CN121021663B - Method for extracting target product from milk raw material - Google Patents
Method for extracting target product from milk raw materialInfo
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- CN121021663B CN121021663B CN202511565367.2A CN202511565367A CN121021663B CN 121021663 B CN121021663 B CN 121021663B CN 202511565367 A CN202511565367 A CN 202511565367A CN 121021663 B CN121021663 B CN 121021663B
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Abstract
The invention discloses a method for extracting a target product from milk raw materials, and relates to the technical field of food processing. The method introduces the synergistic pretreatment step of ultrafiltration and conductivity adjustment before the strong anion exchange chromatography, realizes the acceleration, synergy, quality improvement and cost reduction of the chromatography process, overcomes the technical bottlenecks of slow speed, easy pollution, instability and the like existing in the traditional ion exchange chromatography when the milk raw material is treated, and provides a solution for the efficient, high-purity and large-scale production of the beta-lactoglobulin and the alpha-lactalbumin. The method can also combine strong anion exchange chromatography with weak anion exchange chromatography, further improve the extraction purity of beta-lactoglobulin and alpha-lactalbumin, and synchronously realize the efficient extraction of various target substances such as casein micelle, lactose, milk mineral salt and the like, thereby greatly improving the added value and the resource utilization efficiency of milk source byproducts.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to a method for extracting a target product from milk raw materials.
Background
Dairy products are important nutritional sources for humans, and have a core value of not only providing basic nutrients, but also containing various functional components with high bioactivity and high added value, such as beta-lactoglobulin (beta-Lg), alpha-lactalbumin (alpha-La), casein micelles, lactose, milk mineral salts, and the like. The substances have wide application prospects in the fields of food industry, infant formula food, sports nutrition, pharmacy, health care products and the like. For example, beta-lactoglobulin and alpha-lactalbumin are excellent protein sources with specific amino acid compositions and physiological functions, casein micelles play a significant role in improving the texture of foods due to their unique structures, lactose is an important ingredient for many foods, and milk mineral salts are a natural and high-bioavailability calcium supplement.
At present, a plurality of reports on a technology for separating and extracting the target substances from milk raw materials (such as skim milk, whey and the like) exist, but most of the technologies have certain limitations, and high-efficiency, high-purity and multi-component collaborative extraction is difficult to realize.
Regarding the separation of whey proteins (beta-lactoglobulin and alpha-lactalbumin), the conventional method mainly employs chromatography, particularly ion exchange chromatography and hydrophobic interaction chromatography. At present, the chromatography has the advantages of small treatment capacity, long process flow, large equipment investment and high eluent consumption, so that the production cost is high, and the method is difficult to be suitable for large-scale industrial production. Membrane separation techniques (e.g., ultrafiltration, microfiltration) are also widely used, but conventional ultrafiltration membranes have poor selectivity in separating β -lactoglobulin and α -lactalbumin of similar molecular weights, and it is difficult to obtain a single component of high purity.
Regarding the separation of casein micelles, casein micelles are currently separated from skim milk mainly by ultracentrifugation or microfiltration techniques. However, ultracentrifugation is extremely energy-intensive and there is a risk of destruction of the micelle structure, with limited yields. Although the microfiltration method is mild, the problems of rapid flux decay, short operation period and the like are faced, and the production efficiency and the economic benefit are affected. Meanwhile, how to effectively avoid mixing of whey protein in the separation process and keep the structural integrity of casein micelles is still a technical difficulty.
For lactose and milk mineral salt extraction, lactose is usually obtained by concentrating and crystallizing whey, but the process is easily affected by impurity ions, so that the purity of crystals is not high and the crystal morphology is poor. The extraction of milk mineral salt (mainly milk calcium) mostly adopts acid precipitation or ion exchange method, but the methods possibly introduce chemical reagents, have the problems of low product purity, peculiar smell, or destroyed natural conformation, and the like, and influence the application of the milk mineral salt in high-end food.
The prior art has focused on the separation of single or small amounts of several components from milk raw materials, and lacks an integrated process capable of systematically and continuously efficiently synergistically extracting a plurality of high-value components (including casein micelles, whey proteins, lactose and milk mineral salts) from the same raw material.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for extracting a target product from milk raw materials.
The invention is realized in the following way:
a method of extracting a target product from a milk raw material, the target product comprising beta-lactoglobulin and/or alpha-lactalbumin, comprising the steps of:
obtaining a first permeate after micro-filtration of the skimmed milk;
Ultrafiltering the first permeate to obtain a second permeate and a second retentate;
And adjusting the electric conductivity of the second trapped fluid to 3-8 mS/cm, and then performing strong anion exchange chromatography to obtain the flow-through fluid containing alpha-lactalbumin and the eluent containing beta-lactoglobulin.
The invention has the following beneficial effects:
(1) The application develops a novel extraction method, introduces a synergistic pretreatment step of ultrafiltration and conductivity adjustment before strong anion exchange chromatography, realizes the speed-up, efficiency-enhancement, quality-improvement and cost-reduction of the chromatography process, overcomes the technical bottlenecks of slow speed, easy pollution, instability and the like existing in the traditional ion exchange chromatography when processing milk raw materials, and provides a solution for the efficient, high-purity and large-scale production of beta-lactoglobulin and alpha-lactalbumin.
(2) The extraction purity of beta-lactoglobulin and alpha-lactalbumin can be further improved by combining strong anion exchange chromatography with weak anion exchange chromatography, and the purity reaches more than 98.65 percent and 97.39 percent respectively;
(3) The extraction method can also synchronously realize the efficient extraction of various target products such as casein micelle, lactose, milk mineral salt and the like, thereby greatly improving the added value and the resource utilization efficiency of milk-derived byproducts.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a process flow diagram of a method of extracting a target product from a milk raw material;
FIG. 2 is a flow chart of the chromatography step of FIG. 1;
FIG. 3 shows the target protein spectrum of example 1, wherein A is the spectrum of alpha-lactalbumin and B is the spectrum of beta-lactoglobulin.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The terms "first," "second," "third," "fourth," "1," "2," "3," "4," and the like are used solely for distinguishing between descriptions and not as an indication or suggestion of relative importance.
