Separation and purification method of high-purity lactoferrin in camel milk

Abstract

The invention discloses a method for separating and purifying high-purity lactoferrin in camel milk, which comprises the following steps: taking camel milk, and separating by adopting an isoelectric precipitation method to obtain a camel milk whey protein solution; washing the heparin affinity chromatographic column by adopting a pre-prepared sample loading buffer solution until an ultraviolet absorption baseline and a conductive baseline are stable; loading the camel milk whey protein solution to a heparin affinity chromatographic column, and flushing the heparin affinity chromatographic column with a loading buffer solution to elute non-target protein; washing the heparin affinity chromatographic column by adopting a pre-prepared elution buffer solution, collecting an elution peak of lactoferrin, and dialyzing in distilled water for desalting. According to the method, the lactoferrin with relatively high purity can be separated from the camel whey in one step, and the lactoferrin can be used as a standard substance for detecting the content of the lactoferrin in camel milk and related products.

Description

Technical Field

[0001] The present invention relates to the technical field of camel milk deep processing, and more specifically, to a method for separating and purifying high-purity lactoferrin in camel milk. Background Art

[0002] Lactoferrin is a non-heme iron-binding glycoprotein with a molecular weight of about 80 kD. It was first isolated from bovine milk in the 1930s and can be divided into two homologous halves connected by a short helical segment.

[0003] ——N-lobe and C-lobe, each lobe is about 40kDa in size and can firmly bind an iron ion. It belongs to the transferrin family. It is mainly secreted and expressed by mammalian mammary epithelial cells and exists in natural milk. Especially in the colostrum period of mammals, the content increases significantly. It is the main defense medium for newborn cubs. In addition to enhancing immunity, lactoferrin has also been shown to have multiple biological functions such as antibacterial activity, osteogenic activity, anti-cancer activity, regulating the body's immunity, participating in iron metabolism, promoting cell growth, and regulating the balance of intestinal flora. It can be used as a natural preservative ingredient to extend the shelf life of cosmetics, and it also has anti-inflammatory and whitening effects. Therefore, as a functional factor, lactoferrin has great application prospects in the food, biological, and pharmaceutical industries. Bovine lactoferrin has also been approved by the US Food and Drug Administration and the European Food Safety Authority as a dietary supplement recognized as safe. It is currently mainly used as a food additive in infant formula. According to literature, the lactoferrin content in camel milk is about 0.22 mg / ml, which is about 1.6 times the lactoferrin content in cow milk (0.14 mg / ml). The antibacterial, anti-diabetic, and lipid metabolism regulating functions of camel lactoferrin have also been reported. Therefore, developing lactoferrin in camel milk is an effective way to improve the quality and efficiency of camel milk and its products.

[0004] At present, there are few methods for purifying lactoferrin from camel milk and camel milk whey, and there are few studies on the properties of camel lactoferrin. In addition, the differences in protein composition and environment between different milks make it difficult to directly apply the chromatographic separation conditions of bovine lactoferrin, which may result in poor purification of camel milk lactoferrin, and may cause the loss of the target product or a decrease in product purity. However, the scale of camel breeding has increased year by year, and camel lactoferrin currently has the foundation for deep processing and development of products. High-purity camel lactoferrin is indispensable as a standard for the determination of its content in products. Therefore, there is an urgent need for a method for separating and purifying high-purity lactoferrin from camel milk to achieve the determination of the camel lactoferrin content in related camel milk products. Summary of the invention

[0005] 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.

[0006] In order to achieve these purposes and other advantages according to the present invention, a method for separating and purifying high-purity lactoferrin in camel milk is provided, comprising:

[0007] Step 1, taking camel milk, and separating and obtaining camel milk whey protein solution by isoelectric precipitation method;

[0008] Step 2: flush the heparin affinity chromatography column with a pre-configured loading buffer until the UV absorption baseline and the conductivity baseline are stable;

[0009] Step 3, loading the camel milk whey protein solution onto the heparin affinity chromatography column treated in step 2, and then washing the heparin affinity chromatography column with a loading buffer to elute non-target proteins;

[0010] Step 4: flush the heparin affinity chromatography column treated in step 3 with a pre-configured elution buffer, elute and collect the elution peak of lactoferrin, and dialyze it in distilled water for desalination.

