin, ensuring that bovine lactoferrin does not deform or precipitate during chromatography. The high concentration of salt ions in the flushing solution ensures that the flushing will not affect the binding of bovine lactoferrin to the chromatography column, and only other impurities in the chromatography column are flushed away.

[0027] (3) The eluent used in the elution of the present invention is a 10-50 mmol / L phosphate buffer or a 10-50 mmol / L Tris-HCl buffer, which may also contain 0-0.1 mmol / L additives. The eluent has a low concentration of salt ions, which can weaken the hydrophobic interaction between bovine lactoferrin and the hydrophobic chromatography column. It works synergistically with the equilibrium buffer and flushing liquid of a specific salt ion concentration, and cooperates with the hydrophobic chromatography column of a specific hydrophobic group, thereby achieving efficient elution of bovine lactoferrin and obtaining high-quality bovine lactoferrin. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0030] Example 1

[0031] This embodiment provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0032] S1, after centrifuging the Pichia yeast fermentation broth containing bovine lactoferrin at a speed of 7000rpm for 25min, 5g of Pichia yeast cells were collected, and the cells were resuspended using an equilibrium buffer having a pH value of 7.7, and 15ml of an equilibrium buffer was added to each gram of cells. The equilibrium buffer was a phosphate buffer containing 1.5mol / L ammonium sulfate, and the phosphate buffer was a 20mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0033] S2. The resuspended bacterial solution was homogenized three times at 6°C and 1200 bar, centrifuged at 7000 rpm for 25 min, and the supernatant was collected.

[0034] S3. The supernatant is concentrated by ultrafiltration using an ultrafiltration membrane with a pore size of 40 kDa at a pressure of 0.1 MPa to 1 / 4 of the original volume to obtain a concentrated solution.

[0035] S4, column equilibration: 1 mL of a hydrophobic chromatography column (Lanxiao Monomix MC30-HIC Butyl) was equilibrated using an equilibration buffer having a pH value of 7.7 and 4 column volumes, the equilibration buffer being the same as that used in step S1. The linear velocity of the equilibration buffer was controlled to be 50 cm / h.

[0036] S5, loading: adjust the pH value of the concentrated solution obtained in step S3 to 7.7 with sodium hydroxide solution and load it onto the hydrophobic chromatography column as the loading solution, and control the linear velocity of the loading solution to 75 cm / h; observe the UV280 curve, collect the effluent of the chromatography column after the peak appears, and repeat step S5 with the collected effluent for re-loading after the loading is completed. During this period, the temperature of the loading solution and the effluent is controlled to be 15°C.

[0037] S6. Column flushing: flush the hydrophobic chromatography column with 4 column volumes of flushing solution, the pH value of the flushing solution is 7.7, the flushing solution is the same as the equilibration buffer used in step S1, and the linear velocity of the flushing solution is 50 cm / h.

[0038] S7, elution: using an eluent to elute the bovine lactoferrin on the hydrophobic chromatography column, controlling the linear velocity to 75 cm / h; observing the UV280 curve, starting to collect the eluent when the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily.

[0039] The eluent is a phosphate buffer containing 0.05 mmol / L ammonium chloride, the pH value of the eluent is 6.2, and the phosphate buffer in the eluent is a 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0040] Example 2

[0041] This embodiment provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0042] S1, after centrifuging the Pichia yeast fermentation broth containing bovine lactoferrin at a speed of 10000rpm for 20min, 20g of Pichia yeast cells were collected, and the cells were resuspended using an equilibrium buffer having a pH value of 7.6, and 20ml of an equilibrium buffer was added to each gram of cells. The equilibrium buffer was a phosphate buffer containing 1mol / L ammonium chloride, and the phosphate buffer was a 10mmol / L dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[0043] S2. The resuspended bacterial solution was homogenized at 10°C and 1500 bar for 5 times under high pressure, centrifuged at 10000 rpm for 20 min, and the supernatant was collected.

