Protein separation method and related application thereof

Abstract

The invention discloses a protein separation method and related application thereof, and relates to the technical field of dairy product processing, the method comprises the following steps: heating a whey solution of which the pH value is adjusted to 3.5-4.2 by a citric acid solution, carrying out microfiltration on the heated product, carrying out beta-lactoglobulin and / or alpha-lactalbumin separation on permeate and trapped fluid obtained by microfiltration, the method is based on the membrane filtration technology, high-purity separation of beta-lactoglobulin and alpha-lactalbumin is achieved, the purity can reach 70%-90% or above, the method is compatible with whey from multiple sources, equipment is simple, the method has the advantages of being low in separation cost, high in efficiency, easy to implement, large in flux and the like, and a way is provided for large-scale industrial production of beta-lactoglobulin and alpha-lactalbumin.

Description

Technical Field

[0001] The present invention relates to the technical field of dairy product processing, and in particular, to a method for separating proteins and related applications thereof. Background Art

[0002] Whey protein accounts for about 20% of the total milk protein. Whey is an important by-product during the deep processing of dairy products and the production of cheese, and its output is very abundant. Two protein components, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), account for more than 70% of the protein concentration in whey and exist in a mixed form with a ratio of about 1:4. Both of these proteins are important sources of essential amino acids for the human body. Due to their excellent nutritional and absorption characteristics, they are widely used in sports nutrition and functional foods in their natural mixed or separated forms. In recent years, the research on the separation technology of these two components has received extensive attention because they have different nutritional and physiological characteristics, and these characteristics are caused by the differences in their secondary and tertiary structures.

[0003] α-La contains a relatively high content of tryptophan, which can have a positive impact on enhancing human stress resistance and improving attention by promoting serotonin synthesis. In the infant food industry, separating and purifying the α-La component can reduce the risk of allergy and make its protein composition closer to the characteristics of breast milk - because β-Lg is the main allergen in infant formula milk (this component is almost absent in breast milk). It should be noted that the complex formed by partially denatured α-La and oleic acid has been proven to have the biological activity of inducing apoptosis in human and mouse tumor cells. In addition, the hydrophobic / hydrophilic group distribution characteristics of β-Lg are highly homologous to the lipocalin family, and this structural feature enables it to have the ability to bind and transport small hydrophobic substances, which has important application value in the food industry (such as flavor substance binding).

[0004] The differences in molecular properties between α-La and β-Lg result in their completely different functional characteristics. Since there is no free sulfhydryl group, the separated α-La exhibits high thermal stability even under heating conditions of ultra-high temperature treatment at neutral pH, which makes this protein suitable for application scenarios where gelation is not desired after heat treatment. In contrast, there is a free sulfhydryl group in the structure of β-Lg, which can play its role in applications that require cross-linking and gelation during heat treatment. The amphiphilic nature of β-Lg gives it excellent emulsifying and foaming abilities.

[0005] To better utilize different whey proteins, it is necessary to separate and purify different whey proteins. In the past two decades, technicians have developed many laboratory-scale separation procedures for separating and purifying α-La and β-Lg from whey protein concentrates or isolates. These two protein components can be separated using high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel permeation chromatography, and other chromatographic methods. Although high purity of the separated components can be achieved using the above methods, there are problems such as high equipment costs, low separation efficiency, and great process difficulty, and their applicability in large-scale separation of whey proteins is very limited. In addition, there are also separation methods based on the selective aggregation of β-Lg in the presence of high pH and calcium ions. However, the yield of α-La by this method is very low. In order to make this method applicable to large-scale production of α-La, high-fold diafiltration is required during the membrane filtration process.

[0006] Currently, the purity of separating β-Lg and α-La solely relying on membrane filtration technology is relatively low, usually only reaching 50% - 60%.

[0007] Therefore, there is an urgent need in the market to establish a separation method that can obtain β-Lg and α-La with high purity in large-scale production.

[0008] In view of this, the present invention is specifically proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for separating proteins and its related applications.

