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 preparation method of the bovine lactoferrin freeze-dried powder comprises the following steps: adding a histidine buffer solution into a bovine lactoferrin solution with the mass concentration of 30-40%, and mixing to obtain a mixed solution; adding a freeze-drying auxiliary material accounting for 0.25-7.0% of the volume of the mixed solution into the mixed solution to obtain a bovine lactoferrin freeze-drying solution; performing spray freezing and freeze drying on the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder; the freeze-drying auxiliary materials comprise a surfactant, amino acid, D-chiro-inositol and calcium chloride. Compared with the traditional inositol, the D-chiral inositol in the freeze-dried auxiliary material can more effectively replace water molecules, maintain the natural conformation of protein and prevent the bovine lactoferrin structure from collapsing. The calcium chloride in the freeze-drying auxiliary material can stabilize the iron binding area of the bovine lactoferrin in the freeze-drying process, and the biological activity of the bovine lactoferrin after freeze-drying is guaranteed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein freeze-drying, in particular to a bovine lactoferrin freeze-dried powder and a preparation method thereof. BACKGROUND

[0002] Bovine lactoferrin (BLF) is a natural protein with multiple physiological activities, which participates in the regulation of immune function, anti-microbial, promotion of iron absorption, promotion of intestinal cell proliferation and differentiation and other important physiological processes in the human body. Its efficacy in enhancing immunity and improving iron deficiency anemia has been widely recognized. Therefore, bovine lactoferrin has a very wide application prospect in the fields of infant formula milk powder, health food and clinical nutrition.

[0003] At present, the preparation of bovine lactoferrin powder mainly adopts two means of spray drying and vacuum freeze drying. However, in the spray drying process, the high temperature is easy to cause damage to the heat-sensitive component bovine lactoferrin, resulting in the decrease of its biological activity and the failure to fully exert its functional characteristics. Although the vacuum freeze drying can better preserve the activity of the protein, this technology has the problems of low production efficiency and high energy consumption. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a bovine lactoferrin freeze-dried powder and a preparation method thereof. The preparation method of the bovine lactoferrin freeze-dried powder has low production cost, short time and can fully preserve the biological activity of bovine lactoferrin.

[0005] In a first aspect, the present application provides a preparation method of a bovine lactoferrin freeze-dried powder, comprising the following steps:

[0006] An equal volume of histidine buffer solution with a pH value of 6.0-6.5 is added to the bovine lactoferrin solution to obtain a mixed solution, wherein the mass concentration of the bovine lactoferrin solution is 30-40%;

[0007] A freeze-drying auxiliary material is added to the mixed solution, and the addition amount of the freeze-drying auxiliary material accounts for 0.25%-7.0% of the volume of the mixed solution to obtain a bovine lactoferrin freeze-drying solution;

[0008] The bovine lactoferrin freeze-drying solution is subjected to spray freezing and freeze-drying to obtain a bovine lactoferrin freeze-dried powder;

[0009] The freeze-drying auxiliary material includes a surfactant, an amino acid, D-chiro-inositol and calcium chloride.

[0010] Compared with the prior art, the application firstly adds an equal volume of histidine buffer into the bovine lactoferrin solution to obtain a mixed solution, the histidine buffer can maintain the pH stability of the solution after the subsequent addition of the freeze-drying adjuvant. Then, the freeze-drying adjuvant is added into the mixed solution to obtain a bovine lactoferrin freeze-drying solution, the surfactant in the freeze-drying adjuvant can prevent the aggregation of the bovine lactoferrin in the freeze-drying process; the amino acid can protect the structure of the bovine lactoferrin in the freeze-drying process to prevent the denaturation of the bovine lactoferrin. Meanwhile, the D-chiral inositol in the freeze-drying adjuvant has a boat conformation, the hydroxyl groups are uniformly distributed, the polarity distribution is more uniform, can be combined with multiple polar sites of the bovine lactoferrin at the same time, can replace the water molecules more effectively, can maintain the natural conformation of the protein and prevent the structure of the bovine lactoferrin from collapsing in the freeze-drying process. The calcium chloride in the freeze-drying adjuvant can stabilize the iron binding region of the bovine lactoferrin in the freeze-drying process, thereby further ensuring the biological activity of the bovine lactoferrin after the freeze-drying. The components in the freeze-drying adjuvant can synergistically improve the structural stability and the biological activity of the bovine lactoferrin freeze-drying powder. Finally, the bovine lactoferrin freeze-drying solution is subjected to spray freezing and freeze-drying to obtain the bovine lactoferrin freeze-drying powder, the low-temperature environment of the spray freeze-drying process can significantly reduce the risk of thermal denaturation of the bovine lactoferrin, and the drying time is less than or equal to 12 hours, and the energy consumption can be reduced by more than 40% compared with the vacuum freeze-drying.

[0011] Further, the volume ratio of the addition amounts of the surfactant, the amino acid and the D-chiral inositol in the freeze-drying adjuvant is (0.2-0.5):(0.3-0.5):(2-5).