The method for extracting the target protein from the milk raw material provided by the invention brings remarkable and unexpected synergistic technical effects by introducing the synergistic pretreatment step of ultrafiltration and conductivity adjustment before strong anion exchange chromatography, and mainly comprises the following steps:
The method has the advantages of remarkably improving the chromatographic speed and efficiency, effectively removing the interference of other insoluble impurities except the target product in the milk raw material by ultrafiltration, greatly reducing the blockage and pollution to the chromatographic column, stabilizing the column pressure in the chromatographic process, remarkably improving the flow rate, allowing loading and elution at a higher flow rate, greatly shortening the extraction time, improving the equipment utilization rate, optimizing the conductivity of the raw material liquid to the section with the strongest binding power of the target protein and the filler by accurate conductivity adjustment, and greatly improving the mass transfer efficiency of protein molecules. The combination of ultrafiltration and conductivity adjustment ensures that the target protein can finish the chromatography process with higher flow rate and shorter balance time, and the chromatography speed is improved in a multiplied way, thereby greatly improving the raw material processing amount in unit time.
The purity of the target product is obviously improved, namely, the ultrafiltration pretreatment removes a large amount of soluble impurity proteins which can compete with the target protein for binding sites while removing insoluble impurities, so that the primary purification of the sample is realized. On this basis, accurate conductance adjustment further "sharpens" the difference in charge properties of the target proteins (β -lactoglobulin and α -lactalbumin) and the residual impurity proteins.
The stability of the process is improved by combining the protective effect of ultrafiltration on the filler and the stabilizing effect of conductivity adjustment on the process, so that the repeatability and the stability of the whole purification process are greatly enhanced. The method not only reduces the replacement frequency and the production cost of expensive chromatographic packing, but also ensures the high consistency of the product quality in large-scale production, and lays a solid foundation for industrial application.
In addition, the invention combines the strong anion chromatography and the weak anion chromatography, and combines the membrane filtration technology, thereby obviously improving the chromatography efficiency and flux of the weak anion chromatography, realizing the high-purity extraction of beta-lactoglobulin and alpha-lactalbumin, wherein the purity of the beta-lactoglobulin can reach more than 98.65 percent, and the purity of the alpha-lactalbumin can reach more than 97.39 percent.
Embodiments of the present invention provide a method of extracting a target product from a milk raw material, the target product comprising beta-lactoglobulin and/or alpha-lactalbumin, comprising the steps of:
obtaining a first permeate after micro-filtration of the skimmed milk;
Ultrafiltering the first permeate to obtain a second permeate and a second retentate;
And adjusting the electric conductivity of the second trapped fluid to 3-8 mS/cm, and then performing strong anion exchange chromatography to obtain the flow-through fluid containing alpha-lactalbumin and the eluent containing beta-lactoglobulin.
In an alternative embodiment, the method further comprises micro-filtering the skim milk to obtain a first permeate and a first retentate.
In an alternative embodiment, the pore size of the microfiltration membrane is 0.05-0.2 μm, and specifically may be any one or any range between two of 0.05, 0.1, 0.12, 0.14, 0.16, 0.18 and 0.2 μm.
In an alternative embodiment, the temperature of the microfiltration is 10-60 ℃, and specifically may be any one or any range between 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃.
In an alternative embodiment, the transmembrane pressure difference of the microfiltration is controlled to be 1-2 b ar, and specifically may be any one or any two of 1, 1.2, 1.4, 1.6, 1.8 and 2 bar. The calculation formula of the transmembrane pressure difference is as follows:
。
In an alternative embodiment, the volume concentration multiple of the microfiltration is 2-5 times, and specifically may be any one or any range between two of 2, 3,4 and 5 times.
In an alternative embodiment, the method further comprises washing and filtering the filtered trapped fluid to obtain a final first trapped fluid for a subsequent step, wherein the washing and filtering fluid can be RO water, and the washing and filtering fluid can be 1-5 times of the volume of the skim milk, and specifically can be any one or any range between 1,2,3, 4 and 5 times.
In an alternative embodiment, the method further comprises obtaining a first retentate of skim milk after the microfiltration;
And ultrafiltering the first trapped fluid to obtain a third permeation fluid containing lactose and a third trapped fluid containing casein micelle.
In an alternative embodiment, the molecular cutoff of the filter membrane for ultrafiltration of the first retentate is 3-30 kDa, and specifically may be any one or any two or more of 3,5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 kDa.
In an alternative embodiment, the temperature of the ultrafiltration performed on the first trapped fluid may be 10 to 25 ℃, and specifically may be any one or any two or any range between 10, 12, 14, 16, 18, 20, 22, 24 and 25 ℃.
In an alternative embodiment, the volume concentration multiple of ultrafiltration of the first trapped fluid is 2-5 times, and specifically may be any one or any range between two of 2, 3,4 and 5 times.
In an alternative embodiment, after ultrafiltration of the first retentate, the method comprises washing the ultrafiltered third retentate to obtain a washed third retentate for use in a subsequent step. The washing filtrate of the washing filtration can be RO water, and the washing filtration multiple is 1-5 times of the volume of the skim milk, preferably 1-2 times.
In an alternative embodiment, the method further comprises drying the third retentate to obtain casein micelle powder.
In an alternative embodiment, the drying may be spray drying.
In an alternative embodiment, the molecular cutoff of the filter membrane for ultrafiltering the first permeate is 3-30 kDa, and specifically may be any one or any two or more of 3,5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 kDa.
In an alternative embodiment, the molecular retention of the filter membrane for ultrafiltration of the first permeate is 5-15 kDa.
