A method of extracting a target product from a milk raw material

Abstract

This invention discloses a method for extracting target products from dairy raw materials, relating to the field of food processing technology. Before strong anion exchange chromatography, this method introduces a synergistic pretreatment step of ultrafiltration and conductivity adjustment, achieving "speed increase, efficiency enhancement, quality improvement, and cost reduction" in the chromatography process. It overcomes the technical bottlenecks of traditional ion exchange chromatography in processing dairy raw materials, such as slow speed, susceptibility to contamination, and instability, providing a solution for the efficient, high-purity, and large-scale production of β-lactoglobulin and α-lactalbumin. This method can also combine strong and weak anion exchange chromatography to further improve the extraction purity of β-lactoglobulin and α-lactalbumin. Furthermore, this method can simultaneously achieve efficient extraction of multiple target substances such as casein micelles, lactose, and milk mineral salts, thereby significantly increasing the added value and resource utilization efficiency of dairy by-products.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and more specifically, to a method for extracting a target product from dairy raw materials. Background Technology

[0002] Dairy products are an important source of nutrition for humans. Their core value lies not only in providing basic nutrients but also in containing a variety of highly bioactive and high-value functional components, such as β-lactoglobulin (β-Lg), α-lactalbumin (α-La), casein micelles, lactose, and milk mineral salts. These substances have broad application prospects in the food industry, infant formula, sports nutrition, pharmaceuticals, and health products. For example, β-lactoglobulin and α-lactalbumin are high-quality protein sources with specific amino acid compositions and physiological functions; casein micelles play a significant role in improving food texture due to their unique structure; lactose is an important ingredient in many foods; and milk mineral salts are a natural, highly bioavailable calcium supplement.

[0003] Currently, there are many reports on technologies for separating and extracting the above-mentioned target substances from dairy raw materials (such as skim milk, whey, etc.), but most of them have certain limitations and it is difficult to achieve efficient, high-purity, and multi-component synergistic extraction.

[0004] Regarding the separation of whey proteins (β-lactoglobulin and α-lactalbumin): Traditional methods mainly employ chromatography, especially ion exchange chromatography and hydrophobic interaction chromatography. Currently, chromatography suffers from low throughput, long process flow, high equipment investment, and high eluent consumption, resulting in high production costs and making it unsuitable for large-scale industrial production. Membrane separation technologies (such as ultrafiltration and microfiltration) are also widely used, but conventional ultrafiltration membranes exhibit poor selectivity when separating β-lactoglobulin and α-lactalbumin, which have similar molecular weights, making it difficult to obtain high-purity single components.

[0005] Regarding the separation of casein micelles: Currently, casein micelles are mainly separated from skim milk using ultracentrifugation or microfiltration. However, ultracentrifugation is extremely energy-intensive and carries the risk of damaging the micelle structure, resulting in limited yield. While microfiltration is gentler, it suffers from rapid flux decay and short operating cycles, impacting production efficiency and economic benefits. Furthermore, effectively avoiding whey protein contamination and maintaining the integrity of the casein micelle structure during separation remains a technical challenge.

[0006] Regarding the extraction of lactose and milk mineral salts: Lactose is usually obtained through whey concentration and crystallization, but this process is easily affected by impurity ions, resulting in low crystal purity and poor crystal morphology. Milk mineral salts (mainly milk calcium) are often extracted using acid precipitation or ion exchange methods, but these methods may introduce chemical reagents, leading to problems such as low product purity, off-flavors, or damage to natural conformations, thus affecting their application in high-end foods.

[0007] Most existing technologies focus on separating single or a few components from dairy raw materials, lacking an integrated process that can systematically and continuously extract multiple high-value components (including casein micelles, whey protein, lactose, and milk mineral salts) from the same raw material in a synergistic manner.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a method for extracting a target product from dairy raw materials.

[0010] This invention is implemented as follows:

[0011] A method for extracting a target product from milk raw materials, the target product comprising β-lactoglobulin and / or α-lactalbumin, comprising the following steps:

[0012] Obtain the first permeate of skim milk after microfiltration;

[0013] The first permeate is subjected to ultrafiltration to obtain a second permeate and a second retentate;

[0014] After adjusting the conductivity of the second retentate to 3-8 mS / cm, strong anion exchange chromatography was performed to obtain a flow-through buffer containing α-lactalbumin and an eluent containing β-lactoglobulin.

[0015] The present invention has the following beneficial effects:

[0016] (1) This application develops a novel extraction method. Before strong anion exchange chromatography, a synergistic pretreatment step of ultrafiltration and conductivity adjustment is introduced, which realizes "speeding up, efficiency improving, quality enhancement and cost reduction" of the chromatography process. It overcomes the technical bottlenecks of slow speed, easy contamination and instability in traditional ion exchange chromatography when processing milk raw materials, and provides a solution for the efficient, high-purity and large-scale production of β-lactoglobulin and α-lactalbumin.

[0017] (2) Combining strong anion exchange chromatography and weak anion exchange chromatography can further improve the extraction purity of β-lactoglobulin and α-lactalbumin, with purities reaching 98.65% and 97.39% or higher, respectively;

[0018] (3) This extraction method can also simultaneously achieve efficient extraction of multiple target products such as casein micelles, lactose and milk mineral salts, thereby greatly improving the added value and resource utilization efficiency of dairy by-products. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A process flow diagram for extracting the target product from dairy raw materials;

[0021] Figure 2 for Figure 1 Flowchart of the chromatography steps;

[0022] Figure 3 The mass spectrum of the target protein in Example 1 is shown below; where A is the mass spectrum of α-lactalbumin and B is the mass spectrum of β-lactoglobulin. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The terms “first,” “second,” “third,” “fourth,” “1,” “2,” “3,” “4,” etc., are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0025] The method for extracting target proteins from milk raw materials provided by this invention, particularly through the introduction of a synergistic pretreatment step of ultrafiltration and conductivity adjustment before strong anion exchange chromatography, brings significant and unexpected synergistic technical effects, mainly reflected in the following aspects:

[0026] Significantly improved chromatography speed and efficiency: Ultrafiltration effectively removes interference from insoluble impurities other than the target product in the milk feedstock, greatly reducing column clogging and contamination. This results in stable column pressure and significantly increased flow rate during chromatography, allowing for sample loading and elution at higher flow rates, thereby drastically shortening extraction time and improving equipment utilization. Simultaneously, precise conductivity adjustment optimizes the conductivity of the feed solution to the range where the binding force between the target protein and the packing material is strongest, greatly improving the mass transfer efficiency of protein molecules. The combination of ultrafiltration and conductivity adjustment enables the target protein to complete the chromatography process at a higher flow rate and a shorter equilibration time, resulting in a significant increase in chromatography speed and thus a substantial increase in feedstock throughput per unit time.

[0027] Significantly improves the purity of the target product: Ultrafiltration pretreatment removes not only insoluble impurities but also a large number of soluble contaminating proteins that can compete with the target protein for binding sites, achieving preliminary purification of the sample. Based on this, precise conductivity adjustment further "sharpened" the differences in charge properties between the target proteins (β-lactoglobulin and α-lactalbumin) and residual contaminating proteins.

[0028] Improving process stability: The combination of the "protective effect" of ultrafiltration on the packing material and the "stabilizing effect" of conductivity adjustment on the process greatly enhances the repeatability and stability of the entire purification process. This not only reduces the frequency of expensive chromatography packing material replacement and production costs, but also ensures a high degree of consistency in product quality during large-scale production, laying a solid foundation for industrial applications.

[0029] Furthermore, this invention combines strong anion chromatography and weak anion chromatography with membrane filtration technology, which significantly improves the chromatography efficiency and throughput of weak anion chromatography, achieving high-purity extraction of β-lactoglobulin and α-lactalbumin. The purity of β-lactoglobulin can reach over 98.65%, and the purity of α-lactalbumin can reach over 97.39%.

[0030] Embodiments of the present invention provide a method for extracting a target product from milk raw materials, the target product comprising β-lactoglobulin and / or α-lactalbumin, comprising the following steps:

[0031] Obtain the first permeate of skim milk after microfiltration;

[0032] The first permeate is subjected to ultrafiltration to obtain a second permeate and a second retentate;

[0033] After adjusting the conductivity of the second retentate to 3-8 mS / cm, strong anion exchange chromatography was performed to obtain a flow-through buffer containing α-lactalbumin and an eluent containing β-lactoglobulin.

[0034] In an optional embodiment, the method further includes: microfiltration of skim milk to obtain a first permeate and a first retentate.

[0035] In an optional embodiment, the pore size of the microfiltration membrane is 0.05~0.2 μm, specifically any one or any two of 0.05, 0.1, 0.12, 0.14, 0.16, 0.18 and 0.2 μm.

[0036] In an optional embodiment, the temperature of the microfiltration is 10~60°C, specifically any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60°C.

[0037] In an optional embodiment, the transmembrane pressure difference of the microfiltration is controlled within 1 to 2 bar, specifically within any one or any two of 1, 1.2, 1.4, 1.6, 1.8, and 2 bar. The formula for calculating the transmembrane pressure difference is:

[0038] .

[0039] In an optional embodiment, the volumetric concentration factor of the microfiltration is 2 to 5 times, specifically any one or any two of 2, 3, 4 and 5 times.

[0040] In an optional embodiment, the method further includes washing the microfiltration retentate to obtain a final first retentate for subsequent steps; wherein the washing filtrate can be RO water, and the washing ratio is 1 to 5 times the volume of skim milk, specifically any one or any two of 1, 2, 3, 4 and 5 times.

[0041] In an optional embodiment, the method further includes: obtaining a first retentate of skim milk after microfiltration;

[0042] The first retentate is subjected to ultrafiltration to obtain a third permeate containing lactose and a third retentate containing casein micelles.

[0043] In an optional embodiment, the molecular cutoff of the filter membrane for ultrafiltration of the first retentate is 3~30 kDa, specifically any one or any two of the following: 3, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 kDa.

[0044] In an optional embodiment, the temperature for ultrafiltration of the first retentate can be 10~25°C, specifically any one or any two of 10, 12, 14, 16, 18, 20, 22, 24 and 25°C.

[0045] In an optional embodiment, the volume concentration factor of ultrafiltration of the first retentate is 2 to 5 times, specifically any one or any two of 2, 3, 4 and 5 times.

[0046] In an optional embodiment, after ultrafiltration of the first retentate, the method includes washing the ultrafiltered third retentate to obtain a washed third retentate for subsequent steps. The washing filtrate can be RO water, and the washing ratio is 1 to 5 times the volume of the skim milk, preferably 1 to 2 times.

[0047] In an optional embodiment, the method further includes drying the third retentate to obtain casein micelle powder.

[0048] In an optional embodiment, the drying process can be spray drying.

[0049] In an optional embodiment, the molecular cutoff of the filter membrane for ultrafiltration of the first permeate is 3 to 30 kDa, specifically any one or any two of the following: 3, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 kDa.

[0050] In an optional embodiment, the filter membrane used for ultrafiltration of the first permeate has a molecular cutoff of 5-15 kDa.

[0051] In an optional embodiment, the conditions for ultrafiltration of the first permeate include: a temperature of 10-25°C and a volume concentration factor of 2-5 times. Specifically, the temperature can be any one or any two of 10, 12, 14, 16, 18, 20, 22, 24, and 25°C, and the volume concentration factor can be any one or any two of 2, 3, 4, and 5 times.

[0052] In an optional embodiment, after ultrafiltration of the first permeate, the method further includes washing the second retentate to obtain a washed second retentate for subsequent steps. The washing filtrate can be RO water, and the washing ratio is 1 to 5 times the volume of the skim milk, specifically any one or any combination of 1, 2, 3, 4, and 5 times.