[0012] The above technical solution further limits the volume ratio of the addition amounts of the surfactant, the amino acid and the D-chiral inositol, within the above volume ratio range, the surfactant, the amino acid and the D-chiral inositol cooperate with each other to further improve the protection of the structure of the bovine lactoferrin in the freeze-drying process.

[0013] Further, the final concentration of the calcium chloride after addition is 11-33 mg / L.

[0014] The above technical solution further limits the final concentration of the calcium chloride after addition, within the above concentration range, the stabilizing effect of the calcium chloride on the iron binding region of the bovine lactoferrin is optimal, thereby further ensuring the biological activity of the bovine lactoferrin after the freeze-drying.

[0015] Further, the surfactant includes at least one of polysorbate 80 and poloxamer 188.

[0016] The surfactants polysorbate 80 and poloxamer 188 in the above technical solution can all reduce the surface tension, prevent the aggregation of the bovine lactoferrin in the freeze-drying process and are beneficial to the reconstitution of the bovine lactoferrin freeze-drying powder.

[0017] Further, the amino acid includes at least one of arginine, methionine, and proline.

[0018] The amino acid in the technical solution can improve the stability of the bovine lactoferritin, ensure the biological activity of the bovine lactoferritin after freeze-drying, and improve the shelf life of the bovine lactoferritin freeze-dried powder.

[0019] Further, the freeze-drying auxiliary material further includes rebaudioside M2 and trehalose; wherein,

[0020] The rebaudioside M2 is added in an amount of 0.02% to 0.08% of the volume of the mixed solution.

[0021] The trehalose is added in an amount of 4% to 6% of the volume of the mixed solution.

[0022] Compared with other steviol glycosides, the rebaudioside M2 in the technical solution has more hydroxyl sites, which can replace water molecules and form hydrogen bonds with the polar groups on the surface of the protein during the dehydration process, thereby maintaining the three-dimensional structure of the bovine lactoferritin and preventing the aggregation or denaturation of the bovine lactoferritin. Moreover, when the rebaudioside M2 is used in combination with trehalose, the Tg of the system can be further improved, and the risk of denaturation of the bovine lactoferritin during the desorption drying stage can be reduced.

[0023] Further, the preparation method of the bovine lactoferritin solution includes:

[0024] The bovine lactoferritin extract solution with a mass concentration of 5% to 10% is subjected to ultrafiltration desalination, washed with ultrapure water until the conductivity is less than 5 mS / cm, and concentrated to 1 / 8 to 1 / 5 of the original volume to obtain the bovine lactoferritin solution.

[0025] The above technical solution can effectively remove salt impurities in the bovine lactoferritin extract solution through ultrafiltration desalination and ultrapure water washing, thereby improving the purity of the bovine lactoferritin in the solution.

[0026] Further, the molecular weight of the ultrafiltration cutoff is 10 to 30 kDa; and the control pressure during the ultrafiltration process is 0.1 to 0.3 MPa, and the temperature is 8 to 15°C.

[0027] Further, the spray freezing includes: atomizing the bovine lactoferritin freeze-drying solution by using a high-pressure nozzle with a diameter of 0.3 to 0.5 mm, spraying into a cold trap at -60 to -40°C for freezing, the feeding speed is 5 to 10 mL / min, and the atomization pressure is 0.2 to 0.4 MPa.

[0028] The technical solution atomizes the bovine lactoferritin freeze-dried liquid into tiny droplets through the high-pressure nozzle, and sprays it into a low-temperature environment quickly, so that the droplets are quickly frozen into tiny particles, effectively reducing the growth of ice crystals, thereby protecting the biological activity of bovine lactoferritin. In addition, the diameter of the high-pressure nozzle and the feeding speed are optimized, the atomization efficiency and particle size of the bovine lactoferritin freeze-dried liquid are balanced, and the incomplete freezing or clogging of the droplets can be effectively avoided.

[0029] Further, the freeze-drying includes two stages of sublimation drying and resolution drying:

[0030] Sublimation drying: the temperature is increased to -20℃ to -10℃ at a speed of 1 to 5℃ / min, and maintained for 4 to 8 hours, and the vacuum degree is controlled to be less than or equal to 10 Pa;

[0031] Resolution drying: dried at 20 to 25℃ for 4 to 6 hours.

[0032] The technical solution first sublimates the water in the bovine lactoferritin frozen particles from solid to gas through sublimation drying, removing most of the water in the frozen particles. Then, through resolution drying, the residual water in the frozen particles is further evaporated at a higher temperature, obtaining bovine lactoferritin freeze-dried powder with low water content, loose and non-caking, and good reconstitution. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0034] In a first aspect, the present application provides a preparation method of bovine lactoferritin freeze-dried powder, comprising the following steps:

[0035] S1, adding an equal volume of histidine buffer solution with a pH value of 6.0 to 6.5 to the bovine lactoferritin solution to obtain a mixed solution, wherein the mass concentration of the bovine lactoferritin solution is 30 to 40%. For example, the pH value of the histidine buffer solution can be 6.0, 6.2, 6.4 or 6.5; or a range value composed of any point value.

