Method for simultaneously separating and purifying alpha-lactalbumin and serum albumin from camel milk

Abstract

The invention discloses a method for simultaneously separating and purifying alpha-lactalbumin and serum albumin from camel milk, which comprises the following steps: taking camel milk, and separating to obtain camel milk whey protein by adopting isoelectric precipitation and water system filter membrane filtration methods; sequentially flushing the anion exchange column with a pre-prepared alpha-lactalbumin separation buffer solution and a serum albumin separation buffer solution, and flushing the anion exchange column with a pre-prepared whey combination buffer solution until an ultraviolet absorption base line and a conductive base line are stable; loading a sample of camel milk whey protein into an anion exchange column, and flushing the anion exchange column with whey combined with a buffer solution to elute non-target protein in camel milk whey; washing the anion exchange column by adopting an alpha-lactalbumin separation buffer solution, and eluting and collecting an elution peak of the alpha-lactalbumin; and flushing the anion exchange column by using a serum albumin separation buffer solution, and eluting and collecting an elution peak of the serum albumin. The separation and purification method provided by the invention is simpler in process, lower in cost and more suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of protein extraction, and more specifically, to a method for simultaneously separating and purifying alpha-lactalbumin and serum albumin from camel milk. Background Art

[0002] α-lactalbumin is a small acidic protein with a molecular weight of about 14 kDa secreted by the mammary gland of mammals. It is composed of 123 amino acids and forms a compact globular structure with an isoelectric point of about 4.5-5.5. The tertiary structure of α-lactalbumin is similar to that of lysozyme. 2+ In the presence of α-lactalbumin, it plays an important role in lactose synthesis. The substance formed by α-lactalbumin and cis-unsaturated fatty acids (such as oleic acid) has antitumor activity. The polypeptide produced after hydrolysis of α-lactalbumin has antibacterial activity. A high whey protein diet rich in α-lactalbumin can increase the ratio of insulin to blood glucose in rats. At the same time, α-lactalbumin is rich in tryptophan, which is an important factor in neural development and can regulate sleep, appetite and mood. Compared with bovine α-lactalbumin, camel α-lactalbumin has a more hydrophobic core at positions 25-35 than bovine α-lactalbumin. Camel α-lactalbumin has a high affinity for calcium ions and is more hydrophobic when calcium is depleted. And relative to bovine α-lactalbumin, camel α-lactalbumin has a stronger surface hydrophobicity and higher antioxidant activity.

[0003] Serum albumin is the most abundant single-chain protein in vertebrate plasma that is free of cofactors, glycosylation, and lipid modification. It is usually synthesized by the liver, has high solubility, a molecular weight of about 66kDa, and is negatively charged. The good flexibility of the serum albumin molecular structure makes its structure extremely susceptible to changes in environmental conditions, but the abundant disulfide bonds in serum albumin avoid changes in its molecular structure and determine the rigid structure of serum albumin. As an important component of the body, serum albumin can interact with a variety of compounds due to the binding sites of different types of chemicals on its molecular surface, thereby affecting the distribution of substances in the body in the body's blood circulation and playing a role in storage and transportation. As a natural transport carrier, serum albumin has the advantages of strong coordination binding ability, no immunogenicity, good stability, and strong water solubility. It can be used as an ideal raw material for preparing drug carriers; at the same time, it can bind to the vascular cell matrix and subendothelium to change the permeability of capillaries to macromolecules and solutions, thereby expanding the volume of blood vessels. After transfusion to postoperative patients, normal plasma colloidal osmotic pressure can be restored to avoid tissue edema and affect wound healing. Serum albumin has anti-inflammatory effects, maintains the integrity of the microtubule system, and regulates coagulation.

[0004] Since whey protein accounts for 20% of the weight of milk protein, which is lower than casein, in order to improve the purity and concentration of α-lactalbumin and serum albumin separated from whey protein and to fully exert the biological function of serum albumin, it is necessary to purify α-lactalbumin and serum albumin in milk.

[0005] However, there are few studies on the efficient separation and purification methods of camel-derived α-lactalbumin. The mainstream method for separating and purifying α-lactalbumin and serum albumin from whey is to purify by molecular sieve and multiple chromatography methods. The steps are cumbersome and not conducive to scale up, and it is difficult to apply to industrial production. Therefore, the development of low-cost efficient extraction and separation technology of α-lactalbumin and serum albumin is a technical problem that needs to be solved urgently. Summary of the invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] In order to achieve these objects and advantages according to the present invention, a method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk is provided (the simultaneous mentioned here does not mean separating α-lactalbumin and serum albumin at the same time, but within the time of continuously using the same anion exchange column).

