Bovine lactoferrin freeze-dried powder and preparation method thereof

Abstract

The invention discloses bovine lactoferrin freeze-dried powder and a preparation method thereof, and belongs to the technical field of protein freeze-drying. The bovine lactoferrin freeze-dried powder is obtained by mixing a bovine lactoferrin solution and freeze-drying auxiliary materials and then freeze-drying the mixture, the bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer solution, and the pH value of the citrate buffer solution is 6-7; the freeze-drying auxiliary materials comprise a surfactant, amino acid, stevioside, ferrous sulfate, skim milk powder and glycerol. On one hand, the stevioside prevents the bovine lactoferrin from gathering in the freeze-drying process, and on the other hand, the stevioside and the ferrous sulfate cooperate with each other to prevent the structure of the bovine lactoferrin from changing in the freeze-drying process. Besides, the freeze-dried bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer solution with the pH value of 6-7, the citrate buffer solution is beneficial to maintaining the stability of the bovine lactoferrin structure, and bovine lactoferrin freeze-dried powder with higher purity is obtained after freeze-drying.

Description

Technical Field

[0001] The invention relates to the technical field of protein freeze-drying, and in particular to a bovine lactoferrin freeze-dried powder and a preparation method thereof. Background Art

[0002] Bovine lactoferrin is a multifunctional protein with the characteristics of super activity and low iron saturation. It has broad-spectrum antibacterial and antiviral effects and can regulate the balance of iron in the body. It also has the functions of regulating the formation of bone marrow cells, promoting cell growth, and regulating the body's immunity, thereby enhancing the body's disease resistance.

[0003] At present, bovine lactoferrin is mainly made into finished products through freeze drying, spray drying and other methods. Spray drying can continuously process materials, with a short drying cycle and a large processing capacity, but the shear stress and drying temperature in the process will make biological products such as proteins unstable and reduce their activity. Freeze drying can ensure that the obtained protein products maintain high quality in terms of shape, color, taste, ingredients, etc. by drying at low temperature and vacuum environment. However, the existing bovine lactoferrin freeze-dried powder is easy to form blocks, the yield is not high enough and the purity of bovine lactoferrin will be lost. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a bovine lactoferrin freeze-dried powder and a preparation method thereof, so as to overcome the problems in the prior art that the bovine lactoferrin freeze-dried powder is easy to form agglomerates, the yield is not high enough and the purity of the bovine lactoferrin is lost.

[0005] In a first aspect, the present invention provides a bovine lactoferrin freeze-dried powder, wherein the bovine lactoferrin freeze-dried powder is obtained by mixing a bovine lactoferrin solution and a freeze-drying auxiliary material and then freeze-drying;

[0006] The bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer solution, wherein the pH value of the citrate buffer solution is 6 to 7;

[0007] Freeze-drying excipients include surfactants, amino acids, steviol glycosides, ferrous sulfate, skimmed milk powder and glycerol.

[0008] Compared with the prior art, the present invention adds freeze-drying auxiliary materials when freeze-drying the bovine lactoferrin solution, and the freeze-drying auxiliary materials contain steviol glycoside and ferrous sulfate. On the one hand, steviol glycoside can increase the steric hindrance between protein molecules, thereby preventing bovine lactoferrin from aggregating during freeze-drying; on the other hand, steviol glycoside and ferrous sulfate cooperate with each other, and can further stabilize the secondary and tertiary structures of bovine lactoferrin during freeze-drying, prevent the structure of bovine lactoferrin from changing during freeze-drying, maintain the activity of bovine lactoferrin, and prepare bovine lactoferrin freeze-dried powder that is not easy to agglomerate and has a high yield. In addition, the freeze-dried bovine lactoferrin solution of the present invention is a mixed solution of bovine lactoferrin and a citrate buffer with a pH value of 6 to 7, wherein the citrate buffer is conducive to maintaining the stability of the bovine lactoferrin structure during freeze-drying, thereby obtaining a higher purity bovine lactoferrin freeze-dried powder after freeze-drying.

[0009] Furthermore, the steviol glycoside is any one or both of rebaudioside M and stevioside; or,

[0010] The added amount of stevioside is 0.03% to 0.05% of the volume of the bovine lactoferrin solution.

[0011] The above technical solution further defines the specific type and amount of steviol glycosides. The inventors found that when the steviol glycoside is selected as any one or both of rebaudioside M and stevioside, the structure of bovine lactoferrin can be further stabilized during freeze-drying. When the amount of steviol glycoside added is 0.03% to 0.05% of the volume of the bovine lactoferrin solution, the protective effect on the structure of bovine lactoferrin is the best.

[0012] Furthermore, the surfactant includes any one or more of polysorbate 20, polysorbate 80 or poloxamer; or, the added amount of the surfactant is 0.02% to 0.05% of the volume of the bovine lactoferrin solution.

