A method for separating and preparing milk fat globule membrane protein from buttermilk, a by-product of butter

Abstract

The present invention relates to a method for separating and preparing milk fat globule membrane protein from buttermilk, a by-product of butter, comprising: controlling the temperature of the buttermilk, a by-product of butter, at 25 to 30 °C; adjusting the pH value to 4.5 to 4.7 and centrifuging; performing thermal calcium treatment and centrifuging; performing ultrasonic treatment and then cooling to room temperature; performing dialysis treatment; adjusting the pH value to 6-8, and then performing ultrafiltration treatment, and collecting the solution obtained by the ultrafiltration treatment as the milk fat globule membrane protein. The method of the present invention can increase the removal rate of casein to 95%, avoid the effective protein being dialyzed or filtered out, greatly reduce the loss rate of the milk fat globule membrane protein, make the extraction rate reach more than 70%, and the purity of the prepared milk fat globule membrane reaches 87.7%. Moreover, the raw material selected in the method of the present invention is buttermilk, a by-product of industrial butter production, which is usually discarded or made into animal feed. The present invention reasonably utilizes this raw material, which can not only improve the utilization rate of buttermilk, but also save costs.

Description

Technical Field

[0001] The present invention relates to the technical field of protein separation, and particularly relates to a preparation method for extracting milk fat globule membrane protein from buttermilk, a by-product of butter production. Background Art

[0002] Milk fat globules (MFG) are formed in the epithelial secretory cells of the mammary gland. The formation process is as follows: first, fat globule precursors are formed on the endoplasmic reticulum, then the fat globule precursors pass through the cytoplasmic matrix and form smaller lipid droplets, and then they are wrapped by non-bilayer substances composed of lipids and proteins, and finally, true milk fat globules are formed, with a size of 0.1 - 15 μm.

[0003] The milk fat globule membrane (MFGM) is the membrane on the surface of milk fat globules secreted by mammary cells and is essentially a triple structure. The thickness of the MFGM is 10 - 50 nm, and the three-layer membrane is composed of the single-layer membrane of the endoplasmic reticulum in mammary cells and the double-layer membrane at the top of its cell membrane. The part derived from the endoplasmic reticulum is a single-layer membrane structure composed of proteins and polar lipids, and this layer of membrane covers the surface of the triacylglycerol core before the secretion of lipid droplets. The part of the milk fat globule membrane derived from the apical plasma membrane is the main part of the membrane structure, with the appearance of a typical bilayer membrane and a highly electron-dense inner membrane surface.

[0004] The particularity of the MFGM's source determines the complexity of its composition. Its main components are proteins / glycoproteins (accounting for 20 - 60% of the MFGM components), triglycerides, glycerophospholipids (accounting for 33% of the MFGM components), sphingolipids (mainly sphingomyelin), glycolipids, cholesterol, enzymes, and other trace components.

[0005] In cow milk from different sources, the protein content in the MFGM components varies within the range of 25% - 70%, accounting for only 1 - 2% of the total milk protein. Currently, the components of the isolated MFGM highly depend on the separation methods and analysis procedures used because the connection methods of membrane proteins to the MFGM are not unique, including binding proteins, peripheral membrane proteins, and proteins loosely attached to the membrane surface. In the results of separating MFGM by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 7 - 8 main membrane protein bands can be seen. However, there are still many low-abundance species that have not been identified.

[0006] In fact, milk fat globule membrane protein contains more than 100 kinds of proteins. The eight most abundant proteins are mucin 1 (MUC1), xanthine oxidoreductase (XOR), mucin 15 (MUC15), CD36, butyrophilin (BTN), lactadherin, adipocyte differentiation-related protein, and fatty acid-binding protein. Among them, the specific proteins on the membrane include xanthine oxidoreductase (XDH / XO), adipophilin (ADPH), butyrophilin (BTN), CD 36, tissue glycoprotein periodic acid Schiff 6 / 7 (PAS6 / 7), etc. These specific proteins have received increasing attention due to their nutritional and functional characteristics. Research shows that they can reduce cardiovascular diseases, inflammation, and gastrointestinal infections, and also have a certain positive effect on cholesterol absorption, nervous system myelin formation, and neural development. Milk fat globule membrane protein has rich development and utilization value.