[0053] In an optional embodiment, before adjusting the conductivity, the method further includes: concentrating the second retentate to achieve a protein concentration of 0.4% to 0.8% (w / w), specifically, this concentration can be any one or any two of 0.4%, 0.5%, 0.6%, 0.7%, and 0.8%. The protein concentration of the natural whey obtained after normal microfiltration is 0.2% to 0.3%. With a consistent total protein content, a higher protein concentration results in a smaller total volume of whey. As a direct raw material for chromatography, a smaller volume of whey leads to a shorter time at the same loading flow rate, effectively improving chromatographic efficiency.

[0054] In an optional embodiment, strong anion exchange chromatography is performed after adjusting the conductivity of the second retentate to any one or any two of the ranges of 3, 4, 5, 6, 7, and 8 mS / cm.

[0055] In an optional embodiment, the conductivity adjustment is performed using a sodium chloride solution. The concentration of the sodium chloride solution can be any one or a range between any two of the following: 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, and 30% (w / w).

[0056] In an optional embodiment, the method further includes: nanofiltration of the second permeate and / or the third permeate to obtain a fourth retentate containing lactose and a fourth permeate containing milk mineral salts.

[0057] In an optional embodiment, the nanofiltration membrane has a molecular cutoff of 300~1000 Da, specifically any one or any two of 300, 400, 500, 600, 700, 800, 900, and 1000 Da.

[0058] In an optional embodiment, the nanofiltration temperature can be 10~25°C, specifically any one or any two of 10, 12, 14, 16, 18, 20, 22, 24 and 25°C.

[0059] In an optional embodiment, the volumetric concentration factor of the nanofiltration is 2 to 5 times, specifically any one or any two of 2, 3, 4 and 5 times.

[0060] In an optional embodiment, after nanofiltration, the method further includes washing the fourth retentate after nanofiltration. The washing conditions are the same as those described in any of the foregoing embodiments.

[0061] In an optional embodiment, the method further includes: drying the fourth retentate to obtain lactose powder;

[0062] In an optional embodiment, the method further includes drying the fourth permeate to obtain milk mineral salt powder.

[0063] In an optional embodiment, the chromatography procedure for the strong anion exchange chromatography is: equilibration-sample loading-post-column equilibration-elution 1-elution 2-column CIP-column rinsing.

[0064] In an optional embodiment, the equilibration program for strong anion exchange chromatography is: equilibration of phase A for 2-5 column volumes - equilibration of phase B for 2-5 column volumes - equilibration of phase A for 3-10 column volumes, with a flow rate of 240-500 cm / h; preferably, it is equilibration of phase A for 2 column volumes - equilibration of phase B for 2 column volumes - equilibration of phase A for 3 column volumes, with a flow rate of 400-500 cm / h.

[0065] In an optional embodiment, phase A is a 15-35 mmol / L Tris-HCl solution with a pH of 7.8-8.2.

[0066] In an optional embodiment, phase B is a mixture containing 15–35 mmol / L pH 7.8–8.2 Tris-HCl solution and 0.1–1 mol / L sodium chloride. The preparation of phase B involves adding 0.1–1 mol / L sodium chloride to the 15–35 mmol / L pH 7.8–8.2 Tris-HCl solution, where 0.1–1 mol / L is the concentration of sodium chloride in phase B.

[0067] In an optional embodiment, the 15~35 mmol / L can specifically be any one or any two of 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 35 mmol / L.

[0068] In an optional embodiment, pH 7.8 to 8.2 can specifically be any one of 7.8, 7.9, 8.0, 8.1, 8.2 or any range between two of them.

[0069] In an optional embodiment, the 0.1~1 mol / L can specifically be any one or any two of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mol / L.

[0070] In an optional embodiment, the loading procedure for strong anion exchange chromatography is a concentrated whey solution with a conductivity of 3-8 mS / cm, a flow rate of 500-700 cm / h, preferably 700 cm / h, and a loading volume of 100 times the column volume.

[0071] In an optional embodiment, the post-column equilibration program for strong anion exchange chromatography is to wash phase A at a flow rate of 500-700 cm / h, preferably 700 cm / h, for 2-5 column volumes, preferably 2 column volumes.

[0072] In an optional embodiment, after elution 1, an eluent containing α-lactalbumin is obtained; after elution 2, an eluent containing β-lactoglobulin is obtained.

[0073] In an optional embodiment, the eluent for elution 1 comprises a mixture of phase A (92%–94% v / v) and phase B (6%–8% v / v). Specifically, the 92%–94% can be any one or any two of the following: 92%, 93%, 93.2%, 93.4%, 93.6%, 93.8%, and 94%. Similarly, the 6%–8% can be any one or any two of the following: 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, and 8%.

[0074] In an optional embodiment, elution 1 uses 3 to 8 column volumes of eluent; specifically, 3 to 8 times can be any one or any two of 3, 4, 5, 6, 7 and 8 times.

[0075] In an optional embodiment, the eluent of elution 2 includes phase B.

[0076] In an optional embodiment, elution 2 uses 5 to 10 column volumes of eluent; specifically, 5 to 10 times can be any one or any two of 5, 6, 7, 8, 9 and 10 times.

[0077] In an optional embodiment, the flow rate of elution 1 and / or elution 2 is 240~500 cm / h, specifically it can be any one or any two of 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 cm / h.

[0078] In an optional embodiment, the column CIP program for strong anion exchange chromatography is to wash with 3-5 column volumes of 0.5 mol / L sodium hydroxide solution, preferably 4 column volumes, at a flow rate of 240-500 cm / h, preferably 400-500 cm / h.

[0079] In an optional embodiment, the column washing procedure for strong anion exchange chromatography is 100% B phase elution for 2 to 5 column volumes, preferably 5 column volumes, and a flow rate of 240 to 500 cm / h, preferably 400 to 500 cm / h.

