CN119285755A - Method for extracting lactoferrin from milk - Google Patents
Method for extracting lactoferrin from milk Download PDFInfo
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- CN119285755A CN119285755A CN202411631956.1A CN202411631956A CN119285755A CN 119285755 A CN119285755 A CN 119285755A CN 202411631956 A CN202411631956 A CN 202411631956A CN 119285755 A CN119285755 A CN 119285755A
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- 102000010445 Lactoferrin Human genes 0.000 title claims abstract description 80
- 108010063045 Lactoferrin Proteins 0.000 title claims abstract description 80
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 80
- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 80
- 229940078795 lactoferrin Drugs 0.000 title claims abstract description 80
- 235000013336 milk Nutrition 0.000 title claims abstract description 52
- 239000008267 milk Substances 0.000 title claims abstract description 52
- 210000004080 milk Anatomy 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000005341 cation exchange Methods 0.000 claims abstract description 27
- 238000012856 packing Methods 0.000 claims abstract description 17
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 16
- 238000005406 washing Methods 0.000 claims abstract description 14
- 239000007864 aqueous solution Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 239000002245 particle Substances 0.000 claims abstract description 7
- 239000011148 porous material Substances 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 24
- 239000000945 filler Substances 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 238000004132 cross linking Methods 0.000 claims description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 229920000193 polymethacrylate Polymers 0.000 claims description 4
- 239000007974 sodium acetate buffer Substances 0.000 claims description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 4
- 235000011152 sodium sulphate Nutrition 0.000 claims description 4
- 239000012798 spherical particle Substances 0.000 claims description 4
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 239000012266 salt solution Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 21
- 238000000746 purification Methods 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 6
- 239000012062 aqueous buffer Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000010828 elution Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000020256 human milk Nutrition 0.000 description 3
- 210000004251 human milk Anatomy 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000020247 cow milk Nutrition 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000008133 Iron-Binding Proteins Human genes 0.000 description 1
- 108010035210 Iron-Binding Proteins Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 235000020244 animal milk Nutrition 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 235000020121 low-fat milk Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000020161 semi-skimmed milk Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
A process for extracting lactoferrin from milk comprises (a) passing fresh skim milk directly through a chromatography column packed with a cation exchange packing having a particle size and pore size, (b) washing residual milk and impurities with water and an aqueous solution containing a salt of a first concentration, and (c) eluting with an aqueous solution or buffer containing a salt of a second concentration at a flow rate of 200-1000cm/h to obtain lactoferrin. The invention establishes a method suitable for extracting lactoferrin from milk in industrial grade, and the purity and the yield of the extracted lactoferrin are greatly improved. In addition, the method of the invention avoids the use of large complex equipment, and simultaneously realizes the extraction of low-cost lactoferrin through simple extraction and purification.
Description
Technical Field
The present invention relates to the field of protein extraction and purification, in particular to a method for extracting lactoferrin from milk.
Background
Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in milk, and in human milk, lactoferrin is present at a concentration of about 1.0-3.2mg/ml, providing multiple biological functions for infants, particularly in regulating immunity and resistance to pathogenic microorganisms. For infants fed by non-breast milk, the content of lactoferrin in animal milk is only 0.02-0.35mg/ml, which is far lower than that in human milk, and the feeding requirement of the infants cannot be met.
Based on a plurality of research results and experiments, the addition of the lactoferrin into the infant formula food has important significance, can meet the requirements of the growth and development of infants, and also has a protection effect on the health of infants. Lactoferrin is classified as a nutritional supplement according to the latest national standard GB 14880. Thus, extraction and purification of lactoferrin is particularly important.
