CN115353560A - Separation preparation method of lactoferrin in raw milk - Google Patents
Separation preparation method of lactoferrin in raw milk Download PDFInfo
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- CN115353560A CN115353560A CN202211014115.7A CN202211014115A CN115353560A CN 115353560 A CN115353560 A CN 115353560A CN 202211014115 A CN202211014115 A CN 202211014115A CN 115353560 A CN115353560 A CN 115353560A
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Images
Classifications
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- 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
Abstract
The invention relates to the field of lactoferrin separation, and discloses a method for separating lactoferrin from raw milk, which comprises the following steps: heating fresh raw milk, and separating by a centrifugal machine to remove fat; filtering the skim milk to remove impurities; carrying out heat preservation treatment on the filtrate, then feeding the filtrate into a PP chromatography system, and conveying the filtrate into a protein chromatography column for chromatography treatment; filtering water, eluent and buffer solution with filter membrane; eluting with sodium chloride solution to remove impurities, eluting to separate lactoperoxidase, separating lactoferrin, and eluting target protein; carrying out sample desalting and concentration treatment; the desalted and concentrated eluent is sterilized by a protein solution sterile filtration system; and (3) putting the liquid sample into a low-temperature freeze drying device for freeze drying. The invention solves the problems of low purity of the lactoferrin in raw milk and low activity of the separated protein in the prior art.
Description
Technical Field
The invention belongs to the field of lactoferrin separation, and particularly relates to a method for separating and preparing lactoferrin from raw milk.
Background
Lactoferrin has a wide range of biological activities, such as broad-spectrum antibacterial activity, promotion of iron transfer, anti-inflammation, promotion of cell proliferation, and immunomodulation. Is widely applied to infant milk powder, beverage, skin care products and animal feed at present. In the last 60 th century, the extraction of lactoferrin began by studying various methods. The industrial production process and technical parameters are determined according to the purity, recovery rate, processing process, production cost and other factors of lactoferrin extracted by laboratory experiments. For a long time, the raw material of lactoferrin comes from dairy products manufacturing countries such as the United states, the Netherlands, australia, new Zealand and the like. Due to the difference of industrial production raw materials and processing technology of lactoferrin, the purity of products in various countries is greatly different. Netherlands DMV company is a lactoferrin supplier with the largest European sales volume, most of the lactoferrin added in the milk powder production enterprises in the world is supplied by the supplier, and the purity of the target protein of the product can reach more than 95%. The lactoferrin industrialization technology in China is still in a blank stage, and the main reasons are the limit of various problems such as how to utilize byproducts, immature industrialization separation technology of other milk components, insufficient supply of raw milk, high price, standard regulations and the like. Along with the richness of milk sources, the perfection of laws and regulations, the development of technologies, and the promotion of people on health and international situation awareness in China, the industrialized separation technology of lactoferrin in China is imperative in the future.
The content of lactoferrin in fresh milk is about 0.1mg/mL, the highest content in bovine colostrum is generally 0.8-1g/L, and the content in breast milk is higher than that in bovine colostrum and reaches 1-3 times of the content. Lactoferrin has a molecular weight of about 80KD and is a heat labile protein, and heating above 60 degrees celsius results in a different degree of loss of activity. The fresh raw milk has pH of 6.6 and the isoelectric point of lactoferrin is 7.8-8.2, so that the lactoferrin is positively charged in the milk, the lactoferrin is easily combined with components with acidic characteristics due to high electropositivity, and the molecular weight and the isoelectric point of lactoperoxidase are similar to those of lactoperoxidase, so that the target protein with high purity, high recovery rate and high activity is difficult to separate and prepare. According to research, the separation preparation method comprises ultrafiltration membrane filtration, electrodialysis, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography, membrane and chromatography technology combination and other separation technologies. However, methods such as affinity chromatography have high cost and low yield, purity and recovery rate, and thus industrialization cannot be realized. Ion exchange chromatography combined with membrane desalination and concentration is one of the most effective and widely applied methods for separating and purifying active protein.
Disclosure of Invention
The invention solves the technical problems that the separation purity of lactoferrin in raw milk is low and the activity of the separated protein is low in the prior art.