[0011] Preferably, the loading buffer is a phosphate buffer with a pH of 7.5 to 8.0 and a concentration of 0.15 to 0.2 M;

[0012] The elution buffer is a phosphate buffer containing 0.5-1M NaCl, a pH of 7.0-7.5, and a concentration of 0.02-0.05M.

[0013] Preferably, in step 2, the heparin affinity chromatography column is flushed with 3 to 5 column volumes of loading buffer at a rate of 1.0 to 2.0 mL / min until the ultraviolet absorption baseline and the conductivity baseline are stable.

[0014] Preferably, in step three, the camel milk whey protein solution is filtered with a 0.22 um water filter membrane and then loaded onto the heparin affinity chromatography column treated in step two at a rate of 1.0 to 2.0 mL / min, and then the heparin affinity chromatography column is rinsed with 3 to 5 column volumes of loading buffer until the ultraviolet absorption baseline and the conductivity baseline are stable.

[0015] Preferably, in step 4, the heparin affinity chromatography column treated in step 3 is flushed with 3 to 5 column volumes of elution buffer at a rate of 1.0 to 2.0 mL / min.

[0016] Preferably, in step one, 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 a 40°C water bath for 30 min, the upper whey protein solution and the lower casein precipitate are separated, and the whey protein solution is taken and the pH is adjusted to the above-recorded pH with a saturated sodium bicarbonate solution to obtain a camel milk whey protein solution.

[0017] Preferably, in step three, before loading the camel milk whey protein solution, ammonium sulfate graded precipitation is first used, 50%-80% of the precipitated components are collected, re-dissolved with pure water and dialyzed in distilled water for desalination, and then filtered with a 0.22um water filter membrane, and the filtered solution is used for loading.

[0018] Preferably, the method of ammonium sulfate graded precipitation, collecting 50%-80% of the precipitation components, re-dissolving them with pure water and dialyzing them in distilled water for desalination specifically comprises: slowly adding ammonium sulfate to a camel milk whey protein solution and stirring continuously, after the ammonium sulfate saturation reaches 50%, standing at 4°C for 3 hours, centrifuging at 8000rpm for 15 minutes, discarding the precipitate and collecting the supernatant, continuing to add ammonium sulfate to the supernatant until its saturation reaches 80%, standing at 4°C for 3 hours, centrifuging at 8000rpm for 15 minutes, collecting the precipitate to obtain camel milk lactoferrin components, adding a small amount of ultrapure water to the precipitate to redissolve the precipitate and placing it in a dialysis bag, dialyzing and desalting in distilled water at 4°C, changing the distilled water every 1 hour, using saturated barium chloride to detect whether there is residual ammonium sulfate in the dialysate, and when there is no white precipitate in the dialysate, the dialysis is terminated to obtain crude camel milk lactoferrin.

[0019] The present invention has at least the following beneficial effects:

[0020] 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, 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 the content of lactoferrin in camel milk whey is higher than that in cow milk whey, and it is more economical to separate lactoferrin from camel milk. The method provided in this application meets the current demand for deep processing of camel milk whey. Lactoferrin in camel milk whey is extracted by enrichment and purification methods, so that camel milk whey can be 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.

[0021] In addition, in the prior art, the separation of lactoferrin from camel milk is either by loading and eluting with a buffer having a complex composition, or by using multiple chromatographic columns for separation and purification, which is cumbersome and costly. In the present application, the elution buffer is a phosphate buffer configured with sodium dihydrogen phosphate, and the loading buffer is composed of sodium dihydrogen phosphate and sodium chloride, which has simple composition and is easy to configure, and only one elution is required to separate and purify lactoferrin, and the separation and purification process is simpler, and the purity of the separated lactoferrin can reach 98%, which exceeds the 95% specified in the national standard GB 1903.17-2016, and can be used as a standard product for camel milk lactoferrin to detect the content of lactoferrin in other camel milk products.

[0022] In general, the method provided by the present invention can separate and purify lactoferrin with high purity from camel milk, the process is simpler, and compared with the existing purification process, it is more suitable for large-scale industrial production.