[0010] The present invention is implemented as follows: In the first aspect, an embodiment of the present invention provides a method for separating proteins, which includes the following steps: Heat a whey solution whose pH has been adjusted to 3.5 - 4.2 with a citric acid solution, and then microfilter the heated product using a first filter membrane to obtain a first permeate and a first retentate; the whey solution contains β-lactoglobulin and / or α-lactalbumin; Perform ultrafiltration to separate β-lactoglobulin from the first permeate and / or separate α-lactalbumin from the first retentate; Among them, the separation of β-lactoglobulin includes: performing membrane filtration on the first permeate using a second filter membrane to obtain a second permeate and a second retentate, and adjusting the pH of the second retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution; the separation of α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, and performing membrane filtration using a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution; the cut-off molecular weights of the second filter membrane, the third filter membrane, and the fourth filter membrane are 3 kDa - 10 kDa, 20 kDa - 50 kDa, and 3 kDa - 10 kDa in sequence.

[0011] In a second aspect, the embodiments of the present invention provide an application of the protein separation method as described in the foregoing embodiments in the preparation of a product containing α-lactalbumin and / or β-lactoglobulin.

[0012] The present invention has the following beneficial effects: The present invention establishes a method for separating β-lactoglobulin and / or α-lactalbumin from whey. This method involves heating a whey solution whose pH has been adjusted to 3.5 - 4.2 with a citric acid solution, and further separating and purifying the heated product by combining microfiltration, ultrafiltration techniques, and pH adjustment. Each step synergizes with each other, overcoming the technical limitation of the existing membrane filtration technology that cannot achieve high-purity separation, realizing the high-purity separation of β-lactoglobulin and / or α-lactalbumin, with a purity that can reach over 70% - 90%. Moreover, it is compatible with whey from multiple sources, has simple equipment, and has advantages such as low separation cost, high efficiency, easy implementation, and large throughput, providing a way for the large-scale industrial production of β-lactoglobulin and α-lactalbumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0014] Figure 1 It is a schematic flow chart of the method for separating β-lactoglobulin and α-lactalbumin from whey. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0016] At present, the purity of separating β-Lg and α-La solely relying on membrane filtration technology is relatively low, usually only reaching 50% - 60%. This may be due to the following reasons: (1) The molecular weights of β-Lg and α-La are relatively close. The molecular weight of β-Lg is about 18 kDa, and that of α-La is about 14 kDa. It is difficult to effectively separate the two solely relying on membrane filtration technology; (2) At a pH close to the isoelectric point (pI≈5.2) or at high concentrations, β-Lg is prone to form dimers / octamers. After β-Lg forms dimers, its molecular weight is about 36 kDa, and a 25 - 30 kDa ultrafiltration membrane can be used to separate it from α-La (14 kDa). However, under the existing technology, 36 kDa and 14 kDa are still too close. Limited by the membrane production process, the membrane pore size is not perfectly within the range of 25 - 30 kDa. Generally, to achieve complete retention, the membrane pore size should be one-third of the retention substance. Therefore, to completely retain β-Lg dimers, the membrane pore size should be 10 - 15 kDa, but this pore size will also partially retain α-La; at the same time, under this condition, if the crystallization process is not used, not all β-Lg will form dimers, so the separation effect is poor. When β-Lg forms dimers, some α-La will also be co-retained; (3) β-Lg and α-La in whey may be temporarily bound through hydrophobic interactions or disulfide bonds to form complexes and be co-retained by the membrane; (4) Their charges are similar, and electrostatic repulsion cannot be used to enhance separation. Some membrane materials (such as PVDF) are negatively charged and repel negatively charged proteins (such as β-Lg) at pH > pI, but the charges of α-La are similar, so the separation effect is limited; (5) The complexity of the whey matrix: Whey contains lactose, minerals (Ca²⁺), and lipids, etc., which may bind to proteins (such as Ca²⁺ binding to α-La), change their retention behavior, increase the solution viscosity, and reduce the membrane flux and selectivity. It can be seen that the current membrane filtration technology has limitations and cannot achieve high-purity separation of β-Lg and α-La.