The invention adopts the specific scheme that: a separation preparation method of lactoferrin in raw milk comprises the following steps:
(1) Pretreatment of raw milk: heating fresh raw milk, and separating by a centrifugal machine to remove fat;
(2) Filtering skim milk: filtering the skim milk to remove impurities;
(3) Loading: carrying out heat preservation treatment on the filtrate in the step (2), then feeding the filtrate into a PP chromatography system, and conveying the filtrate into a protein chromatography column for chromatography treatment;
(4) Filtering the buffer solution: filtering water, eluent and buffer solution with filter membrane;
(5) And (3) elution: eluting with sodium chloride solution to remove impurities, eluting to separate lactoperoxidase, separating lactoferrin, and eluting target protein;
(6) Desalting and concentrating: carrying out sample desalting and concentration treatment;
(7) And (3) microfiltration sterilization: the desalted and concentrated eluent is sterilized by a protein solution sterile filtration system;
(8) And (3) freeze drying: and (5) putting the liquid sample in the step (7) into low-temperature freeze drying equipment for freeze drying.
The temperature of the raw milk in the step (1) is 45-55 ℃.
The centrifugal rotating speed in the separation process of the step (1) is 11000rpm.
The freeze-drying temperature of the freeze-drying in the step (8) is-50 ℃ to 40 ℃.
The freeze drying process is divided into two processes, wherein the first stage is a pre-freezing stage, the temperature is reduced from room temperature to minus 50 ℃, vacuum sublimation is carried out to the second stage, the temperature of the second stage is increased from minus 50 ℃ to 40 ℃, and the total freeze drying time of the two stages is 48 hours.
And (4) performing microfiltration sterilization in the step (7) by adopting a protein solution sterile filtration system and a PES (polyether sulfone) membrane with a pore size of 0.22 um.
The desalting and concentrating in the step (6) adopts a full-automatic protein ultrafiltration system, and a PES (polyether sulfone) filter membrane flat ultrafiltration membrane is used for sample desalting and concentrating.
And (3) eluting with 0.1mol/L sodium chloride solution to remove impurities in the elution process in the step (5), eluting and separating lactoperoxidase with 0.5mol/L sodium chloride solution, separating lactoferrin with 1.1mol/L sodium chloride, and eluting the target protein at the elution flow rate of 100-200cm/h.
The step (4) buffer eluent and buffer were filtered through a 10 inch 0.45 micron pore size PES filter membrane.
And (4) enabling the filtrate in the step (3) to enter a chromatography system at room temperature, setting the system pressure to be 0.5MPa, balancing a chromatography column by using more than two-stage purified water, setting the linear flow rate to be 600cm/h, adding sodium chloride into a PB buffer solution to respectively prepare 0.4 eluent and 1.0mol/L eluent with the pH value of 7, filtering the eluates by using a PES (polyether sulfone) filter membrane, and eluting the adsorbed alkaline protein at the elution flow rate of 200cm/h. The invention has the following beneficial effects:
the method takes the raw fresh milk as the raw material, the lactoferrin is prepared by chromatographic separation and membrane concentration, the skim milk is injected at room temperature, the lactoferrin is washed at low speed by adopting the concentration of a buffer solution to remove impurities, the purity of the obtained lactoferrin reaches more than 95 percent, and the yield reaches more than 75 percent.
Drawings
FIG. 1 is a standard curve of lactoferrin in the present invention;
FIG. 2 is a graph showing the influence of temperature on the adsorption rate of a standard solution of a target protein in the present invention;
FIG. 3 is a graph showing the effect of the flow rate of a sample applied on the adsorption rate of a target protein in the present invention;
FIG. 4 is a graph of the effect of sample loading flow rate on system pressure in the present invention;
FIG. 5 is a gradient elution profile of LF standard solution in accordance with the present invention;
FIG. 6 is a graph of the gradient elution of LP standard solution in accordance with the present invention;
FIG. 7 is a graph of the gradient elution of the LP and LF mixed standard solutions of the present invention;
FIG. 8 is a graph of gradient elution of skim milk of the present invention;
FIG. 9 is a graph showing the results of gradient elution gel electrophoresis of skim milk according to the present invention;
FIG. 10 is a graph showing the very poor analysis of the concentration of the eluting salt in the present invention;
FIG. 11 is a very poor analysis of elution flow rate in the present invention;
FIG. 12 is a graph of the pH range analysis of eluents in the present invention;
FIG. 13 is a graph of the lactoferrin isolation preparation in the present invention;
FIG. 14 is an SDS-PAGE gel of flow-through and acceptance peaks according to the invention;
FIG. 15 is a high performance liquid chromatography assay result chart of lactoferrin in the present invention.