[0008] The above methods include:

[0009] Step 1, taking camel milk, and separating camel milk whey protein by isoelectric precipitation and water filter membrane filtration;

[0010] Step 2: flushing the anion exchange column with a pre-configured α-lactalbumin separation buffer and a serum albumin separation buffer in sequence, and then flushing the anion exchange column with a pre-configured whey binding buffer until the ultraviolet absorption baseline and the conductivity baseline are stable;

[0011] Step 3, loading the camel milk whey protein obtained in step 1 onto the anion exchange column treated in step 2, and then washing the anion exchange column with whey binding buffer to elute non-target proteins in the camel milk whey;

[0012] Step 4, washing the anion exchange column treated in step 3 with an α-lactalbumin separation buffer, and eluting and collecting the elution peak of α-lactalbumin;

[0013] Step 5: flush the anion exchange column treated in step 4 with serum albumin separation buffer, and elute and collect the elution peak of serum albumin.

[0014] Preferably, the anion exchange column is a DEAE anion exchange column or a Q anion exchange column, more preferably the DEAE anion exchange column or the Q anion exchange column can be of the Capto series, and the DEAE anion exchange column or the Q anion exchange column can be of the FF (high flow rate) type or the HP (low flow rate) type.

[0015] Preferably, the whey binding buffer is a first buffer having a concentration not higher than 0.02 M and a pH of 6.8 to 8.0, and the first buffer may be any known buffer;

[0016] The α-lactalbumin separation buffer is a whey binding buffer containing less than 0.3M sodium chloride;

[0017] The serum albumin separation buffer is a whey binding buffer containing 0.3-1M sodium chloride.

[0018] Preferably, the first buffer is PBS buffer or Tris buffer.

[0019] Preferably, in step 2, the anion exchange column is first washed with 2 to 10 column volumes of α-lactalbumin separation buffer at a rate of 1 to 2 mL / min, then washed with 2 to 10 column volumes of serum albumin separation buffer at a rate of 1 to 2 mL / min, and then washed with 5 to 20 column volumes of whey binding buffer at a rate of 1 to 2 mL / min until the ultraviolet absorption baseline and the conductivity baseline are stable.

[0020] Preferably, in step three, 1 to 3 mL of camel milk whey protein obtained in step one is taken, and 1 mL is loaded onto the anion exchange column treated in step two at a rate of 1 to 2 mL / min each time, and then the anion exchange column is rinsed with 5 column volumes of whey binding buffer to elute non-target proteins in camel milk whey, and the loading and whey binding buffer rinsing processes are repeated until the camel milk whey protein is completely loaded onto the anion exchange column.

[0021] Preferably, in step 4, the anion exchange column treated in step 3 is washed with 0.5 to 20 column volumes of α-lactalbumin separation buffer at a rate of 1 to 2 mL / min to elute and collect the elution peak of α-lactalbumin.

[0022] Preferably, in step 5, the anion exchange column treated in step 4 is washed with 0.5 to 20 column volumes of serum albumin separation buffer at a rate of 1 to 2 mL / min to elute and collect the elution peak of serum albumin.

[0023] Preferably, in step 1, camel milk is taken, centrifuged at 5000 r / min for 20 min at 4°C, the upper milk fat precipitate is removed, the lower milk protein solution is taken, the pH value of the milk protein solution is measured and recorded, the pH value of the milk protein solution is accurately adjusted to 4.6 using glacial acetic acid, and after being bathed in 40°C water for 30 min, the upper whey protein solution and the lower casein precipitate are separated, the whey protein solution is taken and the pH is adjusted to the above-mentioned recorded pH with saturated sodium bicarbonate solution, and then filtered with a 0.22um water filter membrane to obtain camel milk whey protein.

[0024] The present invention has at least the following beneficial effects:

[0025] In the prior art, the process of deep processing of camel milk into camel milk cheese and camel milk yogurt will produce a large amount of camel milk whey by-products. This by-product contains a variety of whey active proteins and nutrients. If it is discharged directly, it will not only cause a waste of raw milk resources and lose potential economic benefits; direct discharge without treatment will also lead to eutrophication of water bodies, affect water quality, and pollute the natural environment. Existing studies have shown that camel milk whey is different from cow milk whey. It lacks β-lactoglobulin and is rich in α-lactalbumin. It is more economical and feasible to separate α-lactalbumin from camel milk. The method provided in this application meets the current demand for deep processing of camel milk whey. Through a cascade purification method, α-lactalbumin and serum albumin in camel milk whey are extracted at the same time, so that camel milk whey is turned into treasure, which not only provides a by-product recycling solution for the existing dairy processing industry, but also increases the added value of camel milk deep processing products, achieving multiple goals at one stroke.

[0026] In addition, in the prior art, separation and purification of α-lactalbumin and serum albumin from camel milk are usually carried out separately, using different chromatographic columns, respectively, and designing loading buffer and elution buffer for different target proteins, therefore, the process of separation and purification of α-lactalbumin and serum albumin from camel milk is not only cumbersome, but also costly. In the present application, the entire operation process of separation and purification is realized on one chromatographic column, which greatly saves the input of the chromatographic column in the purification process, reduces the cost, and in the separation and purification process, after the loading is completed, α-lactalbumin and serum albumin are eluted and separated in sequence, without the need for re-loading in the middle, and the operation process is simpler and more suitable for industrial production.