For example, CN210841449U discloses a novel natural milk lactoferrin extraction apparatus, including fixed cover, reation kettle, control box, water pump and separator, the control box is installed to the inside bottom of mount, reation kettle is installed to one side of mount inside, one side of fixed cover is provided with the safety tube, the water pump is installed to the bottom of reation kettle, the separator is installed to one side of reation kettle, the inside of separator is provided with the heater strip, install the baffle between the separator, gas pipeline is installed to the bottom of baffle, the below of helical baffle is provided with the filter screen, the discharge port is installed to the bottom of separator, the connector is installed to the bottom of discharge port, the maintenance top cap is installed at the top of separator. The utility model can effectively filter the lactoferrin in the milk by adopting the biological filter membrane at the filter screen at the bottom, thereby collecting the lactoferrin, and has relatively high and complex equipment requirements for large-scale lactoferrin extraction.
For another example, CN106008703a discloses a method for extracting and preparing high-purity low-iron-saturation lactoferrin from cow's milk, which comprises the steps of 1 adsorbing and desorbing lactoferrin in cow's milk by using cation exchange resin, 2 concentrating the desorbed solution obtained in 1, 3 adsorbing Fe 3+ by using insoluble Fe 3+ chelate resin, and concentrating the concentrate. The method can reduce the content of Fe 3+ ions in the lactoferrin, but the extraction level can not meet the extraction and purification requirements under the industrial production condition.
Based on this, there is still a need for a method for extracting lactoferrin from milk which is suitable for industrial grade.
Disclosure of Invention
Aiming at least part of the problems in the prior art, the inventor conducts intensive research to find that a specific extraction step is adopted, and by controlling the chromatographic conditions in the extraction process, a method suitable for extracting the lactoferrin from the milk in industrial grade is established, and the purity and the yield of the extracted lactoferrin are greatly improved. In addition, the method of the invention avoids the use of large complex equipment, and simultaneously realizes the extraction of low-cost lactoferrin through simple extraction and purification. The present invention has been completed based at least in part on this finding. Specifically, the present invention includes the following.
The invention provides a method for extracting lactoferrin from milk, which at least comprises the following steps:
(a) Passing fresh skim milk directly through a chromatographic column packed with cation exchange packing having an average particle size of 60-500 μm and a pore size of 100-500nm;
(b) Washing residual milk and impurities with water and an aqueous solution containing a first concentration of salt;
(c) Eluting with aqueous solution or buffer solution containing salt at a second concentration at a flow rate of 200-1000cm/h to obtain lactoferrin.
The method for extracting lactoferrin from milk according to the present invention preferably controls the column temperature of the chromatography column in the range of 30-60 ℃ in step (a).
The method for extracting lactoferrin from milk according to the present invention, preferably, further comprises (d) washing the chromatography column packed with cation exchange packing with 0.5-1M NaOH solution.
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%, 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%.
The method for extracting lactoferrin from milk according to the present invention, preferably, the cation exchange packing has a packing height of 100-500mm.
According to the method for extracting lactoferrin from milk of the present invention, preferably, the cation exchange filler is porous polymethacrylate spherical particles, and the surface thereof is provided with sulfonate groups.
According to the method for extracting lactoferrin from milk of the present invention, preferably, the degree of cross-linking of the cation exchange filler is 50% -80%.
The method for extracting lactoferrin from milk according to the present invention, preferably, the aqueous salt-containing solution is a sodium chloride solution.
The method for extracting lactoferrin from milk according to the present invention, preferably, the aqueous salt solution is a sodium sulfate solution.
The method according to the invention for extracting lactoferrin from milk, preferably, the second concentration is greater than the first concentration.
The method for extracting lactoferrin from milk according to the present invention is preferably phosphate buffer or sodium acetate buffer with pH 5.0-7.0.
The method according to the invention for extracting lactoferrin from milk, preferably skimmed milk.
The purity and the yield of the extracted lactoferrin are greatly improved by the method. The method is suitable for extracting the lactoferrin from the milk in an industrial level, avoids using large-scale complex equipment, and simultaneously realizes the extraction of the lactoferrin with low cost through simple extraction and purification.
Drawings
FIG. 1 is a purification scheme of an exemplary method according to the present invention.