[0013] The above technical solution further defines the specific type and amount of surfactant added. The type and amount of surfactant added can reduce the surface tension of bovine lactoferrin and reduce the interaction between protein molecules, thereby preventing bovine lactoferrin from aggregating during freeze-drying. In addition, the surfactant can improve the solubility of bovine lactoferrin after freeze-drying, making it easier to re-dissolve.

[0014] Furthermore, the amino acid includes any one or both of arginine and histidine; or, the amount of the amino acid added is 0.05% to 0.08% of the volume of the bovine lactoferrin solution.

[0015] The above technical solution further limits the specific types and addition amounts of amino acids. The addition of arginine and / or histidine can not only prevent bovine lactoferrin from aggregating during freeze-drying, but also maintain the conformation of bovine lactoferrin and prevent bovine lactoferrin from denaturing due to dehydration. In addition, arginine and / or histidine also have a certain antioxidant effect and prevent oxidative damage to bovine lactoferrin.

[0016] Further, the final concentration of ferrous sulfate after addition is 0.1-0.15 mg / L; or, the amount of skimmed milk powder added is 0.06%-0.1% of the volume of the bovine lactoferrin solution; or, the amount of glycerol added is 0.05%-0.07% of the volume of the bovine lactoferrin solution.

[0017] The above technical scheme further limits the added amounts of ferrous sulfate, skimmed milk powder and glycerol. Within the above added amount range, ferrous sulfate and stevioside act synergistically to further enhance the stability of the bovine lactoferrin structure during freeze-drying; the skimmed milk powder can significantly improve the stability and activity of bovine lactoferrin during freeze-drying; glycerol has good permeability and can quickly penetrate into the surface and interior of the bovine lactoferrin molecule, forming hydrogen bonds to replace the water removed by drying, thereby maintaining the original structure of the bovine lactoferrin.

[0018] In a second aspect, the present invention provides a method for preparing a bovine lactoferrin freeze-dried powder, comprising the following steps:

[0019] (1) desalting and replacing the bovine lactoferrin eluate obtained through one or more steps of chromatography, comprising:

[0020] The bovine lactoferrin eluate is ultrafiltered and concentrated to remove salt and water contained in the bovine lactoferrin eluate, and concentrated to 1 / 5 to 1 / 3 of the original volume to obtain a bovine lactoferrin concentrate.

[0021] An equal volume of citrate buffer is added to the bovine lactoferrin concentrate to obtain a mixed solution, the mixed solution is concentrated by ultrafiltration to 1 / 2 of the volume, and salt and water in the mixed solution are continuously removed to obtain a bovine lactoferrin concentrate, and this step is repeated at least twice until a mixed solution of bovine lactoferrin and citrate buffer is obtained.

[0022] (2) freeze-drying the mixed solution obtained by desalting and replacing in step (1), comprising:

[0023] Add freeze-drying auxiliary materials to the mixed solution of bovine lactoferrin and citrate buffer, stir at a speed of 40-60 rpm for 5-10 minutes to obtain bovine lactoferrin freeze-dried liquid, and freeze-dry the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder.

[0024] Compared with the prior art, the present invention uses citrate buffer as a displacement buffer, and realizes desalting displacement of bovine lactoferrin eluate through ultrafiltration concentration. The bovine lactoferrin eluate obtained through one-step or multi-step chromatography has a high content of salt components such as sodium chloride and phosphate, and direct freeze-drying is not conducive to the storage of bovine lactoferrin freeze-dried powder after freeze-drying, and the bovine lactoferrin freeze-dried powder obtained after freeze-drying will contain more salt impurities. The bovine lactoferrin eluate is concentrated by ultrafiltration, and after desalting displacement of citrate, a bovine lactoferrin mixed solution containing citrate buffer is obtained, so that the salt content in the mixed solution is reduced. In addition, when the bovine lactoferrin solution only contains citrate, it is beneficial to maintain the stability of the bovine lactoferrin structure, so that the obtained bovine lactoferrin freeze-dried powder has higher purity and is easy to store.

[0025] Furthermore, the pore size of the ultrafiltration concentration is 10 to 30 kDa, and the pressure of the ultrafiltration concentration is 0.05 to 0.15 MPa.

[0026] The above technical scheme further limits the pore size and pressure of ultrafiltration concentration. Under the pore size and pressure, the salts in the solution can be passed through and the bovine lactoferrin in the solution can be retained. By adding citrate buffer multiple times and performing ultrafiltration concentration multiple times, the salts in the bovine lactoferrin eluate can be completely replaced by citrate, thereby removing other salts contained in the bovine lactoferrin eluate.

[0027] Furthermore, the purity of bovine lactoferrin in the bovine lactoferrin eluate is greater than 95%; or, the bovine lactoferrin eluate comprises bovine lactoferrin, sodium chloride and phosphate buffer.

