Method for extracting lactoferrin from milk

Abstract

The invention discloses a method for extracting lactoferrin from milk, which comprises the following steps: (a) enabling fresh skim milk to directly pass through a chromatographic column filled with cation exchange filler with certain particle size and pore size; (b) cleaning residual milk and impurities with water and an aqueous solution containing salt with a first concentration; and (c) eluting with an aqueous solution or a buffer solution containing a salt with a second concentration at a flow rate of 200-1000 cm / h to obtain lactoferrin. The invention establishes a method suitable for industrial-grade extraction of lactoferrin from milk, and the purity and yield of the extracted lactoferrin are greatly improved. In addition, the method provided by the invention avoids the use of large complex equipment, and realizes low-cost extraction of lactoferrin through simple extraction and purification.

Description

Technical Field

[0001] The invention relates to the field of protein extraction and purification, in particular to a method for extracting lactoferrin, and in particular to a method for extracting lactoferrin from milk. Background Art

[0002] Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in breast milk. The concentration of lactoferrin in human milk is about 1.0-3.2 mg / ml, providing infants with a variety of biological functions, especially regulating immunity and resisting pathogenic microorganisms. For infants who are not breastfed, the lactoferrin content in animal milk is only 0.02-0.35 mg / ml, which is far lower than the lactoferrin content in human milk and cannot meet the feeding requirements of infants.

[0003] Based on numerous research results and experiments, adding lactoferrin to infant formula is of great significance, which can not only meet the needs of infant growth and development, but also protect the health of infants. According to the latest national standard GB14880, lactoferrin is listed as a nutritional enhancer. Therefore, the extraction and purification of lactoferrin is particularly important.

[0004] For example, CN210841449U discloses novel natural milk lactoferrin extraction equipment, including fixed cover, reactor, control box, water pump and separator, the bottom of the fixed frame is equipped with a control box, a reactor is installed on one side of the fixed frame, a safety tube is provided on one side of the fixed cover, a water pump is provided on the bottom of the reactor, a separator is provided on one side of the reactor, a heating wire is provided on the inside of the separator, a baffle is provided between the separators, a gas pipeline is provided on the bottom of the baffle, a filter screen is provided below the spiral baffle, a discharge port is provided on the bottom of the separator, a connector is provided on the bottom of the discharge port, and a maintenance top cover is provided on the top of the separator. The utility model can effectively filter lactoferrin in milk by using a biological filter membrane at the filter screen at the bottom, so that lactoferrin is collected, and for large-scale lactoferrin extraction, equipment requirements are relatively high and complicated.

[0005] For another example, CN106008703A discloses a method for extracting and preparing high-purity low-iron saturation lactoferrin from cow's milk, which comprises the following steps: step 1, using a cation exchange resin to adsorb and desorb lactoferrin in the cow's milk; step 2, concentrating the desorbed solution obtained in step 1; step 3, using an insoluble Fe 3+ Chelating resin for Fe 3+ Adsorb and then concentrate the concentrate. This method can reduce the Fe 3+ion content, but the extraction level still cannot meet the extraction and purification requirements under industrial production conditions.

[0006] Based on this, there is still a need for a method for extracting lactoferrin from milk that is suitable for industrial use. Summary of the invention

[0007] In view of at least some of the problems existing in the prior art, the inventors have conducted in-depth research and found that by adopting a specific extraction step and controlling the chromatographic conditions during the extraction process, a method suitable for extracting lactoferrin from milk at an industrial level is established, and the purity and yield of the extracted lactoferrin are greatly improved. In addition, the method of the present invention avoids the use of large and complex equipment, and at the same time, through simple extraction and purification, low-cost lactoferrin extraction is achieved. The present invention is completed at least in part based on this discovery. Specifically, the present invention includes the following contents.

[0008] The present invention provides a method for extracting lactoferrin from milk, which comprises at least the following steps:

[0009] (a) passing fresh skim milk directly through a chromatography column filled with a cation exchange filler having an average particle size of 60-500 μm and a pore size of 100-500 nm;

[0010] (b) washing the remaining milk and impurities with water and an aqueous solution containing a first concentration of salt;

[0011] (c) eluting with an aqueous solution or buffer solution containing a second salt concentration at a flow rate of 200-1000 cm / h to obtain lactoferrin.

[0012] According to the method for extracting lactoferrin from milk of the present invention, preferably, in step (a), the column temperature of the chromatography column is controlled within the range of 30-60°C.

[0013] The method for extracting lactoferrin from milk according to the present invention preferably further comprises (d) washing and regenerating the chromatography column filled with cation exchange filler with 0.5-1M NaOH solution.

