A method for preparing a target substance and related products and applications thereof

Abstract

The application discloses a preparation method of a target substance and related products and application thereof, and relates to the field of food processing. The application adjusts the pH and conductivity of natural whey to change the whey characteristics, and uses an anion exchange chromatography method to realize high-purity separation of alpha-lactalbumin. The method requires a neutral and slightly alkaline environment, which is beneficial to maintaining the activity of alpha-lactalbumin. The obtained alpha-lactalbumin is rich in alpha-helix structure and has high similarity with the natural structure of alpha-lactalbumin. The separation mode of the anion exchange chromatography is a flow-through mode, which significantly improves the total amount of whey protein treated by ion exchange method, shortens the processing time of each batch, and improves the production efficiency. The method has important significance for the industrialized separation and production of alpha-lactalbumin.

Description

Technical Field

[0001] This invention relates to the field of food processing, and more specifically, to a method for preparing a target substance and related products and applications. Background Technology

[0002] Whey protein is an important nutrient in whey. With the development of comprehensive whey utilization technology, many important functions of whey protein have been gradually discovered. Due to its high nutritional value, easy digestion and absorption, and excellent processing performance, it is widely used in infant formula, health products, and pharmaceuticals.

[0003] Alpha-lactalbumin is widely found in the milk of mammals and humans, and is one of the main components of whey. In cow's milk, the content of alpha-lactalbumin is approximately 1.2 g / L, accounting for 2%–3% of total milk protein; in breast milk, it accounts for 20%–25% of total protein, making it a major protein and a core protein ingredient in infant formula. For infants, alpha-lactalbumin is rich in tryptophan, which can be converted into the neurotransmitter serotonin in the body, promoting the maturation of neurons and synaptic connections, thereby improving infant sleep and enhancing learning and memory abilities. In addition, alpha-lactalbumin exhibits certain functional properties in terms of antibacterial, antioxidant, anticancer, and antitumor activities. Meanwhile, β-lactoglobulin is also an important protein in cow's milk whey, but it is not present in breast milk; therefore, for some infants, β-lactoglobulin is a major allergen. Therefore, to promote the humanization of infant formula, it is necessary to separate alpha-lactalbumin from β-lactoglobulin in cow's milk and purify alpha-lactalbumin.

[0004] Previous studies have shown that α-lactalbumin and β-lactoglobulin are highly similar in molecular weight (14KD and 18KD), isoelectric point (4.7 and 5.3), and hydrolysis characteristics, making them difficult to separate. Common separation methods include enzymatic hydrolysis, selective precipitation, membrane separation, and ion exchange. The first three methods suffer from low recovery rates, high costs, and low purity. In contrast, ion exchange offers higher purity and recovery rates. Most current research on α-lactalbumin focuses on the separation of α-lactalbumin using a binding separation method, which has relatively mature processes. However, this method suffers from weak binding capacity and low daily processing capacity.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a target substance and related products and applications.

[0007] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for preparing a target substance, the target substance including α-lactalbumin, comprising the following steps: obtaining whey from skim milk after removing casein; adjusting the pH of the whey to a first set value, adjusting the conductivity to a second set value, and then performing strong anion exchange chromatography to obtain a flow-through containing α-lactalbumin; wherein the first set value is 7~9, and the second set value is 3~8 ms / cm.

[0008] Secondly, embodiments of the present invention provide products prepared by the preparation method described in any of the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide the application of the preparation method as described in any of the foregoing embodiments or the product as described in any of the foregoing embodiments in the preparation of food or health products.

[0010] The present invention has the following beneficial effects: This invention achieves high-purity separation of α-lactalbumin by adjusting the pH and conductivity of natural whey to alter its properties and using anion exchange chromatography. The required environment for this method is neutral to slightly alkaline, which helps maintain the activity of α-lactalbumin. The resulting α-lactalbumin is rich in α-helical structures and exhibits a high degree of similarity to the natural structure of α-lactalbumin. The flow-through separation mode of the anion exchange chromatography significantly increases the total amount of whey protein processed by ion exchange, shortens the processing time per batch, and improves production efficiency. This is of great significance for the industrial separation and production of α-lactalbumin. Attached Figure Description

[0011] 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.