[0028] Further, freeze drying includes four stages:

[0029] The first stage: freeze the bovine lactoferrin lyophilized liquid at -60℃~-40℃ for 4~6h, and control the vacuum degree to -0.02~0MPa;

[0030] The second stage: quickly heat up to -30℃~-20℃, maintain for 6~8h, and control the vacuum degree to -0.04~-0.02MPa;

[0031] The third stage: After rapidly heating to -10~0℃, maintain for 3~5h, and control the vacuum degree to -0.06~-0.04MPa;

[0032] The fourth stage: After rapidly heating to 20-25°C, maintain for 5-8 hours and control the vacuum degree at -0.09-0.06MPa.

[0033] The above technical scheme further defines the temperature, time and vacuum degree of each stage of freeze-drying of the bovine lactoferrin lyophilized liquid. The present invention controls the temperature and vacuum degree of each stage of freeze-drying, thereby ensuring that the bovine lactoferrin maintains its original structure and activity during the freeze-drying process while removing water.

[0034] Furthermore, the rapid heating rate is 2-3°C / min. DETAILED DESCRIPTION

[0035] 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.

[0036] In a first aspect, an embodiment of the present invention provides a bovine lactoferrin freeze-dried powder, wherein the bovine lactoferrin freeze-dried powder is obtained by mixing a bovine lactoferrin solution and a freeze-drying auxiliary material and then freeze-drying;

[0037] The bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer solution, wherein the pH value of the citrate buffer solution is 6 to 7;

[0038] Freeze-drying excipients include surfactants, amino acids, steviol glycosides, ferrous sulfate, skimmed milk powder and glycerol.

[0039] In the above technical scheme, the bovine lactoferrin freeze-dried powder is obtained by mixing the bovine lactoferrin solution and the freeze-drying auxiliary materials and then freeze-drying. Among them, the bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer with a pH value of 6 to 7, wherein the citrate buffer is conducive to maintaining the stability of the bovine lactoferrin structure during freeze-drying, thereby obtaining a bovine lactoferrin freeze-dried powder with higher purity and easier to store. In addition, freeze-drying auxiliary materials are also added when the bovine lactoferrin solution is freeze-dried, including surfactants, amino acids, steviol glycosides, ferrous sulfate, skimmed milk powder and glycerol. These ingredients all have the effect of protecting the structure of bovine lactoferrin, and can effectively prevent bovine lactoferrin from aggregating and denaturing during freeze-drying by synergizing with each other during freeze-drying. In particular, since bovine lactoferrin contains iron ions, the present invention adds auxiliary materials ferrous sulfate and steviol glycosides during freeze-drying, and the synergistic effect of the two can effectively prevent oxidation and denaturation caused by iron ions and protect the structure and activity of bovine lactoferrin.

[0040] Further, based on the above embodiment, the steviol glycoside in the embodiment of the present invention can be selected as any one or both of rebaudioside M and stevioside. The amount of steviol glycoside added can be 0.03% to 0.05% of the volume of the bovine lactoferrin solution. For example, the amount added can be 0.03%, 0.04% or 0.05% of the volume of the bovine lactoferrin solution.

[0041] The inventors have found that the rebaudioside M and / or stevioside in the above technical solution has a better protective effect on the structure of bovine lactoferrin during freeze-drying compared with sugars such as sucrose and fructose, or compared with other types of steviol glycosides.

[0042] Further, based on the above embodiment, the surfactant in the embodiment of the present invention includes any one or more of polysorbate 20, polysorbate 80 or poloxamer. The added amount of the surfactant can be 0.02% to 0.05% by volume of the bovine lactoferrin solution. For example, the added amount can be 0.02%, 0.03%, 0.04% or 0.05% by volume of the bovine lactoferrin solution, preferably 0.03% to 0.04% by volume of the bovine lactoferrin solution.

[0043] The above-mentioned specific type of surfactant can not only make bovine lactoferrin less likely to aggregate during freeze-drying, but also effectively improve the resolubility of bovine lactoferrin freeze-dried powder.

[0044] Further, based on the above embodiment, the amino acid in the embodiment of the present invention includes any one or two of arginine and histidine. The amount of amino acid added can be 0.05% to 0.08% of the volume of the bovine lactoferrin solution. For example, the amount added can be 0.05%, 0.06%, 0.07% or 0.08% of the volume of the bovine lactoferrin solution, preferably 0.06% to 0.07% of the volume of the bovine lactoferrin solution.

[0045] The arginine and / or histidine in the above technical solution can help bovine lactoferrin maintain its structure and functional activity during the freeze-drying process, thereby improving the yield of bovine lactoferrin after freeze-drying.

[0046] Further, based on the above embodiment, the final concentration of ferrous sulfate after addition in the embodiment of the present invention is 0.1-0.15 mg / L. For example, the final concentration can be 0.1 mg / L, 0.11 mg / L, 0.12 mg / L, 0.13 mg / L, 0.14 mg / L or 0.15 mg / L, preferably 0.12-0.14 mg / L.