[0014] According to the method for extracting lactoferrin from milk of the present invention, preferably, the purity of lactoferrin extracted from milk is not less than 92%, and the yield of lactoferrin is not less than 60%. Preferably, the purity of lactoferrin is 92-96%, and the yield of lactoferrin is 60-90%.

[0015] According to the method for extracting lactoferrin from milk of the present invention, preferably, the filling height of the cation exchange filler is 100-500 mm.

[0016] According to the method for extracting lactoferrin from milk of the present invention, preferably, the cation exchange filler is porous polymethacrylate spherical particles with sulfonate groups on the surface.

[0017] According to the method for extracting lactoferrin from milk of the present invention, preferably, the cross-linking degree of the cation exchange filler is 50%-80%.

[0018] According to the method for extracting lactoferrin from milk of the present invention, preferably, the salt-containing aqueous solution is a sodium chloride solution.

[0019] According to the method for extracting lactoferrin from milk of the present invention, preferably, the salt-containing aqueous solution is a sodium sulfate solution.

[0020] According to the method for extracting lactoferrin from milk of the present invention, preferably, the second concentration is greater than the first concentration.

[0021] According to the method for extracting lactoferrin from milk of the present invention, preferably, the buffer is a phosphate buffer or a sodium acetate buffer at a pH of 5.0-7.0.

[0022] According to the method for extracting lactoferrin from milk of the present invention, preferably, the milk is skim milk.

[0023] The method of the present invention greatly improves the purity and yield of extracted lactoferrin. The method of the present invention is suitable for industrial-grade extraction of lactoferrin from milk, avoids the use of large and complex equipment, and realizes low-cost extraction of lactoferrin through simple extraction and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is an exemplary purification profile according to the method of the present invention.

[0025] Figure 2 FIG. 5 is an exemplary HPLC detection spectrum of the method according to the present invention.

[0026] Figure 3 FIG. 4 is another exemplary HPLC detection spectrum of the method according to the present invention.

[0027] Figure 4 FIG. 4 is another exemplary HPLC detection spectrum of the method according to the present invention.

[0028] Figure 5 This is the HPLC detection spectrum of the product obtained in Comparative Example 1. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0032] In the present invention, purity and content have the same meaning and can be used interchangeably.

[0033] The present invention provides a method for extracting lactoferrin from milk, which comprises at least steps (a)-(c). The milk is preferably skimmed milk, which can be low-fat milk formed by semi-skimming, or fully skimmed milk formed by fully skimming. When it is semi-skimmed milk, it contains 1.0-1.5% fat, and when it is fully skimmed milk, the fat it contains is less than 0.5%. It should be noted that in the present invention, the milk is directly used for subsequent lactoferrin extraction without the need for pretreatment such as removing certain specific proteins, such as globulin, so as to reduce the time and cost of the extraction process.

[0034] Considering that the chromatographic conditions have a greater impact on the extraction and purification of lactoferrin, the chromatographic conditions are described below, and the chromatographic conditions here include, but are not limited to, the selection of chromatography columns, chromatography column fillers, elution flow rates, eluents, etc. It should be noted that lactoferrin extraction is a complicated process, especially when it comes to industrial-grade extraction in large quantities, it is necessary to consider how to achieve effective extraction under the conditions of a large amount of accumulation effects of extracts to be extracted and higher column pressures. Therefore, it is necessary to consider the impact of various factors on the purity and yield of lactoferrin as a whole. It should be noted that the column pressure here refers to the industrial column pressure, which is generally 2-4 bar, preferably 3-4 bar. It is known to those skilled in the art that column pressure refers to the pressure generated by the pressure generated by the milk loading to be extracted and the certain flow rate of the eluent during elution, and the column pressure here is different from the column pressure used in conventional laboratories.

[0035] In step (a) of the present invention, fresh skim milk is directly passed through a chromatography column filled with a cation exchange filler, wherein the average particle size of the cation exchange filler is 60-500 μm and the pore size is 100-500 nm. In the present invention, the inner diameter refers to the diameter of the inner circle of the chromatography column, and the inner diameter of the chromatography column is preferably 10-2000 mm, more preferably 10-1800 mm, and more preferably 600-1800 mm, such as 800 mm, 1000 mm, 1200 mm, 1400 mm, 1600 mm, etc. The filling height is preferably 100-500 mm, more preferably 200-500 mm, and more preferably 250-400 mm.