In an alternative embodiment, the condition of performing ultrafiltration on the first permeate comprises a temperature of 10-25 ℃ and a volume concentration multiple of 2-5 times. The temperature may specifically be in a range between any one or any two of 10, 12, 14, 16, 18, 20, 22, 24 and 25 ℃, and the volume concentration multiple may be in a range between any one or any two of 2, 3,4 and 5.
In an alternative embodiment, after ultrafiltration of the first permeate, the method further comprises washing the second retentate, and obtaining a washed second retentate for use in a subsequent step. The washing filtrate can be RO water, the washing filtration multiple is 1-5 times of the volume of the skim milk, and the washing filtration multiple can be specifically any one or any range between two of 1, 2, 3, 4 and 5 times.
In an alternative embodiment, the method further comprises concentrating the second trapped fluid to a concentration of protein in the second trapped fluid of 0.4% -0.8% (w/w), wherein the concentration may specifically be any one or any range between any two of 0.4%, 0.5%, 0.6%, 0.7%, and 0.8%. The protein concentration of the natural whey liquid obtained after normal microfiltration is 0.2% -0.3%, under the condition that the total protein content is consistent, the higher the protein concentration is, the smaller the total volume of the whey liquid is, the smaller the volume is, the shorter the time is under the condition that the sample loading flow rate is the same, and the chromatographic efficiency can be effectively improved.
In an alternative embodiment, the strong anion exchange chromatography is performed after adjusting the conductance of the second retentate to a range between any one or any two of 3, 4,5, 6, 7, 8 mS/cm.
In an alternative embodiment, the solution used for the conductance adjustment is a sodium chloride solution. The concentration of the sodium chloride solution may be in a range between any one or any two of 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% and 30% (w/w).
In an alternative embodiment, the method further comprises nanofiltration of the second permeate and/or the third permeate to obtain a fourth retentate comprising lactose and a fourth permeate comprising milk mineral salts.
In an alternative embodiment, the molecular retention of the filter membrane used in the nanofiltration is 300-1000 Da, and specifically may be any one or any two of 300, 400, 500, 600, 700, 800, 900, 1000 Da.
In an alternative embodiment, the temperature of the nanofiltration may be 10 to 25 ℃, and specifically may be any one or any range between two of 10, 12, 14, 16, 18, 20, 22, 24 and 25 ℃.
In an alternative embodiment, the volume concentration multiple of the nanofiltration is 2-5 times, and specifically may be any one or any range between two of 2, 3,4 and 5 times.
In an alternative embodiment, after nanofiltration, the method further comprises washing the fourth retentate after nanofiltration. The conditions for washing and filtering are the same as those in any of the previous examples.
In an alternative embodiment, the method further comprises drying the fourth retentate to obtain a milk powder;
in an alternative embodiment, the method further comprises drying the fourth permeate to obtain milk mineral salt powder.
In an alternative embodiment, the chromatography procedure of the strong anion exchange chromatography is equilibration-loading-post column equilibration-elution 1-elution 2-column CIP-column rinse.
In an alternative embodiment, the equilibration procedure for the strong anion exchange chromatography is 2-5 column volumes for phase A-2-5 column volumes for phase B-3-10 column volumes for phase A-240-500 cm/h, preferably 2 column volumes for phase A-2 column volumes for phase B-3 column volumes for phase A-400-500 cm/h;
in an alternative embodiment, the phase A is 15-35 mmol/L Tris-HCl solution with pH of 7.8-8.2.
In an alternative embodiment, the phase B comprises a mixed solution of 15-35 mmol/L of pH 7.8-8.2 Tris-HCl solution and 0.1-1 mol/L of sodium chloride. The preparation of the phase B comprises the steps of adding 0.1-1 mol/L sodium chloride into 15-35 mmol/L pH 7.8-8.2 Tris-HCl solution, wherein 0.1-1 mol/L sodium chloride is the concentration of the sodium chloride in the phase B.
In an alternative embodiment, the 15-35 mmol/L may specifically be any one of 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35mmol/L or any range between any two of them.
In an alternative embodiment, the ph 7.8-8.2 may specifically be any one or any range between 7.8, 7.9, 8.0, 8.1, 8.2.
In an alternative embodiment, the 0.1 to 1 mol/L may specifically be any one or any range between two of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mol/L.
In an alternative embodiment, the loading procedure of the strong anion exchange chromatography is concentrated whey with a conductivity of 3-8 mS/cm, a flow rate of 500-700 cm/h, preferably 700cm/h, and a loading volume of 100 column volumes.
In an alternative embodiment, the post-column equilibration procedure for strong anion exchange chromatography is to flush phase A at a flow rate of 500 to 700cm/h, preferably 700cm/h, 2 to 5 column volumes, preferably 2 column volumes.
In an alternative embodiment, the elution 1 is followed by obtaining an eluate comprising alpha-lactalbumin and the elution 2 is followed by obtaining an eluate comprising beta-lactoglobulin.
In an alternative embodiment, the eluent of the eluent 1 comprises a mixed liquid consisting of 92% -94% (v/v) of phase A and 6% -8% (v/v) of phase B. The 92% -94% may specifically be any one or any two of 92%, 93%, 93.2%, 93.4%, 93.6%, 93.8% and 94%. The content of 6% -8% can be specifically any one or any two of 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7% and 8%.
In an alternative embodiment, the eluting 1 is eluting with 3-8 times of the column volume of the eluent, and the 3-8 times of the eluent can be any one or any two of 3, 4, 5, 6,7 and 8 times of the eluent.
In an alternative embodiment, the eluent of elution 2 includes phase B.
In an alternative embodiment, the eluting 2 uses an eluent with a volume of 5-10 times that of the column, and the 5-10 times may specifically be any one or any two of 5,6, 7, 8, 9 and 10 times.
In an alternative embodiment, the flow rate of the elution 1 and/or the elution 2 may be 240-500 cm/h, specifically, any one or any two of 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 cm/h.
In an alternative embodiment, the column CIP procedure of the strong anion exchange chromatography is to wash 3-5 column volumes, preferably 4 column volumes, with 0.5mol/L sodium hydroxide solution, at a flow rate of 240-500cm/h, preferably 400-500cm/h.