[0036] In the above step, the preparation method of the bovine lactoferritin solution comprises the following steps:

[0037] The bovine lactoferritin extract with a mass concentration of 5% to 10% is subjected to ultrafiltration desalination, washed with ultrapure water until the conductivity is less than 5 mS / cm, and concentrated to 1 / 8 to 1 / 5 of the original volume, so that the mass concentration of the bovine lactoferritin solution obtained after concentration is 30 to 40%. For example, it can be concentrated to 1 / 8, 1 / 7, 1 / 6 or 1 / 5 of the original volume; or a range value composed of any point value.

[0038] The bovine lactoferritin extract in the above step can be obtained after chromatography, and the purity is ≥95%.

[0039] Further, when the bovine lactoferritin extract is desalted by ultrafiltration, the molecular weight cut off by ultrafiltration is 10-30 kDa; the pressure during ultrafiltration is controlled at 0.1-0.3 MPa, and the temperature is 8-15°C.

[0040] For example, the molecular weight cut off by ultrafiltration can be 10 kDa, 20 kDa or 30 kDa; the pressure during ultrafiltration can be 0.1 MPa, 0.2 MPa or 0.3 MPa, or a range value composed of any point value; the temperature during ultrafiltration can be 8°C, 10°C, 12°C or 15°C, or a range value composed of any point value.

[0041] The bovine lactoferritin extract is desalted by ultrafiltration in the above step, which can remove small molecular weight salts or other impurities in the extract, improve the purity of bovine lactoferritin, and then washed with ultrapure water to further remove residual small molecular salt ions, so that the conductivity of the solution decreases to the target value (<5 mS / cm), indicating that the desalination is complete.

[0042] S2, adding freeze-drying adjuvant to the mixed solution, the amount of freeze-drying adjuvant added is 0.25%-7.0% of the volume of the mixed solution, to obtain bovine lactoferritin freeze-dried liquid.

[0043] The freeze-drying adjuvant can be selected from surfactants, amino acids, D-chiro-inositol and calcium chloride. The volume ratio of the added amounts of surfactants, amino acids and D-chiro-inositol is (0.2-0.5):(0.3-0.5):(2-5). For example, the volume ratio of the three can be 0.2:0.5:4, 0.3:0.3:2, 0.3:0.3:5, 0.5:0.4:3 or 0.4:0.4:5; or a range value composed of any point value.

[0044] In some embodiments, the surfactant can be specifically selected from at least one of polysorbate 80 and poloxamer 188; the amount of surfactant added is 0.02%-0.05% of the volume of the mixed solution, for example, 0.02%, 0.03%, 0.04% or 0.05% of the volume of the mixed solution; or a range value composed of any point value. The above surfactants can prevent the aggregation of bovine lactoferritin during freeze-drying, so that the obtained bovine lactoferritin freeze-dried powder is loose, has good flowability and no caking, and the reconstitutability of the bovine lactoferritin freeze-dried powder is improved.

[0045] In some embodiments, the amino acid can be selected from at least one of arginine, methionine, and proline; and the amino acid can be added in an amount of 0.03% to 0.05% of the volume of the mixed solution, for example, 0.03%, 0.04%, or 0.05% of the volume of the mixed solution, or any range formed by any of these points. The above-mentioned amino acid can stabilize the structure and functional activity of the bovine lactoferrin during the freeze-drying process, and reduce the oxidative damage of the bovine lactoferrin during the freeze-drying process.

[0046] In some embodiments, the D-chiro-inositol can be added in an amount of 0.2% to 0.5% of the volume of the mixed solution, for example, 0.2%, 0.3%, 0.4%, or 0.5% of the volume of the mixed solution, or any range formed by any of these points. The above-mentioned D-chiro-inositol has a more uniform polarity distribution than traditional inositol, can simultaneously bind to multiple polar sites of the protein, more effectively replace water molecules, maintain the natural conformation of the protein, and prevent the structure of the bovine lactoferrin from collapsing during the freeze-drying process.

[0047] In other embodiments, the calcium chloride can be added to the mixed solution in a final concentration of 11 to 33 mg / L, for example, 11 mg / L, 22 mg / L, or 33 mg / L, or any range formed by any of these points. The above-mentioned calcium chloride can stabilize the iron binding region of the bovine lactoferrin during the freeze-drying process.

[0048] Preferably, the freeze-drying adjuvant can further include rebaudioside M2 and trehalose, and the freeze-drying adjuvant can be added in an amount of 4.0% to 7.0% of the volume of the mixed solution.

[0049] In the above technical solution, the rebaudioside M2 can be added in an amount of 0.02% to 0.08% of the volume of the mixed solution, for example, 0.02%, 0.04%, 0.06%, or 0.08% of the volume of the mixed solution, or any range formed by any of these points.

[0050] The trehalose can be added in an amount of 4% to 6% of the volume of the mixed solution, for example, 4%, 5%, or 6% of the volume of the mixed solution, or any range formed by any of these points.