Fig. 2 is an exemplary HPLC detection profile of a method according to the present invention.
Fig. 3 is another exemplary HPLC detection profile of a method according to the present invention.
Fig. 4 is another exemplary HPLC detection profile of a method according to the present invention.
FIG. 5 is an HPLC detection chart of the product obtained in comparative example 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
In the present invention, purity and content have the same meaning and are used interchangeably.
The present invention provides a method for extracting lactoferrin from milk, comprising at least steps (a) - (c). The milk is preferably skimmed milk, which may be low-fat milk obtained by semi-degreasing, or full-skimmed milk obtained by full-degreasing. When it is semi-skimmed milk it contains 1.0-1.5% fat, and when it is fully skimmed milk it contains less than 0.5% fat. In the present invention, the milk is directly used for the subsequent extraction of lactoferrin without performing pretreatment such as removal of certain specific proteins such as globulin, etc., to achieve reduction of time and cost of the extraction process.
In view of the large influence of the chromatographic conditions on the extraction and purification of lactoferrin, the chromatographic conditions will be described below, and include, but are not limited to, the selection of a chromatographic column, the packing of the chromatographic column, the elution flow rate, the eluent, and the like. It should be noted that lactoferrin extraction is a complex process, especially when it involves industrial-scale extraction of large quantities, it is necessary to consider how to achieve efficient extraction in the case of a large quantity of accumulation effect of the extract to be extracted and of high column pressure. Therefore, it is necessary to consider the influence of various factors on the purity and yield of lactoferrin as a whole. The column pressure here means an industrial column pressure, generally 2 to 4bar, preferably 3 to 4bar. As is known to the person skilled in the art, the column pressure is meant to include the pressure resulting from the filling of the milk to be extracted and from the pressure resulting from the certain flow rate of the eluent during elution, which is different from the column pressure used in conventional laboratories.
In step (a) of the present invention, fresh skim milk is passed directly through a chromatography column packed with a cation exchange filler having an average particle diameter of 60 to 500 μm and a pore diameter of 100 to 500nm. In the present invention, the inner diameter means the diameter of the inner circle of the column, and the inner diameter of the column is preferably 10 to 2000mm, more preferably 10 to 1800mm, still more preferably 600 to 1800mm, for example 800mm, 1000mm, 1200mm, 1400mm, 1600mm, etc. The filling height is preferably 100 to 500mm, more preferably 200 to 500mm, still more preferably 250 to 400mm.
The average particle diameter of the column packing of the present invention is 60 to 500. Mu.m, preferably 100 to 500. Mu.m, still preferably 100 to 400mm, further preferably 200 to 400mm. The filler preferably has a pore size of 100 to 500nm, more preferably 200 to 500nm, and still more preferably 300 to 400 nm.
Preferably, the filler of the present invention is a cation exchange filler, further preferably porous polymethacrylate spherical particles, and has sulfonate groups on the surface. It is also preferred that the degree of crosslinking of the cation exchange filler is 50% to 80%. Further preferably, the cation exchange filler has a degree of crosslinking of 60% to 75%. Too low a degree of crosslinking, the filler cannot withstand high pressure and deforms, even breaks. On the other hand, if the degree of crosslinking is too high, the flow rate of skim milk is affected, and the exchange is also adversely affected.
The chromatography temperature according to the invention is preferably a temperature of 30-60 ℃, and is also preferably a temperature of 30-45 ℃. For example, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, etc.
In step (b), the residual milk and impurities, in particular the residual milk and impurities adsorbed on the surface of the packing of the chromatographic column, are washed with water and an aqueous solution containing a first concentration of salt. The impurities are not particularly limited herein, and specific components thereof are known to those skilled in the art, and may be, for example, those determined by including national standard GB 5413.30-2016 or the optional national standard IRAM 14 008-1962. The specific step of washing may be, for example, washing the column with water first and then washing the column again with an aqueous solution containing a salt of a first concentration.