[0044] S3. The supernatant was concentrated by ultrafiltration using an ultrafiltration membrane with a pore size of 20 kDa at a pressure of 0.15 MPa to 1 / 5 of the original volume to obtain a concentrated solution.

[0045] S4, column equilibration: 5 mL of a hydrophobic chromatography column (Lanxiao Monomix MC60-HIC Butyl) was equilibrated using an equilibration buffer having a pH value of 7.6 and 2 column volumes, the equilibration buffer being the same as that used in step S1. The linear velocity of the equilibration buffer was controlled to be 100 cm / h.

[0046] S5, loading: adjust the pH value of the concentrated solution obtained in step S3 to 7.6 with sodium hydroxide solution and load it onto the hydrophobic chromatography column as the loading solution, and control the linear velocity of the loading solution to 50 cm / h; observe the UV280 curve, collect the effluent of the chromatography column after the peak appears, and repeat step S5 with the collected effluent after loading. During this period, control the temperature of the loading solution and the effluent to be 20°C.

[0047] S6. Column flushing: flush the hydrophobic chromatography column with 2 column volumes of flushing solution, the pH value of the flushing solution is 7.6, the flushing solution is the same as the equilibration buffer used in step S1, and the linear velocity of the flushing solution is 100 cm / h.

[0048] S7, elution: using an eluent to elute the bovine lactoferrin on the hydrophobic chromatography column, controlling the linear velocity to 50 cm / h; observing the UV280 curve, starting to collect the eluent when the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily.

[0049] The eluent is a phosphate buffer containing 0.1 mmol / L sodium chloride, the pH value of the eluent is 6, and the phosphate buffer in the eluent is a 10 mmol / L dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[0050] Example 3

[0051] This embodiment provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0052] S1, after centrifuging the Pichia yeast fermentation broth containing bovine lactoferrin at a speed of 5000rpm for 30min, 160g of Pichia yeast cells were collected, and the cells were resuspended in an equilibrium buffer having a pH value of 7.8, and 5ml of the equilibrium buffer was added to each gram of cells. The equilibrium buffer was a Tris-HCl buffer containing 1mol / L sodium chloride, and the concentration of the Tris-HCl buffer was 50mmol / L.

[0053] S2. The resuspended bacterial solution was homogenized twice at 2°C and 1000 bar, centrifuged at 5000 rpm for 30 min, and the supernatant was collected.

[0054] S3. The supernatant was concentrated by ultrafiltration using an ultrafiltration membrane with a pore size of 50 kDa at a pressure of 0.05 MPa to 1 / 3 of the original volume to obtain a concentrated solution.

[0055] S4, column equilibration: 40 mL of a hydrophobic chromatography column (Lanxiao Monomix MC60-HIC Butyl) was equilibrated using an equilibration buffer having a pH value of 7.8 and 5 column volumes, the equilibration buffer being the same as that used in step S1. The linear velocity of the equilibration buffer was controlled to be 80 cm / h.

[0056] S5, loading: adjust the pH value of the concentrated solution obtained in step S3 to 7.8 with sodium hydroxide solution and load it onto the hydrophobic chromatography column as the loading solution, and control the linear velocity of the loading solution to be 100 cm / h; observe the UV280 curve, collect the effluent of the chromatography column after the peak appears, and repeat step S5 with the collected effluent after loading. During this period, control the temperature of the loading solution and the effluent to be 20°C.

[0057] S6. Column flushing: flush the hydrophobic chromatography column with 5 column volumes of flushing solution, the pH value of the flushing solution is 7.8, the flushing solution is the same as the equilibration buffer used in step S1, and the linear velocity of the flushing solution is 80 cm / h.

[0058] S7, elution: using an eluent to elute the bovine lactoferrin on the hydrophobic chromatography column, controlling the linear velocity to 100 cm / h; observing the UV280 curve, starting to collect the eluent when the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily.