[0047] The ferrous sulfate in the above technical solution can help maintain the stable state of the bovine lactoferrin solution. During freeze-drying, the ferrous sulfate and stevioside act synergistically to further protect the structural stability of the bovine lactoferrin.

[0048] Further, based on the above embodiment, the amount of skim milk powder added in the embodiment of the present invention is 0.06% to 0.1% by volume of the bovine lactoferrin solution. For example, the amount added can be 0.06%, 0.07%, 0.08%, 0.09% or 0.1% by volume of the bovine lactoferrin solution, preferably 0.07% to 0.09% by volume of the bovine lactoferrin solution. The skim milk powder is further preferably skim milk powder.

[0049] The above technical solution adds an appropriate amount of skimmed milk powder during the freeze-drying process, which can not only provide protection for bovine lactoferrin, but also play a homogenizing role, ensuring the structural stability and uniform distribution of bovine lactoferrin after freeze-drying.

[0050] Further, based on the above embodiment, the amount of glycerol added in the embodiment of the present invention is 0.05% to 0.07% by volume of the bovine lactoferrin solution. For example, the amount added can be 0.05%, 0.06% or 0.07% by volume of the bovine lactoferrin solution.

[0051] The glycerol in the above technical solution increases the viscosity of the bovine lactoferrin solution by interacting with water and other solute molecules, reduces the formation of ice crystals during freeze-drying, and promotes the bovine lactoferrin solution to form a glassy (i.e., amorphous) solid during the drying stage. This glassy state is similar to a rigid protective cage that can "freeze" the protein molecules in their original position and conformation, reduce intermolecular interactions and movements, and thus protect the protein structure from destruction.

[0052] In a second aspect, the present invention provides a method for preparing a lyophilized bovine lactoferrin powder, comprising the following steps:

[0053] S1. Desalting and replacing the bovine lactoferrin eluate obtained by one or more steps of chromatography, comprising:

[0054] S11, ultrafiltration and concentration of the bovine lactoferrin eluate to remove salt and water contained in the bovine lactoferrin eluate, and concentrate the eluate to 1 / 5 to 1 / 3 of the original volume to obtain a bovine lactoferrin concentrate.

[0055] The chromatography in the above steps can be heparin affinity chromatography, and the purity of bovine lactoferrin in the bovine lactoferrin eluate is greater than 95%. The components of the bovine lactoferrin eluate include bovine lactoferrin, sodium chloride and phosphate buffer, etc. Among them, the phosphate buffer can be: disodium hydrogen phosphate and sodium dihydrogen phosphate buffer, or dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer.

[0056] In the above step, the bovine lactoferrin eluate is ultrafiltered and concentrated to 1 / 5 to 1 / 3 of the original volume. On the one hand, a part of the salt components in the bovine lactoferrin eluate can be removed first, and on the other hand, the amount of citrate buffer used in the subsequent steps can be reduced, and a smaller volume of citrate buffer can be used for replacement.

[0057] S12, adding an equal volume of citrate buffer to the bovine lactoferrin concentrate to obtain a mixed solution, concentrating the mixed solution by ultrafiltration to 1 / 2 of the volume, continuing to remove salt and water in the mixed solution to obtain a bovine lactoferrin concentrate, repeating this step at least twice until a mixed solution of bovine lactoferrin and citrate buffer is obtained. Wherein, the citrate buffer may be composed of sodium citrate and citric acid.

[0058] Furthermore, the pore size of the ultrafiltration concentration is 10 to 30 kDa, and the pressure of the ultrafiltration concentration is 0.05 to 0.15 MPa. Ultrafiltration concentration under this pore size and pressure range can pass through the salts in the solution and retain the bovine lactoferrin in the solution in the concentrated solution. The ions passed through the ultrafiltration concentration are: sodium ions, chloride ions, phosphate ions, and citrate ions. It should be understood that when the phosphate buffer is dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer, potassium ions can also pass through.

[0059] The above steps can gradually replace the salt components such as sodium chloride and phosphate in the mixed solution with citrate by adding equal volumes of citrate buffer solution multiple times and performing ultrafiltration concentration multiple times, thereby obtaining a mixed solution of bovine lactoferrin and citrate buffer solution. This step needs to be repeated at least twice, preferably 4 to 6 times.

[0060] After the desalting and replacement, the sodium chloride, phosphate and other salt components with high content in the bovine lactoferrin eluate can be removed to prevent them from affecting the structure and activity of bovine lactoferrin. In addition, after the desalting and replacement, a mixed solution of bovine lactoferrin and citrate buffer can be obtained, wherein the citrate buffer is conducive to maintaining the stability of the bovine lactoferrin structure, thereby further improving the purity of the obtained bovine lactoferrin freeze-dried powder.