Detailed Description
In order that the objects, aspects and advantages of the invention will become more apparent, the invention will be described by way of example only, and in connection with the accompanying drawings. It is to be understood that this description is made only by way of example and not as a limitation on the scope of the invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
The invention discloses a separation preparation method of lactoferrin in raw milk, which comprises the following steps: (1) pretreatment of raw milk: heating fresh raw milk, and separating by a centrifugal machine to remove fat; (2) filtering skim milk: filtering the skim milk to remove impurities; (3) loading: carrying out heat preservation treatment on the filtrate in the step (2), then feeding the filtrate into a PP chromatography system, and conveying the filtrate into a protein chromatography column for chromatography treatment; (4) filtering the buffer solution: filtering water, eluent and buffer solution with filter membrane; (5) elution: eluting with sodium chloride solution to remove impurities, eluting to separate lactoperoxidase, separating lactoferrin, and eluting target protein; (6) desalting and concentrating: carrying out sample desalting and concentration treatment; (7) microfiltration sterilization: the desalted and concentrated eluent is sterilized by a protein solution sterile filtration system; (8) freeze drying: and (5) putting the liquid sample in the step (7) into low-temperature freeze drying equipment for freeze drying. The temperature of the raw milk in the step (1) is 45-55 ℃. The centrifugal rotating speed in the separation process of the step (1) is 11000rpm.
The freeze-drying temperature of the freeze-drying in the step (8) is-50 ℃ to 40 ℃.
The freeze drying process is divided into two processes, wherein the first stage is a pre-freezing stage, the temperature is reduced from room temperature to minus 50 ℃, vacuum sublimation is carried out to the second stage, the temperature of the second stage is increased from minus 50 ℃ to 40 ℃, and the total freeze drying time of the two stages is 48 hours.
And (4) performing microfiltration sterilization in the step (7) by adopting a protein solution sterile filtration system and a PES (polyether sulfone) membrane with a pore size of 0.22 um.
The desalting and concentrating in the step (6) adopts a full-automatic protein ultrafiltration system, and a PES (polyether sulfone) filter membrane flat ultrafiltration membrane is used for sample desalting and concentrating.
And (3) eluting with 0.1mol/L sodium chloride solution to remove impurities in the elution process in the step (5), eluting and separating lactoperoxidase with 0.5mol/L sodium chloride solution, separating lactoferrin with 1.1mol/L sodium chloride, and eluting the target protein at the elution flow rate of 100-200cm/h.
And (3) filtering water, eluent and the buffer solution by using a PES (polyether sulfone) filter membrane with the aperture of 10 inches and the aperture of 0.45 microns in the process of filtering the buffer solution in the step (4).
And (3) enabling the filtrate in the step (3) to enter a chromatography system at room temperature, setting the system pressure to be 0.5MPa, balancing a chromatography column by using more than two-stage purified water, setting the linear flow rate to be 600cm/h, adding sodium chloride into a PB buffer solution to respectively prepare 0.4 eluent and 1.0mol/L eluent with the pH value of 7, filtering the eluates by using a PES (polyether sulfone) filter membrane, and eluting the adsorbed alkaline protein at the elution flow rate of 200cm/h.
Example 1
(1) Pretreatment of raw milk: heating fresh raw milk, and separating by SPX (milk protein) milk fat centrifuge to remove fat, wherein the milk temperature is 45 ℃, the centrifugal rotation speed is 11000rpm, the inlet pressure is lower than 1.2bar, and the outlet pressure is lower than 3.5bar. The skimmed milk has fat content below 0.1%, average particle diameter (D90) less than 1um, and lactoferrin content greater than 0.1mg/ml.
