CN113278063A - Method for obtaining lactoferrin from colostrum by ultrafiltration - Google Patents
Method for obtaining lactoferrin from colostrum by ultrafiltration Download PDFInfo
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- CN113278063A CN113278063A CN202110523613.3A CN202110523613A CN113278063A CN 113278063 A CN113278063 A CN 113278063A CN 202110523613 A CN202110523613 A CN 202110523613A CN 113278063 A CN113278063 A CN 113278063A
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Abstract
The invention discloses a method for obtaining lactoferrin from colostrum by using an ultrafiltration method, and belongs to the technical field of food. The invention discloses a method for obtaining lactoferrin from colostrum by using an ultrafiltration method, which is characterized in that whey containing various protein components such as lactoferrin is obtained from colostrum by a conventional acid precipitation method; the lactoferrin with higher purity is obtained by the ultrafiltration of two cascaded filter membranes. The invention also adopts physiological buffer solution with different pH values to flush the two-stage filter membrane, thereby not only preventing the membrane from being blocked, but also facilitating the automatic integrated assembly of two-stage ultrafiltration equipment, facilitating the separation and removal of immunoglobulin compounds, and simultaneously avoiding the influence of washing solution such as strong alkali and the like added to the separation components; and the separated immunoglobulin compound can be recycled, so that waste is avoided, and the economic benefit is improved.
Description
Technical Field
The invention relates to the technical field of food, in particular to a method for obtaining lactoferrin from mammal colostrum by using an ultrafiltration method.
Background
Lactoferrin (Lactoferrin, LF) is a multifunctional protein that can be used as a nutritional source for humans to supplement iron and amino acids, as well as a drug for the prevention and treatment of various human diseases, for maintaining the flora balance in the intestinal tract, preventing infection and virus resistance, inhibiting tumorigenesis and metastasis, and preventing free radicals produced in the body from damaging the body. Therefore, lactoferrin has been considered as a safe biogenic protein, has no significant side effects, and can be widely used as an additive in the fields of foods, medical treatment, and cosmetics.
Lactoferrin is widely distributed in mammalian milk and in various other tissues and secretions; in human colostrum, the content is as high as 7g/L, and the content in the mature milk is maintained at about 1 g/L. In addition, lactoferrin is present in small amounts in the secretory fluids such as bile, pancreatic juice, intestinal juice, saliva, and tears. Lactoferrin is a single polypeptide chain, with two ends each folded into a ring, and iron ions accompanied by 2 bicarbonate ions (HCO)3-) are bound to the two terminal loop regions, forming a coupling with 4 amino acid residues, including 2 tyrosines, 1 aspartic acid and 1 histidine. The lactoferrin is glycoprotein, the isoelectric point is 8.7, the isoelectric point and the molecular weight fluctuate due to different iron combination and glycosylation degrees, and the molecular weight is maintained between 75000 Da and 82600 Da.
Colostrum is the milk secreted by the mother one week after delivery, and is rich in various functional factors such as immunoglobulin, lactoferrin, lactoperoxidase, lysozyme, nucleotides and various growth factors in addition to lactoferrin. Scientists montereuil.j and johanson.b, equal to 1960, isolated lactoferrin from breast milk, followed by extensive scientific research and gradually discovered various isolation and purification methods. At present, several separation methods mainly include salting out and organic solvent precipitation methods, ion exchange chromatography, immobilized monoclonal antibody affinity adsorption methods, metal ion chelation separation methods, and ultrafiltration methods.
However, the currently established ultrafiltration methods have various problems: (1) the ultrafiltration membrane is easy to block, and a washing liquid containing strong base is introduced into the cleaning method; (2) starting from the filter membrane with small aperture, the lactoferrin is trapped on the upper layer of the membrane, so that the lactoferrin is inconvenient to transfer to the next filter membrane for filtration, and the automatic multi-stage filtration is difficult to realize; (3) because no means such as pH value adjustment is adopted, immunoglobulin polymers are excluded, so that a large amount of immunoglobulin is mixed in the separated lactoferrin.
