CN114478739A

CN114478739A - Method for separating and preparing milk fat globule membrane protein from buttermilk by-product of butter

Info

Publication number: CN114478739A
Application number: CN202011263223.9A
Authority: CN
Inventors: 于景华; 闫可心; 郑姗; 王祎; 杨菲菲; 王梦琪
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2022-05-13

Abstract

The invention relates to a method for separating and preparing milk fat globule membrane protein from buttermilk which is a by-product of butter, comprising the following steps: controlling the temperature of butter by-product buttermilk to be 25-30 ℃; adjusting the pH value to 4.5 to 4.7 and centrifuging; carrying out hot calcium treatment and centrifuging; carrying out ultrasonic treatment and cooling to room temperature; carrying out dialysis treatment; adjusting the pH value to 6-8, then carrying out ultrafiltration treatment, and collecting the solution obtained by ultrafiltration treatment as the milk fat globule membrane protein. The method can improve the removal rate of casein to 95 percent, avoid effective protein from being dialyzed or filtered, greatly reduce the loss rate of milk fat globule membrane protein, ensure that the extraction rate reaches more than 70 percent, and the purity of the prepared milk fat globule membrane reaches 87.7 percent. In addition, the raw materials selected by the method are by-product buttermilk produced in butter industry, and the by-product buttermilk is usually discarded or made into animal feed.

Description

Method for separating and preparing milk fat globule membrane protein from butter by-product buttermilk

Technical Field

The invention relates to the technical field of protein separation, in particular to a preparation method for extracting milk fat globule membrane protein from by-product casein milk in butter production.

Background

Milk Fat Globules (MFG) are formed in epithelial secretory cells of the mammary gland by the process of: firstly, forming a fat globule precursor on an endoplasmic reticulum, then enabling the fat globule precursor to penetrate through a cytoplasm matrix and form smaller fat droplets, and then wrapping the fat globule precursor by a non-bilayer substance consisting of lipid and protein to finally form a real milk fat globule with the size of 0.1-15 mu m.

Milk Fat Globule Membrane (MFGM) is a membrane secreted from mammary gland cells out of the surface of milk fat globules, essentially a triple structure. The MFGM has a thickness of 10-50nm, and the three-layer membrane is composed of a single-layer membrane of endoplasmic reticulum in mammary cells and a double-layer membrane on top of the cell membrane. The portion derived from the endoplasmic reticulum is a single-layered membrane structure composed of proteins and polar lipids, which covers the surface of the triacylglycerol core before 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, having the appearance of a typical bilayer membrane and the inner membrane surface having a high electron density.

The specificity of the MFGM source determines the complexity of its composition. The main components of the composition are protein/glycoprotein (accounting for 20-60% of the MFGM component), triglyceride, glycerophospholipid (accounting for 33% of the MFGM component), sphingolipid (mainly sphingomyelin), glycolipid, cholesterol, enzyme and other trace components.

In cow milk with different sources, the protein content of the MFGM component is changed within the range of 25-70 percent and only accounts for 1-2 percent of the total amount of the milk protein. The composition of MFGM isolated at present is highly dependent on the isolation method and analytical procedure used, since the membrane proteins are not linked to MFGM in a unique manner, including binding proteins, membrane peripherins, and proteins loosely attached to the membrane surface. In the results of separating MFGM by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 7 to 8 major membrane protein bands were observed. However, many species with low abundance have not been identified.

In fact, milk fat globule membrane proteins comprise over 100 proteins, the 8 most abundant proteins being mucin 1(MUCI), Xanthine Oxidoreductase (XOR), mucin 15(MUC15), CD36, cremophil protein (BTN), milk agglutinin, fat differentiation related proteins and fatty acid binding proteins. The specific proteins on the membrane include xanthine oxidoreductase (XDH/XO), fat droplet binding protein (ADPH), cremophil protein (BTN), CD36, tissue glycoprotein periodate Scheff 6/7(PAS6/7), etc. The specific proteins are more and more concerned due to the nutritional and functional characteristics, and researches show that the specific proteins can reduce cardiovascular diseases, inflammations and gastrointestinal tract infection, have certain positive effects on cholesterol absorption, nervous system myelination and neural development, and have rich development and utilization values.