The raw milk comes from Yuling pasture; the lactoferrin standard was from Sigma original leaves.
The instrument comprises the following steps: AKTAprocess10mm PP full-automatic protein chromatography system Cytiva; AKTApure 150M laboratory protein purification system Cytiva;16/200, 200/500 protein chromatography column Cytiva; JHTFF00011G skim milk depth filtration system: jilin Jinhang; SE15X milk fat separator SPX; JHTFF00013G buffer filtration system: jilin Jinhang; LGJ-100FXD low temperature freeze drying system Henan brother instrument equipment.
(2) Filtering skim milk: filtering skim milk with 10 μm-pore size filter membrane by using a skim milk deep filtration system JHTFF00011G to remove impurities such as fat. The recovery rate of the defatted milk protein can reach more than 95 percent.
(3) Loading: heating or keeping the temperature of the filtrate at about 50 ℃, feeding the filtrate into an AKTAprocess10mm PP-system, conveying the filtrate to a protein chromatography column (100/500), setting the pressure of the system to be 0.5MPa, balancing the volume of 3 columns by using more than two levels of purified water, and balancing the volume of the columns according to the conductivity and the ultraviolet absorption spectrum. The sample temperature is about 50 ℃, the linear flow rate is about 200cm/h, and the sample is balanced to have an ultraviolet absorption of 0 in 3 column volumes by purified water or buffer solution after passing through the sample.
(4) Filtering the buffer solution: the water, eluent and buffer solution were filtered through a 10 inch 0.45 micron pore size PES membrane at a flow rate based primarily on the system loading pressure.
(5) And (3) elution: eluting with 0.1mol/L sodium chloride solution to remove impurities, eluting with 0.5mol/L sodium chloride solution to separate lactoperoxidase, eluting with 1.1mol/L sodium chloride to separate lactoferrin, and eluting with target protein at an elution flow rate of 100cm/h.
(6) Desalting and concentrating: the full-automatic protein ultrafiltration system JHTFC 0002 is characterized in that a PES (polyether sulfone) filter membrane flat plate ultrafiltration membrane is made of a 30KD material for sample desalination and concentration, the flow rate of a sample is set according to the bearing pressure of the system, and the volume of purified water washing is 3 times. The volume was 3L collected and the protein recovery was over 95%. The protein solution is concentrated to 8%, and the removal rate of inorganic salt in the sample is higher than 95%.
(7) And (3) microfiltration sterilization: the desalted and concentrated eluate is sterilized by passing through a protein solution sterile filtration system JHTFF00010G and 0.22um pore size PES membrane. The sterilization rate reaches more than 99.9 percent, and the protein recovery rate can reach not less than 95 percent.
(8) And (3) freeze drying: putting the liquid sample into LGJ-100FXD low-temperature freeze drying equipment for freeze drying, wherein the freeze drying temperature is from-50 ℃ to 40 ℃, the first stage is a pre-freezing stage, the temperature is reduced from room temperature to-50 ℃, the vacuum sublimation is started from the second stage to the last stage, the freeze drying time is from-50 ℃ to 40 ℃, and the freeze drying time is 48 hours.
And (3) measuring the content of the lactoferrin prepared by the preparation method: refer to GB1903.17-2016 high performance liquid chromatography.
The lactoferrin prepared by the above preparation method was tested as follows:
selecting a gel according to the target protein, adding 30% of Acr/Bis (29: 1), 1.5M Tris-HCl (pH 8.8), 10% of SDS, distilled water, 10% of PAGE gel coagulant, and PAGE coagulant to prepare a gel. Add 1ml of a water-sealed upper layer. Add 30% Acr/Bis (29), 1.0M Tris-HCl (pH 6.8), 10% SDS, distilled water, 10% PAGE gel coagulant, PAGE gel coagulant to prepare separation gel. Adding SDS-PAGE protein loading buffer (5X) into the sample, wherein the ratio of the two is 4:1, boiling for 3-5min, and adding 5-10ul of sample and Marker into the sample tank.