[0012] Figure 1 The circular dichroism spectrum of the secondary structure of the flow-through liquid in Example 1 is shown. Figure 2 This is a 280µm detection image of the separation process in Example 1; Figure 3 This is a liquid phase detection image of the flow-through liquid in Example 1; Figure 4 This is the SDS-PAGE analysis of the flow-through liquid in Example 1; where, source: natural whey; 5-80: corresponding volumes of flow-through liquid, ①~⑤: low-salt eluent; y1~y4: high-salt eluent, and last 1~last 4: equilibration after salt elution; Figure 5This is an SDS-PAGE image of the flow-through liquid in Example 2; where M: marker; source: emulsion before separation; 5-90: flow-through liquid; ①-⑤: A phase elution solution; y1-y4: high-salt elution solution; and 1-4: equilibration solution. Figure 6 This is a liquid phase detection image of the flow-through liquid in Example 2; Figure 7 The image shows the SDS-PAGE analysis of the flow-through solution in Comparative Example 1; where, source: natural whey; 5-90: corresponding volumes of flow-through solution, ①~⑤: low-salt eluent; y1~y4: high-salt eluent, and last 1~last 4: equilibration after salt elution. Figure 8 The liquid phase detection image is for the flow-through liquid in Comparative Example 1. Figure 9 The circular dichroism spectra of the secondary structures of α-lactalbumin (B) and cationic lactalbumin (A) in Comparative Example 2 are shown. Figure 10 The image shows the SDS-PAGE analysis of the flow-through solution in Comparative Example 3; where M: marker; source: emulsion before separation; 5-90: flow-through solution; ①-⑤: A phase elution solution; y1-y4: high-salt elution solution; and 1-4: equilibration solution. Figure 11 The image shows the SDS-PAGE analysis of the flow-through liquid in Comparative Example 4; where M: marker; source: emulsion before separation; 1-79: flow-through liquid; ①: A phase elution liquid; y1-y2: high-salt elution liquid; and 1-2: equilibration liquid. Detailed Implementation

[0013] 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.

[0014] The terms "first" and "second" are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0015] Ion exchange methods are mainly divided into two modes: binding separation and partial flow-through. Most current research on α-lactalbumin focuses on the binding separation mode, which has a relatively well-established process. However, the binding separation mode has been found to have weak binding capacity and low daily throughput. Research on the flow-through mode for separating α-lactalbumin is relatively limited, and whether stable separation can be achieved remains unknown. The relevant process parameters and separation conditions are also unclear.

[0016] Based on the properties of α-lactalbumin and β-lactoglobulin, and combining the advantages of anion exchange separation, the inventors of this application designed and developed a highly efficient method for separating α-lactalbumin. The product is essentially free of β-lactoglobulin, offering advantages such as high purity and good production efficiency. Furthermore, the obtained α-lactalbumin retains a well-preserved structure, is rich in α-helical structures, and exhibits a high degree of similarity to the natural structure of α-lactalbumin. This invention provides another method for the industrial separation of α-lactalbumin, offering a more suitable raw material for the research and production of infant formula that mimics human milk.

[0017] On one hand, embodiments of the present invention provide a method for preparing a target substance, the target substance comprising α-lactalbumin, comprising the following steps: To obtain whey from skim milk after casein removal; After adjusting the pH of the whey to a first set value and the conductivity to a second set value, strong anion exchange chromatography was performed to obtain a flow-through containing α-lactalbumin. The first setting value is 7~9, and the second setting value is 3~8 ms / cm.

[0018] In an optional embodiment, the step of removing casein from skim milk includes: obtaining the permeate after microfiltration of skim milk, wherein the obtained retentate is whey after casein removal.

[0019] 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.

[0020] In an optional embodiment, the first set value can be any one or any two of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 and 9, and can be selected as 7.5 to 8.5.

[0021] In an optional embodiment, the second set value can be any one or any two of 3, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 7 and 8 ms / cm, and can be selected as 4 to 6 ms / cm.