[0036] The average particle size of the chromatography column filler of the present invention is 60-500 μm, preferably 100-500 μm, further preferably 100-400 mm, and further preferably 200-400 mm. The filler preferably has a pore size of 100-500 nm, further preferably 200-500 nm, and further preferably 300-400 nm.

[0037] Preferably, the filler of the present invention is a cation exchange filler, more preferably a porous polymethacrylate spherical particle with sulfonate groups on the surface. Also preferably, the crosslinking degree of the cation exchange filler is 50%-80%. Further preferably, the crosslinking degree of the cation exchange filler is 60%-75%. If the crosslinking degree is too low, the filler cannot withstand high pressure and deforms or even breaks. On the other hand, if the crosslinking degree is too high, the flow rate of skim milk is affected, which is also not conducive to exchange.

[0038] The chromatography temperature of the present invention is preferably 30-60° C., and is also preferably 30-45° C. For example, 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., etc.

[0039] In step (b), the residual milk and impurities are washed with water and an aqueous solution containing a first concentration of salt, especially the residual milk and impurities adsorbed on the surface of the chromatography column filler. The impurities are not particularly limited here, and the specific components are known to those skilled in the art, for example, they may include those impurities determined by the national standard GB 5413.30-2016 or the non-mandatory national standard IRAM 14 008-1962. The specific steps of washing, for example, may be to wash the column with water first, and then wash the column again with an aqueous solution containing a first concentration of salt.

[0040] In step (c), the lactoferrin is eluted with an aqueous solution or buffer containing a second concentration of salt at a flow rate of 200-1000 cm / h, thereby obtaining the lactoferrin. The flow rate of the elution is preferably 210-1000 cm / h, further preferably 300-900 cm / h, further preferably 400-800 cm / h, such as 500 cm / h, 600 cm / h, 700 cm / h, etc. A too low flow rate will increase the chromatography time, while a too high flow rate will result in an excessively high column pressure and a decreased loading capacity, further affecting the purity and yield of the extraction and purification.

[0041] The saline aqueous solution here can be a sodium chloride solution or a sodium sulfate solution, preferably a sodium chloride solution.

[0042] In the present invention, the second concentration is greater than the first concentration. Preferably, the first concentration is in the range of 0.1-0.5 M. Also preferably, the first concentration is in the range of 0.2-0.4 M. Further preferably, the first concentration is in the range of 0.2-0.3 M.

[0043] Preferably, the second concentration is in the range of 0.6-2 M. Also preferably, the second concentration is in the range of 0.6-1 M. Further preferably, the second concentration is in the range of 0.8-1 M.

[0044] In the present invention, the buffer is a phosphate buffer or sodium acetate buffer with a pH of 5.0-7.0. The pH has an effect on elution, for example, it can affect the time of lactoferrin washing out. Preferably, the pH is 5.5-6.5.

[0045] The method of the present invention further comprises step (d) of washing and regenerating the chromatography column filled with cation exchange filler with 0.5-1M NaOH solution. Step (d) of the present invention is used to elute lactoferrin that has not been eluted in the previous step. Preferably, the NaOH solution is 0.5-0.6M.

[0046] The purity of lactoferrin extracted from milk by the method of the present invention is not less than 92, and the yield of lactoferrin is not less than 60%. Preferably, the purity of lactoferrin extracted from milk is not less than 93%, for example, it can be 94%, 95%, 96%, or even 97%. Preferably, the yield of lactoferrin is not less than 73%, for example, it can be 82%, 83%, 84%, 85%, 86%, 87%, or even 88%, 89% or 90%. In the prior art, for example, the separation and purification process of lactoferrin disclosed in CN1486989, when the flow rate is low and heparin-agarose chromatography column is adopted, the purity and yield of lactoferrin are low. Therefore, the process of the present invention has excellent extraction effect.

[0047] It should be noted that other steps or operations may be included before, during, or between steps (a) to (d) of the present invention, such as further optimizing and / or improving the method of the present invention. For example, a step of balancing the filler with a buffer solution. Another example is a step of washing and regenerating the filler with a higher concentration of NaOH solution.

[0048] Example 1

[0049] The reagents used in the following examples of the present invention were all chromatographically pure, and the other chemicals were all analytically pure, unless otherwise specified.

[0050] This embodiment is a method for extracting lactoferrin from milk, which is as follows:

[0051] 1) using a pump to pass fresh skim milk through a chromatography column filled with a cation exchange filler, wherein the cation exchange filler is a porous polymethacrylate spherical particle with a sulfonate group on the surface, and the cross-linking degree of the cation exchange filler is 50%-80%;

[0052] 2) Wash the remaining milk and impurities with water and saline solution (sodium chloride or sodium sulfate solution);

[0053] 3) washing the lactoferrin with a saline aqueous solution or buffer (such as phosphate buffer, sodium acetate buffer, pH 5.0-7.0), and collecting for later use;

[0054] 4) The chromatography column filled with cation exchange filler is cleaned and regenerated with 0.1-1M NaOH solution.