[0007] Among these major milk fat globule membrane proteins, mucin 1 (MUC1) is a high-molecular-weight transmembrane glycoprotein and an important component of the physical defense of mucous membranes, and may be related to epithelial infections and inflammatory responses. MUC1 is widely expressed in normal glandular epithelial cells, and its expression increases sharply when these cells turn malignant. This has been confirmed in some adenocarcinomas, such as breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, and colon cancer. Recently, MUC1 has also been detected in some squamous cell carcinomas, such as esophageal cancer, pharyngeal cancer, laryngeal cancer, eyelid cancer, and oral mucosal cancer. The upregulated expression of MUC1 in these squamous cell carcinomas suggests that this mucin plays a role in the malignant progression of squamous cell carcinomas.

[0008] PAS6 / 7 is an integral peripheral protein adhering to the surface of milk fat globule membrane, and is an immunogenic lipophilic glycoprotein. It plays multiple functions in vivo, such as facilitating the clearance of apoptotic lymphocytes and other apoptotic cells, promoting the repair of intestinal mucosa, increasing the function of dendritic cell exosomes, promoting the morphological changes of mammary gland branches, promoting angiogenesis, regulating the adhesion of sperm to the periphery of oocytes, etc. Lactadherin can also resist rotavirus infection.

[0009] Butyrophilin (BTN) is a protein related to fat droplets and is a member of the immunoglobulin family. They may be related to the autoimmune regulation of diseases such as autism and multiple sclerosis.

[0010] Xanthine oxidoreductase (XDH / XO) is a metabolic enzyme widely present in the animal body. It belongs to the flavin molybdenum protein family and is most abundant in milk. For many years, XDH / XO was considered to mainly participate in purine metabolism in the body, but more and more evidence shows that XDH / XO has a wide range of biological functions. XDH / XO can inhibit bacterial growth in the body and has antibacterial properties. In vitro, the probability of infants consuming milk rich in XDH / XO suffering from intestinal diseases is lower than that of infants consuming formula milk. XDH / XO also has a certain resistance to oxidative damage of tissues. During ischemia-reperfusion, XDH / XO can catalyze the generation of reactive oxygen free radicals to cause tissue damage, and it plays a certain role in both cardiovascular and heart diseases.

[0011] Fatty acid transport protein (CD36) contains a large amount of carbohydrates (about 24% by mass of neutral sugars) in its structure. The part related to MFGM in CD36 accounts for only 5% or less of its total protein, and since it is an integral protein, most of the protein can be collected in the buttermilk supernatant by centrifugation. As a receptor for collagen and thrombospondin, it can play a certain role in platelet activation and aggregation, and is also an inhibitor of intercellular adhesion and thrombospondin-mediated angiogenesis. CD36 can also bind to apoptotic cells and cell debris, and promote their elimination through phagocytosis. The related scavenger function lies in that CD36 on the surface of macrophages can bind oxidized low-density lipoproteins and eliminate them from the systemic circulation through endocytosis.

[0012] Fatty acid-binding protein (FABP) was initially identified in the screening of breast cancer cell growth inhibitors. This protein is an effective inhibitor of the in vitro growth of breast cancer cells. The molecular weight of the FABP protein was identified to be 13 kDa, and its content accounts for 2% - 3% of the total protein content of MFGM, and its location is between the triacylglycerol core and the three-layer structure of the milk fat globule membrane.

[0013] Adipophilin (ADPH) is insoluble in salt solutions or non-ionic detergent conditions, but can be dissolved by methanol and KOH. Its solubility properties indicate that the connection between fatty acids and the ADPH protein is an ester bond connection.

[19] Therefore, FABP can play a crucial role in the process of fatty acid transport and lipid metabolism.