Preparation of a lactoferrin standard curve: dissolving lactoferrin standard substance with primary pure water to 6 concentrations of 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1.0mg/ml, measuring absorbance at 280nm with pure water as blank by ultraviolet spectrophotometer, and making standard curve.
And (3) measuring the adsorption rate and the elution rate of the target protein: the lactoferrin and lactoperoxidase standard solution enters a chromatographic column under the conditions of certain temperature and flow rate, absorption peaks are observed, flow-through liquid is collected, and the light absorption value of the flow-through liquid is determined by an ultraviolet spectrophotometer. After the chromatographic column is balanced, sodium chloride buffer solution with certain concentration and pH value is used for elution, elution peaks are observed, elution liquid is collected, and the concentration of lactoferrin in the flow-through liquid and the elution liquid is determined by contrasting a standard curve.
Pretreatment of skim milk: heating fresh raw milk to 45-50 deg.C, centrifuging at 11000rpm/min with a milk fat centrifuge to defat, wherein the inlet pressure is lower than 1.2bar, and the outlet pressure is lower than 3.5bar, to reach fat content below 0.1%. Filtering the centrifuged skim milk by a filter membrane with the aperture of 10 microns to remove impurities such as fat and the like. And (3) vertically filtering the skim milk through a filter membrane with the aperture of 10 microns to remove impurities such as fat and the like. The adjustment of the filtration speed is determined by the maximum loading pressure of the filter membrane.
And (3) skim milk ion exchange adsorption and elution: the filtrate enters a chromatography system at room temperature, the pressure of the system is set to be 0.5MPa, the chromatography column is balanced by more than two levels of purified water, and the linear flow rate is set to be 600cm/h. And adding sodium chloride into the PB buffer solution to respectively prepare 0.4 and 1.0mol/L eluents with the pH value of 7, filtering the eluents by a PES (polyether sulfone) filter membrane, and eluting the adsorbed basic protein at the elution flow rate of 200cm/h.
Packing of chromatography columns of the pilot test and the pilot test: the Big Beads cation chromatographic packing is uniformly mixed before use, poured into a container, transferred into a chromatographic column through a funnel and compacted to prevent air bubbles from entering, and the packing length of the pilot chromatographic column packing is 10cm.
And (3) data analysis: all the tests are set for 3 times of repetition, variance analysis is carried out through data statistical analysis software IBM SPSS Statistics 20, the difference is obvious when p is less than 0.05, the statistical significance is achieved, and drawing is carried out through the software.
The result of the analysis of the sample injection temperature by single factor of variance is 14.437, which shows that the influence of temperature on the adsorption of lactoferrin is obvious. From the results in fig. 2, it can be seen that the adsorption rate of the sample has a significant tendency to decrease when the temperature reaches above 30 ℃, which may be related to the increase of the content of dissolved gas in the liquid after the temperature of the sample has increased. The gas affects the adsorption strength of the ion exchange packing to the target protein. The equipment must have the device that the bubble was discharged in the pilot scale production process, prevents that the bubble from getting into the chromatographic column, influences the adsorption efficiency of ion exchange chromatography filler. In addition, the viscosity of the skim milk is slightly reduced along with the rise of the loading temperature of the skim milk, but the difference of the pressure change of the system is not obvious, so that the raw milk can be injected at room temperature after being centrifugally skimmed, and the problem of incomplete equipment discharge caused by the entrance of a large amount of bubbles is prevented.
The production period is one of the key factors to be considered in the large-scale production, and analysis in table 1 shows that the influence of the sample loading flow rate on the lactoferrin adsorption rate is not obvious, because the sample loading flow rate cannot exceed 600cm/h at most under the pressure bearing limit of the system, and the retention time of the sample in the column can sufficiently allow the protein to be subjected to ion exchange with the chromatographic packing at the flow rate. But the sample-feeding flow rate of the skim milk has obvious influence on the pressure of the system, the structure of the agarose gel matrix is softer, the structural stability is obviously reduced after the pores are enlarged, the chromatographic column is blocked by structural breakage after the flow rate of the separation operation is increased, the phenomenon that the column pressure rises suddenly is caused, and in addition, the service life of the filler is reduced due to high pressure. Therefore, the higher flow rate should be selected by comprehensively considering the factors such as the system bearing pressure, the blockage of the continuous product material and the like. In the test, the linear flow rate of the sample is selected to be 400cm/h within the range of 0.5MPa of the highest pressure borne by the system.