[0051] In the above technical solution, the rebaudioside M2 has more hydrogen bonding sites than other steviol glycosides, can replace water molecules to form hydrogen bonds with polar groups on the surface of the protein during the dehydration process, and better maintain the structural stability of the bovine lactoferrin. Furthermore, when used in combination with trehalose, the rebaudioside M2 can further increase the Tg of the bovine lactoferrin freeze-dried liquid, and reduce the risk of denaturation of the bovine lactoferrin during the desorption drying stage.

[0052] S3, spray-freezing and freeze-drying the bovine lactoferrin freeze-dried liquid to obtain a bovine lactoferrin freeze-dried powder.

[0053] In the above step, the spray freezing comprises: atomizing the bovine lactoferrin freeze-dried liquid by using a high-pressure nozzle with a diameter of 0.3-0.5 mm, spraying into a cold trap at-60℃ to-40℃ for freezing, the feeding speed is 5-10 mL / min, and the atomization pressure is 0.2-0.4 MPa.

[0054] For example, the diameter of the high-pressure nozzle can be 0.3 mm, 0.4 mm or 0.5 mm; the freezing temperature of the cold trap can be-60℃, -50℃ or-40℃; the feeding speed can be 5 mL / min, 8 mL / min or 10 mL / min; the atomization pressure can be 0.2 MPa, 0.3 MPa or 0.4 MPa; or a range value composed of any point value.

[0055] The high-pressure nozzle in the above step can atomize the bovine lactoferrin freeze-dried liquid into tiny droplets, and the cold trap at extremely low temperature can quickly freeze the atomized droplets to form fine ice crystals, avoiding mechanical damage to the structure of bovine lactoferrin caused by large ice crystals, and rapid freezing reduces the possibility of denaturation or aggregation of bovine lactoferrin, which helps to better restore its functional activity after freeze-drying.

[0056] In addition, the inventors optimize the nozzle diameter and the feeding speed, and within the above nozzle diameter and feeding speed range, the freezing of the droplets can be effectively avoided to be incomplete or blocked, and uniform frozen particles can be obtained, thereby improving the uniformity and quality stability of the final bovine lactoferrin freeze-dried powder product.

[0057] Further, the freeze-drying comprises two stages of sublimation drying and desorption drying:

[0058] Sublimation drying: warming up to-20℃ to-10℃ at a speed of 1-5℃ / min, keeping for 4-8 h, and controlling the vacuum degree to be≤10 Pa. For example, the warming up can be at a speed of 1℃ / min, 3℃ / min or 5℃ / min to-20℃, -15℃ or-10℃, keeping for 4 h, 5 h, 6 h or 8 h; the vacuum degree can be 8 Pa, 9 Pa or 10 Pa; or a range value composed of any point value.

[0059] Desorption drying: drying at 20-25℃ for 4-6 h. For example, the drying can be at 20℃, 22℃ or 25℃ for 4 h, 5 h or 6 h; or a range value composed of any point value.

[0060] The above step first uses sublimation drying to remove free water in the frozen particles, and then uses desorption drying to further remove bound water in the frozen particles, which effectively reduces the hygroscopicity of the bovine lactoferrin freeze-dried powder during storage, improves the stability of the product, and prolongs the shelf life of the product.

[0061] It should be understood that the raw materials used in the following examples are all commercially available unless otherwise specified.

[0062] Example 1

[0063] The embodiment of the present application provides a preparation method of bovine lactoferritin freeze-dried powder, which comprises the following steps:

[0064] S1, the bovine lactoferritin extract solution with a mass concentration of 5% and a purity of 96.3% is subjected to ultrafiltration desalination, the molecular weight of ultrafiltration retention is 20 kDa, the control pressure in the ultrafiltration process is 0.1 MPa, and the temperature is 10 DEG C. Ultra-pure water is continuously added for washing during the ultrafiltration process, and the washing is stopped until the conductivity of the retained liquid is lower than 5 mS / cm, and the ultrafiltration is continued until the volume of the retained liquid is concentrated to 1 / 8 of the original extract volume, so that the bovine lactoferritin solution with a mass concentration of 40% is obtained. The bovine lactoferritin solution with a mass concentration of 40% is mixed with an equal volume of histidine buffer solution with a pH value of 6.0 to obtain a mixed solution.

[0065] S2, the mixed solution is added with freeze-drying accessories, and the freeze-drying accessories are added in the following amount: 0.03% of the volume of the mixed solution of polysorbate 80, 0.03% of the volume of the mixed solution of methionine, 0.04% of the volume of the mixed solution of rebaudioside M2, 22 mg / L of calcium chloride, 0.5% of the volume of the mixed solution of D-chiro-inositol and 5% of the volume of the mixed solution of trehalose, and the stirring is performed at a rotating speed of 100 rpm until the freeze-drying accessories are completely dissolved, so that the bovine lactoferritin freeze-dried solution is obtained.

[0066] S3, the bovine lactoferritin freeze-dried solution is subjected to spray freezing: the bovine lactoferritin freeze-dried solution is atomized into microdroplets by using a high-pressure nozzle with a diameter of 0.4 mm, and the microdroplets are directly sprayed into a cold trap at-60 DEG C to be frozen to obtain frozen particles, the feeding speed is controlled to be 8 mL / min, and the atomization pressure is 0.3 MPa.