[0014] In addition, another important component of MFGM is MFGM lipids, which not only contain relatively large amounts of polar lipids but also relatively small amounts of neutral lipids. Moreover, among MFGM lipids, the main component phospholipid polar lipids are mainly divided into five categories, namely phosphatidylcholine (PC, accounting for 35% of the total polar lipid content), phosphatidylethanolamine (PE, accounting for 30% of the total polar lipid content), sphingomyelin (SM, accounting for 25% of the total polar lipid content), phosphatidylinositol (PI, accounting for 5% of the total polar lipid content), and phosphatidylserine (PS, accounting for 3% of the total polar lipid content). In addition, the contents of glucosylceramide (GluCer), neutral glycosphingolipids, lactosylceramide (LacCer), and gangliosides (Gang) are relatively small. Among the relatively small amounts of MFGM neutral lipids, triglycerides are the main component, and the proportions of free fatty acids, 1,2-diglyceride, 1,3-diglyceride, monoglyceride, and cholesterol are relatively small. The presence of these complex and diverse lipids causes great difficulties in the separation of MFGM proteins.

[0015] As mentioned above, milk fat globule membrane proteins account for about 1% - 2% of the total proteins. Currently, their extraction processes are all based on the separation of membrane proteins from specific raw materials. If the active ingredients are directly extracted from raw milk, not only will the raw materials be wasted, the utilization rate of raw milk will be low, and the cost will be relatively high. Most current research uses industrial by-products as raw materials for extraction. Some research uses the by-product whey buttermilk from cheese production as the raw material and extracts milk fat globule membrane fragments using the hot calcium treatment method (pH value set at 7.7, temperature at 55 °C, Ca 2+ concentration in whey of 0.205 g / L). Although the casein micelles in the raw material whey buttermilk are removed, the content of MFGM fragments in it is relatively small compared to other raw materials, so the total MFGM content extracted is extremely low. Some research uses the by-product buttermilk from butter production as the raw material, removes casein using rennet, removes residual whey proteins using microfiltration, and removes some residual lactalbumin, lactoglobulin, lactose, and minerals using diafiltration. This method cannot completely remove the casein component, the protein purity of the sample is relatively low, and MFGM protein fragments may adhere to other protein molecules and be filtered out together, resulting in a relatively high MFGM loss rate. In Chinese Patent CN 102863526A, milk fat globule membrane proteins are extracted from raw yak milk. It uses a sodium phosphate buffer solution and a polyethylene glycol octylphenyl ether solution to wash the cream obtained after centrifugation. Although this method has relatively high purity, the raw material utilization rate is relatively low, causing waste, and it is suitable for laboratory mechanism research but not for large-scale factory production. Therefore, in this technical field, there is an urgent need to find a method that can make full use of raw materials, extract with high yield and high purity, and can produce MFGM proteins on a large scale. Summary of the Invention

[0016] To solve the above technical problems, the present invention provides a method for separating and preparing milk fat globule membrane protein from butter by-product buttermilk, and the method comprises the following steps:

[0017] (1) Control the temperature of the by-product buttermilk of butter at 25 to 30 °C;

[0018] (2) Adjust the pH value of the heated buttermilk to 4.5 to 4.7, and then obtain a first supernatant through centrifugation;

[0019] (3) Perform thermal calcium treatment on the first supernatant;

[0020] (4) Centrifuge the first supernatant after thermal calcium treatment to obtain a second supernatant;

[0021] (5) Perform ultrasonic treatment on the second supernatant, and then cool it to room temperature to prepare a sample to be dialyzed;

[0022] (6) Dialyze the sample to be dialyzed to obtain a sample to be ultrafiltered;

[0023] (7) Adjust the pH value of the sample to be ultrafiltered to 6 - 8, and then perform ultrafiltration treatment, and collect the solution obtained by ultrafiltration treatment as the milk fat globule membrane protein.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) By adopting the method of the present invention, the removal rate of casein can reach 95%;

[0026] (2) The method of the present invention can avoid the effective protein being dialyzed or filtered out, greatly reducing the loss rate of milk fat globule membrane protein, and enabling the extraction rate to reach more than 70%.