[0055] The inner diameter of the chromatography column is 10 mm, the height of the cation exchange filler is 30 cm, the linear flow rate is 600 cm / h, 3 L of skim milk is pumped into the chromatography column, washed with water and 0.3 M sodium chloride solution, and then lactoferrin is eluted with 2 M sodium chloride solution to obtain a product with a purity of 95.13% and a yield of 87%. The purification diagram is shown in FIG. Figure 1 As shown, the HPLC detection spectrum is as Figure 2 shown.

[0056] Example 2

[0057] This example illustrates a method for extracting lactoferrin from milk. The difference from Example 1 is that the inner diameter of the chromatography column is 600 mm, the height of the cation exchange filler is 50 cm, the linear flow rate is 800 cm / h, 500 liters of skim milk is pumped into the chromatography column by a pump, and after washing with water and 0.2M sodium chloride solution, lactoferrin is eluted with 1M sodium chloride solution. The remaining steps are the same as those in Example 1. The product purity is 94.40% and the yield is 73%. Its HPLC detection spectrum is as follows: Figure 3 shown.

[0058] Example 3

[0059] This example illustrates a method for extracting lactoferrin from milk. The difference from Example 1 is that the inner diameter of the chromatography column is 1800 mm, the height of the cation exchange filler is 30 cm, the linear flow rate is 700 cm / h, 300 tons of skim milk is pumped into the chromatography column by a pump, and after washing with water and 0.25M sodium chloride solution, lactoferrin is eluted with 0.6M sodium chloride solution. The remaining steps are the same as those in Example 1. The product purity is 92.56% and the yield is 86%. Its HPLC detection spectrum is as follows: Figure 4 shown.

[0060] Comparative Example 1

[0061] The inner diameter of the chromatography column is 16 mm, the height of the cation exchange filler is 20 cm, the linear flow rate is 500 cm / h, and the commercially available large-particle agarose gel matrix cation exchange filler is used. 6L of skim milk is pumped into the chromatography column, and lactoferrin is eluted with 0.4M sodium chloride solution and then with 1.5M sodium chloride solution to obtain a product with a purity of 79.33 and a yield of 79%. The HPLC detection spectrum is as follows Figure 4 shown.

[0062] Comparative Example 2

[0063] The chromatography column has an inner diameter of 10 mm and is filled with commercially available polymer-based cation exchange filler (particle size 80 μm, pore size 100 nm). It has a height of 30 cm and a linear flow rate of 600 cm / h. 1 L of skim milk is pumped into the chromatography column through a pump. The column pressure rises from 4 bar to 7 bar, and the experiment fails because it exceeds the alarm pressure of the chromatography system.

[0064] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for extracting lactoferrin from milk, characterized in that The following steps are involved: (a) passing fresh skim milk directly through a chromatography column filled with a cation exchange filler having an average particle size of 60-500 μm and a pore size of 100-500 nm; (b) washing the remaining milk and impurities with water and an aqueous solution containing a first concentration of salt; (c) eluting with an aqueous solution or buffer solution containing a second salt concentration at a flow rate of 200-1000 cm / h to obtain lactoferrin.

2. The method for extracting lactoferrin from milk according to claim 1, characterized in that The method further comprises (d) cleaning and regenerating the chromatography column filled with cation exchange filler with a 0.5-1 M NaOH solution.

3. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The purity of lactoferrin extracted from milk is not less than 92%, and the yield of lactoferrin is not less than 60%.

4. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The filling height of the cation exchange filler is 100-500 mm.

5. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The cation exchange filler is porous polymethacrylate spherical particles with sulfonate groups on the surface.

6. The method for extracting lactoferrin from milk according to claim 5, characterized in that: The cross-linking degree of the cation exchange filler is 50%-80%.

7. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The salt-containing aqueous solution is a sodium chloride or sodium sulfate solution.

8. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The second concentration is greater than the first concentration.

9. The method for extracting lactoferrin from milk according to claim 1, characterized in that: The buffer is a phosphate buffer or a sodium acetate buffer at pH 5.0-7.

0.

10. The method for extracting lactoferrin from milk according to any one of claims 1 to 9, characterized in that: The milk is skimmed milk.

Images