[0080] In an optional embodiment, the strong anion exchange chromatography uses a TA-QXL BB column.

[0081] In an optional embodiment, the method further includes performing weak anion exchange chromatography on the flow-through liquid and / or the eluent of elution 1.

[0082] In an optional embodiment, the method further includes: subjecting the retentate obtained by ultrafiltration of the flow-through liquid and / or the eluent of elution 1 to the weak anion exchange chromatography.

[0083] In an optional embodiment, the ultrafiltration of the flow-through liquid and / or the eluent of elution 1 uses a molecular cutoff of 3 to 5 kDa, specifically any one or any two of 3, 3.5, 4, 4.5, and 5 kDa.

[0084] In an optional embodiment, the volume concentration factor of the ultrafiltration of the flow-through liquid and / or the eluent of elution 1 is 2 to 5 times, specifically any one or any two of 2, 3, 4, and 5 times.

[0085] In an optional embodiment, after ultrafiltration of the flow-through liquid and / or the eluent of elution 1, the method further includes washing the ultrafiltered retentate. The washing solution used for this washing can be phase A as described in any of the foregoing embodiments, and the washing is performed until the conductivity of the retentate is consistent with that of phase A (1.5 mS / cm).

[0086] In an optional embodiment, the chromatography procedure for the weak anion exchange chromatography is: equilibration-sample loading-post-column equilibration-elution 3-elution 4-column CIP-column rinsing.

[0087] In an optional embodiment, the equilibration program for weak anion exchange chromatography is: equilibration of phase A for 2-5 column volumes - equilibration of phase B for 2-5 column volumes - equilibration of phase A for 3-10 column volumes, with a flow rate of 240-500 cm / h for all phases. Preferably, it is: equilibration of phase A for 2 column volumes - equilibration of phase B for 2 column volumes - equilibration of phase A for 3 column volumes, with a flow rate of 240-360 cm / h for all phases. Phases A and B are the same as described in the aforementioned embodiments.

[0088] In an optional embodiment, the loading procedure for weak anion exchange chromatography is to load all of the flow-through liquid and / or the eluent of elution 1 or the retentate after ultrafiltration, at a flow rate of 500-700 cm / h, preferably 600 cm / h; the post-column equilibration procedure is to wash phase A at a flow rate of 500-700 cm / h, preferably 600 cm / h, for 2-5 column volumes, preferably 2 column volumes.

[0089] In an optional embodiment, after elution 3, an eluent containing α-lactalbumin is obtained, and after elution 4, an eluent containing β-lactoglobulin is obtained.

[0090] In an optional embodiment, the eluent of elution 3 comprises a mixture of phase A (92%–94% v / v) and phase B (6%–8% v / v). Specifically, the 92%–94% can be any one or any two of the following: 92%, 93%, 93.2%, 93.4%, 93.6%, 93.8%, and 94%. Similarly, the 6%–8% can be any one or any two of the following: 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, and 8%.

[0091] In an optional embodiment, the elution 3 uses 3 to 8 column volumes of eluent; specifically, 3 to 8 times can be any one or any two of 3, 4, 5, 6, 7 and 8 times.

[0092] In an optional embodiment, the eluent of elution 4 includes phase B;

[0093] In an optional embodiment, elution 4 uses 5 to 10 column volumes of eluent; specifically, 5 to 10 times can be any one or any two of 5, 6, 7, 8, 9 and 10 times.

[0094] In an optional embodiment, the flow rate of elution 3 and / or elution 4 is 240~500 cm / h, specifically it can be any one or any two of 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 cm / h.

[0095] In an optional embodiment, the column CIP program for weak anion exchange chromatography is to wash with 3-5 column volumes of 0.5 mol / L sodium hydroxide solution, preferably 4 column volumes, at a flow rate of 240-500 cm / h, preferably 240-360 cm / h.

[0096] In an optional embodiment, the column washing procedure for weak anion exchange chromatography is 100% B phase elution for 2-5 column volumes, preferably 5 column volumes, and a flow rate of 240-500 cm / h, preferably 240-360 cm / h.

[0097] In an optional embodiment, the weak anion exchange chromatography column used is a TA-SPXL BB column.

[0098] In an optional embodiment, the method further includes: ultrafiltration and / or drying of the flow-through and / or eluent containing α-lactalbumin obtained by the strong anion exchange chromatography and / or the weak anion exchange chromatography;

[0099] In an optional embodiment, the filter membrane used for ultrafiltration of the flow-through and / or eluent containing α-lactalbumin has a molecular cutoff of 3 to 5 kDa, specifically any one or any two of 3, 3.5, 4, 4.5, and 5 kDa.

[0100] In an optional embodiment, the volume concentration factor of ultrafiltration of the flow-through and / or eluent containing α-lactalbumin is 2 to 5, specifically any one or any two of 2, 3, 4, and 5 times.

[0101] In an optional embodiment, after ultrafiltration of the flow-through and / or eluent containing α-lactalbumin, the retentate after ultrafiltration is further washed with RO water until the conductivity of the retentate is ≤0.1 mS / cm.

[0102] In an optional embodiment, the method further includes: ultrafiltration, pH adjustment, and drying of at least one of the following: the eluent containing β-lactoglobulin obtained by the strong anion exchange chromatography and / or the weak anion exchange chromatography;

[0103] In an optional embodiment, the pH adjustment refers to adjusting the pH of the solution to 2.5-3.5;

[0104] In an optional embodiment, the membrane used for ultrafiltration of the β-lactoglobulin-containing eluent has a molecular cutoff of 3 to 5 kDa, specifically any one or any two of 3, 3.5, 4, 4.5, and 5 kDa.

[0105] In an optional embodiment, the concentration factor of the ultrafiltration of the eluent containing β-lactoglobulin is 2 to 5 times, specifically any one or any two of 2, 3, 4, and 5 times.