In an alternative embodiment, the column wash procedure for strong anion exchange chromatography is 2-5 column volumes, preferably 5 column volumes, for 100% phase B elution, with a flow rate of 240-500cm/h, preferably 400-500 cm/h.
In an alternative embodiment, the strong anion exchange chromatography employs a strong anion exchange chromatography column that is a TA-Q XL BB chromatography column.
In an alternative embodiment, the method further comprises subjecting the flow-through and/or eluate of elution 1 to weak anion exchange chromatography.
In an alternative embodiment, the method further comprises subjecting the retentate obtained after ultrafiltration of the flow-through and/or eluent of elution 1 to the weak anion exchange chromatography.
In an alternative embodiment, the molecular retention amount used for ultrafiltration of the flow-through solution and/or the eluent of the elution 1 is 3-5 kDa, and specifically may be any one or any two of 3, 3.5, 4, 4.5, and 5 kDa.
In an alternative embodiment, the volume concentration multiple of the ultrafiltration of the flowing-through liquid and/or the eluent of the elution 1 is 2-5 times, and specifically may be any one or any range between two of 2, 3, 4 and 5 times.
In an alternative embodiment, after ultrafiltration of the flow-through and/or eluate of elution 1, the method further comprises washing the ultrafiltered retentate. The washing liquid used for the washing and filtering can be phase A, which is described in any embodiment, and the washing and filtering are carried out until the conductivity of the trapped liquid is consistent with that of the phase A (1.5 mS/cm).
In an alternative embodiment, the chromatography procedure of the weak anion exchange chromatography is equilibration-loading-post column equilibration-elution 3-elution 4-column CIP-column rinse.
In an alternative embodiment, the equilibration procedure for weak anion exchange chromatography is 2-5 column volumes for phase A-phase equilibrium 2-5 column volumes for phase B-phase equilibrium 3-10 column volumes for phase A, flow rates of 240-500cm/h each, preferably 2 column volumes for phase A-phase equilibrium 2 column volumes for phase B-phase equilibrium 3 column volumes for phase A, flow rates of 240-360cm/h each. Phases a and B are the same as described in the previous examples.
In an alternative embodiment, the weak anion exchange chromatography loading procedure is such that the flow through and/or the eluent of elution 1 or its post-ultrafiltered retentate is all loaded at a flow rate of 500-700cm/h, preferably 600cm/h, and the post-column equilibration procedure is such that phase A is flushed 2-5 column volumes, preferably 2 column volumes, at a flow rate of 500-700cm/h, preferably 600 cm/h.
In an alternative embodiment, an eluate containing alpha-lactalbumin is obtained after elution 3 and an eluate containing beta-lactoglobulin is obtained after elution 4.
In an alternative embodiment, the eluent of the eluent 3 comprises a mixed liquid consisting of 92% -94% (v/v) of phase A and 6% -8% (v/v) of phase B. The 92% -94% may specifically be any one or any two of 92%, 93%, 93.2%, 93.4%, 93.6%, 93.8% and 94%. The content of 6% -8% can be specifically any one or any two of 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7% and 8%.
In an alternative embodiment, the eluting 3 uses an eluent with a volume of 3-8 times that of the column, and the 3-8 times may be any one or any two of 3, 4, 5, 6,7 and 8 times.
In an alternative embodiment, the eluent of the elution 4 includes the B phase;
in an alternative embodiment, the eluting 4 uses 5-10 times of the column volume of the eluent, and the 5-10 times of the eluent can be any one or any two of 5,6, 7, 8, 9 and 10 times of the eluent.
In an alternative embodiment, the flow rate of the elution 3 and/or the elution 4 may be 240-500 cm/h, specifically, any one or any two of 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 cm/h.
In an alternative embodiment, the column CIP procedure of weak anion exchange chromatography is to wash 3-5 column volumes, preferably 4 column volumes, with 0.5mol/L sodium hydroxide solution, at a flow rate of 240-500cm/h, preferably 240-360cm/h.
In an alternative embodiment, the column wash procedure for weak anion exchange chromatography is 2-5 column volumes, preferably 5 column volumes, for 100% phase B elution, with a flow rate of 240-500cm/h, preferably 240-360cm/h.
In an alternative embodiment, the weak anion exchange chromatography using a TA-SP XL BB column.
In an alternative embodiment, the method further comprises ultrafiltration and/or drying of the alpha-lactalbumin-containing flow-through and/or eluate obtained in the strong anion exchange chromatography and/or the weak anion exchange chromatography;
in an alternative embodiment, the molecular retention of the filter membrane used for ultrafiltration of the alpha-lactalbumin-containing flow-through solution and/or the eluent is 3-5 kDa, and specifically may be any one or any two of 3, 3.5, 4, 4.5 and 5 kDa.
In an alternative embodiment, the volume concentration factor of ultrafiltration of the alpha-lactalbumin-containing flow-through solution and/or the eluent is 2-5, and specifically may be any one or any range between two of 2, 3, 4 and 5 times.
In an alternative embodiment, after ultrafiltration of the alpha-lactalbumin-containing flow-through and/or eluent, the method further comprises washing and filtering the ultrafiltered retentate, wherein the washing and filtering liquid can be RO water, and the electric conductivity of the retentate is less than or equal to 0.1mS/cm.
In an alternative embodiment, the method further comprises at least one of ultrafiltration, pH adjustment and drying of the beta-lactoglobulin-containing eluate obtained in the strong anion exchange chromatography and/or the weak anion exchange chromatography;
in an alternative embodiment, the pH adjustment means that the pH of the solution is adjusted to 2.5-3.5;
in an alternative embodiment, the molecular retention of the filter membrane adopted for ultrafiltration of the eluate containing beta-lactoglobulin is 3-5 kDa, and specifically can be any one or any two of 3, 3.5, 4, 4.5 and 5 kDa.