[0017] Based on the research on whey, β-lactoglobulin, and α-lactalbumin, combined with a large number of experimental verifications, this application innovatively establishes a new separation method. This method heats the whey solution with a pH adjusted to 3.5 - 4.2 by a citrate buffer solution and performs microfiltration on the heating product to obtain a first permeate and a first retentate; based on ultrafiltration technology and pH adjustment, the first permeate and the first retentate are separated respectively, and each step cooperates with each other to achieve high-purity separation of β-lactoglobulin and / or α-lactalbumin. This method is based on membrane filtration technology, is compatible with whey solutions from various sources (containing β-lactoglobulin and / or α-lactalbumin), and is suitable for large-scale industrial production.

[0018] Nomenclature The terms "first", "second", "third", "fourth", etc. in this text are only used for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0019] The term "microfiltration" in this text is a membrane separation technology with physical sieving as the core principle. The pore size range is usually 0.1 - 1 micrometer (μm), and separation is achieved through the uniform microporous structure on the membrane surface.

[0020] The term "ultrafiltration" in this text is a membrane separation technology based on molecular weight cut-off. The molecular weight cut-off (MWCO) range is usually 1,000 - 1,000,000 Da, and it works through the combined action of sieving effect and charge repulsion.

[0021] The term "cross-flow filtration" (also known as cross-flow filtration) in this text is a solid-liquid separation technology that pumps the feed liquid to flow parallel to the membrane surface. Its core principle is that the shear force generated during the flow of the feed liquid continuously flushes the membrane surface, causing the retained particles to be carried away, thus maintaining a relatively thin fouling layer and significantly reducing the membrane fouling problem.

[0022] The term "conventional cross-flow filtration" in this text is the basic form of cross-flow filtration. It relies on an external pumping system to make the feed liquid flow tangentially along the membrane surface, suppressing fouling through hydrodynamic shear force and having no additional mechanical rotating components.

[0023] The term "rotating dynamic cross-flow filtration" in this text is an improved technology of conventional cross-flow filtration. It actively generates high shear force or turbulence on the membrane surface through rotating components (such as rotors, cyclones, or the rotating membrane itself), significantly enhancing the anti-fouling ability.

[0024] It should be noted that, without conflict, the features in the embodiments or implementation manners of the present invention can be combined with each other.

[0025] Technical solution An embodiment of the present invention provides a method for separating proteins, which includes the following steps: Heat the whey solution whose pH is adjusted to 3.5 - 4.2 with a citric acid solution, and then perform microfiltration on the heated product using a first filter membrane to obtain a first permeate and a first retentate; the whey solution contains β-lactoglobulin and α-lactalbumin; Perform separation of β-lactoglobulin on the first permeate and / or separation of α-lactalbumin on the first retentate using ultrafiltration; Among them, the separation of the β-lactoglobulin includes: subjecting the first permeate to membrane filtration using a second filter membrane to obtain a second permeate and a second retentate, and adjusting the pH of the second retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution; the separation of the α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, and subjecting it to membrane filtration using a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution; the molecular weight cut-offs of the second filter membrane, the third filter membrane, and the fourth filter membrane are 3 kDa - 10 kDa, 20 kDa - 50 kDa, and 3 kDa - 10 kDa in sequence.

[0026] Some embodiments also provide a method for separating proteins, specifically including: S1. Prepare a whey solution with a pH adjusted to 3.5 - 4.2 by a citric acid solution.

[0027] Specifically, in some embodiments, the preparation of the whey solution includes the following steps: adjusting the pH of the whey protein solution to 4.5 - 5.2 and then performing solid-liquid separation, and then adjusting the pH of the separated clear liquid to 3.5 - 4.2 again.

[0028] Using a citric acid solution as a pH regulator to adjust the pH of the whey solution within a limited range can obtain a better separation effect of β-lactoglobulin and α-lactalbumin.

[0029] In some embodiments, the pH regulator used for preparing the whey solution is a 0.1 mol / L - 5 mol / L citric acid buffer solution.

[0030] In some embodiments, the concentration of the citric acid buffer solution is 0.1 - 5 mol / L, specifically it can be any one or the range between any two of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mol / L.

[0031] In some embodiments, adjusting the pH to 4.5 - 5.2, the pH can specifically be adjusted to any one or the range between any two of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, and 5.2.