[0061] S2, freeze-drying the mixed solution obtained by desalting and replacing in step S1, comprising:

[0062] Add freeze-drying auxiliary materials to the mixed solution of bovine lactoferrin and citrate buffer, stir at a speed of 40-60 rpm for 5-10 minutes to obtain bovine lactoferrin freeze-dried liquid, and freeze-dry the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder.

[0063] For example, the specific operation can be referred to as follows: pour the bovine lactoferrin freeze-dried liquid into a special freeze-drying plate, maintain the liquid level in the plate at 0.5-1 cm, and then put it into a freeze dryer for freeze drying.

[0064] Furthermore, the freeze drying in the above steps includes four stages:

[0065] The first stage: Freeze the bovine lactoferrin freeze-dried liquid at -60℃~-40℃ for 4~6h, and control the vacuum degree at -0.02~0MPa.

[0066] The first stage can completely freeze the water in the bovine lactoferrin freeze-dried liquid into ice crystals, and make the obtained ice crystals smaller in volume and more evenly distributed, which helps to improve the sublimation efficiency in the subsequent stage and reduce the aggregation and denaturation of bovine lactoferrin.

[0067] The second stage: Rapidly heat up to -30℃~-20℃, maintain for 6~8h, and control the vacuum degree at -0.04~-0.02MPa.

[0068] In the second stage, at a lower temperature, the vacuum degree is increased to promote the sublimation of ice crystals directly from solid to gas, thereby removing most of the water. The lower temperature can reduce the interaction between bovine lactoferrin molecules and prevent aggregation. The appropriate temperature and vacuum degree can also control the drying rate to avoid damage to bovine lactoferrin caused by too fast or too slow drying.

[0069] The third stage: Rapidly heat up to -10~0℃, maintain for 3~5h, and control the vacuum degree to -0.06~-0.04MPa.

[0070] The third stage described above continues to remove residual moisture, especially bound water; the gradually increasing temperature helps to gradually remove moisture while reducing the impact on the structure of bovine lactoferrin; and the higher vacuum degree helps to remove moisture more thoroughly and improve drying efficiency.

[0071] The fourth stage: After rapidly heating to 20-25°C, maintain for 5-8 hours and control the vacuum degree at -0.09-0.06MPa.

[0072] The fourth stage further removes all remaining moisture at a higher temperature to ensure that the product is completely dry; the higher temperature and vacuum degree help to improve the stability of bovine lactoferrin freeze-dried powder during storage and transportation.

[0073] Furthermore, the heating rate of rapid heating during freeze drying is 2-3°C / min, and for example, the heating rate can be 2°C / min or 3°C / min. By controlling the heating rate, the sublimation of water can be accelerated, the drying time can be reduced, and the denaturation and aggregation of bovine lactoferrin caused by local overheating can be avoided, thereby further improving the quality and stability of bovine lactoferrin freeze-dried powder.

[0074] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0075] Example 1

[0076] This embodiment provides a bovine lactoferrin freeze-dried powder, which is prepared according to the following steps:

[0077] S1. Desalting and replacing the bovine lactoferrin eluate obtained by heparin affinity chromatography, comprising:

[0078] S11, ultrafiltration and concentration of bovine lactoferrin eluate under a pressure of 0.1 MPa using an ultrafiltration membrane with a pore size of 10 kDa, removing salt and water contained in the bovine lactoferrin eluate, and concentrating to 1 / 4 of the original volume to obtain a bovine lactoferrin concentrate; wherein the purity of bovine lactoferrin in the bovine lactoferrin eluate is 96%, and the bovine lactoferrin eluate includes bovine lactoferrin, sodium chloride and phosphate buffer, and the phosphate buffer is: disodium hydrogen phosphate and sodium dihydrogen phosphate buffer. The ions that pass through during ultrafiltration and concentration are: sodium ions, chloride ions and phosphate ions.

[0079] S12, add equal volume of citrate buffer with pH value of 6.5 to bovine lactoferrin concentrate to obtain mixed solution, concentrate the mixed solution to 1 / 2 of volume with ultrafiltration membrane of 10kDa pore size under the pressure of 0.1MPa, continue to remove salt and water in the mixed solution to obtain bovine lactoferrin concentrate, repeat this step 5 times to obtain a mixed solution of bovine lactoferrin and citrate buffer. Wherein the components of citrate buffer are sodium citrate and citric acid. The ions that penetrate during ultrafiltration concentration are: sodium ion, chloride ion, phosphate ion and citrate ion.

[0080] S2, freeze-drying the mixed solution obtained by desalting and replacing in step S1, comprising:

[0081] The following lyophilized excipients were added to a mixed solution of 120 mL of bovine lactoferrin and citrate buffer: 0.03% of the mixed solution volume of polysorbate 20, 0.06% of the mixed solution volume of arginine, 0.04% of the mixed solution volume of rebaudioside M, ferrous sulfate with a final concentration of 0.12 mg / L, 0.08% of the mixed solution volume of skim milk powder, and 0.06% of the mixed solution volume of glycerol.