In an alternative embodiment, the concentration multiple of ultrafiltration of the eluate containing beta-lactoglobulin is 2-5 times, and specifically may be any one or any range between two of 2, 3, 4 and 5 times.
In an alternative embodiment, after ultrafiltration of the beta-lactoglobulin-containing eluate, the method further comprises washing the ultrafiltered retentate, and obtaining a washed retentate for subsequent pH adjustment. The washing filtrate can be RO water, and the electric conductivity of the trapped fluid is less than or equal to 0.1mS/cm.
In an alternative embodiment, the target product further comprises at least one of casein micelles, lactose and milk mineral salts.
In an alternative embodiment, the microfiltration, ultrafiltration, nanofiltration membrane of any of the preceding embodiments may be an organic roll membrane and the membrane material may be PES.
The process flow diagram of the extraction method is shown in fig. 1, and the flow diagram of the chromatography step is shown in fig. 2.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The strong anion exchange chromatographic column adopted in the example comprises a filler, namely TA-Q XL BB, wherein the height of a chromatographic column bed is 20cm, the inner diameter of the chromatographic column is 20cm, the length-diameter ratio is 1:1, the compression ratio of the filler is 1:1.15, the volume of the filler is 6.28L, and the working pressure is 0.25MPa.
The weak anion exchange chromatographic column adopted in the example comprises a packing material TA-SP XL BB, a chromatographic column bed height of 20cm, a chromatographic column inner diameter of 40cm, an aspect ratio of 1:2, a packing material compression ratio of 1:1.20, a packing material volume of 25L and a working pressure of 0.28MPa. Phase A is a solution of 25mmol/L Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) at pH 8.0;
Phase B is a mixture of 25mmol/L Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) solution at pH8.0 and 0.5mol/L sodium chloride.
Example 1
A method of extracting a target product from a milk raw material, comprising the following steps.
(1) Step 1:
The skim milk is subjected to microfiltration by using a 0.1-micron PES organic roll membrane to obtain a first permeate (natural whey protein liquid) and a first retentate (casein micelle liquid), wherein the microfiltration temperature is 50 ℃, the transmembrane pressure difference is controlled at 1.4bar, and the concentration multiple is 3 times.
And (3) washing and filtering the first trapped fluid, wherein the washing and filtering liquid is RO water, and the washing and filtering multiple is 1.5 times of the volume of the skim milk, so as to obtain the washed and filtered first trapped fluid (casein micelle fluid).
(2) Step 2:
ultrafiltering the first trapped fluid obtained in the step 1 by using a 10kDa PES organic roll membrane to obtain a third trapped fluid and a third permeate (lactose fluid), wherein the ultrafiltration temperature is 15 ℃, and the concentration multiple is 3 times;
and (3) washing and filtering the third trapped fluid, wherein the washing and filtering liquid is RO water, and the washing and filtering multiple is 1 time of the volume of the skim milk obtained in the step (1), so as to obtain the washed and filtered third trapped fluid (casein micelle fluid).
(3) Step 3:
And (3) spray drying the third trapped liquid (casein micelle liquid) obtained in the step (2) to obtain casein micelle powder.
(4) Step 4:
Ultrafiltering the first permeate (natural whey protein solution) obtained in the step 1 by using a 10kDa PES organic roll membrane to obtain a second retentate and a second permeate, wherein the ultrafiltration temperature is 15 ℃, and the concentration multiple is 3 times;
Washing and filtering the second trapped fluid, wherein the washing and filtering liquid is RO water, and the washing and filtering multiple is 1 time of the volume of the skim milk in the step 1;
Concentrating the second trapped fluid after washing and filtering to make the protein concentration in the feed fluid be 0.6% (w/w), and obtaining the concentrated second trapped fluid (concentrated whey liquid).
(5) Step 5:
Mixing the third permeate in the step 2 and the second permeate in the step 4, and then carrying out nanofiltration by using a 500Da PES organic roll membrane to obtain a fourth trapped fluid (concentrated lactose fluid) and a fourth permeate (milk mineral salt fluid), wherein the nanofiltration temperature is 20 ℃, and the concentration multiple is 3 times;
and (3) washing and filtering the fourth trapped fluid to obtain a fourth trapped fluid after washing and filtering, wherein the washing and filtering liquid is RO water, and the washing and filtering multiple is 2 times of the initial volume.
(6) Step 6:
spray drying the fourth permeate liquid in the step 5 to obtain milk mineral salt powder;
And (3) spray drying the fourth trapped fluid in the step 5 to obtain the lactose powder.
(7) Step 7:
The concentrated whey liquid of step 4 was adjusted to a conductance of 4mS/cm using a sodium chloride solution of 26% (w/w) concentration.
(8) Step 8:
and (3) carrying out chromatography on the concentrated whey liquid with the adjusted electric conduction in the step (7) by using strong anion exchange resin, wherein the chromatography program is balance-loading-post-column balance-elution 1-elution 2-column CIP-column flushing, and the method comprises the following steps:
the balancing procedure is that the volume of the column is 2 times of that of the phase A, 2 times of that of the phase B, 3 times of that of the phase A, and the flow rates are 500cm/h;
the loading procedure is concentrated whey liquid with 4mS/cm conductivity, the flow rate is 700cm/h, and the loading volume is 100 times of column volume;
the post column equilibration procedure was phase A at a flow rate of 700cm/h, 2 column volumes were rinsed;
Elution 1 procedure was 93.4% phase A and 6.6% phase B eluting 3 column volumes at a flow rate of 500cm/h;
The elution 2 procedure is 5 column volumes of 100% phase B elution with a flow rate of 500cm/h;
the column CIP procedure was a 4-fold column volume wash with 0.5mol/L sodium hydroxide solution at a flow rate of 500cm/h;
The column wash procedure was 5 column volumes eluted at 100% phase B with a flow rate of 500cm/h.