[0106] In an optional embodiment, after ultrafiltration of the eluent containing β-lactoglobulin and before pH adjustment, the method further includes washing the ultrafiltered retentate to obtain a washed retentate for subsequent pH adjustment. The washing solution can be RO water, and washing is performed until the conductivity of the retentate is ≤0.1 mS / cm.

[0107] In an optional embodiment, the target product further includes at least one of casein micelles, lactose, and milk mineral salts.

[0108] In optional embodiments, the filter membranes of microfiltration, ultrafiltration, and nanofiltration described in any of the foregoing embodiments can be organic spiral wound membranes, and the membrane material can be PES.

[0109] The process flow diagram of the extraction method is shown in the figure. Figure 1 The flowchart of the chromatography steps is shown in the figure. Figure 2 .

[0110] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0111] The strong anion exchange chromatography column used in the examples has the following characteristics: packing material: TA-Q XL BB; column bed height: 20 cm; column inner diameter: 20 cm; length-to-diameter ratio: 1:1; packing compression ratio: 1:1.15; packing volume: 6.28 L; and operating pressure: 0.25 MPa.

[0112] The weak anion exchange chromatography column used in the examples had the following characteristics: packing material: TA-SP XL BB; column bed height: 20 cm; column inner diameter: 40 cm; aspect ratio: 1:2; packing material compression ratio: 1:1.20; packing material volume: 25 L; operating pressure: 0.28 MPa. Phase A was a 25 mmol / L pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution.

[0113] Phase B is a mixture containing 25 mmol / L Tris-HCl solution at pH 8.0 and 0.5 mol / L sodium chloride.

[0114] Example 1

[0115] A method for extracting a target product from dairy raw materials, comprising the following steps.

[0116] (1) Step 1:

[0117] Skim milk was microfiltered using a 0.1-micron PES organic spiral wound membrane to obtain a first permeate (natural whey protein solution) and a first retentate (casein micelle solution); the microfiltration temperature was 50°C; the transmembrane pressure difference was controlled at 1.4 bar; and the concentration factor was 3 times.

[0118] The first retentate was washed and filtered with RO water at a ratio of 1.5 times the volume of skim milk to obtain the first retentate (casein micelles).

[0119] (2) Step 2:

[0120] The first retentate obtained in step 1 was ultrafiltered using a 10kDa PES organic spiral wound membrane to obtain a third retentate and a third permeate (lactose solution); wherein the ultrafiltration temperature was 15°C and the concentration factor was 3 times;

[0121] The third retentate was washed and filtered with RO water at a ratio of 1 times the volume of the skim milk in step 1 to obtain the washed and filtered third retentate (casein micelles).

[0122] (3) Step 3:

[0123] The third retentate (casein micelles) obtained in step 2 can be spray-dried to obtain casein micelle powder.

[0124] (4) Step 4:

[0125] The first permeate (natural whey protein solution) from step 1 was ultrafiltered using a 10kDa PES organic spiral wound membrane to obtain a second retentate and a second permeate; wherein the ultrafiltration temperature was 15°C and the concentration factor was 3 times;

[0126] The second retentate is washed and filtered, and the washing filtrate is RO water. The washing ratio is 1 times the volume of the skim milk in step 1.

[0127] The second retentate after washing and filtration is concentrated to a protein concentration of 0.6% (w / w) to obtain a concentrated second retentate (concentrated whey).

[0128] (5) Step 5:

[0129] The third permeate from step 2 and the second permeate from step 4 were mixed and then nanofiltered using a 500Da PES organic spiral wound membrane to obtain a fourth retentate (concentrated lactose solution) and a fourth permeate (milk mineral salt solution); wherein the nanofiltration temperature was 20°C and the concentration factor was 3 times.

[0130] The fourth retentate is washed and filtered to obtain the washed and filtered fourth retentate; wherein the washing filtrate is RO water and the washing ratio is twice the initial volume.

[0131] (6) Step 6:

[0132] The fourth permeate from step 5 can be spray-dried to obtain milk mineral salt powder.

[0133] Lactose powder can be obtained by spray drying the fourth retentate from step 5.

[0134] (7) Step 7:

[0135] The conductivity of the concentrated whey from step 4 was adjusted to 4 mS / cm using a 26% (w / w) sodium chloride solution.

[0136] (8) Step 8:

[0137] The concentrated whey solution from step 7, after conductivity adjustment, was subjected to chromatography using a strong anion exchange resin. The chromatography procedure was: equilibration - sample loading - post-column equilibration - elution 1 - elution 2 - column CIP - column washing; wherein:

[0138] The equilibration program was: Phase A equilibration 2 times column volume - Phase B equilibration 2 times column volume - Phase A equilibration 3 times column volume, with a flow rate of 500 cm / h for all phases.

[0139] The loading procedure was a concentrated whey solution with a conductivity of 4 mS / cm, a flow rate of 700 cm / h, and a loading volume of 100 times the column volume.

[0140] The post-column equilibration procedure was to flush phase A at a flow rate of 700 cm / h for 2 times the column volume;

[0141] Elution program 1 consisted of eluting 3 column volumes of 93.4% A phase and 6.6% B phase at a flow rate of 500 cm / h.

[0142] Elution program 2: 100% B phase elution for 5 column volumes, flow rate 500 cm / h;

[0143] The column CIP program was to wash 4 column volumes with 0.5 mol / L sodium hydroxide solution at a flow rate of 500 cm / h.

[0144] The column flushing procedure was 100% B phase elution at 5 column volumes and a flow rate of 500 cm / h.

[0145] (9) Step 9:

[0146] The column flow-through buffer (containing α-lactalbumin) from the loading procedure in step 8 and the eluent from elution 1 (containing α-lactalbumin) were collected and mixed, and ultrafiltration was performed using a 5 kDa PES organic spiral wound membrane; the ultrafiltration temperature was room temperature; and the concentration factor was 3 times.