TABLE 1 analysis of variance of the effect of sample loading flow rate on target protein adsorption Rate
TABLE 2 analysis of variance of the effect of sample loading flow rate on system pressure
In FIGS. 5, 6 and 7, gradient elution curves of lactoferrin, lactoperoxidase and two mixture standard solutions are shown, wherein the elution peaks are absorption values at 412 and 280nm ultraviolet wavelengths respectively. 280nm is the ultraviolet characteristic absorption wavelength of the milk protein. The study showed that 412nm is the UV characteristic absorption wavelength of lactoperoxidase. The comparison of the two graphs in fig. 5 and 6 shows that the concentrations of the elution salts of the two basic proteins are different, and the sequence of the concentrations of the elution buffer solution can be seen from fig. 7, wherein the lactoperoxidase is eluted by using sodium chloride with low concentration firstly, and the lactoferrin is eluted by using sodium chloride with high concentration secondly. The increase in salt concentration increases the dielectric constant D to reduce the interaction force. Although the isoelectric point of lactoferrin is lower than that of lactoperoxidase, that is, lactoferrin is less positively charged than lactoperoxidase. However, the elution results from the above two figures consistently indicate that the band of lactoferrin is more positive than lactoperoxidase at neutral pH, which may be related to the titration curves for both proteins. Through conductivity analysis, the hybrid protein, lactoperoxidase and lactoferrin respectively have maximum absorption peaks at sodium chloride concentrations of about 0.4mol/L and 1.0mol/L.
FIG. 8 is a gradient elution curve of skim milk, which is a graph showing the UV absorption values and conductance changes at 280nm and 412nm, respectively. It can be seen that there are 3 peaks in the elution process, wherein the 2 nd and 3 rd peaks are the same as the elution peaks of the mixed standard solution of lactoperoxidase and lactoferrin, and are absorption peaks at salt concentrations of about 0.4 and 1.0, respectively, and it can be seen that the peak appearing at a salt concentration of 0.4 has a characteristic absorption at 412nm, demonstrating that the second peak is lactoperoxidase, which is consistent with the gradient elution of the mixed standard solution of lactoferrin and lactoperoxidase and the standard solution of lactoperoxidase single protein. FIG. 9 shows SDS-PAGE gel electrophoresis of the eluate, wherein the second and third peaks have a single band with a molecular weight of about 78kD, which is consistent with lactoferrin with a molecular weight of 80kD and lactoperoxidase with a molecular weight of 76 kD. The absorption peak at the salt concentration of 0.15mol/L is other alkaline proteases in milk. The protein molecular weight is about 65KD in SDS electrophoresis analysis result.
The elution salt concentration of 0.8, 1.0 and 1.2mol/L, the PB buffer solution pH of 6.7, 7.0 and 7.3, the elution speed of 100, 200 and 300cm/h are taken as factors, the elution rate is taken as an investigation index, a three-factor three-level orthogonal test is designed, and the optimal process parameters of the elution rate of the lactoferrin standard solution are determined.
TABLE 3L 9 (3 3 ) Orthogonal test table
From the analysis of variance in table 4, it can be seen that the elution salt concentration, the pH of the buffer solution and the elution flow rate all have very significant effects on the elution rate of lactoferrin. According to the F-number analysis, the elution flow rate has the greatest effect on the elution rate, followed by the elution salt concentration and the pH of the buffer solution.
TABLE 4 analysis of variance in orthogonal tests
As can be seen from the results of the differential analyses shown in FIGS. 10-12, the elution rate of lactoferrin was the highest when the pH of the buffer solution was 7, the salt concentration was 1.2mol/L, and the elution flow rate was 100cm/h.