[0082] After the freeze-drying auxiliary materials were added, the mixture was stirred at a speed of 50 rpm for 7 minutes to obtain a bovine lactoferrin freeze-dried liquid. The bovine lactoferrin freeze-dried liquid was poured into a special freeze-drying plate, the liquid level in the plate was maintained at 0.7 cm, and then placed in a freeze dryer for freeze drying to obtain a bovine lactoferrin freeze-dried powder.

[0083] The above freeze drying is divided into four stages:

[0084] The first stage: freeze the bovine lactoferrin lyophilized liquid at -50°C for 5 hours, and control the vacuum degree to -0.01MPa;

[0085] The second stage: quickly raise the temperature to -25°C at a rate of 2°C / min, maintain for 7 hours, and control the vacuum degree to -0.03MPa;

[0086] The third stage: After rapidly heating to -5°C at a rate of 2°C / min, maintain for 4 hours and control the vacuum degree to -0.05MPa;

[0087] The fourth stage: After rapidly heating to 22°C at a rate of 2°C / min, maintain for 6.5h and control the vacuum degree at -0.08MPa.

[0088] Example 2

[0089] This embodiment provides a bovine lactoferrin freeze-dried powder, which is prepared according to the following steps:

[0090] S1. Desalting and replacing the bovine lactoferrin eluate obtained by heparin affinity chromatography, comprising:

[0091] S11, ultrafiltration and concentration of bovine lactoferrin eluate under a pressure of 0.15MPa using an ultrafiltration membrane with a pore size of 20kDa, removing salt and water contained in the bovine lactoferrin eluate, and concentrating to 1 / 5 of the original volume to obtain a bovine lactoferrin concentrate; wherein the purity of bovine lactoferrin in the bovine lactoferrin eluate is 96%, and the components in the bovine lactoferrin eluate include bovine lactoferrin, sodium chloride and phosphate buffer, and the phosphate buffer is: disodium hydrogen phosphate and sodium dihydrogen phosphate buffer. The ions that pass through during ultrafiltration and concentration are: sodium ions, chloride ions and phosphate ions.

[0092] S12, add an equal volume of citrate buffer with a pH value of 6 to the bovine lactoferrin concentrate to obtain a mixed solution, concentrate the mixed solution to 1 / 2 of the volume with an ultrafiltration membrane with a pore size of 20 kDa under a pressure of 0.15 MPa, continue to remove salt and water in the mixed solution to obtain a bovine lactoferrin concentrate, repeat this step 4 times to obtain a mixed solution of bovine lactoferrin and citrate buffer. The components of the citrate buffer are sodium citrate and citric acid. The ions that are permeated during ultrafiltration concentration are: sodium ion, chloride ion, phosphate ion and citrate ion.

[0093] S2, freeze-drying the mixed solution obtained by desalting and replacing in step S1, comprising:

[0094] The following lyophilized excipients were added to a mixed solution of 120 mL of bovine lactoferrin and citrate buffer: 0.02% of the mixed solution volume of poloxamer, 0.05% of the mixed solution volume of histidine, 0.03% of the mixed solution volume of stevioside, ferrous sulfate with a final concentration of 0.1 mg / L, 0.06% of the mixed solution volume of skim milk powder, and 0.05% of the mixed solution volume of glycerol.

[0095] After the freeze-drying auxiliary materials were added, the mixture was stirred at a speed of 40 rpm for 10 min to obtain a freeze-dried bovine lactoferrin solution, which was poured into a freeze-drying tray, the liquid level in the tray was kept at 0.5 cm, and then placed in a freeze dryer for freeze drying to obtain a freeze-dried bovine lactoferrin powder.

[0096] The above freeze drying is divided into four stages:

[0097] The first stage: freeze the bovine lactoferrin lyophilized liquid at -60°C for 4 hours, and control the vacuum degree to -0.02MPa;

[0098] The second stage: quickly raise the temperature to -30°C at a rate of 3°C / min, maintain for 6 hours, and control the vacuum degree to -0.04MPa;

[0099] The third stage: After rapidly heating to -10°C at a rate of 3°C / min, maintain for 3 hours and control the vacuum degree to -0.06MPa;

[0100] The fourth stage: Rapidly heat to 20°C at a rate of 3°C / min, maintain for 5 hours, and control the vacuum degree to -0.09MPa.