[0022] In an optional embodiment, the aspect ratio of the strong anion exchange chromatography column is 0.5~15, specifically 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, The range between any one or any two of the following: 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, 10, 10.2, 10.4, 10.6, 10.8, 11, 11.2, 11.4, 11.6, 11.8, 12, 12.2, 12.4, 12.6, 12.8, 13, 13.2, 13.4, 13.6, 13.8, 14, 14.2, 14.4, 14.6, 14.8, and 15.

[0023] In an optional embodiment, the inner diameter of the strong anion exchange chromatography column is 1-50 mm, specifically any one or any two of 1, 2, 4, 6, 8, 10, 20, 30, 40 and 50 mm; the column height is 50-200 mm, specifically any one or any two of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200 mm.

[0024] In an optional embodiment, the packing material for the strong anion exchange chromatography is cross-linked agarose with dextran chains, and the particle size has an average value of 50-500 μm, specifically any one or any two of the following: 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 and 500 μm.

[0025] In an optional embodiment, the chromatography column for the strong anion exchange chromatography is TA-Q-XL-BB (Chutian Microsphere Biotechnology (Changsha) Co., Ltd.).

[0026] In an optional embodiment, the target substance further includes β-lactoglobulin, and after the strong anion exchange chromatography, an elution buffer containing β-lactoglobulin is obtained.

[0027] In an optional embodiment, the elution step of the β-lactoglobulin-containing eluent is performed using mobile phase B; mobile phase B comprises a mixture of 15–35 mmol / L Tris-HCl solution at pH 7.8–8.2 and 0.1–2 mol / L sodium chloride. Specifically, the 15–35 mmol / L concentration can be any one or any combination of 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 35 mmol / L. The pH 7.8–8.2 concentration can be any one or any combination of 7.8, 7.9, 8.0, 8.1, and 8.2. The 0.1–2 mol / L concentration can be any one or any combination of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2 mol / L.

[0028] In an optional embodiment, the steps of performing the strong anion exchange chromatography include: alternating column equilibration with mobile phase A and mobile phase B, rinsing with mobile phase A until the detection line is stable, and eluting with mobile phase B after sample loading.

[0029] In an optional embodiment, the mobile phase A comprises a Tris-HCl solution containing 15–35 mmol / L at pH 7.8–8.2; specifically, the 15–35 mmol / L concentration can be any one or a range between any two of 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 35 mmol / L. The pH 7.8–8.2 concentration can specifically be any one or a range between any two of 7.8, 7.9, 8.0, 8.1, and 8.2.

[0030] In an optional embodiment, the preparation method further includes at least one of the following: filtering, concentrating, desalting, delactose-reducing, drying, and sterilizing the flow-through and / or eluent obtained after strong anion exchange chromatography.

[0031] In an optional embodiment, the pore size of the filter membrane is 40~60kDa, specifically any one or any two of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60kDa.

[0032] In an optional embodiment, the concentration factor is any one or any two of the factors of 5, 6, 7, 8, 9, 10, 11 and 12.

[0033] In an optional embodiment, the desalting and lactose-removing treatment is performed by ultrafiltration, with a molecular weight cutoff of 3 to 15 kDa, specifically any one or any two of the following: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 kDa.

[0034] In an optional embodiment, the drying includes freeze drying and / or low-temperature spray drying.

[0035] In an optional embodiment, the sterilization includes membrane filtration sterilization or pasteurization. Pasteurization conditions can be 72-75°C for 10-30 seconds.

[0036] On the other hand, embodiments of the present invention also provide products prepared by the preparation method described in any of the foregoing embodiments.

[0037] Furthermore, embodiments of the present invention also provide the application of the preparation method as described in any of the foregoing embodiments or the product as described in any of the foregoing embodiments in the preparation of food or health products.

[0038] In an optional embodiment, the food product includes milk powder.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1 A method for separating α-lactalbumin and β-lactoglobulin, comprising the following steps.