[0032] In some embodiments, the pH of the whey solution is adjusted to any one or the range between any two of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, and 4.2.

[0033] In some embodiments, the solid-liquid separation method includes centrifugation, and the rotational speed of centrifugation is 1000 - 14000 rpm, specifically it can be any one or the range between any two of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000 and 14000 rpm.

[0034] In some embodiments, the whey protein solution includes at least one of whey permeate separated from fresh milk by membrane separation, acid whey discharged from cheese production, and reconstituted solution of whey protein powder, and the protein content of the whey protein solution is ≤ 15% (w / w).

[0035] In some embodiments, the acid whey discharged from cheese production refers to the by-product separated after rennet or lactic acid fermentation during the cheese production process.

[0036] In some embodiments, the whey permeate separated from fresh milk by membrane separation refers to the liquid component that passes through the membrane pores and enters the filtrate side when fresh milk passes through membrane separation technology (such as ultrafiltration, microfiltration).

[0037] In some embodiments, the fresh milk includes at least one of cow milk, goat milk, camel milk, horse milk, donkey milk, and yak milk.

[0038] In some embodiments, the reconstituted solution of whey protein powder is obtained by dissolving whey protein powder in pure water.

[0039] In some embodiments, the protein content of the whey protein solution is 5% - 10%, specifically it can be any one or the range between any two of 5%, 6%, 7%, 8%, 9% and 10%.

[0040] S2. Heat the whey solution with a pH adjusted to 3.5 - 4.2 by citric acid solution, and then microfilter the heated product with a first filter membrane to obtain a first permeate and a first retentate.

[0041] Specifically, in some embodiments, the temperature for heating the whey solution is 45 - 55 °C, specifically it can be any one or the range between any two of 45, 46, 48, 50, 52, 54 or 55 °C.

[0042] In some embodiments, the time for heating the whey solution is 1 - 4 h, specifically it can be any one or the range between any two of 1, 1.5, 2, 2.5, 3, 3.5 and 4 h.

[0043] In some embodiments, the pore size of the first filter membrane is any one or in the range between any two of 100, 150, 200, 250, 300, 350, 400, 450, and 500 nm.

[0044] In some embodiments, the structural form of the first filter membrane is selected from: flat sheet membrane, hollow fiber membrane, hollow tube membrane, and spiral wound membrane.

[0045] In some embodiments, the membrane material of the first filter membrane is selected from: ceramic membrane, composite membrane, and polymer membrane. Among them, ceramic membranes (such as alumina, zirconia, titanium dioxide) belong to inorganic membranes; composite membranes include organic-inorganic composite membranes and pure inorganic composite membranes (such as ceramic-metal composite membranes); polymer membranes belong to organic membranes, for example, polyethersulfone (PES), polysulfone (PS), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), regenerated cellulose (RC), and polyamide (PA).

[0046] In some embodiments, the first filter membrane is selected from: microfiltration membrane (such as ceramic), hollow fiber membrane, or hollow tube membrane.

[0047] In some embodiments, the operating mode of the microfiltration is cross-flow filtration, and the cross-flow filtration is selected from traditional cross-flow filtration or rotary dynamic cross-flow filtration.

[0048] In some embodiments, the washing filtrate for microfiltration using the first filter membrane includes: the second permeate and / or 0.1 - 5 mol / L citric acid-sodium citrate buffer solution with a pH of 3.5 - 4.2; and / or, the washing filtration multiple is 1 - 5 times the volume of the raw material liquid, specifically, it can be any one or in the range between any two of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 times.

[0049] S3. The separation of the β-lactoglobulin includes: performing membrane filtration on the first permeate using a second filter membrane to obtain a second permeate and a second retentate, and adjusting the pH of the second retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution.

[0050] Specifically, in some embodiments, the molecular weight cut-off of the second filter membrane is any one or in the range between any two of 3, 4, 5, 6, 7, 8, 9, and 10 kDa.

[0051] In some embodiments, the structural form and membrane raw material of the second filter membrane are the same as those of the first filter membrane described in the foregoing embodiments, and will not be elaborated herein.