Among these major milk fat globule membrane proteins, mucin 1(MUCI) is a high molecular weight transmembrane glycoprotein that is an important component of mucosal physical defense, possibly associated with epithelial infections and inflammatory responses. MUC I is widely expressed in normal glandular epithelial cells, and its expression increases dramatically when these cells turn malignant. These have been demonstrated in several adenocarcinomas such as breast, ovarian, pancreatic, prostate, gastric and colon. MUC I has also recently been detected in several squamous cell carcinomas such as esophageal, pharyngeal, laryngeal, eyelid, and oral mucosal, and upregulated MUCI expression in these squamous carcinomas suggests a role for this mucin in the malignant progression of squamous cell carcinoma.

PAS6/7 is a mosaic type peripherin attached to the surface of milk fat globule membrane, a lipophilic glycoprotein with immunogenicity. Plays a plurality of functions in vivo, such as being beneficial to the elimination of apoptotic lymphocytes and other apoptotic cells, promoting the repair of intestinal mucosa, increasing the function of dendritic cell exosome, promoting the morphological change of mammary gland branches, promoting the formation of blood vessels, regulating the adhesion of sperms and the periphery of ova, and the like. Milk agglutinin may also be resistant to rotavirus infection.

Cremophilic protein (BTN) is a protein associated with fat droplets and is a member of the immunoglobulin family. They may be involved in the autoimmune regulation of autism, multiple sclerosis, etc.

Xanthine oxidoreductase (XDH/XO) is a metabolic enzyme widely existing in animal bodies, belongs to the flavin molybdenum protein family, and is most abundant in milk. For many years, XDH/XO has been thought to be primarily involved in purine metabolism in organisms, but there is increasing evidence that XDH/XO has a wide range of biological functions. XDH/XO can inhibit bacterial growth in vivo, and has antibacterial property; in vitro, infants fed XDH/XO-rich breast milk have a lower incidence of intestinal disease than infants fed formula milk. The XDH/XO also has certain resistance to the oxidative damage of tissues, can catalyze to generate active oxygen free radicals to cause tissue damage during ischemia reperfusion, and plays a certain role in cardiovascular and heart diseases.

The fatty acid transporter (CD36) has a large amount of carbohydrates (about 24 mass% neutral sugars) present in the structure. The MFGM-related fraction of CD36 is only 5% or less of its total protein and, because it is a whole protein, the majority of the protein can be collected in the butter milk supernatant by centrifugation. As a receptor for collagen and thrombospondin, it plays a role in platelet activation and aggregation, and is also an inhibitor of intercellular adhesion and thrombospondin-mediated angiogenesis. CD36 can also promote its elimination through phagocytosis by binding to apoptotic cells and cell debris. The relevant scavenger function is that CD36 on the macrophage surface can bind oxidized low density lipoproteins and eliminate them from the systemic circulation by endocytosis.

Fatty Acid Binding Proteins (FABPs) were originally identified in the screening of inhibitors of breast cancer cell growth. The protein is a potent inhibitor of breast cancer cell growth in vitro. The molecular weight of FABP protein is identified to be 13kDa, the content of the FABP protein accounts for 2% -3% of the total protein content of MFGM, and the FABP protein is positioned between three-layer structures of a triacylglycerol kernel and a milk fat globule membrane.

The lipid drop associated protein (ADPH) is not soluble in salt solution or nonionic detergent, but can be dissolved by methanol and KOH, and its solubility property indicates lipidThe connection mode between the fatty acid and the ADPH protein is ester bond connection^[19]. Thus, FABPs can play a crucial role in fatty acid transport processes and lipid metabolism.