[0023] 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

[0024] Figure 1 The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 1 of the present invention;

[0025] Figure 2 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 1 of the present invention;

[0026] Figure 3 The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 2 of the present invention;

[0027] Figure 4 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 2 of the present invention;

[0028] Figure 5 The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 3 of the present invention;

[0029] Figure 6 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 3 of the present invention;

[0030] Figure 7 The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 4 of the present invention;

[0031] Figure 8 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 4 of the present invention;

[0032] Fig. 9 This is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 5 of the present invention;

[0033] Fig.10 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 5 of the present invention;

[0034] Fig.11 This is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 6 of the present invention;

[0035] Fig.12 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 6 of the present invention;

[0036] Fig.13 This is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 7 of the present invention;

[0037] Fig.14 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 7 of the present invention;

[0038] Fig.15 This is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 8 of the present invention;

[0039] Fig.16 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 8 of the present invention;

[0040] Fig.17 This is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Example 9 of the present invention;

[0041] Fig.18 The SDS-PAGE pattern of the camel milk lactoferrin purified in Example 9 of the present invention;

[0042] Fig.19 The SDS-PAGE pattern of camel milk lactoferrin separated from the camel milk whey protein solution to be tested in Example 10 of the present invention;

[0043] Fig. 20 1 is a chromatogram of camel milk lactoferrin standard solution of different concentrations in Example 10 of the present invention;

[0044] Fig.21 The camel milk lactoferrin standard curve obtained by preparing the standard solution of camel milk lactoferrin obtained in Example 1 in Example 10 of the present invention;

[0045] Fig. 22The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Comparative Example 3 of the present invention;

[0046] Fig.23 The SDS-PAGE pattern of the camel milk lactoferrin purified in Comparative Example 3 of the present invention;

[0047] Fig.24 The chromatogram is a chromatogram of the camel milk whey protein solution eluted through a heparin affinity chromatography column in Comparative Example 4 of the present invention;

[0048] Fig.25 The figure is the SDS-PAGE pattern of the camel milk lactoferrin purified in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] <Example 1>

[0052] ① A method for separating and purifying high-purity lactoferrin in camel milk, comprising:

[0053] Step 1, taking camel milk, and separating and obtaining camel milk whey protein solution by isoelectric precipitation method;

[0054] 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 water bathed at 40°C 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 to obtain a camel milk whey protein solution.

[0055] Step 2: flush the heparin affinity chromatography column with a pre-configured loading buffer until the UV absorption baseline and the conductivity baseline are stable;

[0056] Specifically, first prepare the loading buffer.

[0057] The loading buffer can be configured in the following manner:

[0058] Mix NaH2PO4·2H2O and water at a solid-liquid ratio of 31.2 g / L, and adjust the pH to 8.0 with NaOH to obtain the product.

[0059] Specifically, the process of washing the heparin affinity chromatography column with the loading buffer is as follows:

[0060] The heparin affinity chromatography column was flushed with 5 column volumes of loading buffer at a rate of 1 mL / min until the UV absorption baseline and conductivity baseline were stable.

[0061] The heparin affinity chromatography column is a HiTrap Heparin heparin affinity chromatography column.

[0062] Step 3, the camel milk whey protein solution obtained in step 1 is first subjected to ammonium sulfate graded precipitation, 50%-80% of the precipitated components are collected and re-dissolved with pure water and dialyzed in distilled water for desalination, and then filtered with a 0.22um water filter membrane, and then the filtered solution is loaded onto the heparin affinity chromatography column treated in step 2, and then the heparin affinity chromatography column is rinsed with a loading buffer to elute non-target proteins;

[0063] Specifically, the method of ammonium sulfate graded precipitation, collecting 50%-80% of the precipitation components, re-dissolving them with pure water and dialyzing them in distilled water for desalination specifically includes: slowly adding ammonium sulfate to a camel milk whey protein solution and constantly stirring, after the ammonium sulfate saturation reaches 50%, standing at 4°C for 3 hours, centrifuging at 8000 rpm for 15 minutes, discarding the precipitate and collecting the supernatant, continuing to add ammonium sulfate to the supernatant until its saturation reaches 80%, standing at 4°C for 3 hours, centrifuging at 8000 rpm for 15 minutes, collecting the precipitate to obtain the camel milk lactoferrin component, adding a small amount of ultrapure water to the precipitate to redissolve the precipitate and placing it in a dialysis bag, dialyzing and desalting in distilled water at 4°C, changing the distilled water every 1 hour, using saturated barium chloride to detect whether there is residual ammonium sulfate in the dialysate, and when there is no white precipitate in the dialysate, the dialysis is terminated to obtain crude camel milk lactoferrin.