[0027] In general, the method provided by the present invention can simultaneously separate and purify α-lactalbumin and serum albumin from camel milk, the process is simpler, and the protein content and purity purified by the method of the present invention basically meet the requirements of industrial production. In addition, the method of the present invention has lower economic cost than the existing purification process, and the operation process does not involve the use of ammonium sulfate, which is more suitable for large-scale industrial production.

[0028] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 1 of the present invention;

[0030] Figure 2 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 1 of the present invention;

[0031] Figure 3 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 1 of the present invention;

[0032] Figure 4 This is a graph showing the contents of α-lactalbumin and serum albumin purified from 1 mg of camel milk whey protein in Example 1 of the present invention;

[0033] Figure 5 This is a chromatogram of camel milk whey protein eluted through a Q anion exchange column in Example 2 of the present invention;

[0034] Figure 6 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 2 of the present invention;

[0035] Figure 7 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 2 of the present invention;

[0036] Figure 8 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 3 of the present invention;

[0037] Fig. 9 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 3 of the present invention;

[0038] Fig.10 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 3 of the present invention;

[0039] Fig.11 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 4 of the present invention;

[0040] Fig.12 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 4 of the present invention;

[0041] Fig.13 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 4 of the present invention;

[0042] Fig.14 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 5 of the present invention;

[0043] Fig.15 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 5 of the present invention;

[0044] Fig.16 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 5 of the present invention;

[0045] Fig.17 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 6 of the present invention;

[0046] Fig.18 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 6 of the present invention;

[0047] Fig.19 This is the IMAEG J band grayscale image of the α-lactalbumin and serum albumin purified in Example 6 of the present invention.

[0048] Fig. 20 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 7 of the present invention;

[0049] Fig.21 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 7 of the present invention;

[0050] Fig. 22 This is the IMAEG J band grayscale image of the α-lactalbumin and serum albumin purified in Example 7 of the present invention.

[0051] Fig.23 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 8 of the present invention;

[0052] Fig.24 The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 8 of the present invention;

[0053] Fig.25 This is a chromatogram of camel milk whey protein eluted through a DEAE anion exchange column in Example 9 of the present invention;

[0054] Fig.26The SDS-PAGE pattern of α-lactalbumin and serum albumin purified in Example 9 of the present invention;

[0055] Fig. 27 This is the IMAEG J band grayscale image of α-lactalbumin and serum albumin purified in Example 9 of the present invention;

[0056] Fig.28 This is an ultra high performance liquid chromatography (UHPLC) diagram of α-lactalbumin and standard α-lactalbumin obtained in Example 9 of the present invention. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0058] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified; in the description of the present invention, the terms "lateral", "longitudinal", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0059] <Example 1>

[0060] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, comprising:

[0061] Step 1, taking camel milk, and separating camel milk whey protein by isoelectric precipitation and water filter membrane filtration;

[0062] Specifically, camel milk is taken, centrifuged at 5000 r / min for 20 min at 4°C, the upper milk fat precipitate is removed, the lower milk protein solution is taken, the pH value of the milk protein solution is measured and recorded, the pH value of the milk protein solution is accurately adjusted to 4.6 with glacial acetic acid, and after being bathed in 40°C water for 30 min, the upper whey protein solution and the lower casein precipitate are separated, the whey protein solution is taken and the pH is adjusted to the above-mentioned recorded pH with a saturated sodium bicarbonate solution, and then filtered with a 0.22 um water filter membrane to obtain camel milk whey protein.

[0063] Step 2, using a pre-configured α-lactalbumin separation buffer and a serum albumin separation buffer to sequentially rinse the DEAE anion exchange column, and then using a pre-configured whey binding buffer to rinse the DEAE anion exchange column until the ultraviolet absorption baseline and the conductivity baseline are stable;

[0064] Specifically, a whey binding buffer, an α-lactalbumin separation buffer and a serum albumin separation buffer are first prepared.

[0065] The whey binding buffer can be configured in the following manner:

[0066] Na2HPO4·12H2O and water were mixed at a solid-liquid ratio of 71.6 g / L to obtain a 0.2M Na2HPO4 solution. The 0.2M Na2HPO4 solution was mixed with water at a volume ratio of 1:9 to obtain a 0.02M Na2HPO4 solution.

[0067] NaH2PO4·2H2O and water were mixed at a solid-liquid ratio of 31.2 g / L to obtain a 0.2 M NaH2PO4 solution. The 0.2 M NaH2PO4 solution was mixed with water at a volume ratio of 1:9 to obtain a 0.02 M NaH2PO4 solution.

[0068] Take 0.02M Na2HPO4 solution and mix it with 0.02M NaH2PO4 solution, adjust the pH to 8, add 0.02MNa2HPO4 if it is acidic, and add 0.02M NaH2PO4 if it is alkaline, to obtain whey binding buffer - PBS buffer with a concentration of 0.02M and a pH of 8.0.

[0069] The α-lactalbumin separation buffer can be configured by the following method:

[0070] The α-lactalbumin separation buffer, i.e., the whey binding buffer containing 0.1 M sodium chloride, is obtained by mixing NaCl and the whey binding buffer at a solid-liquid ratio of 5.85 g / L.