In addition, another important class of compositions of MFGM is MFGM lipids, which contain not only relatively large amounts of polar lipids, but also relatively small amounts of neutral lipids. Moreover, in MFGM lipids, the main components phospholipid polar lipids are mainly divided into five major groups, namely lecithin (PC, 35% of the total polar lipid content), phosphatidylethanolamine (PE, 30% of the total polar lipid content), sphingomyelin (SM, 25% of the total polar lipid content), phosphatidylinositol (PI, 5% of the total polar lipid content), and phosphatidylserine (PS, 3% of the total polar lipid content). In addition, glucosylceramide (GluCer), neutral sphingoglycolipid, lacylceramide (LacCer) and ganglioside (Gang) are present in lesser amounts. In MFGM neutral lipids in relatively small amounts, triglycerides account for the major component, and the proportions of free fatty acids, 1, 2-diglycerides, 1, 3-diglycerides, monoglycerides, and cholesterol are small. The presence of these complex and diverse lipids poses a great challenge to the isolation of MFGM proteins.

As mentioned above, milk fat globule membrane proteins account for about 1% -2% of the total protein, and at present, their extraction processes are all based on the separation of membrane proteins from specific raw materials, and if active ingredients are directly extracted from raw milk, not only raw materials are wasted, but also the raw milk has low utilization rate and high cost, and most of the current researches are carried out by taking industrial byproducts as raw materials. There have been studies on the use of a calcium heat treatment method (pH 7.7, temperature 55 ℃ C., Ca) using whey buttermilk, a by-product of cheese production, as a raw material²⁺Whey at a concentration of 0.205 g/L) was extracted, and although casein micelles in the raw material whey casein were removed, the amount of MFGM fragments contained therein was small relative to other raw materials, so that the total MFGM content extracted was extremely low. The research uses the by-product buttermilk of butter production as raw material, uses rennin to remove casein, uses microfiltration method to remove residual lactalbumin, uses percolation method to remove some residual lactalbumin, lactoglobulin, lactose and mineral substanceThe method cannot completely remove casein components, the purity of the sample protein is low, and MFGM protein fragments can be attached to other protein molecules and filtered together, so that the loss rate of MFGM is high. In the Chinese patent CN 102863526A, milk fat globule membrane protein is extracted from raw yak milk, dilute cream obtained after centrifugation is washed by using sodium ion phosphate buffer solution and polyethylene glycol octyl phenyl ether solution, and the method has high purity but low raw material utilization rate, causes waste, is suitable for mechanism research in laboratories, and is not suitable for large-scale production in factories. Therefore, there is an urgent need in the art to find a method capable of extracting MFGM proteins with high yield and high purity by fully utilizing raw materials, and capable of mass production.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for separating and preparing milk fat globule membrane protein from butter by-product buttermilk, which comprises the following steps:

(1) controlling the temperature of buttermilk which is the by-product of butter to be 25-30 ℃;

(2) adjusting the pH of the heated buttermilk to 4.5 to 4.7, and then obtaining a first supernatant by centrifugation;

(3) subjecting the first supernatant to calcium heat treatment;

(4) centrifuging the first supernatant subjected to the calcium heat treatment to obtain a second supernatant;

(5) carrying out ultrasonic treatment on the second supernatant, and then cooling to room temperature to obtain a sample to be dialyzed;

(6) carrying out dialysis treatment on the sample to be dialyzed to obtain a sample to be ultrafiltered;

(7) and adjusting the pH value of the sample to be ultrafiltered to 6-8, then carrying out ultrafiltration treatment, and collecting the solution obtained by ultrafiltration treatment as the milk fat globule membrane protein.

Compared with the prior art, the invention has the following advantages:

(1) by adopting the method, the removal rate of casein can reach 95 percent;

(2) the method can avoid effective protein from being dialyzed or filtered, greatly reduce the loss rate of milk fat globule membrane protein and ensure that the extraction rate reaches more than 70 percent.

(3) The raw materials are selected as by-product buttermilk in the industrial production of butter, and the by-product buttermilk is usually discarded or made into animal feed.

Detailed Description

In order to make the objects, 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 with reference to the embodiments of the present invention, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

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

(1) controlling the temperature of butter by-product buttermilk to be 25-30 ℃;

(3) subjecting the first supernatant to calcium heat treatment;

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

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

More preferably, in the step (3), calcium chloride is added to the first supernatant liquid, and then the first supernatant liquid is heated to a pH of 7 to 7.7 (preferably 7.0) and 40 to 60 ℃ (for example, 50 ℃), sufficiently mixed, and left to stand for 20 to 40min (for example, 30min) in the hot calcium treatment.