[0041] (1) Sample preparation At room temperature, natural whey was obtained by sequentially removing milk fat and casein from cow's milk through microfiltration (Kornet membrane, USA) with a pore size of 0.1 μm. The protein content of the natural whey obtained by membrane separation was determined to be 0.5% using the Kjeldahl method, and the conductivity was determined to be 3.3 mS / cm. The sample concentration was adjusted to pH 7.7 with hydrochloric acid, and the sample conductivity was adjusted to 5 mS / cm with phosphate solution. (2) Ion exchange separation and purification Materials and reagents: The strong anion exchange column packing type is: 5ml TA-Q-XL-BB pre-packed column (Chutian Microsphere Biotechnology (Changsha) Co., Ltd.), with an inner diameter of 8.9mm and a column height of 100mm. The packing material is cross-linked agarose with dextran chains and a particle size of 100-300μm; Mobile phase A: 25mM Tris-HCl, pH=8; Mobile phase B: 25mM Tris-HCl, 1M NaCl, pH=8; Elution and collection method: Column equilibration was performed using alternating mobile phases A and B. The column was washed with mobile phase A until the detection line stabilized. Once the detection line stabilized, 450 mL of the prepared sample was loaded onto the ion exchange column at a flow rate of 700 cm / h. The flow-through was collected every 5 column volumes and numbered sequentially, for a total of 90 column volumes. 80 column volumes of sample were collected. After collection, unbound proteins were washed with a low-salt solution. Then, bound proteins were washed with high-salt mobile phase B, and the eluent was collected. Once all proteins had been eluted and the baseline stabilized, other proteins bound to the column were washed off with 0.5 M NaOH at a flow rate of 700 cm / h, activating the strong anion exchange packing material. This completed one purification cycle.

[0042] The collected flow-through was rich in α-lactalbumin, and the eluent was rich in β-lactoglobulin.

[0043] (3) Sample preparation: Each of the above samples (flow-through and eluent) was used to remove impurity proteins using a membrane with a pore size of 50 kDa. The solution was then concentrated 10 times and ultrafiltration (molecular flow rate of 10 kDa) was used to remove lactose and milk mineral salts from the protein solution. The solution was then concentrated 10 times to obtain high-purity protein raw materials. The liquid obtained from the ultrafiltration desalination was freeze-dried to obtain a powdered whey protein sample.

[0044] (4) Detection method The collected whey protein samples were analyzed by SDS-PAGE. Based on the SDS-PAGE results, the samples were mixed and then analyzed by RP-HPLC.

[0045] SDS-PAGE analysis was performed to determine the protein content of each sample. First, 10 μl of each numbered sample was mixed with 10 μl of 5× protein loading buffer (Solepro) and diluted with water to 50 μl. Then, 10 μl of each numbered sample eluted from 100% phase B was mixed with 10 μl of 5× protein loading buffer and diluted with pure water to 200 μl. The mixed sample was heated at 100℃ for 10 min. After natural cooling, 4 μL of each sample was loaded onto a 4%-20% precast gel (GenScript) and electrophoresed at 180V for 30 min. After electrophoresis, staining and destaining were performed using a staining instrument (GenScript).

[0046] RP-HPLC analysis. The contents of α-lactalbumin and β-lactoglobulin in natural whey, flow-through, and eluent, as well as the ratio of α-lactalbumin to the sum of the two, were analyzed using RP-HPLC external standard method. A C4 column was used for the determination of α-lactalbumin and β-lactoglobulin.

[0047] Sample preparation for RP-HPLC: Dilute the samples sequentially according to their protein concentrations. Dilute natural whey 5 times, dilute α-mix 5 times with pure water, and dilute β-mix 50 times with pure water. Then filter the samples using a 0.22 μm filter and place them into sample vials for analysis.

[0048] The RP-HPLC detection method was set as follows: injection volume 10 μL, column temperature 60℃, flow rate 0.8 mL / min, and detection wavelength 210 nm. Mobile phase A was prepared as 0.1% pure water, and mobile phase B was prepared as 0.1% acetonitrile. The gradient (rate of change) of mobile phase B was as follows: elution from 25% to 45% for 25 min, elution from 45% to 70% for 2 min, holding at 70% B for 3 min, elution from 70% to 25% for 1 min, and finally holding at 25% B for 7 min.

[0049] (5) Test results The secondary structure of the flow-through liquid was determined by circular dichroism spectroscopy, and compared with the tertiary structure of α-lactalbumin to obtain the following results.