[0071] The serum albumin separation buffer can be configured in the following manner:

[0072] The serum albumin separation buffer, namely, the whey binding buffer containing 0.5 M sodium chloride, is obtained by mixing NaCl and the whey binding buffer at a solid-liquid ratio of 29.25 g / L.

[0073] Specifically, the process of washing the DEAE anion exchange column with α-lactalbumin separation buffer and serum albumin separation buffer in sequence, and then washing the DEAE anion exchange column with whey binding buffer is as follows:

[0074] First, the DEAE anion exchange column was flushed with 5 column volumes of α-lactalbumin separation buffer at a rate of 1 mL / min, then with 5 column volumes of serum albumin separation buffer at a rate of 1 mL / min, and then with 10 column volumes of whey binding buffer at a rate of 1 mL / min until the UV absorption baseline and conductivity baseline were stable.

[0075] Step 3, loading the camel milk whey protein obtained in step 1 onto the DEAE anion exchange column treated in step 2, and then washing the DEAE anion exchange column with whey binding buffer to elute non-target proteins in the camel milk whey;

[0076] Specifically, 1 mL of camel milk whey protein obtained in step 1 is loaded onto the DEAE anion exchange column treated in step 2 at a rate of 1 mL / min, and then the DEAE anion exchange column is washed with 5 column volumes of whey binding buffer to elute non-target proteins in camel milk whey;

[0077] Step 4, washing the DEAE anion exchange column treated in step 3 with an α-lactalbumin separation buffer, and eluting and collecting the elution peak of α-lactalbumin;

[0078] Specifically, the DEAE anion exchange column treated in step 3 is washed with 5 column volumes of α-lactalbumin separation buffer at a rate of 1 mL / min to elute and collect the elution peak of α-lactalbumin.

[0079] Step 5: flush the DEAE anion exchange column treated in step 4 with serum albumin separation buffer, and elute and collect the elution peak of serum albumin.

[0080] Specifically, the DEAE anion exchange column treated in step 4 is flushed with 2 column volumes of serum albumin separation buffer at a rate of 1 mL / min to elute and collect the elution peak of serum albumin.

[0081] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Figure 1 As shown, from Figure 1 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0082] ②Component analysis and purity testing:

[0083] SDS-PAGE gel electrophoresis was used to analyze the components of α-lactalbumin and serum albumin:

[0084] The protein elution peak collected in Example 1 was mixed with SDS-PAGE loading buffer (5X) at a volume ratio of 1:4, and then cooled in a 100°C boiling water bath for 10 min to obtain a sample for standby use. A Tris-SDS-PAGE protein separation gel system with 5% concentrated gel and 12% separation gel was prepared. The sample was loaded into the Tris-SDS-PAGE protein separation gel system, and electrophoresis was performed at a constant voltage of 90V for 30 min. After the sample migrated to the separation gel, the voltage was adjusted to 120V, and the electrophoresis was stopped after running for 80 min until the bromophenol blue indicator reached about 0.5 cm from the bottom of the separation gel. The gel was taken out and immersed in Coomassie brilliant blue staining solution for overnight staining. After complete staining, it was decolorized with a decolorizing solution until the gel background was crystal clear, and then detected and photographed using a gel imager. During the electrophoresis, a low molecular weight standard molecular weight marker was also added. The protein molecular weight was judged by observing the relative position of the protein band and the marker band on the gel. The results are as follows: Figure 2 shown.

[0085] Figure 2 In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0086] from Figure 2 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0087] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Figure 3 As shown in Table 1:

[0088] Table 1

[0089]

[0090] It is not difficult to see from Table 1 that the purity of α-lactalbumin reaches 83.31%, and the purity of serum albumin reaches 80.88%, while the purity of commercially available camel-derived α-lactalbumin and serum albumin is about 85%. The purity of α-lactalbumin and serum albumin obtained in the present application basically meets the requirements of industrial production.

[0091] ③Content detection

[0092] The BCA method was used to quantify α-lactalbumin and serum albumin respectively:

[0093] 1) Preparation of working reagent (WR): Mix BCA reagent and Cu reagent in a volume ratio of 50:1 to make WR working solution, which is stable at room temperature for 4 hours;

[0094] 2) Preparation of standard protein solutions: Use PBS buffer (1X) to prepare the protein standards into the following concentration solutions: 1, 0.75, 0.5, 0.25, 0.125, 0.025 mg / mL;

[0095] 3) Determination of standards and samples: 20uL of protein standard solutions and samples of different concentrations were added to different wells of a 96-well plate, and 200uL of WR working solution was added to each well. The plates were placed at 37°C for 10 min, and then the absorbance (OD) value of each well at 562nm was measured using an ELISA reader. The protein concentration of the standard and its corresponding OD were then calculated. 562 Draw a standard curve with the value, and then use the OD 562 Substitute the value into the standard curve to calculate the concentration of the sample to be tested. The result is as follows Figure 4 shown.

[0096] from Figure 4 It is not difficult to see that, by using the method provided in the present application, about 0.19 mg of α-lactalbumin and about 0.15 mg of serum albumin can be separated and purified from each mg of whey.