More preferably, the addition amount of the calcium chloride is 0.02-0.16 g/100g of the emulsion; it is also preferred that the pH is adjusted with sodium hydroxide.

In some embodiments, in step (3), the temperature of the hot calcium treatment is 60 ℃, the Ph is adjusted to 7, CaCl₂The addition amount is 0.16g/100g emulsion, and the standing time is 40 min.

More preferably, in the step (4), the centrifugation is performed at 3000 to 4000rpm (preferably 4000rpm) for 20 to 30min (for example, 20 min).

In addition, in the step (5), the ultrasonic frequency of the ultrasonic treatment is 45 to 80MHz (for example, 50, 60 or 70MHz), the ultrasonic time is 1 to 2 hours (for example, 1, 1.5 or 2 hours), and the ultrasonic temperature is 45 to 55 ℃ (for example, 45, 50 or 55 ℃).

Still more preferably, in the step (6), the dialysis is performed by a reverse osmosis treatment using a dialysis bag made of cellulose, and the reverse osmosis treatment is performed at a pH of 6.

Still more preferably, the reverse osmosis treatment is carried out in a thermostatic water bath at 25 ℃; preferably, the reverse osmosis treatment is performed while continuing stirring with a magnetic stirrer for a dialysis time of 18 to 30 hours.

Still more preferably, the dialysis bag has a molecular cut-off of 14 KDa.

In addition, more preferably, in the step (7), the ultrafiltration treatment is performed by using a cross-flow filtration device, a filtration membrane is a polysulfone membrane, and the molecular cut-off amount of a filtration plate is 30-100 KDa, preferably 100 KDa; the ratio of the material to the liquid is 1: 2-1: 8 (for example, 1:2, 1:3, 1:4, 1:5, 1:6 or 1: 7); transmembrane pressure of 0.14 to 0.16MPa (e.g., 0.15 MPa); the flow rate is 450 to 500L/h.

In some specific embodiments, the method comprises the steps of:

(1) taking fresh butter by-product buttermilk from a factory, and controlling the temperature to be 25-30 ℃;

(2) adjusting the pH value of the heated buttermilk in the step (1) to 4.6 by using 2mol/L hydrochloric acid solution, then centrifuging the milk at the rotating speed of 3000-4000 rpm for 20-30 min, and collecting supernatant;

(3) performing hot calcium treatment on the supernatant obtained in the step (2), adding calcium chloride into the emulsion, adjusting the pH to 7-7.7 by using sodium hydroxide, heating to 40-50 ℃, adding 0.02-0.16 g of calcium chloride into 100g of the emulsion, fully mixing uniformly, and standing for 20-40 min;

(4) centrifuging the sample subjected to the calcium heat treatment in the step (3) at the rotating speed of 3000-4000 rpm for 20-30 min, and collecting supernatant;

(5) carrying out ultrasonic treatment on the supernatant collected in the step (4), wherein the ultrasonic frequency is 45-80 MHz, the ultrasonic time is 1-2 h, the ultrasonic temperature is 45-55 ℃, and cooling the ultrasonically treated sample to room temperature;

(6) taking the sample cooled to room temperature in the step (5), and performing reverse osmosis treatment by using a dialysis bag (molecular cut-off is 14KDa) made of cellulose, performing constant-temperature water bath at 25 ℃, and continuously stirring for 24 hours by using a magnetic stirrer;

(7) and (3) adjusting the pH value of the sample subjected to reverse osmosis treatment in the step (6) to 6-8, and then performing ultrafiltration treatment: the cross-flow type filtering device is characterized in that a filtering membrane is a polysulfone membrane, a filtering plate is selected to have a molecular cut-off of 30-100 KDa, a material-liquid ratio is 1: 2-1: 8, a trans-membrane pressure is 0.15MPa, a flow rate is 500L/h, and filtered solution is collected to obtain milk fat globule membrane protein with high purity.