The basic reaction process of ion exchange chromatography is the exchange between the ion exchanger counter ions and the substance to be separated and the ions in the buffer solution, so the selection of the ionic strength and pH of the counter buffer and the elution buffer in the ion exchange layer system has a great influence on the separation effect. Since the test uses fresh milk as raw material and the by-product is reprocessed, the purified water is selected to balance the sample injection chromatographic column. The elution process is followed by the increase of the pH of the buffer solution, and the elution rate is increased and then decreased. The buffer pH will not only affect the release of the target protein but also the dissociation of the charged groups bound to the filler matrix. The ion exchange chromatography medium can dissociate hydrogen ions, and then interact with the ionized amino on the surface of the protein molecule to adsorb the protein so as to realize separation. When the pH value is higher, the adsorption strength of the target protein with positive charges and the filler is weakened, but hydroxide anions added in the solution are not beneficial to the combination of the chromatography filler matrix and hydrogen ions, so that the dissociation degree is reduced; at lower pH, although the electrostatic interaction between the medium and the adsorbate is increased, the strength of the binding action between the positive charge of the target protein and the filler group is increased, which is not beneficial to the release of the target protein.
The elution rate of the lactoferrin is obviously increased along with the increase of the concentration of the elution salt, and sodium chloride ions in the eluent can gradually exchange with various positive charge groups bound on the ion exchanger to replace the various positive charge groups. The positive electrode groups with small binding force with the ion exchanger are displaced first, and then the strong groups are displaced, thereby achieving the separation. The increase of sodium chloride not only increases the preparation cost, but also causes the difficulty of desalting and concentrating at the later stage, and as can be seen from the figure, when the concentration of sodium chloride exceeds 1.0mol/L, the increase of the elution rate is relatively smooth. Therefore, the salt concentration should be selected within a reasonable range of elution rate, and in the present invention, the salt concentration is selected to be 1.0mol/L.
The elution rate has obvious influence difference on the elution effect, the slower the elution speed is, the longer the action time of the eluent with the adsorbed target protein and the chromatographic packing is, the more favorable the exchange of the adsorbed positive charge protein and the positive ions is, but the too slow elution speed can cause overlong elution time and broadening of a peak spectrum, so that the proper elution rate is selected according to the production condition.
FIG. 13 shows the lactoferrin preparation curve by ion exchange chromatography under the optimal conditions for screening, peak 1 is flow-through, peak 2 is heteroprotein, and peak 3 is received protein. FIG. 14 is an SDS-PAGE gel electrophoresis of a received target sample. The results showed that the flow-through had a molecular weight of 17-35kD, and the received peak 2 was a protein with a molecular weight of about 78 kD. Fig. 15 shows the results of high performance liquid chromatography detection of proteins in the product, and combining the above results, the collected sample was determined to be lactoferrin, with a purity of 96.5% and a yield of 79.5%.
Example 2
This example differs from example 1 in that the raw milk in step (1) has a temperature of 55 ℃.
Example 3
This example differs from example 1 in that the raw milk in step (1) is at a temperature of 50 ℃.
Example 4
The difference between the present example and example 1 is that in the elution process in step (5), 0.1mol/L sodium chloride solution is used for eluting and removing impurities, 0.5mol/L sodium chloride solution is used for eluting and separating lactoperoxidase, 1.1mol/L sodium chloride is used for separating lactoferrin, and elution of target protein is performed, wherein the elution flow rate is 200cm/h.
Although the production process of the lactoferrin is mature in recent years, and researches show that a plurality of cations can realize the large-scale production of the lactoferrin, the production process and technical parameters of the lactoferrin are different due to different factors such as raw materials, byproducts, production equipment, chromatographic fillers, filling length of the fillers and the like. The lactoferrin is prepared by taking fresh milk as a raw material and utilizing AKTAProcess10mm PP pilot plant processing equipment through chromatographic separation and membrane concentration process separation. The sample injection is carried out on the skim milk at room temperature, the linear flow rate is 400cm/h, the concentration of sodium chloride PB buffer solution with the pH value of 7 is 0.4mol/L, the hybrid protein and the lactoferrin are eluted at a low speed of 1.0mol/L, the purity of the prepared lactoferrin is more than 95%, and the yield is more than 75%. The purity of the lactoferrin prepared by the method meets the requirements of national food safety standard food nutrition enhancer lactoferrin GB1903.17-2016, the physicochemical properties of byproducts are not affected, and the product yield is high.