[0147] The retentate obtained by ultrafiltration was washed and filtered until the conductivity of the retentate was the same as that of phase A (1.5 mS / cm).

[0148] (10) Step 10:

[0149] The retentate from step 9 was subjected to weak anion exchange chromatography. The chromatography procedure was: equilibration-sample loading-post-column equilibration-elution 3-elution 4-column CIP-column washing, wherein:

[0150] The equilibration program was: equilibration of phase A by 2 column volumes - equilibration of phase B by 2 column volumes - equilibration of phase A by 3 column volumes, with a flow rate of 360 cm / h. Phase A was a 25 mmol / L pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution, and phase B was a mixture containing a 25 mmol / L pH 8.0 tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution and 0.5 mol / L sodium chloride.

[0151] The loading procedure is to load all the retentate obtained in step 9 at a flow rate of 600 cm / h.

[0152] The post-column equilibration procedure was to flush phase A at a flow rate of 600 cm / h for 2 times the column volume;

[0153] The elution program was 6 column volumes of 93.4% A phase and 6.6% B phase, with a flow rate of 360 cm / h.

[0154] Elution program 4: 100% B phase elution for 5 column volumes, flow rate 360 ​​cm / h;

[0155] The column CIP program was to wash 4 column volumes with 0.5 mol / L sodium hydroxide solution at a flow rate of 240 cm / h.

[0156] The column flushing program was 100% B-phase elution at 5 column volumes and a flow rate of 360 cm / h.

[0157] (11) Step 11

[0158] The eluent from step 10, elution 3, was ultrafiltered using a 5 kDa PES organic spiral wound membrane to obtain a retentate; the ultrafiltration temperature was room temperature; and the concentration factor was 3 times.

[0159] The retentate was washed and filtered with RO water until the conductivity of the retentate was ≤0.1mS / cm.

[0160] (12) Step 12:

[0161] The eluent from elution 2 in step 8 and the eluent from elution 4 in step 10 were mixed and then ultrafiltered using a 5kDa PES organic spiral wound membrane to obtain the retentate; wherein, the ultrafiltration temperature was room temperature and the concentration factor was 3 times;

[0162] The retentate was washed and filtered with RO water until the conductivity of the retentate was ≤0.1mS / cm.

[0163] (13) Step 13:

[0164] The retentate obtained in step 11 was spray-dried to obtain high-purity α-lactalbumin powder.

[0165] (14) Step 14:

[0166] The retentate obtained in step 12 was adjusted to pH 3 with 3 mol / L hydrochloric acid and then spray-dried to obtain high-purity β-lactoglobulin powder.

[0167] Example 2

[0168] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0169] (1) Do not perform steps 9 and 10;

[0170] (2) Step 11: Replace “elution buffer of elution 3 in step 10” with a mixture of column flow-through buffer (flow-through buffer containing α-lactalbumin) and elution buffer of elution 1 (elution buffer containing α-lactalbumin) in the sample loading procedure in step 8;

[0171] (3) Step 12: Omit the scheme involving "elution solution of elution 4 in step 10".

[0172] Example 3

[0173] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0174] (1) Do not perform steps 7, 8 and 9;

[0175] (2) Step 10: Replace “the retentate in step 9” with “the second retentate (concentrated whey) obtained in step 4”;

[0176] (3) Step 12: Omit the scheme involving "elution solution of elution 2 in step 8".

[0177] Example 4

[0178] A method for extracting a target product from milk raw materials is largely the same as in Experimental Example 1, except that step 14 does not involve pH adjustment.

[0179] Example 5

[0180] The target products were extracted from milk raw materials according to the methods in Examples 1-4, and α-lactalbumin and β-lactoglobulin were detected using high performance liquid chromatography-mass spectrometry (HPLC-MS). The specific methods are as follows:

[0181] 1. Standards: Bovine alpha-lactalbumin specific peptide standard (Sequence: VGINYWLAHK, molecular weight 1200.4 Da, purity >99%); Bovine alpha-lactalbumin isotope-labeled specific peptide standard (Sequence: VGI*NYWL*AHK, molecular weight 1214.4 Da, purity >95%); Bovine alpha-lactalbumin isotope-labeled internal standard (Sequence: KIDKVGI*NYWL*AHKALCSEK, molecular weight 2443.9 Da, purity >95%); Bovine β-lactoglobulin specific peptide standard (Sequence: IDALNENK, molecular weight 916.0 Da, purity >99%); Bovine β-lactoglobulin isotope-labeled specific peptide standard (Sequence: I*DAL*NENK, molecular weight 930.0 Da). Da (purity ≥ 95%)); Bovine β-lactoglobulin isotope-labeled internal standard (sequence: KIPAVFKI*DAL*NENKVIVLDTDYK, molecular weight 2761.2 Da, purity > 95%); Note: Amino acids marked with * in the peptide sequences shown above are isotope amino acids.

[0182] 2. Weigh approximately 2 g of solid sample or approximately 10 g of liquid sample (accurate to 0.01 g, containing approximately 200 mg of protein) into a 500 mL beaker. Dissolve the sample thoroughly in 900 mL of water in portions. Transfer the solution to a 1000 mL volumetric flask and dilute to the mark with water. Vortex the solution until fully dissolved and set aside. Accurately transfer 200 μL of the above sample solution and 50 μL of the isotope internal standard intermediate mixture into a 2 mL centrifuge tube. Mix well, then add 150 μL of ammonium bicarbonate solution and 10 μL of dithiothreitol solution. Mix well and incubate at 75°C for 30 min. Cool to room temperature, add 30 μL of iodoacetamide solution, and incubate in the dark for 30 min. Then add 10 μL of calcium chloride solution and 50 μL of trypsin solution. Mix thoroughly and incubate at 37°C for 5 hours for enzymatic hydrolysis. Mix with 10 μL of formic acid, let stand at room temperature for 15 min, add 490 μL of water, vortex mix, filter through a 0.22 μm filter membrane for detection by liquid chromatography-tandem mass spectrometry.