The particularity of the MFGM source makes the proteins and lipids contained therein have unique nutritional properties and can play a role in system stability. The enrichment, separation and purification of MFGM in industry has been a difficult point. The inventors carried out comparative analysis on the content and species of MFGM protein in different industrial byproducts. Butter whey is found to be a byproduct in the production process of anhydrous cream, but is rich in MFGM protein, and can be used as a raw material for further separating and enriching MFGM protein. In the prior art, the separation and extraction process of MFGM protein and the functional characteristics of MFGM materials are deeply analyzed, and different membrane component analysis methods are also reported. Of these, microfiltration is the only separation method used in industry at present, but causes some loss of small molecule free protein during the separation process. The inventor finds that the pore size of the ultrafiltration membrane is just large enough to separate the macromolecular MFGM protein from the milk protein, and the research on the separation of the MFGM protein by the ultrafiltration method is few and only stays at the macroscopic level for research, so that the research on the change of the MFGM protein and the separation effect of different membrane components in the enrichment process by combining the quantitative analysis method is very meaningful.

Examples

The invention will be further illustrated by the following examples, without limiting the scope of the invention to these examples.

Example 1

In this example, the specific steps for extracting milk fat globule membrane protein from the butter by-product casein are as follows:

firstly, taking fresh butter by-product buttermilk from a factory, and heating the buttermilk to 30 ℃;

secondly, regulating the pH value of the heated buttermilk in the step one to 4.6 by using 2mol/L hydrochloric acid solution, then centrifuging the milk at the rotating speed of 4000r for 20min, and collecting supernatant;

thirdly, the supernatant liquor obtained in the second step is taken for calcium heating treatment, the temperature is heated to 60 ℃, hydrochloric acid solution is used for adjusting the pH value to 7, and CaCl is added₂Adding 0.16g/100g of emulsion into the emulsion, mixing, and standing for 40 min;

fourthly, centrifuging the sample subjected to the calcium heat treatment in the third step at the rotating speed of 4000 for 20min, and collecting supernatant;

fifthly, taking the supernatant collected in the step four for ultrasonic treatment, wherein the ultrasonic frequency is 80MHz, the ultrasonic time is 1h, and the ultrasonic temperature is 45 ℃, and cooling the sample after ultrasonic treatment to room temperature;

sixthly, taking the sample cooled to room temperature in the step five, and performing reverse osmosis treatment on the sample by using a dialysis bag (the molecular interception amount is 14KDa) made of cellulose, performing constant-temperature water bath at 25 ℃, and continuously stirring the sample for 24 hours by using a magnetic stirrer;

seventhly, taking the sample subjected to reverse osmosis treatment in the step six, adjusting the pH value to 6, and then performing ultrafiltration treatment: frame filter equipment, filtration membrane are the polysulfone membrane, and the filter selects for use the molecular cut-off to be 50KDa, and the feed liquid ratio is 1:2, selecting 0.15MPa transmembrane pressure and 500L/h flow, and collecting the filtered solution to obtain the milk fat globule membrane protein with high purity.

Example 2

fifthly, taking supernatant obtained in the fourth step, performing reverse osmosis treatment by using a dialysis bag (molecular cut-off is 14KDa) made of cellulose, performing constant-temperature water bath at 25 ℃, and continuously stirring for 24 hours by using a magnetic stirrer;

sixthly, taking the sample subjected to reverse osmosis treatment in the fifth step, adjusting the pH value to 6, and then performing ultrafiltration treatment: the cross-flow type filtering device is characterized in that a filtering membrane is a polysulfone membrane, a filtering plate selects 100KDa molecular cut-off, the material-liquid ratio is 1:2, the transmembrane pressure selects 0.15MPa, the flow rate is 500L/h, and filtered solution is collected, namely the milk fat globule membrane protein with high purity is obtained.