[0097] <Example 2>

[0098] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is substantially the same as that in Example 1, except that a Q anion exchange column is used for elution in this example.

[0099] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Figure 5 As shown, from Figure 5 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0100] ②Component analysis and purity testing:

[0101] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Figure 6 shown.

[0102] Figure 6 In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0103] from Figure 6 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0104] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Figure 7 As shown in Table 2:

[0105] Table 2

[0106]

[0107] <Example 3>

[0108] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is basically the same as that in Example 1, except that in this embodiment: the whey binding buffer is a PBS buffer with a concentration of 0.02M and a pH of 6.8; the α-lactalbumin separation buffer is a whey binding buffer containing 0.1M sodium chloride; and the serum albumin separation buffer is a whey binding buffer containing 0.5M sodium chloride.

[0109] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Figure 8 As shown, from Figure 8 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0110] ②Component analysis and purity testing:

[0111] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig. 9 shown.

[0112] Fig. 9In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0113] from Fig. 9 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0114] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig.10 As shown in Table 3:

[0115] Table 3

[0116]

[0117] <Example 4>

[0118] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, wherein the process is substantially the same as that in Example 1, except that in this embodiment: the whey binding buffer is a PBS buffer having a concentration of 0.02 M and a pH of 7.5; the α-lactalbumin separation buffer is a whey binding buffer containing 0.1 M sodium chloride; and the serum albumin separation buffer is a whey binding buffer containing 0.5 M sodium chloride.

[0119] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.11 As shown, from Fig.11 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0120] ②Component analysis and purity testing:

[0121] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.12 shown.

[0122] Fig.12In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0123] from Fig.12 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0124] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig.13 As shown in Table 4:

[0125] Table 4

[0126]

[0127] <Example 5>

[0128] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, wherein the process is substantially the same as that in Example 1, except that in this embodiment, the speeds of washing the DEAE anion exchange column with the α-lactalbumin separation buffer, the serum albumin separation buffer, and the whey binding buffer are all 1.5 mL / min.

[0129] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.14 As shown, from Fig.14 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0130] ②Component analysis and purity testing:

[0131] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.15 shown.

[0132] Fig.15 In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0133] from Fig.15 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0134] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig.16 As shown in Table 5:

[0135] Table 5

[0136]

[0137] <Example 6>

[0138] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is substantially the same as that in Example 1, except that in this embodiment: the speeds of washing the DEAE anion exchange column with the α-lactalbumin separation buffer, the serum albumin separation buffer, and the whey binding buffer are all 2 mL / min.

[0139] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.17 As shown, from Fig.17 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0140] ②Component analysis and purity testing:

[0141] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.18 shown.

[0142] Fig.18 In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0143] from Fig.18It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0144] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig.19 As shown in Table 6:

[0145] Table 6

[0146]

[0147] <Example 7>

[0148] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is basically the same as that in Example 1, except that in this embodiment: Step 3, taking 3 mL of the camel milk whey protein obtained in Step 1, loading 1 mL at a speed of 1 mL / min onto the DEAE anion exchange column treated in Step 2 each time, then washing the DEAE anion exchange column with 5 column volumes of whey binding buffer to elute non-target proteins in the camel milk whey, and then repeating the loading and washing the anion exchange column with whey binding buffer for 3 times until 3 mL of camel milk whey protein is loaded.

[0149] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig. 20 As shown, from Fig. 20 It is not difficult to see that under the online detection conditions of the 280nm UV detector, 3 flow-through peaks and 2 elution peaks are obtained. This is because during the loading process, non-target proteins were eluted 3 times, so 3 flow-through peaks were produced, and the elution of the two target proteins was carried out once each, so there was one elution peak for each of the two target proteins. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0150] ②Component analysis and purity testing:

[0151] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.21 shown.

[0152] Fig.21In which M is a marker, 1 is CWP, 2 to 4 are flow-through components, 5 is an elution component of α-lactalbumin separation buffer, and 6 is an elution component of serum albumin separation buffer.

[0153] from Fig.21 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0154] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig. 22 As shown in Table 7:

[0155] Table 7

[0156]

[0157] <Example 8>

[0158] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is substantially the same as that in Example 1, except that in this embodiment:

[0159] The whey binding buffer is a PBS buffer with a concentration of 0.02M and a pH of 8.

[0160] The α-lactalbumin separation buffer is configured in 6 kinds, each containing a different concentration of sodium chloride, which are:

[0161] α-Lactalbumin Isolation Buffer 1—Whey Binding Buffer containing 0.1 M sodium chloride;

[0162] α-Lactalbumin Isolation Buffer II—Whey Binding Buffer containing 0.11 M sodium chloride;

[0163] α-Lactalbumin Isolation Buffer III—Whey Binding Buffer containing 0.115 M sodium chloride;

[0164] α-Lactalbumin Isolation Buffer IV—Whey Binding Buffer containing 0.125 M sodium chloride;

[0165] α-Lactalbumin Isolation Buffer V—Whey Binding Buffer containing 0.15 M sodium chloride;

[0166] α-Lactalbumin Isolation Buffer VI - Whey Binding Buffer containing 0.2 M sodium chloride.

