Method for separating and purifying alpha-lactalbumin in whey

Abstract

The invention belongs to the technical field of protein purification processes, and particularly relates to a method for separating and purifying alpha-lactalbumin in whey. Comprising the following steps: S1, obtaining skimmed milk; s2, performing microfiltration separation on the skimmed milk obtained in the step S1 to obtain whey protein liquid containing alpha-lactalbumin; s3, loading the whey protein liquid containing alpha-lactalbumin obtained in S2 into a balanced strong cation chromatographic column, and collecting a flow-through liquid to obtain separated and purified alpha-lactalbumin; according to the method, the SP Purose 6XL filler is combined with the specific 10-60mM acetic acid buffer solution with the pH value of 4-6, so that the method is particularly suitable for separating the alpha-lactalbumin from other proteins in the whey, and the alpha-lactalbumin is collected in a flow-through manner, so that large-volume sample loading can be realized, and more importantly, the obtained alpha-lactalbumin is high in yield and purity; in addition, the process is simple in extraction step and free of a tedious elution process, a large amount of time can be saved, the production cost is reduced, and industrial production is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of purification process, and in particular relates to a method for separating and purifying alpha-lactalbumin in whey. Background Art

[0002] α-Lactalbumin is a natural whey protein, a globular protein composed of 123 amino acids, mainly found in the milk of mammals, but also in the body fluids of other animals and plants.

[0003] It is reported that α-lactalbumin in human milk is an important protein component that promotes infant growth, and it has many important biological functions in the body, such as carrying oxygen, regulating the immune system, maintaining cell structure, etc. In addition, α-lactalbumin is rich in tryptophan (an important factor in neural development) and has the function of regulating sleep, appetite and mood.

[0004] α-Lactalbumin is found in both human milk and bovine milk. The molecular weight of α-lactalbumin in human milk is 14070Da, which is close to the molecular weight of α-lactalbumin in bovine milk (14178Da). Studies have shown that the sequence homology of α-lactalbumin in human milk and bovine milk is very high, with about 74% conservative amino acid sequence homology between the two, of which 6% of the amino acids are extremely similar.

[0005] Therefore, fortifying α-lactalbumin in infant formula can make the formula closer to human milk, thereby narrowing the gap between formula feeding and breastfeeding. Therefore, research on isolating α-lactalbumin from bovine milk has attracted much attention.

[0006] In the prior art, α-lactalbumin and β-lactoglobulin are separated by gel chromatography or membrane filtration combined with anion exchange column chromatography to prepare high-purity α-lactalbumin. However, this method has a low processing capacity and can generally only achieve a sample load within 10 column volumes. At the same time, the yield is low, which is not conducive to large-scale industrial production. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art such as difficulty in extracting α-lactalbumin from bovine milk / complex extraction steps, low sample volume, low yield, etc., and a method for separating and purifying α-lactalbumin with large sample volume, high efficiency, high yield and high purity is developed and designed.

[0008] In order to achieve the above object, the first aspect of the present invention discloses a method for separating and purifying α-lactalbumin in whey, comprising the following steps:

[0009] S1. Obtain skim milk;

[0010] S2, microfiltration separation of the skimmed milk obtained in S1 to obtain a whey protein solution with an α-lactalbumin content of 0.5 to 1.5 mg / ml;

[0011] S3, using a buffer to balance a strong cation exchange chromatography column; loading the whey protein solution obtained in S2 onto the balanced strong cation exchange chromatography column, and after loading, washing the strong cation exchange chromatography column with the buffer, and collecting the flow-through liquid; the strong cation exchange chromatography column uses SP Purose 6XL filler; the buffer is 10-60mM pH 4-6 acetate buffer;

[0012] The collected flow-through liquid is the separated and purified α-lactalbumin.

[0013] Preferably, the loading volume of the whey protein solution in S3 is 30-300CV.

[0014] Preferably, in S3, the whey protein solution has an α-lactalbumin content of 0.5 to 1.5 mg / ml.

[0015] Preferably, in S2, the microfiltration separation uses a ceramic membrane with a particle size of 0.1 μm, and the operating transmembrane pressure difference is 0.05-0.20 MPa.

[0016] Preferably, the collected flow-through liquid is desalted, concentrated and dried.

[0017] Preferably, in S1, raw milk is added to a centrifugal skimmer, and the skimmed milk is obtained by centrifugation at a speed of 8000 to 15000 r / min to remove fat.