In step (c), lactoferrin is obtained by eluting with an aqueous solution or buffer containing a salt at a second concentration at a flow rate of 200-1000 cm/h. The flow rate of elution is preferably 210 to 1000cm/h, more preferably 300 to 900cm/h, still more preferably 400 to 800cm/h, for example 500cm/h, 600cm/h, 700cm/h, etc. Too small a flow rate increases the time of chromatography, and too large a flow rate results in too high column pressure and reduced loading, further affecting the purity and yield of extraction and purification.
The aqueous solution containing salt may be a sodium chloride solution or a sodium sulfate solution. Sodium chloride solution is preferred.
In the present invention, the second concentration is greater than the first concentration. Preferably, the first concentration refers to a concentration range of 0.1-0.5M. Also preferably, the first concentration refers to a concentration range of 0.2-0.4M. Further preferably, the first concentration refers to a concentration range of 0.2-0.3M.
Preferably, the second concentration refers to a concentration range of 0.6-2M. Also preferably, the second concentration refers to a concentration range of 0.6-1M. Further preferably, the second concentration refers to a concentration range of 0.8-1M.
In the present invention, the buffer is phosphate buffer or sodium acetate buffer having a pH of 5.0 to 7.0. The pH has an influence on the elution, for example it may influence the time of lactoferrin elution. Preferably, the pH is from 5.5 to 6.5.
The process of the present invention further comprises the step (d) of washing the regenerated chromatography column packed with cation exchange packing with 0.5-1M NaOH solution. Step (d) of the present invention is used to elute lactoferrin that has not been eluted by the previous step. Preferably, the NaOH solution is 0.5-0.6M.
The purity of the lactoferrin extracted from the milk by the method is not lower than 92, and the yield of the lactoferrin is not lower than 60%. Preferably, the purity of the lactoferrin extracted from the milk is not lower than 93%, for example 94%, 95%, 96% and even 97%. Preferably, the yield of lactoferrin is not lower than 73%, for example 82%, 83%, 84%, 85%, 86%, 87%, even 88%, 89% or 90%. In the prior art, for example, the separation and purification process of lactoferrin disclosed in CN1486989 is low in purity and yield when the flow rate is low and heparin-agarose chromatography column is used. Therefore, the process of the present invention has excellent extraction effect.
It is noted that other steps or operations may be included before, after, or between steps (a) - (d) of the present invention, such as further optimizing and/or improving the methods described herein. For example, a buffer is used to equilibrate the packing. As another example, a step of washing the regenerated filler with a higher concentration NaOH solution.
Example 1
The reagents used in the following examples of the present invention were all chromatographically pure and the remaining drugs were analytically pure unless otherwise indicated.
This example is a method for extracting lactoferrin from milk, specifically as follows:
1) Passing fresh skimmed milk through a chromatographic column filled with cation exchange filler, wherein the cation exchange filler is porous polymethacrylate spherical particles, the surface of the cation exchange filler is provided with sulfonate groups, and the crosslinking degree of the cation exchange filler is 50-80%;
2) Cleaning residual milk and impurities (sodium chloride or sodium sulfate solution) with water and saline solution;
3) Washing lactoferrin with aqueous solution or buffer solution (such as phosphate buffer solution and sodium acetate buffer solution, pH of 5.0-7.0), and collecting for use;
4) The chromatographic column filled with the cation exchange packing is washed and regenerated by 0.1-1M NaOH solution.
Wherein, the inner diameter of the chromatographic column is 10mm, the height of the filled cation exchange filler is 30cm, the linear flow rate is 600cm/h, 3L of skim milk is pumped into the chromatographic column by a pump, and after washing impurities by water and 0.3M sodium chloride solution, the lactoferrin is eluted by 2M sodium chloride solution, thus obtaining the product with the purity of 95.13 percent and the yield of 87 percent. The purification pattern is shown in figure 1, and the HPLC detection pattern is shown in figure 2.