It should be noted that, in the above embodiments, as long as the technical solutions can be aligned and combined without contradiction, those skilled in the art can exhaust all possibilities according to the mathematical knowledge of the alignment and combination, and therefore, the present invention does not describe the technical solutions after alignment and combination one by one, but it should be understood that the technical solutions after alignment and combination have been disclosed by the present invention.
The present embodiments are merely exemplary and do not limit the scope of the patent, and those skilled in the art can make modifications to the parts thereof without departing from the spirit and scope of the patent.
Claims (10)
1. A method for separating and preparing lactoferrin from raw milk is characterized by comprising the following steps:
(1) Pretreatment of raw milk: heating fresh raw milk, and separating by a centrifugal machine to remove fat;
(2) Filtering skim milk: filtering the skim milk to remove impurities;
(3) Loading: carrying out heat preservation treatment on the filtrate in the step (2), then feeding the filtrate into a PP chromatography system, and conveying the filtrate into a protein chromatography column for chromatography treatment;
(4) Filtering the buffer solution: filtering water, eluent and buffer solution with filter membrane;
(5) And (3) elution: eluting with sodium chloride solution to remove impurities, eluting to separate lactoperoxidase, separating lactoferrin, and eluting target protein;
(6) Desalting and concentrating: carrying out sample desalination and concentration treatment;
(7) And (3) microfiltration sterilization: the desalted and concentrated eluent is sterilized by a protein solution sterile filtration system;
(8) And (3) freeze drying: and (5) putting the liquid sample in the step (7) into low-temperature freeze drying equipment for freeze drying.
2. The method for separating and preparing lactoferrin from raw milk according to claim 1, wherein the temperature of raw milk in step (1) is 45-55 ℃.
3. The method for separating and preparing lactoferrin from raw milk according to claim 1, wherein the centrifugal speed in the separation process in step (1) is 11000rpm.
4. The method for separating and preparing lactoferrin from raw milk according to claim 1, wherein the freeze-drying temperature for freeze-drying in the step (8) is-50 ℃ to 40 ℃.
5. The method for separating and preparing lactoferrin from raw milk according to claim 4, wherein the freeze-drying process is divided into two processes, the first process is a pre-freezing process, the temperature is reduced from room temperature to-50 ℃, vacuum sublimation is carried out to the second process, the temperature of the second process is increased from-50 ℃ to 40 ℃, and the total time of freeze-drying in the two processes is 48 hours.
6. The method for separating and preparing lactoferrin from raw milk according to claim 5, wherein the step (7) of microfiltration sterilization adopts a protein solution sterile filtration system and adopts a PES membrane with a pore size of 0.22um for sterilization.
7. The method for separating and preparing lactoferrin from raw milk according to claim 6, wherein the desalting and concentrating in step (6) is performed by using a full-automatic protein ultrafiltration system, and PES (polyether sulfone) membrane flat ultrafiltration membrane is used for sample desalting and concentrating.
8. The method for separating and preparing lactoferrin from raw milk according to claim 7, wherein in the step (5), elution is performed to remove impurities by using 0.1mol/L sodium chloride solution, lactoperoxidase is eluted by using 0.5mol/L sodium chloride solution, lactoferrin is eluted by using 1.1mol/L sodium chloride, and elution of the target protein is performed at an elution flow rate of 100-200cm/h.
9. The method for separating and preparing lactoferrin from raw milk according to claim 7, wherein the eluent of the buffer solution and the buffer solution in step (4) are filtered through PES filter membrane with 10-inch 0.45 μm pore size.
10. The method for separating and preparing lactoferrin from raw milk according to any one of claims 1 to 9, wherein the filtrate in step (3) is fed into a chromatography system at room temperature, the pressure of the system is set to 0.5MPa, the chromatography column is balanced with more than two levels of purified water, the linear flow rate is set to 600cm/h, the PB buffer solution is added with sodium chloride to respectively prepare 0.4 to 1.0mol/L of eluent with pH 7, the eluent is filtered by PES membrane, and the adsorbed basic protein is eluted, and the elution flow rate is 200cm/h.
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