[0059] The eluent is a Tris-HCl buffer, the pH value of the eluent is 6.4, and the concentration of the Tris-HCl buffer in the eluent is 50 mmol / L.

[0060] Example 4

[0061] This embodiment provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0062] S1, after centrifuging the Pichia yeast fermentation broth containing bovine lactoferrin at a speed of 7000rpm for 25min, 160g of Pichia yeast cells were collected, and the cells were resuspended using a balance buffer having a pH value of 7.7, and 15ml of a balance buffer was added to each gram of cells. The balance buffer was a phosphate buffer containing additives of ammonium sulfate and imidazole, and the phosphate buffer was a 20mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. The total amount of additives added in the balance buffer was 1mol / L, and the molar ratio of ammonium sulfate to imidazole was 2:1.

[0063] S2. The resuspended bacterial solution was homogenized three times at 6°C and 1200 bar, centrifuged at 7000 rpm for 25 min, and the supernatant was collected.

[0064] S3. The supernatant is concentrated by ultrafiltration using an ultrafiltration membrane with a pore size of 40 kDa at a pressure of 0.1 MPa to 1 / 4 of the original volume to obtain a concentrated solution.

[0065] S4, column equilibration: 40 mL of a hydrophobic chromatography column (Lanxiao Monomix MC30-HIC Butyl) was equilibrated using an equilibration buffer having a pH value of 7.7 and 3 column volumes, the equilibration buffer being the same as that used in step S1. The linear velocity of the equilibration buffer was controlled to be 80 cm / h.

[0066] S5, loading: adjust the pH value of the concentrated solution obtained in step S3 to 7.7 with sodium hydroxide solution and load it onto the hydrophobic chromatography column as the loading solution, and control the linear velocity of the loading solution to 60 cm / h; observe the UV280 curve, collect the effluent of the chromatography column after the peak appears, and repeat step S5 with the collected effluent for re-loading after the loading is completed. During this period, the temperature of the loading solution and the effluent is controlled to be 15°C.

[0067] S6. Column flushing: flush the hydrophobic chromatography column with 3 column volumes of flushing solution, the pH value of the flushing solution is 7.7, the flushing solution is the same as the equilibration buffer used in step S1, and the linear velocity of the flushing solution is 80 cm / h.

[0068] S7, elution: using an eluent to elute the bovine lactoferrin on the hydrophobic chromatography column, controlling the linear velocity to 60 cm / h; observing the UV280 curve, starting to collect the eluent when the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily.

[0069] The eluent is a phosphate buffer containing ammonium sulfate and imidazole as additives, the pH value of the eluent is 6.2, and the phosphate buffer in the eluent is a 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. The total amount of additives added to the eluent is 0.1 mmol / L, wherein the molar ratio of ammonium sulfate to imidazole is 1:1.

[0070] Example 5

[0071] This embodiment provides a method for purifying bovine lactoferrin using hydrophobic chromatography technology, comprising the following steps:

[0072] S1, after centrifuging the Pichia yeast fermentation broth containing bovine lactoferrin at a speed of 8000rpm for 25min, 160g of Pichia yeast cells were collected, and the cells were resuspended using a balance buffer having a pH value of 7.7, and 15ml of a balance buffer was added to each gram of cells. The balance buffer was a phosphate buffer containing additives of sodium chloride and ammonium chloride, and the phosphate buffer was a 20mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. The total amount of the additive added in the balance buffer was 2mol / L, and the molar ratio of sodium chloride to ammonium chloride was 1:1.

[0073] S2. The resuspended bacterial solution was homogenized at 8°C and 1200 bar for 4 times under high pressure, centrifuged at 8000 rpm for 25 min, and the supernatant was collected.

[0074] S3. The supernatant was concentrated by ultrafiltration using an ultrafiltration membrane with a pore size of 30 kDa at a pressure of 0.1 MPa to 1 / 4 of the original volume to obtain a concentrated solution.