[0067] S4, the obtained frozen particles are transferred to a drying chamber for freeze-drying:

[0068] The first stage of sublimation drying: the temperature is increased to-15 DEG C at a speed of 1 DEG C / min, and the temperature is kept for 8 h, and the vacuum degree is controlled to be 10 Pa.

[0069] The second stage of analytical drying: the temperature is increased to 25 DEG C at a speed of 0.2 DEG C / min after the first stage is completed, and the drying is performed for 4 h.

[0070] Example 2

[0071] The embodiment of the present application provides a preparation method of bovine lactoferritin freeze-dried powder, which comprises the following steps:

[0072] S1, the mass concentration of 5%, purity of 96.3% of bovine lactoferritin extract solution is desalted by ultrafiltration, the molecular weight of ultrafiltration is 10kDa, the control pressure is 0.2MPa during the ultrafiltration process, and the temperature is 10 DEG C. Ultrafiltration is continuously washed with ultrapure water, until the conductivity of the retentate is less than 5mS / cm, then stop washing, continue to ultrafiltration, until the volume of the retentate is concentrated to 1 / 6 of the original extract solution, the mass concentration of bovine lactoferritin solution is 30%. To the mass concentration of 30% bovine lactoferritin solution, add equal volume of pH 6.0 histidine buffer to mix, get the mixed solution.

[0073] S2, add freeze-drying accessories to the mixed solution, the amount of freeze-drying accessories is as follows: 0.05% of the volume of the mixed solution of poloxamer 188, 0.04% of the volume of the mixed solution of methionine and proline (1:1 volume ratio), 0.06% of the volume of the mixed solution of rebaudioside M2, 33mg / L of calcium chloride, 0.3% of the volume of the mixed solution of D-chiro-inositol and 6% of the volume of the mixed solution of trehalose, stirring at 100rpm until the freeze-drying accessories are completely dissolved, to get the bovine lactoferritin freeze-dried solution.

[0074] S3, the bovine lactoferritin freeze-dried solution is spray frozen: the bovine lactoferritin freeze-dried solution is atomized into microdroplets by a high-pressure nozzle with a diameter of 0.3mm, and is directly sprayed into a cold trap at-40 DEG C to freeze to obtain frozen particles, the feeding speed is controlled at 5mL / min, and the atomization pressure is 0.3MPa.

[0075] S4, the obtained frozen particles are transferred to a drying chamber for freeze-drying:

[0076] The first stage of sublimation drying: the temperature is increased to-20 DEG C at a rate of 1 DEG C / min, and kept for 6h, and the vacuum degree is controlled at 9Pa.

[0077] The second stage of analytical drying: after the first stage is completed, the temperature is increased to 25 DEG C at a rate of 0.2 DEG C / min, and dried for 4h.

[0078] Example 3

[0079] S1, the mass concentration of 5%, purity of 96.3% of bovine lactoferritin extract solution is desalted by ultrafiltration, the molecular weight of ultrafiltration is 30kDa, the control pressure is 0.2MPa during the ultrafiltration process, and the temperature is 10 DEG C. Ultrafiltration is continuously washed with ultrapure water, until the conductivity of the retentate is less than 5mS / cm, then stop washing, continue to ultrafiltration, until the volume of the retentate is concentrated to 1 / 8 of the original extract solution, the mass concentration of bovine lactoferritin solution is 40%. To the mass concentration of 40% bovine lactoferritin solution, add equal volume of pH 6.0 histidine buffer to mix, get the mixed solution.

[0080] S2, add lyophilization adjuvant to the mixed solution, the amount of lyophilization adjuvant added is as follows: 0.03% of the volume of the mixed solution of polysorbate 80 and poloxamer 188 (mixed at a volume ratio of 1:1), 0.03% of the volume of the mixed solution of methionine and arginine (mixed at a volume ratio of 1:2), 0.03% of the volume of the mixed solution of rebaudioside M2, 11 mg / L of calcium chloride, 0.2% of the volume of the mixed solution of D-chiro-inositol, and 4% of the volume of the mixed solution of trehalose, stir at a speed of 100 rpm until the lyophilization adjuvant is completely dissolved, to obtain a bovine lactoferritin lyophilized solution.

[0081] S3, spray freeze the bovine lactoferritin lyophilized solution: use a high-pressure nozzle with a diameter of 0.5 mm to atomize the bovine lactoferritin lyophilized solution into droplets, directly spray into a cold trap at -50°C for freezing to obtain frozen particles, the feeding speed is controlled at 5 mL / min, and the atomization pressure is 0.4 MPa.

[0082] S4, transfer the obtained frozen particles to a drying chamber for freeze-drying:

[0083] First stage sublimation drying: increase the temperature to -10°C at a rate of 1°C / min and maintain for 5 h, and control the vacuum degree to be 8 Pa.

[0084] Second stage desorption drying: after the first stage is completed, increase the temperature to 25°C at a rate of 0.2°C / min and dry for 4 h.