[0064] Specifically, the crudely extracted camel milk lactoferrin was loaded onto the heparin affinity chromatography column treated in step 2 at a rate of 1 mL / min, and then the heparin affinity chromatography column was washed with 5 column volumes of loading buffer until the ultraviolet absorption baseline and the conductivity baseline were stable.

[0065] Step 4: flush the heparin affinity chromatography column treated in step 3 with a pre-configured elution buffer, elute and collect the elution peak of camel milk lactoferrin, and dialyze it in distilled water for desalination.

[0066] Specifically, first prepare an elution buffer.

[0067] The elution buffer can be configured in the following manner:

[0068] Weigh a certain amount of NaH2PO4·2H2O and NaCl and dissolve them in water, the solid-liquid ratio of NaH2PO4·2H2O to water is 7.8 g / L, and the solid-liquid ratio of NaCl to water is 58.44 g / L. Adjust the pH to 7.0 with NaOH to obtain the product.

[0069] Specifically, the process of washing the heparin affinity chromatography column with the elution buffer is as follows:

[0070] The heparin affinity chromatography column treated in step 3 was washed with elution buffer of 5 column volumes at a rate of 1 mL / min to elute and collect the elution peak of camel milk lactoferrin.

[0071] The collected camel milk lactoferrin is dialyzed in distilled water for desalination to obtain separated and purified camel milk lactoferrin.

[0072] 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 one elution peak 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.

[0073] ②Component analysis and purity testing:

[0074] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis:

[0075] 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 2shown.

[0076] Figure 2 M is a marker, 1 is whey, 2 is a precipitated component with an ammonium sulfate saturation of 50% to 80% in the whey, 3 is a flow-through component, and 4 is an eluted component with an elution buffer.

[0077] 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0078] It can also be seen from the UPLC chromatogram obtained by elution with a C4 chromatographic column in Example 7 below that the purity of the camel milk lactoferrin separated and purified in Example 1 is as high as 98% (the ratio of the main peak area to the sum of all peak areas in the elution spectrum where it is located), and can be used as a standard to draw a standard curve.

[0079] <Example 2>

[0080] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the pH of the loading buffer in this embodiment is 7.5.

[0081] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Figure 3 As shown, from Figure 3 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and one elution peak 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] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Figure 4 shown.

[0084] Figure 4 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0085] from Figure 4It 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0086] <Example 3>

[0087] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the pH of the loading buffer in this embodiment is 7.7.

[0088] 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 one elution peak 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.

[0089] ②Component analysis and purity testing:

[0090] The components of camel milk lactoferrin 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.

[0091] Figure 6 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0092] 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0093] <Example 4>

[0094] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the concentration of NaH2PO4 in the loading buffer in this embodiment is 0.15 M.

[0095] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Figure 7 As shown, from Figure 7 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and one elution peak 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.

[0096] ②Component analysis and purity testing:

[0097] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Figure 8 shown.

[0098] Figure 8 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0099] from Figure 8 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0100] <Example 5>

[0101] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the speed of washing the heparin affinity chromatography column with the loading buffer and the elution buffer in this embodiment is 1.5 mL / min.

[0102] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig. 9 As shown, from Fig. 9 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and one elution peak 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.

[0103] ②Component analysis and purity testing:

[0104] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.10 shown.

[0105] Fig.10 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0106] from Fig.10 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0107] <Example 6>

[0108] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate graded precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and in this embodiment, the speed of washing the heparin affinity chromatography column with the loading buffer and the elution buffer is 2.0 mL / min.

[0109] 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 one elution peak 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 camel milk lactoferrin 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.

[0112] Fig.12 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0113] 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0114] <Example 7>

[0115] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the pH of the elution buffer in this embodiment is 7.5.

[0116] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.13 As shown, from Fig.13 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and one elution peak 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.

[0117] ②Component analysis and purity testing:

[0118] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.14 shown.

[0119] Fig.14 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0120] from Fig.14 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0121] <Example 8>

[0122] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the concentration of NaH2PO4 in the elution buffer in this embodiment is 0.02 M.

[0123] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.15 As shown, from Fig.15 It is not difficult to see that under the online detection conditions of the 280nm UV detector, one flow-through peak and one elution peak 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.

[0124] ②Component analysis and purity testing:

[0125] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.16 shown.