[0075] S4, column equilibration: 40 mL of a hydrophobic chromatography column (Lanxiao Monomix MC30-HIC Butyl) was equilibrated using an equilibration buffer having a pH value of 7.7 and 3 column volumes, the equilibration buffer being the same as that used in step S1. The linear velocity of the equilibration buffer was controlled to be 100 cm / h.

[0076] S5, loading: adjust the pH value of the concentrated solution obtained in step S3 to 7.7 with sodium hydroxide solution and load it onto the hydrophobic chromatography column as the loading solution, and control the linear velocity of the loading solution to 90 cm / h; observe the UV280 curve, collect the effluent of the chromatography column after the peak appears, and repeat step S5 with the collected effluent after loading. During this period, control the temperature of the loading solution and the effluent to be 10°C.

[0077] S6. Column flushing: flush the hydrophobic chromatography column with 3 column volumes of flushing solution, the pH value of the flushing solution is 7.7, the flushing solution is the same as the equilibration buffer used in step S1, and the linear velocity of the flushing solution is 100 cm / h.

[0078] S7, elution: using an eluent to elute the bovine lactoferrin on the hydrophobic chromatography column, controlling the linear velocity to 90 cm / h; observing the UV280 curve, starting to collect the eluent when the main peak of bovine lactoferrin appears, and stopping the collection when the baseline drops steadily.

[0079] The eluent is a phosphate buffer containing sodium chloride and ammonium chloride as additives, the pH value of the eluent is 6.2, and the phosphate buffer in the eluent is a 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. The total amount of additives added to the eluent is 0.1 mmol / L, wherein the molar ratio of sodium chloride to ammonium chloride is 2:1.

[0080] Comparative Example 1

[0081] Bovine lactoferrin was purified according to the method of Example 1, except that the pH value of the eluent was set to 7 in step S7.

[0082] Comparative Example 2

[0083] Bovine lactoferrin was purified according to the method of Example 1, except that the pH value of the eluent was set to 5.5 in step S7.

[0084] Comparative Example 3

[0085] Bovine lactoferrin was purified according to the method of Example 1, except that the pH value of the equilibration buffer was set to 8.

[0086] Comparative Example 4

[0087] Bovine lactoferrin was purified according to the method of Example 1, except that the pH value of the equilibration buffer was set to 7.

[0088] Comparative Example 5

[0089] Bovine lactoferrin was purified according to the method of Example 1, except that the concentration of ammonium sulfate in the equilibration buffer was 3 mol / L.

[0090] Comparative Example 6

[0091] Bovine lactoferrin was purified according to the method of Example 1, except that the concentration of ammonium sulfate in the equilibration buffer was 0.5 mol / L.

[0092] Comparative Example 7

[0093] Bovine lactoferrin was purified according to the method of Example 1, except that the concentration of ammonium chloride in the eluent was 0.2 mmol / L.

[0094] Test Case

[0095] The content and purity of bovine lactoferrin in the bovine lactoferrin eluates collected after elution in Examples 1 to 5 and Comparative Examples 1 to 7 were tested. The results are shown in Table 1.

[0096] Table 1

[0097]