[0085] Example 4

[0086] S1, subject the bovine lactoferritin extract solution with a mass concentration of 7% and a purity of 96.3% to ultrafiltration desalination, the molecular weight cut off during ultrafiltration is 20 kDa, the pressure during ultrafiltration is controlled at 0.3 MPa, and the temperature is 8°C. During the ultrafiltration process, ultrapure water is continuously added for washing, and after the conductivity of the cut-off solution is lower than 5 mS / cm, the washing is stopped, and the ultrafiltration is continued until the volume of the cut-off solution is concentrated to 1 / 5 of the original extract solution, to obtain a bovine lactoferritin solution with a mass concentration of 35%. Add an equal volume of histidine buffer with a pH value of 6.5 to the bovine lactoferritin solution with a mass concentration of 35% to mix, to obtain a mixed solution.

[0087] S2, add lyophilization adjuvant to the mixed solution, the amount of lyophilization adjuvant added is as follows: 0.02% of the volume of the mixed solution of polysorbate 80, 0.05% of the volume of the mixed solution of methionine and arginine (mixed at a volume ratio of 2:3), 0.08% of the volume of the mixed solution of rebaudioside M2, 22 mg / L of calcium chloride, 0.4% of the volume of the mixed solution of D-chiro-inositol, and 5% of the volume of the mixed solution of trehalose, stir at a speed of 100 rpm until the lyophilization adjuvant is completely dissolved, to obtain a bovine lactoferritin lyophilized solution.

[0088] S3, spray-freezing the bovine lactoferritin freeze-dried liquid: using a high-pressure nozzle with a diameter of 0.4 mm to atomize the bovine lactoferritin freeze-dried liquid into microdroplets, directly spraying into a cold trap at -60 DEG C for freezing to obtain frozen particles, the feeding speed is controlled at 10 mL / min, and the atomization pressure is 0.2 MPa.

[0089] S4, transferring the obtained frozen particles to a drying chamber for freeze-drying:

[0090] First-stage sublimation drying: increasing the temperature to -10 DEG C at a speed of 5 DEG C / min, keeping for 4 h, and controlling the vacuum degree at 8 Pa.

[0091] Second-stage desorption drying: increasing the temperature to 22 DEG C at a speed of 0.2 DEG C / min after the first stage is completed, and drying for 6 h.

[0092] Example 5

[0093] The embodiment of the present application provides a preparation method of bovine lactoferritin freeze-dried powder, which comprises the following steps:

[0094] S1, desalting the bovine lactoferritin extract liquid with a mass concentration of 5% and a purity of 96.3% by ultrafiltration, the molecular weight cut off in the ultrafiltration is 20 kDa, the pressure in the ultrafiltration process is controlled at 0.1 MPa, and the temperature is 15 DEG C. Ultrafiltration is continuously carried out while adding ultrapure water for washing until the conductivity of the cut-off liquid is lower than 5 mS / cm, then the washing is stopped, and the ultrafiltration is continuously carried out until the volume of the cut-off liquid is concentrated to 1 / 7 of the original extract liquid, thereby obtaining a bovine lactoferritin solution with a mass concentration of 35%. The bovine lactoferritin solution with a mass concentration of 35% is mixed with an equal volume of histidine buffer with a pH value of 6.2, thereby obtaining a mixed liquid.

[0095] S2, adding freeze-drying accessories to the mixed liquid, the amount of the freeze-drying accessories added is as follows: 0.04% of the volume of the mixed liquid of polysorbate 80, 0.04% of the volume of the mixed liquid of methionine, 0.5% of the volume of the mixed liquid of D-chiro-inositol, and the stirring speed is 100 rpm until the freeze-drying accessories are completely dissolved, thereby obtaining a bovine lactoferritin freeze-dried liquid.

[0096] S3, spray-freezing the bovine lactoferritin freeze-dried liquid: using a high-pressure nozzle with a diameter of 0.4 mm to atomize the bovine lactoferritin freeze-dried liquid into microdroplets, directly spraying into a cold trap at -60 DEG C for freezing to obtain frozen particles, the feeding speed is controlled at 10 mL / min, and the atomization pressure is 0.2 MPa.

[0097] S4, transferring the obtained frozen particles to a drying chamber for freeze-drying:

[0098] First stage sublimation drying: temperature raised to -15°C at a rate of 3°C / min, held for 4h, vacuum controlled at 10 Pa.

[0099] Second stage desorption drying: temperature raised to 20°C at a rate of 0.2°C / min after the first stage, dried for 5h.

[0100] Comparative Example 1

[0101] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 1, with the only difference that the D-chiro-inositol was replaced with the traditional myo-inositol in equal amounts, and the rest was unchanged.

[0102] Comparative Example 2

[0103] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 1, with the only difference that the calcium chloride was replaced with sodium chloride in equal amounts, and the rest was unchanged.

[0104] Comparative Example 3

[0105] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 1, with the only difference that the rebaudioside M2 was replaced with rebaudioside M in equal amounts, and the rest was unchanged.