[0052] In some embodiments, the second filter membrane is a spiral wound membrane (polymer).

[0053] In some embodiments, the pH is adjusted to 6.9 - 8.0, specifically, it can be any value within the range between any one or any two of 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[0054] In some embodiments, the washing filtrate for membrane filtration using the second filter membrane includes reverse osmosis water (RO water) and / or a 0.1 - 5 mol / L citric acid - sodium citrate buffer solution with a pH of 3.5 - 4.2, and / or the washing filtration multiple is 1 - 3 times the volume of the raw material liquid, specifically, it can be any value within the range between any one or any two of 1, 1.5, 2, 2.5, and 3 times.

[0055] In some embodiments, the pH of the citric acid - sodium citrate buffer solution can be any value within the range between any one or any two of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, and 4.2, and the concentration can be any value within the range between any one or any two of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 mol / L.

[0056] S4. The separation of the α - lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, and performing membrane filtration using a third filter membrane and / or a fourth filter membrane.

[0057] Specifically, in some embodiments, the molecular weight cut - off of the third filter membrane is any value within the range between any one or any two of 20, 25, 30, 35, 40, 45, and 50 kDa.

[0058] In some embodiments, the molecular weight cut - off of the fourth filter membrane is any value within the range between any one or any two of 3, 4, 5, 6, 7, 8, 9, and 10 kDa.

[0059] In some embodiments, the structural form and membrane raw material of the third filter membrane and / or the fourth filter membrane are the same as those of the first filter membrane described in the foregoing embodiments, and will not be elaborated herein.

[0060] In some embodiments, the third filter membrane and / or the fourth filter membrane is a spiral wound membrane (polymer).

[0061] In some embodiments, the separation of the α - lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, performing membrane filtration using the third filter membrane to obtain a third permeate and a third retentate; the third permeate is an α - lactalbumin solution.

[0062] In some embodiments, the separation of the α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, and subjecting the third permeate to membrane filtration using the fourth filter membrane to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution.

[0063] In some embodiments, the separation of the α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, subjecting it to membrane filtration using a third filter membrane to obtain a third permeate and a third retentate; subjecting the third permeate to membrane filtration using the fourth filter membrane to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution. Separating and purifying simultaneously using the third filter membrane and the fourth filter membrane results in a higher purity.

[0064] In some embodiments, the separation method further includes: adjusting the pH of the third retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution. The third retentate contains β-lactoglobulin, which can be mixed with the second retentate and then the pH can be adjusted uniformly to obtain a β-lactoglobulin solution, or the pH of the third retentate can be adjusted to obtain a β-lactoglobulin solution and then mixed with the β-lactoglobulin solution obtained based on the second retentate to obtain the final β-lactoglobulin solution.

[0065] In some embodiments, adjusting the pH to 6.9 - 8.0 can specifically be any one or the range between any two of 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.

[0066] In some embodiments, the pH regulator used to adjust the pH to 6.9 - 8.0 includes a 1 - 5 mol / L sodium hydroxide solution.

[0067] In some embodiments, the concentration of the sodium hydroxide solution can be any one or the range between any two of 1, 2, 3, 4, and 5 mol / L.

[0068] In some embodiments, the washing filtrate for membrane filtration using the third filter membrane and / or the fourth filter membrane includes reverse osmosis water (RO water), and / or the washing filtration multiple is 1 - 3 times the volume of the raw material liquid, and specifically can be any one or the range between any two of 1, 1.5, 2, 2.5, and 3 times.

[0069] S5. Spray-drying the β-lactoglobulin solution and / or the α-lactalbumin solution to obtain β-lactoglobulin powder and / or α-lactalbumin powder.

[0070] Specifically, in some embodiments, the spray-drying is electrostatic spray-drying.

[0071] In some embodiments, the purity of α-lactalbumin in the α-lactalbumin solution and / or α-lactalbumin powder can reach 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or above 98%.

[0072] In some embodiments, the purity of β-lactoglobulin in the β-lactoglobulin solution and / or β-lactoglobulin powder can reach 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or above 98%.

[0073] In addition, the embodiments of the present invention also provide the application of the protein separation method as described in any of the foregoing embodiments in the preparation of products containing α-lactalbumin and / or β-lactoglobulin.