(9) Step 9:
Collecting and mixing the column flow through liquid (flow through liquid containing alpha-lactalbumin) and the eluent (eluent containing alpha-lactalbumin) of the elution 1 in the loading procedure in the step 8, and performing ultrafiltration by using a 5kDa PES organic roll-type membrane, wherein the ultrafiltration temperature is normal temperature, and the concentration multiple is 3 times;
washing and filtering the trapped fluid obtained by ultrafiltration, wherein the washing and filtering liquid is phase A, and washing and filtering until the conductance of the trapped fluid is consistent with the conductance of phase A (1.5 mS/cm).
(10) Step 10:
Subjecting the retentate from step 9 to weak anion exchange chromatography, wherein the chromatography procedure is equilibration-loading-post column equilibration-elution 3-elution 4-column CIP-column rinse, wherein:
The equilibration procedure was 2 times the column volume of phase A-2 times the column volume of phase B-3 times the column volume of phase A, the flow rates were 360cm/h, the phase A was 25mmol/L Tris (Tris-HCl) solution at pH8.0, and the phase B was a mixture containing 25mmol/L Tris (Tris-HCl) solution at pH8.0 and 0.5mol/L sodium chloride;
The loading procedure is that all the trapped fluid obtained in the step 9 is loaded, and the flow rate is 600cm/h;
the post column equilibration procedure was phase A at a flow rate of 600cm/h, 2 column volumes were rinsed;
The elution 3 procedure was 93.4% phase A and 6.6% phase B eluting 6 column volumes at a flow rate of 360cm/h;
The elution 4 procedure was 5 column volumes for 100% phase B elution with a flow rate of 360cm/h;
The column CIP procedure was a 4-fold column volume wash with 0.5mol/L sodium hydroxide solution at a flow rate of 240cm/h;
the column wash procedure was 5 column volumes eluted at 100% phase B with a flow rate of 360cm/h.
(11) Step 11
Ultrafiltering the eluent eluted by the step (3) by using a 5kDa PES organic roll membrane to obtain trapped fluid, wherein the ultrafiltration temperature is normal temperature, and the concentration multiple is 3 times;
washing and filtering the trapped fluid, wherein the washing and filtering liquid is RO water, and the electric conductivity of the trapped fluid is less than or equal to 0.1mS/cm.
(12) Step 12:
mixing the eluent of the eluent 2 in the step 8 and the eluent of the eluent 4 in the step 10, and then carrying out ultrafiltration by using a 5kDa PES organic roll membrane to obtain trapped fluid, wherein the ultrafiltration temperature is normal temperature, and the concentration multiple is 3 times;
washing and filtering the trapped fluid, wherein the washing and filtering liquid is RO water, and the electric conductivity of the trapped fluid is less than or equal to 0.1mS/cm.
(13) Step 13:
and (3) spray drying the trapped liquid obtained in the step 11 to obtain the high-purity alpha-lactalbumin powder.
(14) Step 14:
And (3) adjusting the pH of the trapped fluid obtained in the step (12) to 3 by using 3mol/L hydrochloric acid, and then performing spray drying to obtain the high-purity beta-lactoglobulin powder.
Example 2
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
(1) Step 9 and step 10 are not performed;
(2) Step 11, replacing the eluent of the eluent 3 in the step 10 with a mixed solution of the column flow-through liquid (alpha-lactalbumin-containing flow-through liquid) and the eluent of the eluent 1 (alpha-lactalbumin-containing eluent) in the loading procedure in the step 8;
(3) Step 12. The protocol relating to "eluent for elution 4 in step 10" is omitted.
Example 3
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
(1) Step 7, step 8 and step 9 are not performed;
(2) Step 10, replacing the trapped fluid in the step 9 with the second trapped fluid (concentrated whey fluid) obtained in the step 4;
(3) Step 12. The protocol relating to "eluent for elution 2 in step 8" is omitted.
Example 4
A method for extracting a target product from a milk raw material is substantially the same as in Experimental example 1, except that the pH adjustment is not performed in step 14.
Example 5
The method according to examples 1 to 4 extracts a target product from a milk raw material, and detects alpha-lactalbumin and beta-lactoglobulin by using a high performance liquid chromatography-mass spectrometry method, and the specific method is as follows:
1. The standard substance comprises a bovine alpha-lactalbumin specific peptide standard substance (sequence: VGINYWLAHK, molecular weight 1200.4Da, purity > 99%), a bovine alpha-lactalbumin isotope labeling specific peptide standard substance (sequence: VGI NYWL AHK, molecular weight 1214.4Da, purity > 95%), a bovine alpha-lactalbumin isotope labeling internal standard substance (sequence: KIDKVGI X AHKALCSEK, molecular weight 2443.9 Da, purity > 95%), a bovine beta-lactoglobulin specific peptide standard substance (sequence: IDALNENK, molecular weight 916.0Da, purity > 99%), a bovine beta-lactoglobulin isotope labeling specific peptide standard substance (sequence: I X NENK, molecular weight 930.0 Da, purity > 95%), a bovine beta-lactoglobulin isotope labeling internal standard substance (sequence: KIPAVFKI X NEN KVIVLDTDYK, molecular weight 2761.2 Da, purity > 95%) and amino acids in the peptide sequence indicated by the amino acid isotope.
2. About 2g for solid samples or about 10 g for liquid samples (accurate to 0.01 g, about 200 mg for protein) were weighed into a 500 mL beaker, the samples were thoroughly dissolved in 900 mL water in portions, transferred to 1000 mL, volumetric flask and scaled with water, and placed on a vortex mixer for sufficient dissolution. Accurately transferring 200 mu L of the sample solution and 50 mu L of the intermediate mixed solution of the isotope internal standard into a 2mL centrifuge tube, adding 150 mu L of ammonium bicarbonate solution and 10 mu L of dithiothreitol solution after uniformly mixing, placing in a constant-temperature water bath at 75 ℃ for 30min, cooling to room temperature, adding 30 mu L of iodoacetamide solution, standing in dark for 30min, adding 10 mu L of calcium chloride solution and 50 mu L of trypsin solution, fully mixing, and placing in a constant-temperature water bath at 37 ℃ for enzymolysis for 5 hours. Mixing with 10. Mu.L formic acid, standing at room temperature for 15 min%, adding 490. Mu.L water, mixing by vortex, and filtering with 0.22 μm filter membrane for detection by liquid chromatography tandem mass spectrometer.