[0106] Comparative Example 4

[0107] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 1, with the only difference that the trehalose was replaced with rebaudioside M2 in equal amounts, and the rest was unchanged.

[0108] Comparative Example 5

[0109] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 2, with the only difference that the high-pressure nozzle with a diameter of 0.3 mm was replaced with a high-pressure nozzle with a diameter of 0.2 mm.

[0110] Comparative Example 6

[0111] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 3, with the only difference that the high-pressure nozzle with a diameter of 0.5 mm was replaced with a high-pressure nozzle with a diameter of 0.7 mm.

[0112] Comparative Example 7

[0113] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 2, with the only difference that the feed rate during spray freezing was reduced from 5 mL / min to 3 mL / min.

[0114] Comparative Example 8

[0115] The lyophilized powder of bovine lactoferritin was prepared according to the method of Example 4, with the only difference that the feed rate during spray freezing was increased from 10 mL / min to 12 mL / min.

[0116] Test Example

[0117] 1. The tissue state, moisture content, purity of lactoferrin and reconstitution of the lactoferrin lyophilized powder prepared in the above Examples 1-5 and Comparative Examples 1-8 were detected, respectively, and the results are shown in Table 1.

[0118] Table 1

[0119] Organizational state Moisture content Bovine lactoferrin purity Reconstitution Example 1 Loose, free-flowing, no clumping 2.77％ 96.5％ Clear and transparent Example 2 Loose, free-flowing, no clumping 3.14％ 96.8％ Clear and transparent Example 3 Loose, free-flowing, no clumping 2.91％ 96.2％ Clear and transparent Example 4 Loose, free-flowing, no clumping 2.98％ 97.1％ Clear and transparent Example 5 Loose, free-flowing, no clumping 4.65％ 95.6％ Clear and transparent Comparative Example 1 Loose, free-flowing, no clumping 3.95％ 93.9％ Clear and transparent Comparative Example 2 Loose, free-flowing, no clumping 3.35％ 91.4％ Clear and transparent Comparative Example 3 Loose, free-flowing, no clumping 4.24％ 95.4％ Slightly hazy Comparative Example 4 Slightly clumped 3.76％ 94.7％ Clear and transparent Comparative Example 5 Slightly clumped 2.93％ 94.5％ Clear and transparent Comparative Example 6 Slightly clumped 6.74％ 93.6％ Clear and transparent Comparative Example 7 Loose, free-flowing, no clumping 4.21％ 92.8％ Clear and transparent Comparative Example 8 Slightly clumped 7.18％ 92.7％ Slightly hazy

[0120] 2. Biological activity detection

[0121] The lactoferrin lyophilized powder prepared in the above Examples 1-5 and Comparative Examples 1-8 was dissolved in PBS buffer (pH 7.4) to prepare 13 lactoferrin solutions with a concentration of 2 mg / mL.

[0122] An excess of iron ion solution (FeCl3 solution) was added to the above 13 lactoferrin solutions to make the molar ratio of iron ion to lactoferrin 3:1. The resulting mixed solution was stirred at 50°C for 1 hour to allow the iron ion to fully react with the lactoferrin, and then centrifuged to separate the unbound iron ion. The supernatant was taken and detected by UV spectrophotometry to calculate the iron binding capacity of the lactoferrin lyophilized powder, and the results are shown in Table 2.

[0123] Table 2

[0124] Iron binding capacity Example 1 97.5％ Example 2 97.8％ Example 3 97.2％ Example 4 98.3％ Example 5 95.8％ Comparative Example 1 93.2％ Comparative Example 2 85.7％ Comparative Example 3 95.3％ Comparative Example 4 92.1％ Comparative Example 5 91.8％ Comparative Example 6 86.3％ Comparative Example 7 88.6％ Comparative Example 8 80.5％

[0125] From the above, it can be seen that after the D-chiro-inositol is replaced with an equal amount of traditional myo-inositol in Comparative Example 1, the purity of lactoferrin in the lyophilized powder decreases, and the iron binding capacity decreases, indicating that compared with traditional myo-inositol, the D-chiro-inositol in the present application, in combination with other lyophilization adjuvants, can better protect the structure and biological activity of lactoferrin during the lyophilization process.

[0126] After calcium chloride is replaced with an equal amount of sodium chloride in Comparative Example 2, the purity of lactoferrin in the lyophilized powder decreases, and the iron binding capacity decreases, indicating that the calcium chloride in the present application, in combination with other lyophilization adjuvants, can better stabilize the iron binding region of lactoferrin during the lyophilization process, thereby ensuring the biological activity of lactoferrin after lyophilization.

[0127] After rebaudioside M2 is replaced with an equal amount of rebaudioside M in Comparative Example 3, the reconstituted lyophilized powder is slightly turbid compared with the lyophilized powder of Example 1, the purity of lactoferrin in the lyophilized powder decreases, and the iron binding capacity decreases, indicating that the rebaudioside M2 in the present application, in coordination with trehalose and in combination with other lyophilization adjuvants, can improve the reconstitution of the lyophilized powder and better protect the structure and biological activity of lactoferrin during the lyophilization process.