[0018] The second aspect of the present invention discloses α-lactalbumin, which is obtained by the above method.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention can separate whey protein and casein by microfiltration operation, so that the casein content in the prepared whey protein solution is extremely low. The microfiltration operation membrane filtration particle size and transmembrane pressure difference are adjusted so that the shear force generated by the microfiltration operation separation will not cause significant damage to the protein activity, and the viscosity and turbidity of the raw milk sample are greatly reduced, so that the subsequent chromatography process is easy to implement.

[0021] 2. The present invention further utilizes a method combining strong cation exchange chromatography with a flow-through mode; the inventors of the present application adopted a strong cation exchange chromatography filler (SP Purose 6XL filler) developed and produced by their unit, and found through experiments that the combination of SP Purose 6XL filler and a specific 10-60mM pH 4-6 acetate buffer is particularly suitable for the separation of "α-lactalbumin" from other proteins in whey. The α-lactalbumin is collected by flow-through mode, which can not only achieve large volume loading (the loading volume can reach 30-300cv, with high loading capacity), but more importantly, the yield and purity of α-lactalbumin are high; in addition, the process has simple extraction steps and no complicated elution process, which can save a lot of time, reduce production costs, and is easy to industrialize, achieving unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph of the chromatography results of Example 1, wherein F1 represents α-lactalbumin obtained directly through flow-through, and F2 represents other eluted proteins.

[0023] Figure 2 This is the SDS-PAGE gel image of Example 1, wherein Y1 represents the whey before loading on the column, Y2 represents the α-lactalbumin component obtained directly through flow-through, and Y3 represents other eluted proteins.

[0024] Figure 3 This is the HPLC curve chart of detecting α-lactalbumin in Example 1.

[0025] Figure 4 This is a standard curve diagram of the HPLC method for determining the mass concentration of α-lactalbumin in Example 1.

[0026] Figure 5 This is a graph of the chromatography results of Example 3, wherein F1 represents α-lactalbumin obtained directly through flow-through, and F2 represents other eluted proteins.

[0027] Figure 6 This is the HPLC curve chart of detecting α-lactalbumin in Example 3.

[0028] Figure 7 This is a graph of the chromatography results of Example 4, wherein F1 represents α-lactalbumin obtained directly through flow-through, and F2 represents other eluted proteins.

[0029] Figure 8 This is the HPLC curve chart of Example 4 detecting α-lactalbumin. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The process parameters not specified in the embodiments of the present application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0031] Example 1

[0032] S1. Obtain skim milk;

[0033] In a specific embodiment of the present application, in S1, raw milk is added to a centrifugal skimmer, and the skimmed milk is obtained by centrifugation at a speed of 8000 to 15000 r / min to remove fat.

[0034] In this embodiment, raw milk is added into a centrifugal skimmer and centrifuged at a speed of 10000 r / min to remove fat and obtain skimmed milk.

[0035] S2, obtaining whey protein solution;

[0036] In a specific embodiment of the present application, the skimmed milk obtained in S1 is subjected to microfiltration separation to obtain a whey protein solution having an α-lactalbumin content of 0.5 to 1.5 mg / ml.

[0037] Preferably, the α-lactalbumin content in the whey protein solution is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 mg / ml.

[0038] In a preferred embodiment of the present application, in S2, the microfiltration separation uses a ceramic membrane with a particle size of 0.1 μm, and the operating transmembrane pressure difference is 0.05-0.20 MPa.

[0039] In this example, the skimmed milk obtained in S1 was microfiltered using a 0.1 μm ceramic membrane with an operating transmembrane pressure difference of 0.10 MPa, and the concentration of α-lactalbumin in the resulting whey protein solution was 1.14 mg / ml.

[0040] S3. Separation and purification of α-lactalbumin by strong cation exchange column chromatography

[0041] In a specific embodiment of the present application, S3 uses a buffer to equilibrate a strong cation exchange chromatography column, the strong cation exchange chromatography column uses SP Purose 6XL filler, and the buffer is 10-60mM pH 4-6 acetate buffer.

[0042] In this example, 10 mL of SP Purose 6XL (Dongfulong Qianchun Biotechnology Co., Ltd.) suspension was placed in a beaker, replaced with deionized water three times to remove the 20% ethanol preservative solution, and then loaded into a chromatography column. Deionized water was used to compact the column at a flow rate of 5 mL / min. After the filler was compacted, the column volume was measured to be 5 mL.

[0043] In a specific embodiment of the present application, the column is fully equilibrated with 10-60 mM pH 4-6 acetate buffer before loading, with a flow rate of 1 mL / min.

[0044] In this embodiment, the equilibration buffer is 50 mM acetate buffer at pH 4.5.