3. The chromatographic column comprises a silane-based C18 column with a column length of 100 mm, a column inner diameter of 2.1 mm, a filler with a particle diameter of 1.7 um and a pore diameter of 30 nm (300)) Or equivalent column effect.
4. Mobile phase A0.1% aqueous formic acid solution, mobile phase B0.1% acetonitrile formic acid solution, gradient elution according to the reference conditions given in Table 1.
6. The flow rate of the mobile phase is 0.3 mL/min.
7. Column temperature of chromatographic column is 40 ℃;
8. the temperature of the test solution is 10 ℃.
9. The sample injection volume is 10 mu L.
10. Mass spectrum reference conditions
ESI, mass spectrum scanning mode, multi-reaction monitoring (MRM), capillary voltage of 3.5kV, cone hole voltage of 35V, desolvation temperature of 500 ℃, desolvation gas flow of 800L/h, cone hole back-blowing gas flow of 30L/h, collision chamber pressure of 3.0×mbar。
11. The mass spectrometry conditions are shown in table 2:
the results of the detection are shown in FIG. 3 and the following table.
TABLE 3 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 1
TABLE 4 detection results of Casein micelle powder, milk sugar powder and milk mineral salt powder of example 1
TABLE 5 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 2
TABLE 6 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 3
Note that the results of example 3 are after 5 replicates.
TABLE 7 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 4
Table 8 flow rate comparison of ion exchange chromatography for various examples
From the results, it can be seen that:
As is clear from comparative examples 1 and 2, only 1-step strong anion chromatography can give alpha-lactalbumin and beta-lactoglobulin having purities 86.97% and 97.32%, which are significantly reduced in purity as compared with the two-step chromatography of experimental example 1, but can be used as a process for producing alpha-lactalbumin having ordinary purity.
As is clear from comparative examples 1 and 3, the results of experiments conducted only with weak anion chromatography revealed that the results were greatly affected by the fluctuation of the raw material, the fluctuation of the alpha-lactalbumin purity was large, and the production of a product of purity of 95% or more was impossible, the overall chromatographic flow rate of the weak anion chromatography was about 1/3 of that of experimental example 1, and the loading of the weak anion chromatography was about 7/10 of that of experimental example 1 in 70 column volumes.
As can be seen from comparative examples 1 and 4, the absence of pH adjustment in step 14 of example 1 resulted in a significant decrease in the solubility of beta-lactoglobulin.
Example 6
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
(1) Step 9 is not performed;
(2) And 10, replacing the trapped liquid in the step 9 with the mixed liquid of the flow-through liquid and the elution 1 in the step 8.
The target product was detected as described in example 5, with the following results.
TABLE 9 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 6
From the results, it was found that the absence of step 9 resulted in too high a conductivity of the feed solution for the second chromatography, and the ability of the α -lactalbumin to pass through the column was reduced, resulting in a reduced recovery rate, and the proteins were not easily separated during elution. And without step 9 the feed was not concentrated, an increase in feed from about 40L to about 120L in example 1 would result in a 3-fold increase in chromatography time in step 10.
Example 7
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
the raw material liquid of the strong anion chromatography was changed to the first permeate (natural whey liquid) of step 1.
The target product was detected as described in example 5, with the following results.
TABLE 10 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 7
As shown by the results, the recovery rate and the protein purity of the raw material prepared by directly using the natural whey are obviously reduced, the flow rate during the loading of the strong anion chromatography is reduced from 700cm/h to 500cm/h, otherwise, the column pressure exceeds the working pressure, the loading time is increased by about 25% -35%, the raw material liquid with the protein concentration of 0.6% in the embodiment 1 is loaded with about 630 CV, the total protein amount is about 3.78kg, the protein concentration of the natural whey in the embodiment is about 0.3%, the protein amount of the loaded 100CV is about 630L, the total protein amount is about 1.89kg, the batch treatment amount is reduced by half, and the process time is increased by 1.25-1.35 times when the same protein treatment amount is achieved.
Example 8
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
omitting the elution 1 in the chromatography of step 8;
Elution 3 in step 10 chromatography was omitted.
The target product was detected as described in example 5, with the following results.
TABLE 11 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 8
From the results, it was found that the recovery rate of α -lactalbumin was reduced as compared with example 1, and the purity of β -lactoglobulin was reduced.
Example 9
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
Omitting the column flushing in the chromatography of the step 8;
column washing in step 10 chromatography was omitted.
The target product was detected as described in example 5, with the following results.
The purity and recovery rate of the target protein are not obviously affected after omitting the column flushing step in the two-step chromatography procedure, but the column balancing time of the next batch is greatly prolonged, the balancing procedure in the step 8 of the second batch is that the phase A is balanced by 5 times of column volume, the flow rate is 360 cm/h-B phase is balanced by 3 times of column volume, the flow rate is 120 cm/h-A phase is balanced by 7 times of column volume, the flow rate is 360cm/h, the balancing procedure in the step 10 of the second batch is that the phase A is balanced by 5 times of column volume, the flow rate is 360 cm/h-B phase is balanced by 3 times of column volume, the flow rate is 240 cm/h-A phase is balanced by 7 times of column volume, and the flow rate is 360cm/h.
Example 10
A method for extracting a target product from a milk raw material is substantially the same as in experimental example 1, except that:
omitting post-column equilibrium in the chromatography of the step 8;
Post-column equilibration in step 10 chromatography was omitted.
The target product was detected as described in example 5, with the following results.