[0126] Fig.16 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0127] from Fig.16 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0128] <Example 9>

[0129] ① A method for separating and purifying high-purity lactoferrin in camel milk is basically the same as Example 1, except that: in step 3 of this embodiment, the camel milk whey protein solution is not subjected to ammonium sulfate fractional precipitation, but is directly filtered through a 0.22 um water filter membrane before loading, and the concentration of NaCl in the elution buffer in this embodiment is 0.5 M.

[0130] 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 one elution peak 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.

[0131] ②Component analysis and purity testing:

[0132] The components of camel milk lactoferrin 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.

[0133] Fig.18 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0134] from Fig.18 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 elution buffer is about 80kDa. Therefore, it can be preliminarily confirmed that the component is the target protein - lactoferrin. The above protein band is obvious and single, with basically no impurity protein, and the purification effect is good.

[0135] <Example 10>

[0136] A method for detecting lactoferrin in camel milk, comprising:

[0137] ①Qualitative testing

[0138] The camel milk to be tested was treated in step 1 of Example 1 to obtain a camel milk whey protein solution to be tested, which was basically the same as Example 1, except that in step 3 of this example, the camel milk whey protein solution was not subjected to ammonium sulfate fractionation precipitation, but was directly filtered with a 0.22 um water filter membrane before loading. The camel milk lactoferrin separated and purified in this example was qualitatively characterized by SDS-PAGE. The results were as follows: Fig.19 shown.

[0139] Fig.19 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0140] from Fig.19 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 the elution buffer is about 80kDa, so it can be preliminarily confirmed that this component is the target protein - lactoferrin.

[0141] ②Quantitative detection

[0142] Step A, taking the camel milk lactoferrin obtained in Example 1 and using the BCA method to quantify the protein, and then preparing camel milk lactoferrin standard solutions of different concentrations within a concentration range of 50 to 1000 mg / L. The concentrations of the camel milk lactoferrin standard solutions prepared in this embodiment are 0.05, 0.1, 0.25, 0.5, 0.75, and 1 mg / mL respectively;

[0143] Step B: Using a C4 chromatographic column (ACQUITY UPLC ProteinBEH C4 column, 1.7μm, 21mm×100mm, 1 / pkg) were subjected to gradient elution of camel milk lactoferrin standard solutions of different concentrations, wherein the flow rate of the gradient elution was 0.2mL / min, mobile phase A was 0.1% trifluoroacetic acid water, mobile phase B was acetonitrile, the detection wavelength was 280nm, the injection volume was 10μL, the sample temperature was 25°C, the column temperature was 30°C, and the mobile phase elution gradient was as shown in Table 1 below:

[0144] Table 1

[0145] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 2.08 45 55 4.17 40 60 5.00 70 30 6.67 70 30

[0146] Step C, according to step B, obtain the chromatogram of camel milk lactoferrin standard solution of different concentrations (such as Fig. 20 ), a standard curve between the concentration and peak area of ​​camel milk lactoferrin standard was established (as shown in Fig.21 The standard curve regression equation is Y = 156564X-7318.9 (R 2 =0.9968, linear range is 0.05-1 mg / mL);

[0147] Step D, treating the camel milk to be tested by step 1 in Example 1 to obtain a camel milk whey protein solution to be tested, filtering the camel milk whey protein solution to be tested with a 0.22um water filter membrane, and then eluting by the gradient elution method in step B to obtain a chromatogram of the camel milk whey protein to be tested, and then obtaining the peak area of ​​camel milk lactoferrin therefrom, and then calculating the concentration of camel milk lactoferrin in the camel milk whey protein solution to be tested according to the standard curve in step C.

[0148] <Comparative Example 1>

[0149] The T / TDSTIA006-2019 standard discloses the "Liquid Chromatography Method for the Determination of Lactoferrin in Milk and Dairy Products", in which the purification process with a heparin affinity column includes: activating the heparin affinity column with 10 mL of phosphate buffer II, accurately transferring 10 mL of supernatant through the column, rinsing with 10 mL of phosphate buffer II, eluting with 4.5 mL of phosphate buffer III, collecting the eluate, making the volume up to 5.0 mL with phosphate buffer III, vortex mixing, and passing through a microporous filter membrane for testing.

[0150] <Comparative Example 2>

[0151] In the article "Isolation, Purification and Antibacterial Activity of Alashan Bactrian Camel Milk Lactoferrin", the process of obtaining camel milk lactoferrin with a purity of 91% through two ammonium sulfate precipitations and two Sephadex G-100 column chromatography separations is disclosed.