[0128] After the trehalose was replaced by rebaudioside M2 in the same amount in Comparative Example 4, a small amount of lumps appeared in the freeze-dried powder, the purity of lactoferrin in the freeze-dried powder was reduced, and the iron binding capacity was reduced, indicating that rebaudioside M2 in the application cooperates with trehalose, and cooperates with other freeze-drying adjuvants, which can avoid the appearance of lumps in the freeze-dried powder, and better protect the structure and biological activity of lactoferrin in the freeze-drying process.

[0129] After the diameter of the high-pressure nozzle was reduced to 0.2 mm in Comparative Example 5 and increased to 0.7 mm in Comparative Example 6, a small amount of lumps appeared in the freeze-dried powder, the purity of lactoferrin in the freeze-dried powder was reduced, and the iron binding capacity was reduced, and the water content in the freeze-dried powder of Comparative Example 6 was increased, indicating that the high-pressure nozzle with a specific diameter in the application, combined with the feeding speed and other conditions, can obtain more uniform frozen particles, reduce the loss of protein activity during freezing and subsequent drying process, and improve the uniformity and quality stability of the final lactoferrin freeze-dried powder.

[0130] After the feeding speed during spray freezing was reduced from 5 mL / min to 3 mL / min in Comparative Example 7 and increased from 10 mL / min to 12 mL / min in Comparative Example 8, the purity of lactoferrin in the freeze-dried powder was reduced, and the iron binding capacity was reduced, and a small amount of lumps appeared in the freeze-dried powder of Comparative Example 8, the water content was increased, and the reconstitution was slightly turbid. It is indicated that the specific feeding speed during spray freezing in the application, combined with the high-pressure nozzle with a suitable diameter and other conditions, can reduce the damage to protein activity during freezing, obtain completely and uniformly frozen particles, which is beneficial to the removal of water during subsequent drying, and improve the uniformity and quality stability of the final lactoferrin freeze-dried powder.

[0131] The above only describes the preferred embodiments of the application and is not intended to limit the application. Although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A method for preparing bovine lactoferrin freeze-dried powder, characterized in that: The following steps are involved: adding an equal volume of histidine buffer having a pH value of 6.0 to 6.5 to the bovine lactoferrin solution to obtain a mixed solution, wherein the mass concentration of the bovine lactoferrin solution is 30 to 40%; Adding a freeze-dried auxiliary material to the mixed solution, wherein the amount of the freeze-dried auxiliary material added accounts for 0.25% to 7.0% of the volume of the mixed solution, to obtain a bovine lactoferrin freeze-dried solution; spray freezing and freeze-drying the bovine lactoferrin freeze-dried liquid to obtain bovine lactoferrin freeze-dried powder; The freeze-drying auxiliary materials include surfactant, amino acid, D-chiro-inositol and calcium chloride.

2. The preparation method according to claim 1, characterized in that In the freeze-dried excipient, the volume ratio of the added amounts of the surfactant, the amino acid and the D-chiro-inositol is (0.2-0.5):(0.3-0.5):(2-5).

3. The preparation method according to claim 1 or 2, characterized in that The final concentration of the calcium chloride after addition is 11-33 mg / L.

4. The preparation method according to any one of claims 1 to 3, characterized in that The surfactant includes at least one of polysorbate 80 and poloxamer 188.

5. The preparation method according to any one of claims 1 to 4, characterized in that The amino acid includes at least one of arginine, methionine, and proline.

6. The preparation method according to any one of claims 1 to 5, characterized in that The freeze-dried excipients also include rebaudioside M2 ​​and trehalose; wherein, The amount of rebaudioside M2 ​​added is 0.02% to 0.08% of the volume of the mixed solution; The added amount of trehalose is 4% to 6% of the volume of the mixed solution.

7. The preparation method according to claim 1, characterized in that The preparation method of the bovine lactoferrin solution comprises: The bovine lactoferrin extract with a mass concentration of 5% to 10% is subjected to ultrafiltration desalination, washed with ultrapure water until the conductivity is less than 5mS / cm, and concentrated to 1 / 8 to 1 / 5 of the original volume to obtain the bovine lactoferrin solution.

8. The preparation method according to claim 7, characterized in that The molecular weight of the ultrafiltration cutoff is 10-30 kDa; the pressure is controlled at 0.1-0.3 MPa and the temperature is controlled at 8° C.-15° C. during the ultrafiltration process.

9. The preparation method according to claim 1, characterized in that The spray freezing comprises: using a high-pressure nozzle with a diameter of 0.3-0.5 mm to atomize the bovine lactoferrin freeze-dried liquid, spraying it into a cold trap at -60°C to -40°C for freezing, with a feed rate of 5-10 mL / min and an atomization pressure of 0.2-0.4 MPa.

10. The preparation method according to claim 1, characterized in that The freeze drying includes two stages: sublimation drying and desorption drying: Sublimation drying: Raise the temperature to -20℃~-10℃ at a rate of 1~5℃ / min, maintain for 4~8h, and control the vacuum degree ≤10Pa; Desorption drying: Dry at 20-25°C for 4-6 hours.