[0167] The serum albumin separation buffer is configured as: a whey binding buffer containing 0.3M sodium chloride.

[0168] In step 4, the DEAE anion exchange column is rinsed sequentially using 8 column volumes of alpha-lactalbumin separation buffer 1, 0.5 column volume of alpha-lactalbumin separation buffer 2, 0.5 column volume of alpha-lactalbumin separation buffer 3, 2 column volumes of alpha-lactalbumin separation buffer 4, 1.5 column volumes of alpha-lactalbumin separation buffer 5, and 1.5 column volumes of alpha-lactalbumin separation buffer 6, at a rinsing rate of 1 mL / min, and the elution peak of each rinse is collected;

[0169] In step 5, the DEAE anion exchange column treated in step 4 is washed with 1.5 column volumes of serum albumin separation buffer at a rate of 1 mL / min to elute and collect the elution peak of serum albumin.

[0170] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.23 As shown, concB is the sodium chloride concentration change curve in the eluent, and cond is the conductivity change curve. Fig.23 It is not difficult to see that there are 7 steps in the change of sodium chloride concentration in the eluent, which correspond to α-lactalbumin separation buffers 1 to 6 and serum albumin separation buffer, respectively. During the elution process of α-lactalbumin separation buffer 1 and serum albumin separation buffer, a relatively obvious elution peak appeared.

[0171] ②Component analysis and purity testing:

[0172] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.24 shown.

[0173] Fig.24 Wherein M is Marker, 1 is CWP, 5 to 8 are elution components of α-lactalbumin separation buffer, and 11 is elution components of serum albumin separation buffer.

[0174] from Fig.24It can be seen that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak (including bands 5 to 8) obtained by elution with α-lactalbumin separation buffer is about 13 kDa, so it can be preliminarily confirmed that the component is the target protein - α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak (including band 11) obtained by elution with serum albumin separation buffer is about 70 kDa, so it can be preliminarily confirmed that the component is the target protein - serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0175] In addition, it can be seen from this embodiment that when the sodium chloride concentration in the α-lactalbumin separation buffer reaches 0.3 M or more, serum albumin will be eluted from the anion exchange column, which is not conducive to the separation of α-lactalbumin and serum albumin. Therefore, the sodium chloride concentration in the α-lactalbumin separation buffer is required to be lower than 0.3 M in this application.

[0176] <Example 9>

[0177] ① A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, the process of which is substantially the same as that in Example 1, except that in this embodiment:

[0178] The whey binding buffer is a Tris buffer with a concentration of 0.02M and a pH of 8.0;

[0179] The α-lactalbumin separation buffer is a whey binding buffer containing less than 0.1 M sodium chloride;

[0180] The serum albumin separation buffer is a whey binding buffer containing 0.5 M sodium chloride.

[0181] The whey binding buffer can be configured in the following manner:

[0182] Tris and water were mixed at a solid-liquid ratio of 24.228 g / L to obtain a 0.2 M Tris solution. The 0.2 M Tris solution was mixed with water at a volume ratio of 1:9 to obtain a 0.02 M Tris solution.

[0183] Take 0.02M Tris solution and mix it with 0.02M HCl solution, adjust the pH to 8, add 0.02M Tris if it is acidic, and add 0.02M HCl if it is alkaline, to obtain whey binding buffer - Tris buffer with a concentration of 0.02M and a pH of 8.

[0184] The α-lactalbumin separation buffer can be configured by the following method:

[0185] The α-lactalbumin separation buffer, which is a 0.02M Tris buffer with a pH of 8 and a concentration of 0.1M sodium chloride, can be obtained by mixing NaCl with a Tris buffer with a concentration of 0.02M and a pH of 8 at a solid-liquid ratio of 5.85 g / L.

[0186] The serum albumin separation buffer can be configured in the following manner:

[0187] The serum albumin separation buffer, a 0.02M Tris buffer with a pH of 8 and a concentration of 0.5M sodium chloride, can be obtained by mixing NaCl with a 0.02M Tris buffer with a pH of 8 at a solid-liquid ratio of 29.25 g / L.

[0188] In this embodiment, the elution was performed using the AKTA automatic purification system. The obtained chromatogram is as follows: Fig.25 As shown, cond is the conductivity change curve, from Fig.25 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and two elution peaks are obtained. It can also be seen from the conductivity curve that the conductivity change is basically consistent with the elution peak, indicating that the elution peak material is indeed eluted.

[0189] ②Component analysis and purity testing:

[0190] The components of α-lactalbumin and serum albumin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.26 shown.

[0191] Fig.26 In which M is Marker, 1 is whey, 2 is flow-through component, 3 is elution component of α-lactalbumin separation buffer, and 4 is elution component of serum albumin separation buffer.