[0152] <Comparative Example 3>

[0153] A method for separating and purifying lactoferrin in camel milk comprises the following steps: treating camel milk according to step 1 of embodiment 1 to obtain a camel milk whey protein solution; filtering the camel milk whey protein solution with a 0.22 um water filter membrane; and separating and purifying the camel milk lactoferrin in the camel milk whey protein solution according to steps 3 to 5 of embodiment 1; in the present embodiment, the loading buffer is a 0.2 M tris buffer with a pH of 8.0, and the elution buffer is a 0.05 M tris buffer with a pH of 7.0 containing 1 M NaCl.

[0154] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig. 22 As shown, from Fig. 22 It is not difficult to see that under the online detection conditions of the 280nm UV detector, 1 flow-through peak and 2 elution peaks were obtained, which indicates that multiple substances were eluted.

[0155] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.23 shown.

[0156] Fig.23In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0157] from Fig.23 It is not difficult to see that there are two SDS-PAGE gel electrophoresis protein bands corresponding to the elution peak obtained by elution with elution buffer, one with a molecular weight of about 80 kDa and the other with a molecular weight of 18 kDa. Therefore, it can be preliminarily confirmed that the elution component contains other components besides lactoferrin, and it is impossible to separate and purify lactoferrin.

[0158] <Comparative Example 4>

[0159] A method for separating and purifying lactoferrin in camel milk comprises the following steps: treating camel milk according to step 1 of embodiment 1 to obtain a camel milk whey protein solution; filtering the camel milk whey protein solution with a 0.22 um water filter membrane; and separating and purifying the camel milk lactoferrin in the camel milk whey protein solution according to steps 3 to 5 of embodiment 1; in the present embodiment, the loading buffer is a 0.4 M tris buffer with a pH of 8.0, and the elution buffer is a 0.05 M tris buffer with a pH of 7.0 containing 1 M NaCl.

[0160] The elution was carried out using the AKTA automatic purification system, and the chromatographic peaks were as follows: Fig.24 As shown, from Fig.24 It is not difficult to see that under the online detection conditions of the 280nm UV detector, 1 flow-through peak and 2 elution peaks were obtained, which indicates that multiple substances were eluted.

[0161] The components of camel milk lactoferrin were analyzed by SDS-PAGE gel electrophoresis, and the process was basically the same as in Example 1. The results are as follows: Fig.25 shown.

[0162] Fig.25 In which M is Marker, 1 is whey, 2 is flow-through component, and 3 is elution buffer elution component.

[0163] from Fig.25 It is not difficult to see that there are two SDS-PAGE gel electrophoresis protein bands corresponding to the elution peak obtained by elution with elution buffer, one with a molecular weight of about 80 kDa and the other with a molecular weight of 18 kDa. Therefore, it can be preliminarily confirmed that the elution component contains other components besides lactoferrin, and it is impossible to separate and purify lactoferrin.

[0164] It can be seen from the above comparative examples that the buffers recommended for the heparin affinity chromatography column in comparative example 1 are all composed of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride, with many components and complicated configuration process. In the present application, the elution buffer is a phosphate buffer prepared from sodium dihydrogen phosphate, and the loading buffer is a phosphate buffer containing sodium chloride, which has simple components and is easy to configure. Moreover, only one elution is required to separate and purify lactoferrin, and the separation and purification process is simpler. The purity of the separated lactoferrin can reach 98%, which exceeds the 95% specified in the national standard GB 1903.17-2016.

[0165] In Comparative Example 2, two Sephadex G-100 column chromatography separations are required, and the purity of the camel milk lactoferrin obtained is 91%, while in the present application, only one heparin affinity chromatography column elution is required, the process is simpler, and the purity of the lactoferrin obtained by separation and purification in the present application is 98%, which is much higher than the purity of the camel milk lactoferrin obtained in Comparative Example 2.

[0166] Comparative Examples 3 and 4 adopt the same method of the present application, except that the phosphate buffer in the loading buffer and the elution buffer is replaced with a tris buffer. From the test results, it can be seen that in addition to lactoferrin, there are other components in the final eluted fraction, and it is impossible to separate and purify lactoferrin. Therefore, in the separation and purification method of the present application, the selected loading buffer and elution buffer have their uniqueness.