[0027] (3) The selected raw material is the by-product buttermilk from industrial butter production, which is usually discarded or made into animal feed. The present invention rationally utilizes this raw material, which can not only improve the utilization rate of buttermilk but also save costs. Detailed Embodiments

[0028] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] As described above, the present invention provides a method for separating and preparing milk fat globule membrane protein from the by-product buttermilk of butter, and the method comprises the following steps:

[0030] (1) Controlling the temperature of the butter by-product buttermilk at 25 to 30 °C;

[0031] (2) Adjusting the pH value of the heated buttermilk to 4.5 to 4.7, and then obtaining a first supernatant through centrifugation;

[0032] (3) Performing heat calcium treatment on the first supernatant;

[0033] (4) Centrifuging the first supernatant after heat calcium treatment to obtain a second supernatant;

[0034] (5) Performing ultrasonic treatment on the second supernatant, and then cooling to room temperature to obtain a sample to be dialyzed;

[0035] (6) Dialyzing the sample to be dialyzed to obtain a sample to be ultrafiltered;

[0036] (7) Adjusting the pH value of the sample to be ultrafiltered to 6 - 8, and then performing ultrafiltration treatment, and collecting the solution obtained from the ultrafiltration treatment as the milk fat globule membrane protein.

[0037] Preferably, in step (2), the centrifugation speed is 3000 to 4000 rpm (preferably 4000 rpm), the centrifugation time is 20 to 30 min, preferably 20 min, and the standing time is 20 to 30 min, for example, 25 min.

[0038] More preferably, in step (2), a 2 mol / L hydrochloric acid solution is used to adjust the pH value; preferably, the pH value is adjusted to 4.6.

[0039] Still more preferably, in step (3), when performing the heat calcium treatment, calcium chloride is added to the first supernatant, then the pH is adjusted to 7 - 7.7 (preferably 7.0), and then heated to 40 - 60 °C (such as 50 °C), fully mixed, and allowed to stand for 20 - 40 min (such as 30 min).

[0040] Still more preferably, the addition amount of calcium chloride is 0.02 - 0.16 g / 100 g of emulsion; preferably, the pH is adjusted with sodium hydroxide.

[0041] In some embodiments, in step (3), the temperature of the heat calcium treatment is 60 °C, the pH value is adjusted to 7, the addition amount of CaCl₂ is 0.16 g / 100 g of emulsion, and the standing time is 40 min.

[0042] More preferably, in step (4), the centrifugation speed is 3000 - 4000 rpm (preferably 4000 rpm), and the centrifugation time is 20 - 30 min (e.g., 20 min).

[0043] More preferably, in step (5), the ultrasonic frequency of the ultrasonic treatment is 45 - 80 MHz (e.g., 50, 60 or 70 MHz), the ultrasonic time is 1 - 2 h (e.g., 1, 1.5 or 2 h), and the ultrasonic temperature is 45 - 55 °C (e.g., 45, 50 or 55 °C).

[0044] More preferably, in step (6), the dialysis is carried out by reverse osmosis treatment using a dialysis bag made of cellulose, and the reverse osmosis treatment is carried out under the condition of pH 6.

[0045] More preferably, the reverse osmosis treatment is carried out in a constant temperature water bath at 25 °C; preferably, while the reverse osmosis treatment is carried out, continuous stirring is carried out by a magnetic stirrer, and the dialysis time is 18 to 30 h.

[0046] More preferably, the molecular cut-off of the dialysis bag is 14 KDa.

[0047] More preferably, in step (7), the ultrafiltration treatment is carried out by a cross-flow filtration device, the filter membrane is a polysulfone membrane, the molecular cut-off of the filter plate is 30 - 100 KDa, preferably 100 KDa; the feed-liquid ratio is 1:2 - 1:8 (e.g., 1:2, 1:3, 1:4, 1:5, 1:6 or 1:7); the transmembrane pressure is 0.14 to 0.16 MPa (e.g., 0.15 MPa); the flow rate is 450 to 500 L / h.