Example 3

thirdly, the supernatant liquor obtained in the second step is taken for calcium heating treatment, the temperature is heated to 60 ℃, hydrochloric acid solution is used for adjusting the pH value to 7, and CaCl is added₂Adding 0.16g/100g of emulsion into the emulsion, mixing, and standing for 5 min;

fifthly, taking supernatant collected in the fourth step for ultrasonic treatment, wherein the ultrasonic frequency is 80MHz, the ultrasonic time is 1h, the ultrasonic temperature is 45 ℃, and cooling the ultrasonic treated sample to room temperature;

sixthly, taking the sample subjected to ultrasonic treatment in the step five, adjusting the pH value to 6, and then performing ultrafiltration treatment: the frame type filtering device is characterized in that a filtering membrane is a polysulfone membrane, a filtering plate selects 100KDa molecular interception amount, the material-liquid ratio is 1:2, the transmembrane pressure selects 0.15MPa, the flow rate is 500L/h, and filtered solution is collected, namely the milk fat globule membrane protein with high purity is obtained.

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 LC-MS (liquid chromatography-Mass Spectrometry) technique. As a result, it was found that the milk fat globule membrane protein produced in example 1 had a darker color in the gel-electrophoresed band than in examples 2 and 3. And as can be seen from LC/MS, the relative abundance values of several major proteins BTN in the milk fat globule membrane prepared in example 1 are 6.3E +010, the relative abundance value of ADPH is 4.2E +010, and the relative abundance value of PAS6/7 is 6.8E + 010; in contrast, in the milk fat globule membrane proteins prepared in example 2 and example 3, the relative abundance values of BTN were 4.1E +010, 3.9E +010, respectively; ADPH is respectively 3.7E +010 and 3.2E + 010; PAS6/7 is 3.8E +010, 4.3+010 respectively. It can be seen by high performance liquid chromatography that the peak area of the hetero peak obtained in example 1 is only 60% of that of example 2 and 63% of that of example 3, and α can be seen₃₂The low-CN and kappa-CN casein components have essentially disappeared. The purity of the milk fat globule membrane prepared in example 1 was calculated to be 94.7%, whereas the purity of example 2 was 85.6% and that of example 3 was only 82.3%.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A method for separating and preparing milk fat globule membrane protein from buttermilk, a by-product of butter, comprising the steps of:

(3) subjecting the first supernatant to calcium heat treatment;

2. The method of claim 1, wherein:

in the step (2), the centrifugal speed is 3000-4000 rpm, the centrifugal time is 20-30 min, and the standing time is 20-30 min.

3. The method according to claim 1 or 2, characterized in that:

in the step (2), 2mol/L hydrochloric acid solution is used for adjusting the pH value; preferably, the pH is adjusted to 4.6.

4. The method according to any one of claims 1 to 3, characterized in that:

in the step (3), during the hot calcium treatment, calcium chloride is added into the first supernatant, then the pH is adjusted to 7-7.7, the mixture is heated to 40-60 ℃, and the mixture is fully mixed and kept stand for 20-40 min.

5. The method of claim 4, wherein:

the addition amount of the calcium chloride is 0.02-0.16 g/100g of the emulsion; it is also preferred that the pH is adjusted with sodium hydroxide.

6. The method according to any one of claims 1 to 5, characterized in that:

in the step (4), the centrifugal speed is 3000-4000 rpm, and the centrifugal time is 20-30 min.

7. The method according to any one of claims 1 to 6, characterized in that:

in the 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 ℃.

8. The method according to any one of claims 1 to 7, characterized in that:

in the step (6), the dialysis is performed by reverse osmosis treatment by using a dialysis bag made of cellulose, and the reverse osmosis treatment is performed under the condition that the pH value is 6;

preferably, the reverse osmosis treatment is carried out in a thermostatic water bath at 25 ℃; preferably, the reverse osmosis treatment is performed while continuing stirring with a magnetic stirrer for a dialysis time of 18 to 30 hours.

9. The method of claim 8, wherein:

the molecular cut-off of the dialysis bag was 14 kDa.

10. The method according to any one of claims 1 to 9, characterized in that:

in the step (7), the ultrafiltration treatment is carried out by adopting a cross-flow type filtration device, a filtration membrane is a polysulfone membrane, and the molecular interception amount of a filtration plate is 30-100 KDa, preferably 100 KDa; the ratio of the material to the liquid is 1: 2-1: 8; the transmembrane pressure is 0.14 to 0.16 MPa; the flow rate is 450 to 500L/h.