[0045] In a specific embodiment of the present application, in S3, the whey protein solution obtained in S2 is loaded onto a equilibrated strong cation exchange chromatography column, and after loading, the strong cation exchange chromatography column is rinsed with the buffer solution, and the flow-through is collected to obtain separated and purified α-lactalbumin.

[0046] In a specific embodiment of the present invention, the loading volume of the whey protein solution in S3 is 30-300CV, preferably, the loading volume is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300CV.

[0047] In a specific embodiment of the present application, the buffer used in S4 is the same as the buffer used for equilibration in S3.

[0048] In this embodiment, a chromatography column with a column volume of 5 mL is used, and in S3, the whey protein solution is loaded at a flow rate of 0.5-2 mL / min; preferably 1 mL / min. In this embodiment, a chromatography column with a column volume of 5 mL is used, and in S4, the buffer solution is passed through the strong cation exchange chromatography at a flow rate of 0.5-2 mL / min.

[0049] In the present embodiment, 750 mL (150 column volumes) of the whey protein solution in step 1 were sampled and loaded at 1 mL / min, and the flow-through liquid was collected; after the sample flow was completed, the remaining α-lactalbumin component was washed with a buffer (same as the equilibrium buffer, using 50 mM acetate buffer of pH 4.5) at a flow rate of 1 mL / min, and the flow-through liquid was continued to be collected; after the protein peak was washed out and the baseline was stable, the other proteins bound to the chromatography column were washed with an acetate buffer containing 1 M NaCl at a flow rate of 1 mL / min.

[0050] In this example, the collected flow-through liquid is adjusted to neutrality, desalted, concentrated and dried to obtain the α-lactalbumin product.

[0051] In this example, after the flow-through was collected, the strong cation exchange chromatography column was rinsed with a buffer solution to which 1 M NaCl was added (1 M NaCl, 50 mM acetate buffer, pH 4.5).

[0052] Detection of α-lactalbumin products

[0053] The content of α-lactalbumin was determined by high performance liquid chromatography (HPLC). The conditions of high performance liquid chromatography were as follows:

[0054] Chromatographic column: Prosphere 300C4 Column (5 μm, 4.6 mm × 150 mm);

[0055] Mobile phase: A is an aqueous solution containing 0.1% trifluoroacetic acid, and B is an acetonitrile solution containing 0.1% trifluoroacetic acid.

[0056] The purity of α-lactalbumin was calculated according to the following formula:

[0057]

[0058] x represents the purity of α-lactalbumin

[0059] Among them, the HPLC chart of the component to be tested is as follows Figure 3 As shown, it can be seen that high-purity α-lactalbumin is contained therein.

[0060] In the embodiments of the present application, the α-lactalbumin standard product was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with batch number 9051-29-0 and a purity of ≥85%.

[0061] Drawing of working curve (external standard method for determining substance content):

[0062] Accurately weigh α-lactalbumin standard (its HPLC curve is shown in Figure 3 ), prepare α-lactalbumin solution, measure the solution by high performance liquid chromatography, and draw standard curve based on the absorption peak at 210nm. Figure 4 .

[0063] The chromatogram of the collected flow-through fraction is as follows: Figure 1 The collected components were subjected to SDS-PAGE electrophoresis for qualitative analysis, and the results were as follows Figure 2 The HPLC curve for detecting α-lactalbumin is shown in Figure 3 shown.

[0064] Combined with the above test results, as shown in Table 1.

[0065] Table 1

[0066] Concentration mg / mL Volume mL Protein content mg purity Yield Raw whey protein liquid 1.14 750 853.34 α-Lactalbumin 0.95 775 735.93 86.10％ 86.24％

[0067] From the above results, it can be seen that the purity of α-lactalbumin in Example 1 reaches more than 85%, and the yield reaches more than 85%.

[0068] Comparative Example 1

[0069] This comparative example provides a method for purifying α-lactalbumin, and the specific steps are different from those in Example 1 in that:

[0070] In S2, a whey protein solution with an α-lactalbumin content of 1.09 mg / ml is obtained;

[0071] In S3 and S4, the buffer used was 70 mM pH 6.6 acetate buffer.

[0072] The test results are shown in Table 2, where the original represents whey protein solution.

[0073] Table 2

[0074] Concentration mg / mL Volume mL Protein content mg purity Yield Raw whey protein liquid 1.09 750 819.85 α-Lactalbumin 0.86 790 681.98 36.12％ 83.18％

[0075] From the above results, it can be seen that when the acetate buffer with pH 6.6 is used instead, the purity of the obtained α-lactalbumin is lower. This is because the binding force between other impurities and the filler becomes weaker and flows out; therefore, compared with Example 1, the purity of α-lactalbumin is significantly reduced.