[0048] In some specific embodiments, the method comprises the following steps:

[0049] (1) Take fresh butter by-product buttermilk from the factory, and control the temperature at 25 - 30 °C;

[0050] (2) Adjust the pH value of the buttermilk heated in step (1) to 4.6 with 2 mol / L hydrochloric acid solution, then centrifuge the emulsion at a speed of 3000 - 4000 rpm for 20 - 30 min, and collect the supernatant;

[0051] (3) Take the supernatant of step (2) for thermal calcium treatment, add calcium chloride to the emulsion, adjust the pH to 7 - 7.7 with sodium hydroxide, heat to 40 - 50 °C, the addition amount is 0.02 - 0.16 g / 100 g emulsion, mix well, and let stand for 20 - 40 min;

[0052] (4) Centrifuge the sample after the hot calcium treatment in step (3) at a rotational speed of 3000 - 4000 rpm for 20 - 30 min, and collect the supernatant;

[0053] (5) Subject the supernatant collected in step (4) to ultrasonic treatment at an ultrasonic frequency of 45 - 80 MHz for 1 - 2 h at an ultrasonic temperature of 45 - 55 °C, and cool the sample after ultrasonic treatment to room temperature;

[0054] (6) Subject the sample cooled to room temperature in step (5) to reverse osmosis treatment using a dialysis bag made of cellulose (molecular cut-off volume of 14 KDa) in a constant temperature water bath at 25 °C, and continuously stir for 24 h using a magnetic stirrer;

[0055] (7) Adjust the pH value of the sample after reverse osmosis treatment in step (6) to 6 - 8, and then perform ultrafiltration treatment: a cross-flow filtration device with a polysulfone membrane as the filtration membrane, a filtration plate with a molecular cut-off volume of 30 - 100 KDa, a feed liquid ratio of 1:2 - 1:8, a transmembrane pressure of 0.15 MPa, and a flow rate of 500 L / h, and collect the filtered solution, which is the milk fat globule membrane protein with higher purity.

[0056] The particularity of the MFGM source endows the proteins and lipids it contains with unique nutritional characteristics and can play a certain role in the stability of the system. The enrichment, separation, and purification of MFGM in the industry have always been a difficult point. The present inventor conducted a comparative analysis of the content and types of MFGM proteins in different industrial by-products. It was found that although butter whey is a by-product in the production process of anhydrous butter, it contains rich MFGM proteins and can be used as a raw material for further separating and enriching MFGM proteins. In the prior art, there have also been in-depth analyses of the separation and extraction process of MFGM proteins and the functional characteristics of MFGM materials, and different membrane component analysis methods have also been reported. Among them, microfiltration is the only separation method currently applied in the industry, but some small molecule free proteins will be lost during the separation process. The present inventor found that the pore size of the ultrafiltration membrane is just right to separate the large molecule MFGM proteins from the milk proteins, and currently, there are few studies on the separation of MFGM proteins by ultrafiltration method and all of them only stay at the macroscopic level. Therefore, it is very meaningful to study the changes of MFGM proteins during the enrichment process and the separation effects of different membrane components by combining quantitative analysis methods.

[0057] Examples

[0058] The present invention will be further described below through examples, but the protection scope of the present invention is not limited to these examples.

[0059] Example 1

[0060] In this embodiment, milk fat globule membrane protein is extracted from butter by-product buttermilk, and the specific steps are as follows:

[0061] I. Take fresh butter by-product buttermilk from the factory and heat it to 30°C;

[0062] II. Adjust the pH value of the buttermilk heated in step I to 4.6 with 2 mol / L hydrochloric acid solution, then centrifuge the emulsion at a speed of 4000 r, for a centrifugation time of 20 min, and collect the supernatant;

[0063] III. Take the supernatant from step II for heat calcium treatment, heat it to 60°C, adjust the pH to 7 with hydrochloric acid solution, add CaCl2 to the emulsion, with an addition amount of 0.16 g / 100 g emulsion, mix well, and let it stand for 40 min;

[0064] IV. Take the sample after heat calcium treatment in step III for centrifugation at a speed of 4000 and a centrifugation time of 20 min, and collect the supernatant;

[0065] V. Take the supernatant collected in step IV for ultrasonic treatment, with an ultrasonic frequency of 80 MHz, an ultrasonic time of 1 h, and an ultrasonic temperature of 45°C, and cool the sample after ultrasonic treatment to room temperature;