Therefore, the problem to be solved by those skilled in the art is to provide a method for obtaining lactoferrin from colostrum by ultrafiltration.
Disclosure of Invention
In view of the above, the present invention provides a method for obtaining lactoferrin from colostrum by ultrafiltration, and lactoferrin with higher purity is obtained by ultrafiltration with cascaded two-stage filter membranes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for obtaining lactoferrin from colostrum by using an ultrafiltration method comprises the following specific steps:
(1) degreasing: centrifuging the primary emulsion, removing upper fat, and collecting skim milk;
(2) obtaining a whey protein sample by an acid precipitation method; adjusting the pH of the skim milk to 4.6;
(3) and (3) secondary ultrafiltration purification of lactoferrin:
firstly, washing a whey protein sample with physiological saline to obtain a mixed solution; then the pH value of the mixed solution is adjusted to 6.8-7.4 by 1mol/L NaOH (food grade); filtering the mixed solution by using a filter membrane for intercepting the molecular weight of 90-120kDa to obtain primary filtrate, wherein the molecular weight of protein in the intercepted solution is less than 90 kDa; the addition amount of the normal saline is 0.5-3.0 times of the volume of the lactalbumin sample, and the pressure difference parameter of two sides of the membrane is 0.04-0.15 Mpa; aims at filtering and removing macromolecular proteins such as immunoglobulin polymer and the like;
flushing the primary filtrate with normal saline to obtain a mixed solution, adjusting the pH of the mixed solution to 7.0-8.5, and filtering the mixed solution through a filter membrane with the molecular weight cutoff of 60-80KDa to obtain a concentrated cutoff solution; the molecular weight range of the protein in the trapped fluid is concentrated at 80 +/-5 kDa; the addition amount of normal saline is 0.5-2.0 times of the volume of primary filtrate, and the pressure difference parameter at two sides of the membrane is 0.04-0.10 Mpa; the aim is to remove whey proteins of small molecular weight;
(4) freeze drying of lactoferrin: drying the concentrated trapped fluid by a freeze dryer at the temperature of minus 40 ℃ to 10 ℃ to obtain the lactoferrin.
Further, the centrifugation condition in the step (1) is 3000-.
Further, the specific steps of obtaining the whey protein sample by the acid precipitation method in the step (2) are as follows: adjusting pH of the skim milk to 4.6 with 1mol/L HCl (food grade), centrifuging at 4 deg.C for 20min at 5000r/min, and collecting supernatant as whey protein sample obtained by acid precipitation.
Further, in the step (3), the mixed solution is filtered by a filter membrane with the molecular weight of 100kDa, and primary filtrate is obtained.
Further, filtering with a filter membrane with the molecular weight cutoff of 70KDa in the step (3) to obtain concentrated cutoff liquid.
The separation principle of the ultrafiltration method is as follows: the molecular weight range of lactoferrin is maintained at 80 ± 5kDa under specific pH conditions to distinguish between other protein components. Based on the method, the liquid can pass through filter membranes with different continuous pore sizes to separate and obtain the lactoferrin.
According to the technical scheme, compared with the prior art, the invention discloses a method for obtaining lactoferrin from colostrum by using an ultrafiltration method, whey is obtained from colostrum by using a conventional acid precipitation method, and the whey contains a plurality of protein components such as lactoferrin; the lactoferrin with higher purity is obtained by the ultrafiltration of two cascaded filter membranes. The invention mainly improves the prior method that the retained component is obtained by a filter membrane with small aperture and then the lactoferrin is obtained from the retained component. The invention innovatively firstly obtains the filtered components through a large-aperture filter membrane, and then obtains the lactoferrin from the filtered solution, thereby facilitating two-step filtration cascade in the process to form the automation of industrial production.