Example 2
This example illustrates a process for extracting lactoferrin from milk, unlike example 1, in which the column inner diameter is 600mm, the height of the packed cation exchange packing is 50cm, the linear flow rate is 800cm/h, 500 liters of skim milk is pumped into the column by a pump, after washing with water and 0.2M sodium chloride solution, lactoferrin is eluted with 1M sodium chloride solution, and the remaining steps are the same as in example 1. The purity of the obtained product is 94.40 percent and the yield is 73 percent. The HPLC detection pattern is shown in FIG. 3.
Example 3
This example illustrates a process for extracting lactoferrin from milk, unlike example 1, in which the column inner diameter is 1800mm, the height of the packed cation exchange packing is 30cm, the linear flow rate is 700cm/h, 300 tons of skim milk are pumped into the column by a pump, after washing with water and 0.25M sodium chloride solution, lactoferrin is eluted with 0.6M sodium chloride solution, and the rest of the steps are the same as in example 1. The purity of the obtained product is 92.56 percent and the yield is 86 percent. The HPLC detection pattern is shown in FIG. 4.
Comparative example 1
The inner diameter of the chromatographic column is 16mm, the height of the filled cation exchange filler is 20cm, the linear flow rate is 500cm/h, the cation exchange filler of the commercial agarose gel matrix with large particle size is adopted, 6L of skimmed milk is pumped into the chromatographic column, the skimmed milk is subjected to 0.4M sodium chloride solution, and then 1.5M sodium chloride solution is used for eluting lactoferrin, so that the product purity 79.33 is obtained, the yield is 79%, and the HPLC detection spectrum is shown in figure 4.
Comparative example 2
The column inner diameter is 10mm, the commercial polymer matrix cation exchange filler (particle diameter is 80 microns, aperture is 100 nm) is filled, the height is 30cm, linear flow rate is 600cm/h, 1L of skimmed milk is pumped into the column by a pump, the column pressure is increased from 4bar to 7bar, and the experiment fails due to exceeding the alarm pressure of the chromatography system.
While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. A method for extracting lactoferrin from milk, comprising the steps of:
(a) Passing fresh skim milk directly through a chromatographic column packed with cation exchange packing having an average particle size of 60-500 μm and a pore size of 100-500nm;
(b) Washing residual milk and impurities with water and an aqueous solution containing a first concentration of salt;
(c) Eluting with aqueous solution or buffer solution containing salt at a second concentration at a flow rate of 200-1000cm/h to obtain lactoferrin.
2. The method for extracting lactoferrin from milk as in claim 1, further comprising (d) washing the regenerated cation exchange packed chromatography column with 0.5-1M NaOH solution.
3. The method for extracting lactoferrin from milk as claimed in claim 1, wherein the purity of the lactoferrin extracted from milk is not less than 92% and the yield of the lactoferrin is not less than 60%.
4. The method for extracting lactoferrin from milk as in claim 1, wherein the packing height of the cation exchange packing is 100-500mm.
5. The method for extracting lactoferrin from milk as in claim 1, wherein the cation exchange filler is porous polymethacrylate spherical particles with sulfonate groups on the surface.
6. The method for extracting lactoferrin from milk as claimed in claim 5, wherein the cross-linking degree of the cation exchange packing is 50-80%.
7. The method for extracting lactoferrin from milk as in claim 1, wherein the aqueous salt solution is sodium chloride or sodium sulfate solution.
8. The method for extracting lactoferrin from milk as in claim 1, wherein the second concentration is greater than the first concentration.
9. The method for extracting lactoferrin from milk as in claim 1, wherein the buffer is phosphate buffer or sodium acetate buffer with pH 5.0-7.0.
10. Method for extracting lactoferrin from milk as in any one of the claims 1 to 9, characterized in that the milk is skimmed milk.
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