[0066] VI. Take the sample cooled to room temperature in step V and perform reverse osmosis treatment with a dialysis bag made of cellulose (molecular cut-off amount is 14 KDa), in a constant temperature water bath at 25°C, and continuously stir with a magnetic stirrer for 24 h;

[0067] VII. Take the sample after reverse osmosis treatment in step VI, adjust the pH value to 6, and then perform ultrafiltration treatment: a frame type filtration device, with a filtration membrane of polysulfone membrane, a filtration plate with a molecular cut-off amount of 50 KDa, a feed liquid ratio of 1:2, a transmembrane pressure of 0.15 MPa, a flow rate of 500 L / h, and collect the filtered solution, which is the milk fat globule membrane protein with higher purity.

[0068] Example 2

[0069] I. Take fresh butter by-product buttermilk from the factory and heat it to 30°C;

[0070] II. Adjust the pH value of the buttermilk heated in step I to 4.6 with 2 mol / L hydrochloric acid solution, then centrifuge the emulsion at a speed of 4000 r, for a centrifugation time of 20 min, and collect the supernatant;

[0071] III. Take the supernatant from step II for heat calcium treatment, heat it to 60°C, adjust the pH to 7 with hydrochloric acid solution, add CaCl2 to the emulsion, with an addition amount of 0.16 g / 100 g emulsion, mix well, and let it stand for 40 min;

[0072] IV. Centrifuge the sample after the hot calcium treatment in Step III at a speed of 4000 for 20 min, and collect the supernatant;

[0073] V. Perform reverse osmosis treatment on the supernatant in Step IV using a dialysis bag made of cellulose (molecular cut-off 14KDa), in a constant temperature water bath at 25°C, and continuously stir with a magnetic stirrer for 24 h;

[0074] VI. Adjust the pH value of the sample after the reverse osmosis treatment in Step V to 6, and then perform ultrafiltration: a cross-flow filtration device, with a polysulfone membrane as the filtration membrane, a filtration plate with a molecular cut-off of 100KDa, a feed-liquid ratio of 1:2, a transmembrane pressure of 0.15 MPa, a flow rate of 500 L / h, and collect the filtered solution, which is the milk fat globule membrane protein with a higher purity.

[0075] Example 3

[0076] I. Take fresh butter by-product buttermilk from the factory and heat it to 30°C;

[0077] II. Adjust the pH value of the buttermilk heated in Step I to 4.6 with 2 mol / L hydrochloric acid solution, and then centrifuge the emulsion at a speed of 4000 r for 20 min, and collect the supernatant;

[0078] III. Perform hot calcium treatment on the supernatant in Step II, heat it to 60°C, adjust the pH to 7 with hydrochloric acid solution, add CaCl2 to the emulsion, with an addition amount of 0.16 g / 100 g emulsion, mix well, and let it stand for 5 min;

[0079] IV. Centrifuge the sample after the hot calcium treatment in Step III at a speed of 4000 for 20 min, and collect the supernatant;

[0080] V. Perform ultrasonic treatment on the supernatant collected in Step IV, with an ultrasonic frequency of 80 MHz, an ultrasonic time of 1 h, and an ultrasonic temperature of 45°C, and cool the sample after ultrasonic treatment to room temperature;

[0081] VI. Adjust the pH value of the sample after the ultrasonic treatment in Step V to 6, and then perform ultrafiltration: a frame-type filtration device, with a polysulfone membrane as the filtration membrane, a filtration plate with a molecular cut-off of 100KDa, a feed-liquid ratio of 1:2, a transmembrane pressure of 0.15 MPa, a flow rate of 500 L / h, and collect the filtered solution, which is the milk fat globule membrane protein with a higher purity.