In addition, the pH of the skim milk is adjusted to 4.6, which is important for obtaining lactoferrin through ultrafiltration separation in the later period; the invention also adopts physiological buffer solution with different pH values to flush the two-stage filter membrane, thereby not only preventing the membrane from being blocked, but also facilitating the automatic integrated assembly of two-stage ultrafiltration equipment, facilitating the separation and removal of immunoglobulin compounds, and simultaneously avoiding the influence of washing solution such as strong alkali and the like added to the separation components; and the separated immunoglobulin compound can be recycled, so that waste is avoided, and the economic benefit is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a process for the isolation of lactoferrin from colostrum in accordance with example 1 of the present invention;
FIG. 2 is a schematic diagram showing the composition and ultrafiltration separation of colostrum whey protein according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for obtaining lactoferrin from colostrum by ultrafiltration (see figure 1 for process flow) comprises the following steps:
(1) degreasing: centrifuging bovine colostrum at 4 deg.C for 20min at 3000r/min, removing upper layer fat, and collecting skimmed milk;
(2) obtaining a whey protein sample by an acid precipitation method: adjusting the pH of the skim milk to 4.6 by using 1mol/L HCl, centrifuging the skim milk at the temperature of 4 ℃ for 20min at 5000r/min, and collecting supernatant, namely a whey protein sample obtained by an acid precipitation method;
(3) and (3) secondary ultrafiltration purification of lactoferrin:
firstly, washing a whey protein sample with physiological saline to obtain a mixed solution; then adjusting the pH value of the mixed solution to 6.8; filtering the mixed solution by using a filter membrane with the molecular weight of 100kDa to obtain primary filtrate; the addition amount of the normal saline is 0.5 times of the volume of the whey protein sample, and the pressure difference parameter at two sides of the membrane is 0.04 Mpa;
flushing the primary filtrate with normal saline to obtain a mixed solution, adjusting the pH of the mixed solution to 7.0, and filtering the mixed solution through a filter membrane with the molecular weight cutoff of 70KDa to obtain a concentrated cutoff solution; the addition amount of the normal saline is 0.5 times of the volume of the primary filtrate, and the pressure difference parameter at two sides of the membrane is 0.04 Mpa; the composition and ultrafiltration separation protocol of the proteins in colostrum whey is shown in fig. 2;
(4) freeze drying of lactoferrin: drying the concentrated trapped fluid with a freeze dryer at-40 deg.C to obtain lactoferrin.
Wherein, the yield of each liter of bovine colostrum immunoglobulin and the yield of each liter of bovine colostrum lactoferrin are respectively shown in table 1 and table 2.
TABLE 1 yield of bovine colostrum immunoglobulin per liter
TABLE 2 yield of bovine colostrum lactoferritin per liter
Example 2
A method for obtaining lactoferrin from colostrum by using an ultrafiltration method comprises the following specific steps:
(1) degreasing: centrifuging bovine colostrum at 4 deg.C for 15min at 5000r/min, removing upper layer fat, and collecting skimmed milk;
(2) obtaining a whey protein sample by an acid precipitation method: adjusting the pH of the skim milk to 4.6 by using 1mol/L HCl, centrifuging the skim milk at the temperature of 4 ℃ for 20min at 5000r/min, and collecting supernatant, namely a whey protein sample obtained by an acid precipitation method;
(3) and (3) secondary ultrafiltration purification of lactoferrin:
firstly, washing a whey protein sample with physiological saline to obtain a mixed solution; then adjusting the pH value of the mixed solution to 7.4; filtering the mixed solution by using a filter membrane with the molecular weight of 90kDa to obtain primary filtrate; the addition amount of the normal saline is 2.0 times of the volume of the whey protein sample, and the pressure difference parameter at two sides of the membrane is 0.1 Mpa;
flushing the primary filtrate with normal saline to obtain a mixed solution, adjusting the pH of the mixed solution to 8.0 ℃, and filtering the mixed solution through a filter membrane with the molecular weight cutoff of 60KDa to obtain a concentrated cutoff solution; the addition amount of the normal saline is 1.0 time of the volume of the primary filtrate, and the pressure difference parameter at two sides of the membrane is 0.08 Mpa;
(4) freeze drying of lactoferrin: drying the concentrated trapped fluid with a freeze dryer at-20 deg.C to obtain lactoferrin.