[0076] Example 2

[0077] This embodiment provides a method for purifying α-lactalbumin, and the specific steps are different from those in Example 1:

[0078] In S1, the skimmed milk is obtained by centrifuging at a speed of 12000 r / min to remove fat;

[0079] In S2, a whey protein solution having an α-lactalbumin content of 1.12 mg / ml is obtained;

[0080] In S3, the loading volume of whey protein solution was 300 ml (60 column volumes), and other conditions remained the same.

[0081] In S3 and S4, the buffer used was 30 mM pH 5.5 acetate buffer.

[0082] The chromatogram of the collected flow-through fraction is as follows: Figure 5 The collected components were subjected to high performance liquid chromatography quantitative analysis, and the results were shown in Figure 6 shown.

[0083] Combined with the above test results, as shown in Table 3, original represents whey protein liquid.

[0084] Table 3

[0085] Concentration mg / mL Volume mL Protein content mg purity Yield Raw whey protein liquid 1.12 300 336.33 α-Lactalbumin 0.82 350 289.47 85.90％ 86.06％

[0086] Example 3

[0087] This embodiment provides a method for purifying α-lactalbumin, and the specific steps are different from those in Example 1:

[0088] In S1, the skimmed milk is obtained by centrifuging at a speed of 8000 r / min to remove fat;

[0089] In S2, a whey protein solution having an α-lactalbumin content of 1.08 mg / ml is obtained;

[0090] In S3, the loading volume of whey protein solution was 225 ml (45 column volumes), and other conditions remained the same.

[0091] In S3 and S4, the buffer used was 20 mM pH 5 acetate buffer.

[0092] The chromatogram of the collected flow-through fraction is as follows: Figure 7 The collected components were subjected to high performance liquid chromatography quantitative analysis, and the results were shown in Figure 8 shown.

[0093] Combined with the above test results, as shown in Table 4, original represents whey protein liquid.

[0094] Table 4

[0095]

[0096]

[0097] Combining the results of Example 2, Example 3, and Example 1, and comparing the results of Example 1 with those of Comparative Example 1, it can be seen that the use of a specific SP Purose 6XL filler and a specific 10-60mM pH 4-6 acetate buffer in combination is particularly suitable for the separation of "α-lactalbumin" from other proteins in whey, and α-lactalbumin is collected by flow-through, which can not only achieve large volume loading (the loading volume can reach 30-300cv, with high loading capacity), but more importantly, obtain α-lactalbumin with high yield and purity, greatly reducing production costs and facilitating industrial production.

[0098] The present invention is not limited to the above-mentioned embodiments. The protection scope of the present invention is defined by the attached claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principle and essence of the present invention, which should all be equivalent replacement methods and fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying α-lactalbumin in whey, characterized in that: The steps include: S1. Obtain skim milk; S2, microfiltration separation of the skimmed milk obtained in S1 to obtain a whey protein solution with an α-lactalbumin content of 0.5 to 1.5 mg / ml; S3, using a buffer to balance a strong cation exchange chromatography column; loading the whey protein solution obtained in S2 onto the balanced strong cation exchange chromatography column, and after loading, washing the strong cation exchange chromatography column with the buffer, and collecting the flow-through liquid; the strong cation exchange chromatography column uses SP Purose 6XL filler; the buffer is 10-60mM pH 4-6 acetate buffer; The collected flow-through liquid is the separated and purified α-lactalbumin.

2. The method for separating and purifying α-lactalbumin in whey according to claim 1, characterized in that: The loading volume of the whey protein solution in S3 is 30-300CV.

3. The method for separating and purifying α-lactalbumin in whey according to claim 1, characterized in that: In S3, the whey protein solution has an α-lactalbumin content of 0.5 to 1.5 mg / ml.

4. A method for separating and purifying α-lactalbumin in whey according to any one of claims 1 to 3, characterized in that: In S2, the microfiltration separation uses a ceramic membrane with a particle size of 0.1 μm, and the operating transmembrane pressure difference is 0.05-0.20 MPa.

5. The method for separating and purifying α-lactalbumin in whey according to any one of claims 1 to 3, characterized in that: The collected flow-through solution is desalted, concentrated and dried.

6. The method for separating and purifying α-lactalbumin in whey according to any one of claims 1 to 3, characterized in that: In S1, raw milk is added into a centrifugal skimmer, and the skimmed milk is obtained by centrifugal defatting at a speed of 8000-15000 r / min.

7. An α-lactalbumin, characterized in that: The α-lactalbumin is obtained by the method according to any one of claims 1 to 6.

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