[0050] Table 1. Distribution of secondary structure proportions in flow-through fluid

[0051] The results showed that the flow-through protein (α-lactalbumin) contained a large number of α-helical structures, indicating that after the method was optimized, the isolated protein largely retained the native structure of α-lactalbumin.

[0052] The flow-through liquids (1-80 CV) were mixed and denoted as the flow-through liquid mixture, and the high-salt eluted samples (y1-y4) were denoted as the elution liquid mixture. The purity and recovery of the samples were determined by high performance liquid chromatography, and the results are shown in Table 2.

[0053] Table 2 Purity and Recovery Ratio of Flow-through and Eluent

[0054] The circular dichroism spectrum of the secondary structure of the flow-through liquid is shown below. Figure 1 The separation process of Example 1, measured at 280µm, is shown in the image. Figure 2 The liquid phase detection image of the flow-through fluid is shown below. Figure 3 The SDS-PAGE analysis results for the flow-through fluid are shown below. Figure 4 .

[0055] The results in Table 2 show that the flow-through mixture under these process conditions is rich in high-purity α-lactalbumin, with a purity ≥90%. Furthermore, this process exhibits high recovery rates in the separation and purification of α-lactalbumin; in this example, the recovery rate of α-lactalbumin is ≥90%, achieving high-purity and high-recovery separation. Simultaneously, this process can obtain high-purity α-la raw material while also producing high-purity β-lactoglobulin samples (purity ≥95%, recovery rate ≥80%).

[0056] Example 2 A method for separating α-lactalbumin and β-lactoglobulin is similar to that in Example 1, except that in step (1) sample preparation, the pH is adjusted to 8 and the loading volume is 90 times the column volume to ensure that the maximum loading volume is reached.

[0057] The product was tested using the same method as in Example 1.

[0058] SDS-PAGE analysis of the flow-through fluid is shown below. Figure 5 The liquid phase analysis results for the flow-through fluid are shown below. Figure 6 .

[0059] Under the same sample loading volume, in this embodiment, natural whey with pH adjusted to 8 was used as raw material for separation. At the same time, the flow-through liquid rich in high-purity α-lactalbumin was collected, and the α-lactalbumin in the flow-through liquid was detected by SDS-PAGE and RP-HPLC. It can be seen that the purity and recovery rate of the flow-through liquid with pH adjusted to 8 and collected in the same volume as in Example 1 were 83.8% and 87.9% respectively by RP-HPLC analysis.

[0060] Comparative Example 1 A method for separating α-lactalbumin and β-lactoglobulin is generally the same as in Example 1, except that: in step (1) sample preparation, pH and conductivity are not adjusted, and the product is detected using the same detection method as in Example 1.

[0061] SDS-PAGE analysis of the flow-through fluid is shown below. Figure 7 The liquid phase analysis results for the flow-through fluid are shown below. Figure 8 .

[0062] Under the same sample loading volume, natural whey without adjusting conductivity and pH was used as the raw material for separation. At the same time, the flow-through liquid rich in high-purity α-lactalbumin was collected and the α-lactalbumin in the flow-through liquid was detected by SDS-PAGE and RP-HPLC. It can be seen that the high-purity flow-through volume of the sample without adjusting conductivity and pH is only 50CV (50 column volumes), which is far lower than the level of Example 1. The purity and recovery rate of the flow-through liquid collected with the same volume as in Example 1 were only 77% and 51%, respectively, according to RP-HPLC analysis.

[0063] Comparative Example 2 Commercially available α-lactalbumin (α-La) products and cationic isolates were collected as comparative example 2.

[0064] The commercially available α-La was sourced from Agropur in the United States, model number Bipro Alpha 9000. Its α-lactalbumin purity is ≥90%. The cationic product was primarily prepared by laboratory cation exchange chromatography, and its α-lactalbumin purity was ≥90% as determined by RP-HPLC. The secondary structure was compared with that of the sample obtained in Example 1. The circular dichroism spectrum of the cationic lactalbumin secondary structure of Comparative Example 2 is shown below. Figure 9 .

[0065] Table 3. Secondary structure distribution of domestically produced α-lactalbumin and cationic isolated lactalbumin.