Example 3
A method for obtaining lactoferrin from colostrum by using an ultrafiltration method comprises the following specific steps:
(1) degreasing: centrifuging bovine colostrum at 4 deg.C at 4000r/min for 18min, removing upper layer fat, and collecting skimmed milk;
(2) obtaining a whey protein sample by an acid precipitation method: adjusting the pH of the skim milk to 4.6 by using 1mol/L HCl, centrifuging the skim milk at the temperature of 4 ℃ for 20min at 5000r/min, and collecting supernatant, namely a whey protein sample obtained by an acid precipitation method;
(3) and (3) secondary ultrafiltration purification of lactoferrin:
firstly, washing a whey protein sample with physiological saline to obtain a mixed solution; then adjusting the pH value of the mixed solution to 7.1; filtering the mixed solution by using a filter membrane with the molecular weight of 120kDa to obtain primary filtrate; the addition amount of the normal saline is 3.0 times of the volume of the whey protein sample, and the pressure difference parameter at two sides of the membrane is 0.15 Mpa;
flushing the primary filtrate with normal saline to obtain a mixed solution, adjusting the pH of the mixed solution to 8.5, and filtering the mixed solution through a filter membrane with the molecular weight cutoff of 80KDa to obtain a concentrated cutoff solution; the addition amount of the normal saline is 2.0 times of the volume of the primary filtrate, and the pressure difference parameter at two sides of the membrane is 0.10 Mpa;
(4) freeze drying of lactoferrin: drying the concentrated trapped solution at 10 deg.C with a freeze dryer to obtain lactoferrin.
EXAMPLES 1-3 bacteriostatic action of Lactoferrin (LF) samples isolated
The oxford cup method is adopted. Inoculating the slant strain into broth peptone culture solution for amplification for 16-20 h, counting nutrient agar culture medium plates, and adjusting the concentration of the bacterial suspension to 106～108CFU/ml. Camp inAnd inoculating a bacterial suspension of the test strain in the logarithmic growth phase on the agar culture medium, and uniformly coating. A sterile Oxford cup was placed in the center of the petri dish, and 0.1ml of LF solution (pH 7.0) was added to the cup in different dilutions with sterile microfiltration, against sterile water. After the culture dish is placed in a constant-temperature incubator at 37 ℃ for 24 hours, the diameter (mm) of the inhibition zone is measured. The lowest sample concentration greater than the diameter (8mm) of the zone of inhibition of the control group is the minimum inhibitory concentration (MIC, g/ml).
Preparing a lactoferrin test solution: respectively weighing 50mg of the lactoferrin sample separated in the embodiment 1-3, adding 50mL of PBS buffer solution (0.24 g of monopotassium phosphate, 1.44g of disodium phosphate, 8g of sodium chloride and 0.2g of potassium chloride) into about 800mL of deionized water, fully stirring and dissolving, then adding concentrated hydrochloric acid to adjust the pH value to 7.4, finally fixing the volume to 1L, dissolving at the pH value of 7.4, and preparing a mother solution (the concentration is 1 mg/mL); when the bacteriostatic test is carried out, deionized water is used for diluting to a certain highest concentration to be measured, namely 200g/ml, and then 2 times of deionized water is used for sequentially diluting.
The results of the bacteriostatic action of the Lactoferrin (LF) samples isolated in examples 1-3 are shown in tables 3-5.
TABLE 3 diameter (mm) of zone of inhibition of LF on different microorganisms isolated in example 1
Note: MIC is the minimum inhibitory concentration; the diameter of the control group bacteriostasis zone is 8 mm.
TABLE 4 diameter of zone of inhibition (mm) of LF against different microorganisms isolated in example 2
Note: MIC is the minimum inhibitory concentration; the diameter of the control group bacteriostasis zone is 8 mm.