[0167] 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 the content of lactoferrin in camel milk whey is higher than that in cow milk whey, and it is more economical to separate lactoferrin from camel milk. The method provided in this application meets the current demand for deep processing of camel milk whey. Lactoferrin in camel milk whey is extracted by enrichment and purification methods, so that camel milk whey can be turned into treasure, which not only provides a by-product recycling solution for the existing dairy processing industry, but also improves the added value of camel milk deep processing products, achieving multiple goals at one stroke.

[0168] 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 methods. 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 patent 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 separating and purifying high-purity lactoferrin in camel milk, characterized in that: include: Step 1, taking camel milk, and separating and obtaining camel milk whey protein solution by isoelectric precipitation method; Step 2: flush the heparin affinity chromatography column with a pre-configured loading buffer until the UV absorption baseline and the conductivity baseline are stable; Step 3, loading the camel milk whey protein solution onto the heparin affinity chromatography column treated in step 2, and then washing the heparin affinity chromatography column with a loading buffer to elute non-target proteins; Step 4: flush the heparin affinity chromatography column treated in step 3 with a pre-configured elution buffer, elute and collect the elution peak of lactoferrin, and dialyze it in distilled water for desalination.

2. The method for separating and purifying high-purity lactoferrin in camel milk according to claim 1, characterized in that: The loading buffer adopts a phosphate buffer with a pH of 7.5 to 8.0 and a concentration of 0.15 to 0.2 M; The elution buffer is a phosphate buffer containing 0.5-1M NaCl, a pH of 7.0-7.5, and a concentration of 0.02-0.05M.

3. The method for separating and purifying high-purity lactoferrin in camel milk as claimed in claim 2, characterized in that: In step 2, the heparin affinity chromatography column is flushed with 3 to 5 column volumes of loading buffer at a rate of 1.0 to 2.0 mL / min until the ultraviolet absorption baseline and the conductivity baseline are stable.

4. The method for separating and purifying high-purity lactoferrin in camel milk according to claim 3, characterized in that: In step three, the camel milk whey protein solution is filtered with a 0.22 um water filter membrane and then loaded onto the heparin affinity chromatography column treated in step two at a rate of 1.0 to 2.0 mL / min, and then the heparin affinity chromatography column is rinsed with 3 to 5 column volumes of loading buffer until the ultraviolet absorption baseline and the conductivity baseline are stable.

5. The method for separating and purifying high-purity lactoferrin in camel milk according to claim 4, characterized in that: In step 4, the heparin affinity chromatography column treated in step 3 is washed with 3 to 5 column volumes of elution buffer at a rate of 1.0 to 2.0 mL / min.

6. The method for separating and purifying high-purity lactoferrin in 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 at 4°C for 20 minutes, and the whey protein solution is taken and the pH is adjusted to the above-recorded pH with saturated sodium bicarbonate solution to obtain a camel milk whey protein solution.

7. The method for separating and purifying high-purity lactoferrin in camel milk according to claim 1, characterized in that: In step three, before loading the camel milk whey protein solution, ammonium sulfate fractionation precipitation is first used, 50%-80% of the precipitated components are collected, re-dissolved with pure water and dialyzed in distilled water for desalination, and then filtered with a 0.22um water filter membrane, and the filtered solution is used for loading.

8. The method for separating and purifying high-purity lactoferrin in camel milk according to claim 7, characterized in that: The specific method of ammonium sulfate graded precipitation, collecting 50%-80% precipitation components, re-dissolving with pure water and dialyzing in distilled water for desalination comprises: slowly adding ammonium sulfate to a camel milk whey protein solution and stirring continuously, standing at 4°C for 3 hours after the ammonium sulfate saturation reaches 50%, centrifuging at 8000 rpm for 15 minutes, discarding the precipitate and collecting the supernatant, continuing to add ammonium sulfate to the supernatant until its saturation reaches 80%, standing at 4°C for 3 hours, centrifuging at 8000 rpm for 15 minutes, collecting the precipitate to obtain camel milk lactoferrin components, adding a small amount of ultrapure water to the precipitate to redissolve the precipitate and placing it in a dialysis bag, dialyzing in distilled water at 4°C for desalination, changing the distilled water every 1 hour, using saturated barium chloride to detect whether there is residual ammonium sulfate in the dialysate, and when there is no white precipitate in the dialysate, the dialysis is terminated to obtain crude camel milk lactoferrin.

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