[0192] from Fig.26 It is not difficult to see that the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with α-lactalbumin separation buffer is about 13kDa, so it can be preliminarily confirmed that the component is the target protein α-lactalbumin, and the molecular weight of the SDS-PAGE gel electrophoresis protein band corresponding to the elution peak obtained by elution with serum albumin separation buffer is about 70kDa, so it can be preliminarily confirmed that the component is the target protein serum albumin. The above two protein bands are obvious and single, basically free of impurity proteins, and the purification effect is good.

[0193] In order to further clarify the purity of the protein, this application used Image J software for analysis, and the results are as follows Fig. 27 As shown in Table 8:

[0194] Table 8

[0195]

[0196] In order to further confirm that the separated protein is the target protein α-lactalbumin, the present application uses ammonium sulfate precipitation method and liquid chromatography to separate α-lactalbumin from camel milk whey protein solution as a standard product, and uses ultra-high performance liquid chromatography to detect the α-lactalbumin standard product and compare it with the α-lactalbumin prepared in this example.

[0197] The process of separating α-lactalbumin from camel milk whey protein solution by using ammonium sulfate precipitation method and liquid chromatography includes: 1) taking whey protein solution (the preparation method of whey protein solution is the same as that in step 1 of Example 1), slowly adding ammonium sulfate to 80% saturation, stirring with a magnetic stirrer, standing at 4°C for 1h after adding, centrifuging at 8000rpm for 20min at 4°C, taking the supernatant and continuing to add ammonium sulfate to 100% saturation, centrifuging at 8000rpm for 20min at 4°C, i.e., CWP8 component precipitation, re-dissolving with phosphate buffer, and dialyzing in pH7.4 PBS buffer. The dialysate is replaced every 2h. A saturated barium chloride solution is used to detect whether the dialysate after dialyzation has sulfate ion precipitation, until the dialyzate is dialyzed until there is no precipitation, and the dialysis is completed. The solution in the dialysis bag is the CWP8 component, which is collected for standby use; 2) the CWP8 component is further separated using a medium- and high-pressure chromatography system AKTA Pure to obtain an α-lactalbumin standard product. The chromatographic conditions are as follows: chromatographic column: HiLoad16 / 600Superdex 75Prep Grade, mobile phase: 0.05M PBS+0.15M NaCl (Ph7.4), injection volume: 1mL, flow rate: 1mL / min, detection wavelength: 280nm. The fraction collected when the elution volume is 80mL is α-lactalbumin.

[0198] When the α-lactalbumin standard product and the α-lactalbumin prepared in this embodiment were detected by ultra-high performance liquid chromatography, the conditions of ultra-high performance liquid chromatography were as follows: Chromatographic column: ACQUITY UPL C Protein BEH C4 column ( 1.7um, 21mm*100mm), mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile, injection volume 10uL, flow rate 0.3mL / min, detection wavelength 214nm, running 10.24min, mobile phase elution gradient settings are shown in Table 9 below:

[0199] Table 9 Mobile phase elution gradient table

[0200]

[0201] Test results such as Fig.28As shown in the figure, it can be seen that the standard product and the protein purified in this example only have one peak, and the retention time of the two peaks is 5.389 min. It can be concluded that the protein purified in this example is α-lactalbumin.

[0202] <Comparative Example 1>

[0203] The patent application number 202310306013.0 discloses a method for separating and purifying α-lactalbumin in camel milk, comprising the following steps: (1) recording the initial pH value of the milk protein solution after centrifugation of camel milk, adjusting the pH value to 4.6, and then keeping it in a 40°C water bath for 30 minutes, and then standing at 4°C to obtain a precipitated milk sample; (2) centrifuging the milk sample to separate the upper whey protein solution and the lower casein precipitate, adjusting the pH value of the whey protein solution to the initial pH value, and obtaining a whey protein component; (3) precipitating the whey protein component with an ammonium sulfate solution, dissolving the obtained precipitate in water, and dialyzing. The milk protein solution retained during the dialysis process is centrifuged, and the obtained lower liquid is freeze-dried to obtain camel milk α-lactalbumin freeze-dried powder.

[0204] <Comparative Example 2>

[0205] The technical document "Isolation, Purification and Structural Characterization of Serum Albumin in Bactrian Camel Milk" was published in "Food Industry Science and Technology (vol. 39, No. 17, 2018)", which used DEAE-FF ion exchange chromatography and Sephacryl S-100 gel filtration chromatography to separate and purify serum albumin in camel milk.

[0206] As can be seen from the above comparative example 1, in the prior art, α-lactalbumin in camel milk is separated and purified by ammonium sulfate graded precipitation, and as can be seen from the above comparative example 2, in the prior art, serum albumin in camel milk whey is separated and purified by DEAE-FF ion exchange chromatography and Sephacryl S-100 gel filtration chromatography in sequence. Therefore, in the prior art, α-lactalbumin and serum albumin in camel milk are generally separated and purified separately, and the process is not only cumbersome, but also costly. The applicant performs cost statistics on comparative examples 1 and 2 respectively, and then adds up to obtain the cost of purifying 100mL camel whey by its purification process (excluding the cost of instruments and camel milk itself) (calculating column materials, reagents and consumables), which is about 674 yuan.