TABLE 5 diameter (mm) of zone of inhibition of LF on different microorganisms isolated in example 3
Note: MIC is the minimum inhibitory concentration; the diameter of the control group bacteriostasis zone is 8 mm.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A method for obtaining lactoferrin from colostrum by using an ultrafiltration method is characterized by comprising the following specific steps:
(1) degreasing: centrifuging the primary emulsion, removing upper fat, and collecting skim milk;
(2) obtaining a whey protein sample by an acid precipitation method; adjusting the pH of the skim milk to 4.6;
(3) and (3) secondary ultrafiltration purification of lactoferrin:
firstly, washing a whey protein sample with physiological saline to obtain a mixed solution; then adjusting the pH value of the mixed solution to 6.8-7.4; filtering the mixed solution by using a filter membrane for intercepting the molecular weight of 90-120kDa to obtain primary filtrate; the addition amount of the normal saline is 0.5-3.0 times of the volume of the lactalbumin sample, and the pressure difference parameter of two sides of the membrane is 0.04-0.15 Mpa;
flushing the primary filtrate with normal saline to obtain a mixed solution, adjusting the pH of the mixed solution to 7.0-8.5, and filtering the mixed solution through a filter membrane with the molecular weight cutoff of 60-80KDa to obtain a concentrated cutoff solution; the addition amount of normal saline is 0.5-2.0 times of the volume of primary filtrate, and the pressure difference parameter at two sides of the membrane is 0.04-0.10 Mpa;
(4) freeze drying of lactoferrin: drying the concentrated trapped fluid by a freeze dryer at the temperature of minus 40 ℃ to 10 ℃ to obtain the lactoferrin.
2. The method for extracting lactoferrin from colostrum by ultrafiltration according to claim 1, wherein the centrifugation conditions in step (1) are 3000-.
3. The method for extracting lactoferrin from colostrum by ultrafiltration according to claim 1, wherein the acid precipitation method in step (2) is to obtain a whey protein sample by the following steps: adjusting the pH of the skim milk to 4.6 by using 1mol/L HCl, centrifuging the skim milk at the temperature of 4 ℃ for 20min at the speed of 5000r/min, and collecting supernatant, namely the whey protein sample obtained by the acid precipitation method.
4. The method for extracting lactoferrin from colostrum by ultrafiltration according to claim 1, wherein in step (3), the mixture is filtered with a filter membrane that retains 100kDa molecular weight to obtain a primary filtrate.
5. The process of claim 1, wherein step (3) is performed by filtration through a filter membrane having a cut-off molecular weight of 70KDa to obtain a concentrated retentate.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724013A (en) * | 2008-10-24 | 2010-06-09 | 哈尔滨康普乳品有限公司 | Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way |
| US20150315264A1 (en) * | 2012-10-08 | 2015-11-05 | Murray Goulburn Co-Operative Co. Limited | Process for purifying lactoferrin from milk and products thereof |
| CN110964095A (en) * | 2019-12-26 | 2020-04-07 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in raw fresh milk |
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2021
- 2021-05-13 CN CN202110523613.3A patent/CN113278063A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724013A (en) * | 2008-10-24 | 2010-06-09 | 哈尔滨康普乳品有限公司 | Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way |
| US20150315264A1 (en) * | 2012-10-08 | 2015-11-05 | Murray Goulburn Co-Operative Co. Limited | Process for purifying lactoferrin from milk and products thereof |
| CN110964095A (en) * | 2019-12-26 | 2020-04-07 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in raw fresh milk |
Non-Patent Citations (3)
| Title |
|---|
| RONG RONG LU等: "Isolation of lactoferrin from bovine colostrum by ultrafiltration coupled with strong cation exchange chromatography on a production scale", 《JOURNAL OF MEMBRANE SCIENCE》 * |
| 于长青等: "超滤技术制备乳铁蛋白制品工艺的研究", 《食品工业》 * |
| 刘江丽: "牛初乳中乳铁蛋白分离纯化的产业化技术参数研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
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