[0074] In some embodiments, the products containing α-lactalbumin and / or β-lactoglobulin include any one or more of: milk powder, fresh milk, yogurt, cheese, whey protein powder, nutritional supplements and purified protein products.

[0075] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0076] Materials in the examples: The preparation method of 1 mol / L citric acid buffer solution is: dissolve 210.14 g of citric acid monohydrate in 1 L of distilled water.

[0077] The preparation method of 1 mol / L citric acid-sodium citrate buffer solution with pH 4.2 is: weigh 210.14 g of citric acid, dissolve it in an appropriate amount of distilled water, transfer it to a 1 L volumetric flask, and make up the volume to obtain 1 mol / L citric acid solution; weigh 63.53 g of trisodium citrate dihydrate, dissolve it in an appropriate amount of distilled water, transfer it to a 1 L volumetric flask, and make up the volume to obtain 0.216 mol / L trisodium citrate dihydrate solution; take 784 mL of the above 1 mol / L citric acid solution and 216 mL of the above 0.216 mol / L trisodium citrate dihydrate solution, mix them evenly, and obtain 1 mol / L citric acid-sodium citrate buffer solution with pH 4.2.

[0078] The preparation method of 4 mol / L sodium hydroxide solution is: dissolve 160 g of sodium hydroxide in 1 L of water.

[0079] Example 1 This example provides a method for α-lactalbumin and β-lactoglobulin, including the following steps.

[0080] A. Preparation of working whey: The whey permeate obtained by membrane separation of fresh cow milk is ultrafiltered and concentrated to a protein content of 9% (w / w). B. pH adjustment and centrifugation: After adjusting the pH of the working whey to 4.6 using 1 mol / L citric acid buffer (pH regulator), centrifugal separation is carried out using a tubular centrifuge at 10,000 rpm, and the supernatant is taken. C. pH adjustment and membrane filtration: After adjusting the pH of the supernatant to 4.2 (acidic pH) using 1 mol / L citric acid buffer (pH regulator), it is left standing at 50 °C (heating temperature) for 2 hours, and then traditional cross-flow filtration is carried out using a 100 nm ceramic microfiltration membrane (the first filter membrane). After microfiltering the raw liquid to the minimum volume, 1 mol / L citric acid-sodium citrate buffer (washing filtrate) with a pH of 4.2 is used to wash and filter the raw liquid, and the washing and filtering multiple is 2 times the volume of the raw liquid, obtaining the first permeate and the first retentate. D. Separation of β-lactoglobulin: The first permeate is ultrafiltered using a 10 kDa ultrafiltration membrane (the second filter membrane, polyethersulfone (PES) spiral wound membrane). After ultrafiltering the raw liquid to the minimum volume, RO water is used, and the washing and filtering multiple is 1.5 times the volume of the raw liquid, obtaining the second permeate and the second retentate. The pH of the second retentate is adjusted to 7.0 using 4 mol / L sodium hydroxide solution to obtain β-lactoglobulin solution. E. Separation of α-lactalbumin: After adjusting the pH of the first retentate to 7.0 using 4 mol / L sodium hydroxide solution, it is ultrafiltered using a 30 kDa ultrafiltration membrane (the third filter membrane, polyethersulfone spiral wound membrane). After ultrafiltering the raw liquid to the minimum volume, RO water (washing filtrate) is used to wash and filter the raw liquid, and the washing and filtering multiple is 2 times the volume of the raw liquid, obtaining the third permeate and the third retentate. The third permeate is ultrafiltered using a 10 kDa ultrafiltration membrane (the fourth filter membrane, polyethersulfone spiral wound membrane). After ultrafiltering the raw liquid to the minimum volume, RO water (washing filtrate) is used to wash and filter the raw liquid, and the washing and filtering multiple is 1.5 times the volume of the raw liquid, obtaining the fourth permeate and the fourth retentate. Among them, the fourth retentate is α-lactalbumin solution. F. Spray drying: The α-lactalbumin solution and β-lactoglobulin solution are prepared into α-lactalbumin powder and β-lactoglobulin powder using electrostatic spray drying method (60 °C).