[0101] Example 3

[0102] This embodiment provides a bovine lactoferrin freeze-dried powder, which is prepared according to the following steps:

[0103] S1. Desalting and replacing the bovine lactoferrin eluate obtained by heparin affinity chromatography, comprising:

[0104] S11, ultrafiltration and concentration of bovine lactoferrin eluate under a pressure of 0.05MPa using an ultrafiltration membrane with a pore size of 30kDa, removing salt and water contained in the bovine lactoferrin eluate, and concentrating to 1 / 3 of the original volume to obtain a bovine lactoferrin concentrate; wherein the purity of bovine lactoferrin in the bovine lactoferrin eluate is 96%, and the components in the bovine lactoferrin eluate include bovine lactoferrin, sodium chloride and phosphate buffer, and the phosphate buffer is: dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer. The ions that pass through during ultrafiltration and concentration are: sodium ions, chloride ions, phosphate ions and potassium ions.

[0105] S12, add equal volume of citrate buffer with pH value of 7 to bovine lactoferrin concentrate to obtain mixed solution, concentrate the mixed solution to 1 / 2 of volume with ultrafiltration membrane of 30kDa pore size under the pressure of 0.05MPa, continue to remove salt and water in the mixed solution to obtain bovine lactoferrin concentrate, repeat this step 6 times to obtain a mixed solution of bovine lactoferrin and citrate buffer. Wherein the components of citrate buffer are sodium citrate and citric acid. The ions that penetrate during ultrafiltration concentration are: sodium ion, chloride ion, phosphate ion, potassium ion and citrate ion.

[0106] S2, freeze-drying the mixed solution obtained by desalting and replacing in step S1, comprising:

[0107] The following lyophilized excipients were added to a mixed solution of 120 mL of bovine lactoferrin and citrate buffer: 0.05% of the mixed solution volume of polysorbate 80, 0.08% of the mixed solution volume of arginine, 0.05% of the mixed solution volume of rebaudioside M, ferrous sulfate with a final concentration of 0.15 mg / L, 0.1% of the mixed solution volume of skim milk powder, and 0.07% of the mixed solution volume of glycerol.

[0108] After the freeze-drying auxiliary materials were added, the mixture was stirred at a speed of 60 rpm for 5 minutes to obtain a bovine lactoferrin freeze-dried liquid. The bovine lactoferrin freeze-dried liquid was poured into a special freeze-drying plate, the liquid level in the plate was kept at 1 cm, and then placed in a freeze dryer for freeze drying to obtain a bovine lactoferrin freeze-dried powder.

[0109] The above freeze drying is divided into four stages:

[0110] The first stage: freeze the bovine lactoferrin lyophilized liquid at -40°C for 6 hours, and control the vacuum degree to 0 MPa;

[0111] The second stage: quickly raise the temperature to -20°C at a rate of 2°C / min, maintain for 8 hours, and control the vacuum degree to -0.02MPa;

[0112] The third stage: quickly raise the temperature to 0°C at a rate of 2°C / min, maintain for 5 hours, and control the vacuum degree to -0.04MPa;

[0113] The fourth stage: rapidly raise the temperature to 25°C at a rate of 2°C / min, maintain for 8 hours, and control the vacuum degree to -0.06MPa.

[0114] Comparative Example 1

[0115] The bovine lactoferrin freeze-dried powder was prepared according to the method of Example 1, except that the citrate buffer in step S1 was replaced by an equal amount of Tris-HCl buffer.

[0116] Comparative Example 2

[0117] The bovine lactoferrin freeze-dried powder was prepared according to the method of Example 1, except that the freeze-dried auxiliary material rebaudioside M was replaced by sucrose in an equal amount.

[0118] Comparative Example 3

[0119] The bovine lactoferrin freeze-dried powder was prepared according to the method of Example 1, except that the freeze-drying auxiliary material ferrous sulfate was replaced by ammonium sulfate in equal amount.

[0120] Comparative Example 4

[0121] The bovine lactoferrin lyophilized powder was prepared according to the method of Example 1, except that the bovine lactoferrin eluate obtained by heparin affinity chromatography was desalted and replaced in step S1 without using citrate buffer, and lyophilization auxiliary materials were directly added to the bovine lactoferrin eluate before lyophilization.

[0122] Test Example 1

[0123] The purity, yield and morphology of the bovine lactoferrin freeze-dried powder prepared in Examples 1 to 3 and Comparative Examples 1 to 4 were tested, and the results are shown in Table 1.

[0124] Table 1

[0125]

[0126]

[0127] From the above test results, it can be seen that in Comparative Example 1, after the citrate buffer in step S1 is replaced by Tris-HCl buffer in equal amounts, the purity and yield of the bovine lactoferrin freeze-dried powder are both reduced, and there is a little agglomeration, indicating that the citrate buffer is beneficial to maintaining the stability of the bovine lactoferrin structure. In Comparative Example 2, after the freeze-drying auxiliary material rebaudioside M is replaced by sucrose in equal amounts, and in Comparative Example 3, after the ferrous sulfate is replaced by ammonium sulfate in equal amounts, the purity and yield of the bovine lactoferrin freeze-dried powder are both reduced, and a little agglomeration occurs in Comparative Example 3, indicating that stevioside and ferrous sulfate cooperate with each other to further protect the structure and activity of bovine lactoferrin during freeze-drying. Comparative Example 4: In step S1, the bovine lactoferrin eluate obtained by heparin affinity chromatography was desalted and replaced without using citrate buffer. The lyophilization auxiliary material was directly added to the bovine lactoferrin eluate and then lyophilized. The purity and yield of the obtained bovine lactoferrin lyophilized powder were both reduced, and there was a little agglomeration, indicating that if the bovine lactoferrin eluate is not desalted and replaced, the salt components such as sodium chloride and phosphate contained in the bovine lactoferrin eluate will affect the stability of the bovine lactoferrin structure.