[0066] The results show that the secondary structures of commercially available α-lactalbumin and α-lactalbumin isolated by the cationic method contain a large number of random coils, indicating that their secondary structures have undergone significant changes and pose a risk to their function.

[0067] Comparative Example 3 A method for separating α-lactalbumin and β-lactoglobulin is similar to that in Example 1, except that the pH is adjusted to 9.5 during sample preparation in step (1).

[0068] SDS-PAGE analysis of the flow-through fluid is shown below. Figure 10 .

[0069] Under the same sample loading volume, natural whey with pH adjusted to 9.5 was used as the raw material for separation. Simultaneously, a flow-through rich in high-purity α-lactalbumin was collected, and the α-lactalbumin in the flow-through was detected by SDS-PAGE. It was found that under these conditions, the raw material could not achieve separation of α-lactalbumin and β-lactoglobulin via the chromatography column; α-lactalbumin and β-lactoglobulin aggregated and eluted simultaneously. This is a significant difference compared to Example 1.

[0070] Comparative Example 4 A method for separating α-lactalbumin and β-lactoglobulin is roughly the same as in Example 1, except that: in step (2) ion exchange separation and purification, the chromatography column is Q purose 6XL (Qianchun Biotechnology) with an inner diameter of 7.8 mm and a column height of 100 mm; the packing material is cross-linked agarose with dextran chains and an average particle size of 90 μm.

[0071] SDS-PAGE analysis of the flow-through fluid is shown below. Figure 11 .

[0072] Under the same sample loading volume, Q purose 6XL (Qianchun Bio) was used for separation in the comparative example. At the same time, the flow-through solution rich in high-purity α-lactalbumin was collected and the α-lactalbumin in the flow-through solution was detected by SDS-PAGE. Under these conditions, the raw materials could achieve a high level of separation of α-lactalbumin and β-lactoglobulin through the chromatography column. However, compared with the example, the flow-through point of α-lactalbumin was delayed, and the optimal flow rate of this packing material was 400 cm / h, compared with 700 cm / h in Example 1.

[0073] 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 preparing a target substance, wherein the target substance comprises α-lactalbumin, characterized in that, It includes the following steps: To obtain whey from skim milk after casein removal; After adjusting the pH of the whey to a first set value and the conductivity to a second set value, strong anion exchange chromatography was performed to obtain a flow-through containing α-lactalbumin. The first setting value is 7~9, and the second setting value is 3~8 ms / cm.

2. The preparation method according to claim 1, characterized in that, The first setting value is 7.5~8.5, and / or the second setting value is 4~6 ms / cm.

3. The preparation method according to claim 1, characterized in that, The strong anion exchange chromatography column has a height-to-diameter ratio of 0.5 to 15, and the packing material is cross-linked agarose with dextran chains and a particle size of 100 to 300 μm.

4. The preparation method according to claim 3, characterized in that, The chromatography column for the strong anion exchange chromatography was TA-Q-XL-BB, manufactured by Chutian Microsphere Biotechnology (Changsha) Co., Ltd.

5. The preparation method according to claim 1, characterized in that, The target substance also includes β-lactoglobulin, and after the strong anion exchange chromatography, an elution buffer containing β-lactoglobulin is obtained.

6. The preparation method according to claim 5, characterized in that, The preparation method further includes at least one of the following: filtering, concentrating, desalting, delactose-reducing, drying, and sterilizing the flow-through liquid and / or the eluent.

7. The preparation method according to claim 5, characterized in that, The elution step of the β-lactoglobulin-containing eluent is carried out using mobile phase B; mobile phase B comprises a mixture of 15-35 mmol / L Tris-HCl solution at pH 7.8-8.2 and 0.1-2 mol / L sodium chloride.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The step of removing casein from skim milk includes: obtaining the permeate from skim milk after microfiltration, which is whey after removing casein; The microfiltration membrane has a pore size of 0.05~0.2 μm.

9. The product obtained by the preparation method according to any one of claims 1 to 8.

10. The preparation method according to any one of claims 1 to 8 or the product according to claim 9 in the preparation of food or health products.

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