[0081] The process schematic diagram can be referred to Figure 1 。

[0082] Example 2 This example provides a method for α-lactalbumin and β-lactoglobulin, which is generally the same as Example 1, except that: Step B is omitted, that is, the working whey prepared in Step A is directly subjected to Step C.

[0083] Example 3 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that: the step of filtering with the third filter membrane in step E is omitted, that is, the first retentate with the pH adjusted to 7.0 is directly ultrafiltered with the fourth filter membrane.

[0084] Example 4 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that this example further includes: after mixing the third retentate in step E and the second retentate in step D, the pH is adjusted to 7.0 with 4 mol / L sodium hydroxide solution to obtain a β-lactoglobulin solution.

[0085] Example 5 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that: the pore size of the first filter membrane is 500 nm.

[0086] Example 6 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that the cut-off molecular weight of the third filter membrane in step E is 50 kDa.

[0087] Example 7 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that the cut-off molecular weight of the fourth filter membrane in step E is 5 kDa.

[0088] Comparative Example 1 This example provides a method for α-lactalbumin and β-lactoglobulin, which is substantially the same as Example 1, except that: Step C: Microfiltration is not performed, the first filter membrane uses a 30 kDa ultrafiltration membrane, and the washing filtrate is RO water; Step D: The first retentate is washed and filtered with the second filter membrane, and the pH of the second retentate is not adjusted; Step E: The step of membrane filtration with the third filter membrane is omitted, and the first permeate is membrane-filtered with the fourth filter membrane.

[0089] Steps C, D, and E of Comparative Example 1 are specifically as follows.

[0090] Step C: After adjusting the pH of the supernatant obtained in Step B to 4.2 (acidic pH) using 1 mol / L citric acid buffer (pH regulator), ultrafiltration is carried out using a 30 kDa ultrafiltration membrane (the first membrane, polyethersulfone spiral wound membrane) at around 35 °C (30 - 40 °C). First, the feed liquid is ultrafiltered to the minimum volume, and then RO water (washing filtrate) is used to wash and filter the feed liquid. The washing and filtering multiple is 1.5 times the volume of the feed liquid, obtaining the first permeate and the first retentate; Step D: Ultrafilter the first retentate using a 10 kDa ultrafiltration membrane (the second membrane, polyethersulfone spiral wound membrane). First, the feed liquid is ultrafiltered to the minimum volume, and then RO water (washing filtrate) is used to wash and filter the feed liquid. The washing and filtering multiple is 1.5 times the volume of the feed liquid, obtaining the second permeate and the second retentate, where the second retentate is β-lactoglobulin solution; Step E: Ultrafilter the first permeate using a 10 kDa ultrafiltration membrane (the fourth membrane, polyethersulfone spiral wound membrane). First, the feed liquid is ultrafiltered to the minimum volume, and then RO water (washing filtrate) is used to wash and filter the feed liquid. The washing and filtering multiple is 1.5 times the volume of the feed liquid, obtaining the fourth permeate and the fourth retentate, where the fourth retentate is α-lactalbumin solution.

[0091] Comparative Example 2 This example provides a method for α-lactalbumin and β-lactoglobulin, which is generally the same as Example 1, except that the pore size of the first membrane is 1000 nm.

[0092] After the start of microfiltration, α-lactalbumin particles will quickly clog the microfiltration membrane, and the process cannot proceed.

[0093] Comparative Example 3 This example provides a method for α-lactalbumin and β-lactoglobulin, which is generally the same as Example 1, except that the pH regulator in Step C is replaced with 1 mol / L hydrochloric acid.

[0094] Comparative Example 4 This example provides a method for α-lactalbumin and β-lactoglobulin, which is generally the same as Example 1, except that the heating temperature in Step C is set to 65 °C. Protein denaturation and flocculation occur after heating.