[0082] The milk fat globule membrane proteins obtained in Example 1 and those obtained in Examples 2 and 3 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with liquid chromatography-mass spectrometry. The results showed that the color of the bands of the milk fat globule membrane proteins prepared in Example 1 was darker than those in Examples 2 and 3 in the gel electrophoresis. Moreover, it can be seen from LC / MS that the relative abundance values of several major proteins BTN in the milk fat globule membrane prepared in Example 1 were 6.3E+010, those of ADPH were 4.2E+010, and those of PAS6 / 7 were 6.8E+010; while in the milk fat globule membrane proteins prepared in Example 2 and Example 3, the relative abundance values of BTN were 4.1E+010 and 3.9E+010 respectively; those of ADPH were 3.7E+010 and 3.2E+010 respectively; and those of PAS6 / 7 were 3.8E+010 and 4.3+010 respectively. It can be seen by high performance liquid chromatography that the peak areas of the impurity peaks obtained in Example 1 were only 60% of those in Example 2 and 63% of those in Example 3, and it can be seen that α 32 -CN and κ-CN low-content casein components had basically disappeared. After calculation, the purity of the milk fat globule membrane prepared in Example 1 was 94.7%, while the purity of Example 2 was 85.6%, and that of Example 3 was only 82.3%.

[0083] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention in each example.

Claims

1. A method for separating and preparing milk fat globule membrane protein from buttermilk, a by-product of butter, characterized in that, The method includes the following steps: (1) Control the temperature of the butter by-product buttermilk at 25 to 30 °C; (2) Adjust the pH value of the heated buttermilk to 4.5 to 4.7, and then obtain the first supernatant by centrifugation; (3) Perform thermal calcium treatment on the first supernatant; (4) Centrifuge the first supernatant after thermal calcium treatment to obtain a second supernatant; (5) Perform ultrasonic treatment on the second supernatant, and then cool it to room temperature to obtain a sample to be dialyzed; (6) Perform dialysis treatment on the sample to be dialyzed to obtain a sample to be ultrafiltered; (7) Adjust the pH value of the sample to be ultrafiltered to 6 - 8, and then perform ultrafiltration treatment, and collect the solution obtained by ultrafiltration treatment as the milk fat globule membrane protein; Wherein: In step (2), the centrifugation speed is 3000 to 4000 rpm, the centrifugation time is 20 to 30 min, and the standing time is 20 to 30 min; In step (3), when performing the thermal calcium treatment, add calcium chloride to the first supernatant, then adjust the pH to 7 - 7.7, and then heat to 40 - 60 °C, mix well, and stand for 20 - 40 min; In step (4), the centrifugation speed is 3000 - 4000 rpm, and the centrifugation time is 20 - 30 min; In step (5), the ultrasonic frequency of the ultrasonic treatment is 45 - 80 MHz, the ultrasonic time is 1 - 2 h, and the ultrasonic temperature is 45 - 55 °C; In step (6), the dialysis is carried out by reverse osmosis using a dialysis bag with a molecular cut-off of 14 KDa, and the reverse osmosis treatment is carried out at a pH of 6 and a temperature of 25 °C; In step (7), the ultrafiltration treatment is carried out using a cross-flow filtration device, the filter membrane is a polysulfone membrane, the molecular cut-off of the filter plate is 30 - 100 KDa; the liquid-to-solid ratio is 1:2 - 1:8; the transmembrane pressure is 0.14 to 0.16 MPa; the flow rate is 450 to 500 L / h.

2. The method according to claim 1, wherein: In step (2), a 2 mol / L hydrochloric acid solution is used to adjust the pH value.

3. The method according to claim 2, wherein: In step (2), the pH value is adjusted to 4.

6.

4. The method according to claim 1, wherein: In step (3), the addition amount of calcium chloride is 0.02 - 0.16 g / 100 g of emulsion.

5. The method according to claim 4, wherein: In step (3), the pH is adjusted using sodium hydroxide.

6. The method according to any one of claims 1 to 5, wherein: The reverse osmosis treatment is carried out in a constant temperature water bath at 25 °C.

7. The method according to claim 6, wherein: During the reverse osmosis treatment, continuously stir using a magnetic stirrer, and the dialysis time is 18 to 30 h.

8. The method according to claim 1, wherein: The dialysis bag is made of cellulose.

9. The method according to any one of claims 1 to 5, wherein: The molecular cut-off of the filter plate is 100 KDa.