[0098] From the above results, it can be seen that after the pH value of the eluent was changed in Comparative Examples 1 and 2, the content and purity of bovine lactoferrin in the bovine lactoferrin eluent were reduced, indicating that the specific pH value of the eluent in the present invention is helpful for eluting bovine lactoferrin. After Comparative Examples 3 and 4 changed the pH value of the equilibration buffer, the content and purity of bovine lactoferrin in the bovine lactoferrin eluent were reduced, indicating that the specific pH value of the equilibration buffer of the present invention is helpful for making bovine lactoferrin better adsorbed on the hydrophobic chromatography column when loading. After Comparative Examples 5 and 6 changed the concentration of the additive in the equilibration buffer, the content and purity of bovine lactoferrin in the bovine lactoferrin eluent were reduced, indicating that the addition of a specific concentration of additives in the equilibration buffer of the present invention is helpful for promoting the binding of bovine lactoferrin to the hydrophobic chromatography column when loading. After Comparative Example 7 changed the concentration of the additive in the eluent, the content and purity of bovine lactoferrin in the bovine lactoferrin eluent were reduced, indicating that the specific concentration of additives in the eluent of the present invention is helpful for the efficient elution of bovine lactoferrin.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for purifying bovine lactoferrin using hydrophobic chromatography, characterized in that: The following steps are involved: (1) centrifuging the fermentation broth containing bovine lactoferrin, collecting the bacterial cells, and resuspending the bacterial cells in an equilibrium buffer having a pH value of 7.6 to 7.8; (2) high-pressure homogenization and centrifugation of the bacterial solution obtained after resuspension, and collecting the supernatant; (3) ultrafiltration and concentration of the supernatant to 1 / 5 to 1 / 3 of the original volume to obtain a concentrated solution; (4) Column equilibration: Use equilibration buffer with a pH value of 7.6 to 7.8 and 2 to 5 column volumes to equilibrate the hydrophobic chromatography column; (5) Loading: The pH value of the concentrated solution obtained in step (3) is adjusted to 7.6 to 7.8 and then loaded onto a hydrophobic chromatography column as a loading solution, and the linear velocity of the loading solution is controlled to be 50 to 100 cm / h; the UV280 curve is observed, and after the peak appears, the effluent of the chromatography column is collected, and after the loading is completed, the collected effluent is repeated with step (5) and loaded again; (6) Column flushing: flushing the hydrophobic chromatography column with 2 to 5 column volumes of a flushing solution, wherein the pH value of the flushing solution is 7.6 to 7.8; (7) Elution: Elute the bovine lactoferrin on the hydrophobic chromatography column using an eluent at a linear speed of 50 to 100 cm / h; observe the UV280 curve, start collecting the eluent when the main peak of bovine lactoferrin appears, and stop collecting when the baseline drops steadily; The eluent is a 10-50 mmol / L phosphate buffer or a 10-50 mmol / L Tris-HCl buffer; the pH value of the eluent is 6-6.

4.

2. The method according to claim 1, characterized in that: The bacterial body is Pichia pastoris.

3. The method according to claim 1, characterized in that When resuspending the bacterial cells in step (1), 5 to 20 ml of equilibration buffer is added for each gram of bacterial cells for resuspending.

4. The method according to claim 1, characterized in that: In step (2), the high pressure homogenization is performed at a pressure of 1000 to 1500 bar, a temperature of 2 to 10° C., and homogenization is performed 2 to 5 times.

5. The method according to claim 1, characterized in that The centrifugal speed is 5000-10000 rpm, and the centrifugal time is 20-30 min.

6. The method according to claim 1, characterized in that In step (3), the pore size of the ultrafiltration membrane is 20-50 kDa, and the ultrafiltration pressure is 0.05-0.15 MPa.

7. The method according to claim 1, characterized in that During the step (5) of loading the sample, the temperature of the loading liquid and the effluent liquid is controlled to be 4 to 20°C.

8. The method according to claim 1, characterized in that The balancing buffer and the flushing solution have the same composition, namely: 10-50 mmol / L phosphate buffer or 10-50 mmol / L Tris-HCl buffer, the phosphate buffer or the Tris-HCl buffer contains 1-2 mol / L of an additive, and the additive is any one or more of ammonium sulfate, ammonium chloride, sodium chloride, and imidazole.

9. The method according to claim 1, characterized in that: The eluent in step (7) also contains 0-0.1 mmol / L of an additive, wherein the additive is any one or more of ammonium sulfate, ammonium chloride, sodium chloride, and imidazole.

10. The method according to claim 1, characterized in that The filler of the hydrophobic chromatography column is a matrix filler containing a hydrophobic group; the matrix filler includes an agarose-based filler, a polyacrylate filler or a silica-based filler; the hydrophobic group includes any one or more of phenyl, butyl, pentyl, ethyl, benzyl, and a long-chain alkyl.

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