TABLE 12 detection results of alpha-lactalbumin powder and beta-lactoglobulin powder of example 10
From the results, the purity of the target protein was reduced but the recovery rate was improved after omission.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (22)
1. A method of extracting a target product from a milk raw material, wherein the target product comprises β -lactoglobulin and/or α -lactalbumin, the method comprising the steps of:
obtaining a first permeate after micro-filtration of the skimmed milk;
Ultrafiltering the first permeate to obtain a second permeate and a second retentate;
The conductivity of the second trapped fluid is regulated to 3-8 mS/cm, then strong anion exchange chromatography is carried out, so that alpha-lactalbumin-containing flow-through fluid and beta-lactoglobulin-containing eluent are obtained, the elution step of the strong anion exchange chromatography comprises eluting 1 and eluting 2, the eluting 1 is carried out so that alpha-lactalbumin-containing eluent is obtained, the eluting 2 is carried out so that beta-lactoglobulin-containing eluent is obtained, the eluting 1 is a mixed solution composed of 92% -94% (v/v) of phase A and 6% -8% (v/v) of phase B, the eluting 2 is the phase B, wherein the phase A is 15-35 mmol/L of Tris-HCl solution with pH of 7.8-8.2, and the phase B is a mixed solution containing 15-35 mmol/L of pH 7.8-8. 8.2 Tris-HCl solution and 0.1-1 mol/L of sodium chloride;
Ultrafiltering the flowing through liquid and/or the eluent of the elution 1 to collect trapped fluid, and adopting the relative trapped fluid of A to carry out washing filtration until the conductance of the trapped fluid is consistent with the conductance of the phase A;
The method comprises the steps of performing weak anion exchange chromatography on trapped liquid after washing and filtering, wherein the eluting step of the weak anion exchange chromatography comprises eluting 3 and eluting 4, eluting 3 to obtain an eluent containing alpha-lactalbumin, eluting 4 to obtain an eluent containing beta-lactoglobulin, the eluent of eluting 3 adopts a mixed liquid consisting of 92% -94% (v/v) of phase A and 6% -8% (v/v) of phase B, the eluent of eluting 4 is phase B, wherein the phase A is 15-35 mmol/L of Tris-HCl solution with pH of 7.8-8.2, and the phase B is a mixed liquid containing 15-35 mmol/L of Tris-HCl solution with pH of 7.8-8.2 Tris-HCl solution and 0.1-1 mol/L of sodium chloride.
2. The method according to claim 1, wherein the molecular retention of the membrane for ultrafiltration of the first permeate is 5-20 kDa.
3. The method of claim 1, wherein prior to adjusting the conductance, the method further comprises concentrating the second retentate such that the concentration of protein in the second retentate is 0.4% -0.8% (w/w).
4. The method according to claim 1, wherein the strong anion exchange chromatography is performed after the second retentate is adjusted to a conductivity of 3 to 5 mS/cm.
5. The method of claim 1, wherein the strong anion exchange chromatography employs a column that is a TA-Q XL BB column.
6. The method of claim 1, wherein the chromatography procedure of the strong anion exchange chromatography is equilibration-loading-post column equilibration-elution 1-elution 2-column CIP-column rinse.
7. The method of claim 1, wherein the chromatography procedure of weak anion exchange chromatography is equilibrium-loading-post column equilibrium-elution 3-elution 4-column CIP-column rinse.
8. The method according to claim 1, characterized in that the ultrafiltration of the flow-through and/or elution 1 eluate adopts a molecular cut-off of 3-5 kda.
9. The method according to claim 1, further comprising performing at least one of ultrafiltration, pH adjustment and drying of the beta-lactoglobulin-containing eluate obtained in the strong anion exchange chromatography and/or the weak anion exchange chromatography.
10. The method of claim 9, wherein the pH adjustment is to adjust the pH of the solution to 2.5-3.5.
11. The method according to claim 9, wherein the beta-lactoglobulin-containing eluate is ultrafiltered with a molecular cutoff of 3-5 kDa.
12. The method according to claim 1, wherein the method further comprises ultrafiltration and/or drying of the alpha-lactalbumin-containing flow-through and/or eluate obtained in the strong anion exchange chromatography and/or the weak anion exchange chromatography.
13. The method according to claim 12, characterized in that the molecular retention of the filter membrane used for ultrafiltration of the alpha-lactalbumin-containing flow-through and/or eluate is 3-5 kda.
14. The method of claim 1, wherein the target product further comprises at least one of casein micelles, lactose and milk mineral salts.
15. The method of claim 1, wherein the microfiltration membrane has a pore size of 0.05-0.2 μm.
16. The method of claim 1, further comprising obtaining a first retentate of skim milk after the microfiltration;
And ultrafiltering the first trapped fluid to obtain a third permeation fluid containing lactose and a third trapped fluid containing casein micelle.
17. The method of claim 16, wherein the first retentate is ultrafiltered with a molecular cutoff of 3-30 kDa.
18. The method of claim 16, further comprising drying the third retentate to obtain casein micelle powder.
19. The method of claim 16, further comprising nanofiltration of the second permeate and/or the third permeate to obtain a fourth retentate comprising lactose and a fourth permeate comprising milk mineral salts.
20. The method of claim 19, wherein the nanofiltration employs a membrane molecular cutoff of 300-1000 Da.
21. The method according to claim 19, wherein and/or the method further comprises: and drying the fourth trapped fluid to obtain the lactose powder.
22. The method of claim 19, further comprising drying the fourth permeate to obtain milk mineral salt powder.
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| CN118909086A (en) * | 2024-10-12 | 2024-11-08 | 安徽天凯生物科技有限公司 | Method for separating alpha-lactalbumin and beta-lactoglobulin |
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| CN118909086A (en) * | 2024-10-12 | 2024-11-08 | 安徽天凯生物科技有限公司 | Method for separating alpha-lactalbumin and beta-lactoglobulin |
| CN120607599A (en) * | 2025-01-26 | 2025-09-09 | 黑龙江飞鹤乳业有限公司 | Protein separation and preparation method |
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