[0183] 3. Chromatographic column: silane-based C18 column, 100 mm long, 2.1 mm inner diameter; packing material particle size 1.7 μm, pore size 30 nm (300 (or those with equivalent column effects)

[0184] 4. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution; Gradient elution: Elute according to the reference conditions given in Table 1.

[0185]

[0186] 6. Mobile phase flow rate: 0.3 mL / min.

[0187] 7. Column temperature: 40℃;

[0188] 8. Test solution temperature: 10 ℃.

[0189] 9. Injection volume: 10 μL.

[0190] 10. Mass Spectrometry Reference Conditions

[0191] Electrospray ionization (ESI) mode; mass spectrometry mode: multiple reaction monitoring (MRM); capillary voltage: 3.5 kV, orifice voltage: 35 V, desolventizing temperature: 500 °C, desolventizing gas flow rate: 800 L / h, orifice backflush gas flow rate: 30 L / h, collision chamber pressure: 3.0 × 10⁻⁶ kJ / h mbar.

[0192] 11. Mass spectrometry detection conditions are shown in Table 2:

[0193]

[0194] Test results as follows Figure 3 As shown in the table below.

[0195] Table 3. Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 1

[0196]

[0197] Table 4. Detection results of casein micelle powder, lactose powder, and milk mineral salt powder in Example 1.

[0198]

[0199] Table 5. Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 2

[0200]

[0201] Table 6. Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 3

[0202]

[0203] Note: The results of Example 3 are the results of 5 repeated experiments.

[0204] Table 7. Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 4

[0205]

[0206] Table 8. Comparison of flow rates for ion exchange chromatography in various embodiments

[0207]

[0208] The results show that:

[0209] Comparing Examples 1 and 2, it can be seen that performing only one strong anion chromatography step can yield α-lactalbumin and β-lactoglobulin with purities of 86.97% and 97.32%, respectively. The purity of α-lactalbumin is significantly lower than that of the two-step chromatography in Example 1, but it can still be used as a process for producing α-lactalbumin of ordinary purity.

[0210] Comparing Examples 1 and 3, it can be seen from multiple experiments that weak anion chromatography alone is greatly affected by fluctuations in raw material, resulting in significant fluctuations in the purity of α-lactalbumin, and it is impossible to produce a product with a purity of over 95%. The overall flow rate of weak anion chromatography is about 1 / 3 of that of weak anion chromatography in Example 1, and the sample loading volume of weak anion chromatography is 70 times the column volume, which is about 7 / 10 of the sample loading volume of strong anion chromatography in Example 1.

[0211] Comparing Examples 1 and 4, it can be seen that not performing the pH adjustment in step 14 of Example 1 will result in a significant decrease in the solubility of β-lactoglobulin.

[0212] Example 6

[0213] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0214] (1) Do not perform step 9;

[0215] (2) Step 10: Replace “the retentate in step 9” with the mixture of the flow-through solution in step 8 and the elution solution in step 1.

[0216] The target product was tested using the method described in Example 5, and the results are as follows.

[0217] Table 9. Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 6

[0218]

[0219] The results show that omitting step 9 results in excessively high conductivity of the feed solution, making it unsuitable for the second step of chromatography. This reduces the column-attachment ability of α-lactalbumin, leading to a lower recovery rate, and makes it difficult for the proteins to separate during elution. Furthermore, without step 9, the feed solution is not concentrated, increasing from approximately 40 L in Example 1 to approximately 120 L, which will increase the chromatography time in step 10 by a factor of 3.

[0220] Example 7

[0221] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0222] The feed solution for strong anion chromatography was changed to the first permeate solution (natural whey) from step 1.

[0223] The target product was tested using the method described in Example 5, and the results are as follows.

[0224] Table 10 Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 7

[0225]

[0226] The results show that using natural whey directly as a raw material significantly reduces both the recovery rate and protein purity. The flow rate during strong anion exchange chromatography loading was reduced from 700 cm / h to 500 cm / h; otherwise, the column pressure would exceed the working pressure, increasing the loading time by approximately 25%-35%. In Example 1, the raw material solution with a protein concentration of 0.6% was loaded at 100 CV, yielding approximately 630 L and a total protein content of approximately 3.78 kg. In this example, the protein concentration of the natural whey solution was approximately 0.3%, and the loading was approximately 100 CV, yielding approximately 630 L and a total protein content of approximately 1.89 kg. The batch processing capacity was reduced by half. To achieve the same protein processing capacity, the process time would need to be increased by 1.25-1.35 times.

[0227] Example 8

[0228] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0229] Elution 1 in step 8 of the chromatography is omitted;

[0230] Elution 3 in step 10 of the chromatography is omitted.

[0231] The target product was tested using the method described in Example 5, and the results are as follows.

[0232] Table 11 Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 8

[0233]

[0234] The results show that the recovery rate of α-lactalbumin was lower than that of Example 1, and the purity of β-lactoglobulin was reduced.

[0235] Example 9

[0236] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0237] Omit column washing in step 8 of the chromatography process;

[0238] The column rinsing in step 10 of the chromatography is omitted.

[0239] The target product was tested using the method described in Example 5, and the results are as follows.

[0240] Omitting the column washing step in the two-step chromatography procedure has no significant impact on the purity and recovery rate of the target protein, but it will significantly prolong the column equilibration time of the next batch. The equilibration procedure for the second batch in step 8 is: equilibration of phase A with 5 column volumes at a flow rate of 360 cm / h - equilibration of phase B with 3 column volumes at a flow rate of 120 cm / h - equilibration of phase A with 7 column volumes at a flow rate of 360 cm / h; the equilibration procedure for the second batch in step 10 is: equilibration of phase A with 5 column volumes at a flow rate of 360 cm / h - equilibration of phase B with 3 column volumes at a flow rate of 240 cm / h - equilibration of phase A with 7 column volumes at a flow rate of 360 cm / h.