[0207] In the present application, the separation and purification operation is performed on only one chromatographic column from beginning to end, which greatly saves the chromatographic column, reduces the cost, and reduces the process of replacing the chromatographic column. At the same time, during the separation and purification process, after the sample is loaded, α-lactalbumin and serum albumin are eluted and separated in sequence, and no re-loading is required in the middle. The operation process is simpler, and the method used in the present application does not contain ammonium sulfate, which is more suitable for industrial production. According to statistics, the method used in the present application can reduce the cost to about 549 yuan, effectively reducing the cost of purification.

[0208] In addition, the current camel milk deep processing process to make camel milk cheese and camel milk yogurt will produce a large amount of camel milk whey by-products, which contain a variety of whey active proteins and nutrients. If it is discharged directly, it will not only cause a waste of raw milk resources and lose potential economic benefits; direct discharge without treatment will also lead to eutrophication of water bodies, affect water quality, and pollute the natural environment. Existing studies have shown that camel milk whey is different from cow milk whey. It lacks β-lactoglobulin and is rich in α-lactalbumin. It is more economical and feasible to separate α-lactalbumin from camel milk. The method provided in this application just meets the current demand for camel milk whey deep processing. By simultaneously extracting α-lactalbumin and serum albumin from camel milk whey, camel milk whey is turned into treasure, which not only provides a by-product recycling solution for the existing dairy processing industry, but also increases the added value of camel milk deep processing products, achieving multiple goals at one stroke.

[0209] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk, characterized in that: include: Step 1, taking camel milk, and separating camel milk whey protein by isoelectric precipitation and water filter membrane filtration; Step 2: flushing the anion exchange column with a pre-configured α-lactalbumin separation buffer and a serum albumin separation buffer in sequence, and then flushing the anion exchange column with a pre-configured whey binding buffer until the ultraviolet absorption baseline and the conductivity baseline are stable; Step 3, loading the camel milk whey protein obtained in step 1 onto the anion exchange column treated in step 2, and then washing the anion exchange column with whey binding buffer to elute non-target proteins in the camel milk whey; Step 4, washing the anion exchange column treated in step 3 with an α-lactalbumin separation buffer, and eluting and collecting the elution peak of α-lactalbumin; Step 5: flush the anion exchange column treated in step 4 with serum albumin separation buffer, and elute and collect the elution peak of serum albumin.

2. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 1, characterized in that: The anion exchange column is a DEAE anion exchange column or a Q anion exchange column.

3. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 2, characterized in that: The whey binding buffer is a first buffer with a concentration not higher than 0.02M and a pH of 6.8 to 8.0; The α-lactalbumin separation buffer is a whey binding buffer containing less than 0.3M sodium chloride; The serum albumin separation buffer is a whey binding buffer containing 0.3-1M sodium chloride.

4. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 3, characterized in that: The first buffer is PBS buffer or Tris buffer.

5. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 4, characterized in that: In step 2, the anion exchange column is firstly washed with 2 to 10 column volumes of α-lactalbumin separation buffer at a speed of 1 to 2 mL / min, and then washed with 2 to 10 column volumes of serum albumin separation buffer at a speed of 1 to 2 mL / min, and then washed with 5 to 20 column volumes of whey binding buffer at a speed of 1 to 2 mL / min until the ultraviolet absorption baseline and the conductivity baseline are stable.

6. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 5, characterized in that: In step 3, 1 to 3 mL of the camel milk whey protein obtained in step 1 is taken, and 1 mL is loaded onto the anion exchange column treated in step 2 at a speed of 1 to 2 mL / min each time, and then the anion exchange column is rinsed with 5 column volumes of whey binding buffer to elute non-target proteins in the camel milk whey, and the loading and whey binding buffer washing processes are repeated until the camel milk whey protein is completely loaded onto the anion exchange column.

7. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 6, characterized in that: In step 4, 0.5 to 20 column volumes of α-lactalbumin separation buffer are used to wash the anion exchange column treated in step 3 at a rate of 1 to 2 mL / min to elute and collect the elution peak of α-lactalbumin.

8. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 7, characterized in that: In step 5, the anion exchange column treated in step 4 is washed with 0.5 to 20 column volumes of serum albumin separation buffer at a rate of 1 to 2 mL / min to elute and collect the elution peak of serum albumin.

9. The method for simultaneously separating and purifying α-lactalbumin and serum albumin from camel milk according to claim 1, characterized in that: In step 1, camel milk is taken, centrifuged at 5000 r / min for 20 minutes at 4°C, the upper milk fat precipitate is removed, the lower milk protein solution is taken, the pH value of the milk protein solution is measured and recorded, and the pH value of the milk protein solution is accurately adjusted to 4.6 using glacial acetic acid. After being water bathed at 40°C for 30 minutes, the upper whey protein solution and the lower casein precipitate are separated by centrifugation at 5000 r / min for 20 minutes at 4°C, the whey protein solution is taken and the pH is adjusted to the above-recorded pH with a saturated sodium bicarbonate solution, and then filtered with a 0.22 um water filter membrane to obtain camel milk whey protein.

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