[0128] 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 bovine lactoferrin freeze-dried powder, characterized in that: The bovine lactoferrin freeze-dried powder is obtained by mixing a bovine lactoferrin solution and a freeze-drying auxiliary material and then freeze-drying; The bovine lactoferrin solution is a mixed solution of bovine lactoferrin and a citrate buffer solution, wherein the pH value of the citrate buffer solution is 6 to 7; The freeze-drying auxiliary materials include surfactant, amino acid, steviol glycoside, ferrous sulfate, skimmed milk powder and glycerol.

2. The bovine lactoferrin freeze-dried powder according to claim 1, characterized in that The steviol glycoside is any one or two of rebaudioside M and stevioside; or, The added amount of the stevioside is 0.03% to 0.05% of the volume of the bovine lactoferrin solution.

3. The bovine lactoferrin freeze-dried powder according to claim 1, characterized in that The surfactant includes any one or more of polysorbate 20, polysorbate 80 or poloxamer; or, The added amount of the surfactant is 0.02% to 0.05% of the volume of the bovine lactoferrin solution.

4. The bovine lactoferrin freeze-dried powder according to claim 1, characterized in that The amino acids include any one or both of arginine and histidine; or, The amount of the amino acid added is 0.05% to 0.08% of the volume of the bovine lactoferrin solution.

5. The bovine lactoferrin freeze-dried powder according to claim 1, characterized in that The final concentration of the ferrous sulfate after addition is 0.1-0.15 mg / L; or, The amount of skimmed milk powder added is 0.06% to 0.1% of the volume of the bovine lactoferrin solution; or, The added amount of the glycerol is 0.05% to 0.07% of the volume of the bovine lactoferrin solution.

6. A method for preparing bovine lactoferrin freeze-dried powder, characterized in that: The steps include: (1) desalting and replacing the bovine lactoferrin eluate obtained through one or more steps of chromatography, comprising: The bovine lactoferrin eluate is subjected to ultrafiltration and concentration to remove salt and water contained in the bovine lactoferrin eluate, and concentrated to 1 / 5 to 1 / 3 of the original volume to obtain a bovine lactoferrin concentrate; Adding an equal volume of citrate buffer to the bovine lactoferrin concentrate to obtain a mixed solution, concentrating the mixed solution by ultrafiltration to 1 / 2 of the volume, continuing to remove salt and water in the mixed solution to obtain a bovine lactoferrin concentrate, and repeating this step at least twice until a mixed solution of bovine lactoferrin and citrate buffer is obtained; (2) freeze-drying the mixed solution obtained by desalting and replacing in step (1), comprising: Add freeze-drying auxiliary materials to the mixed solution of bovine lactoferrin and citrate buffer, stir at a speed of 40-60 rpm for 5-10 minutes to obtain bovine lactoferrin freeze-dried liquid, and freeze-dry the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder.

7. The method for preparing the bovine lactoferrin freeze-dried powder according to claim 6, characterized in that: The pore size of the ultrafiltration concentration is 10-30 kDa, and the pressure of the ultrafiltration concentration is 0.05-0.15 MPa.

8. The method for preparing the bovine lactoferrin freeze-dried powder according to claim 6, characterized in that: The purity of bovine lactoferrin in the bovine lactoferrin eluate is greater than 95%; or, The bovine lactoferrin eluent comprises bovine lactoferrin, sodium chloride and phosphate buffer.

9. The method for preparing the bovine lactoferrin freeze-dried powder according to claim 6, characterized in that: The freeze drying comprises four stages: The first stage: freezing the bovine lactoferrin lyophilized liquid at -60°C to -40°C for 4 to 6 hours, and controlling the vacuum degree to be -0.02 to 0 MPa; The second stage: quickly heat up to -30℃~-20℃, maintain for 6~8h, and control the vacuum degree to -0.04~-0.02MPa; The third stage: After rapidly heating to -10~0℃, maintain for 3~5h, and control the vacuum degree to -0.06~-0.04MPa; The fourth stage: After rapidly heating to 20-25°C, maintain for 5-8 hours and control the vacuum degree at -0.09-0.06MPa.

10. The method for preparing bovine lactoferrin freeze-dried powder according to claim 9, characterized in that: The rapid heating rate is 2-3°C / min.

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