[0241] Example 10

[0242] A method for extracting the target product from dairy raw materials is largely the same as in Example 1, with the following differences:

[0243] The post-column equilibration in step 8 of the chromatography is omitted;

[0244] Post-column equilibration in step 10 of the chromatography is omitted.

[0245] The target product was tested using the method described in Example 5, and the results are as follows.

[0246] Table 12 Detection results of α-lactalbumin powder and β-lactoglobulin powder in Example 10

[0247]

[0248] The results show that omitting the target protein reduces its purity but increases its recovery rate.

[0249] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting a target product from dairy raw materials, characterized in that, The target product includes β-lactoglobulin and / or α-lactalbumin, and the method includes the following steps: Obtain the first permeate of skim milk after microfiltration; The first permeate is subjected to ultrafiltration to obtain a second permeate and a second retentate; After adjusting the conductivity of the second retentate to 3-8 mS / cm, strong anion exchange chromatography is performed to obtain a flow-through buffer containing α-lactalbumin and an eluent containing β-lactoglobulin. The elution steps of the strong anion exchange chromatography include elution 1 and elution 2. Elution 1 yields an eluent containing α-lactalbumin, and elution 2 yields an eluent containing β-lactoglobulin. The eluent for elution 1 is a mixture of 92%-94% (v / v) phase A and 6%-8% (v / v) phase B. The eluent for elution 2 is phase B. Phase A is a 15-35 mmol / L Tris-HCl solution with pH 7.8-8.2, and phase B is a mixture of 15-35 mmol / L Tris-HCl solution with pH 7.8-8.2 and 0.1-1 mol / L sodium chloride. The flow-through liquid and / or the eluent of elution 1 are subjected to ultrafiltration to collect the retentate, and the retentate is washed with phase A until the conductivity of the retentate is consistent with that of phase A. The retentate after elution was subjected to weak anion exchange chromatography; the elution steps of the weak anion exchange chromatography included elution 3 and elution 4. After elution 3, an eluent containing α-lactalbumin was obtained, and after elution 4, an eluent containing β-lactoglobulin was obtained; the eluent for elution 3 was a mixture of 92%~94% (v / v) of phase A and 6%~8% (v / v) of phase B; the eluent for elution 4 was phase B; wherein phase A was a 15~35 mmol / L Tris-HCl solution with pH 7.8~8.2; and phase B was a mixture containing 15~35 mmol / L Tris-HCl solution with pH 7.8~8.2 and 0.1~1 mol / L sodium chloride.

2. The method according to claim 1, characterized in that, The membrane used for ultrafiltration of the first permeate has a molecular cutoff of 5-20 kDa.

3. The method according to claim 1, characterized in that, Before adjusting the conductivity, the method further includes: concentrating the second retentate to make the protein concentration in the second retentate 0.4%~0.8% (w / w).

4. The method according to claim 1, characterized in that, After adjusting the conductivity of the second retentate to 3-5 mS / cm, the strong anion exchange chromatography was performed.

5. The method according to claim 1, characterized in that, The strong anion exchange chromatography used a TA-Q XL BB column.

6. The method according to claim 1, characterized in that, The chromatography procedure for the strong anion exchange chromatography is: equilibration-sample loading-post-column equilibration-elution 1-elution 2-column CIP-column rinsing.

7. The method according to claim 1, characterized in that, The chromatography procedure for the weak anion exchange chromatography is: equilibration - sample loading - post-column equilibration - elution 3 - elution 4 - column CIP - column rinsing.

8. The method according to claim 1, characterized in that, The ultrafiltration of the flow-through fluid and / or the eluent of elution 1 uses a molecular cutoff of 3-5 kDa.

9. The method according to claim 1, characterized in that, The method further includes at least one of ultrafiltration, pH adjustment, and drying of the eluent containing β-lactoglobulin obtained by the strong anion exchange chromatography and / or the weak anion exchange chromatography.

10. The method according to claim 9, characterized in that, The pH adjustment refers to adjusting the pH of the solution to 2.5~3.

5.

11. The method according to claim 9, characterized in that, The membrane used for ultrafiltration of eluent containing β-lactoglobulin has a molecular cutoff of 3-5 kDa.

12. The method according to claim 1, characterized in that, The method further includes ultrafiltration and / or drying of the flow-through and / or eluent containing α-lactalbumin obtained by the strong anion exchange chromatography and / or the weak anion exchange chromatography.

13. The method according to claim 12, characterized in that, The filter membrane used for ultrafiltration of flow-through and / or eluent containing α-lactalbumin has a molecular cutoff of 3-5 kDa.

14. The method according to claim 1, characterized in that, The target product also includes at least one of casein micelles, lactose, and milk mineral salts.

15. The method according to claim 1, characterized in that, The microfiltration membrane has a pore size of 0.05~0.2 μm.

16. The method according to claim 1, characterized in that, The method further includes: obtaining a first retentate of skim milk after microfiltration; The first retentate is subjected to ultrafiltration to obtain a third permeate containing lactose and a third retentate containing casein micelles.

17. The method according to claim 16, characterized in that, The molecular cutoff of the filter membrane used for ultrafiltration of the first retentate is 3~30 kDa.

18. The method according to claim 16, characterized in that, The method further includes drying the third retentate to obtain casein micelle powder.

19. The method according to claim 16, characterized in that, The method further includes: nanofiltration of the second permeate and / or the third permeate to obtain a fourth retentate containing lactose and a fourth permeate containing milk mineral salts.

20. The method according to claim 19, characterized in that, The nanofiltration membrane used has a molecular cutoff of 300~1000 Da.

21. The method according to claim 19, characterized in that, And / or, the method further includes: drying the fourth retentate to obtain lactose powder.

22. The method according to claim 19, characterized in that, The method further includes drying the fourth permeate to obtain milk mineral salt powder.

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