[0095] Verification Example 1 Obtain the working whey, β-lactoglobulin solution, α-lactalbumin solution, α-lactalbumin powder and β-lactoglobulin powder of Examples 1 to 7 and Comparative Examples 1 and 3, and detect at least one of the total solids, moisture, ash, protein, α-lactalbumin content, β-lactoglobulin content, total casein, immunoglobulin IgG, bovine serum albumin, and caseinoglycomacropeptide therein. Among them, the total solids and moisture are detected according to the first method of GB 5009.3-2016, the ash is detected according to the first method of GB 5009.4-2016, the protein is detected according to the first method of GB 5009.5-2016, and the contents of α-lactalbumin and β-lactoglobulin are detected by ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS / MS) method. The detection conditions are as follows: Chromatographic column: Acquity UPLC BEH 300 C18 column (particle size 1.7μm, 2.1×100mm) Chromatographic conditions: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile aqueous solution containing 0.1% formic acid. The injection volume is 5 μL, and the column temperature is 40°C. The LC elution gradient program is: starting with 5% mobile phase B for 1.2 min, then linearly increasing to 60% mobile phase B within 1.8 min, then increasing to 80% mobile phase B within 1 min, increasing to 100% mobile phase B within 0.2 min, equilibrating at 100% mobile phase B for 1.4 min, and then returning to 5% mobile phase B within 2.2 min. The total running time for each injection is 8 min.

[0096] Ion source: Electrospray ionization source (ESI); Mass spectrometry conditions: The capillary voltage is 3.50 kV, the cone voltage is 30 V, the source temperature is 150°C, the desolvation gas temperature is 350°C, the cone gas flow rate is 50 L / h nitrogen, the desolvation gas flow rate is 900 L / h nitrogen, and the argon collision gas pressure during MS / MS analysis is 3×10 -3 mbar; Calculate the purity of α-La and β-Lg according to the following formula: ; ; Among them, x represents the purity of α-La, and y represents the purity of β-Lg.

[0097] Table 1 Partial detection results of Example 1

[0098] Table 2 Partial detection results of Example 1

[0099] Table 3 Detection Results of Example 2

[0100] Table 4 Detection Results of Example 3

[0101] Table 5 Detection Results of Example 4

[0102] Table 6 Detection Results of Example 5

[0103] Table 7 Detection Results of Example 6

[0104] Table 8 Detection Results of Example 7

[0105] Table 9 Detection Results of Comparative Example 1

[0106] Table 10 Detection Results of Comparative Example 3

[0107] From the above results, it can be seen that: Omitting step B (Example 2), the purity of α-lactalbumin and β-lactoglobulin decreases compared with Example 1, but the purity of α-lactalbumin is still relatively high; Omitting the step of ultrafiltration using the third filter membrane in step E (Example 3), the purity of α-lactalbumin decreases compared with Example 1, but the purity of β-lactoglobulin is still relatively high; After recycling the third retentate for the preparation of β-lactoglobulin solution (Example 4), the purity of β-lactoglobulin decreases compared with Example 1, but the utilization rate of raw materials can be improved, the generation of waste in the production process can be reduced, and resources and production costs can be saved; Replacing the pore size of the first filter membrane with 500 nm (Example 5) will cause part of the α-lactalbumin to pass through the microfiltration membrane, reducing the purity of β-lactoglobulin. At the same time, the α-lactalbumin particles will block the microfiltration membrane pores in the later stage of microfiltration, reducing the separation efficiency; After replacing the microfiltration membrane in step C with a 30 kDa ultrafiltration membrane (Comparative Example 1), the purity of α-lactalbumin and β-lactoglobulin decreases significantly compared with Example 1.

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for separating a protein, characterized in that, It includes the following steps: Heat the whey solution with a pH adjusted to 3.5 - 4.2 by a citric acid solution, and then microfilter the heated product using a first filter membrane to obtain a first permeate and a first retentate; the whey solution contains β-lactoglobulin and α-lactalbumin; Use ultrafiltration to separate β-lactoglobulin from the first permeate and / or separate α-lactalbumin from the first retentate; Among them, the separation of β-lactoglobulin includes: subjecting the first permeate to membrane filtration using a second filter membrane to obtain a second permeate and a second retentate, and adjusting the pH of the second retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution; the separation of α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0 and subjecting it to membrane filtration using a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution; the cut-off molecular weights of the second filter membrane, the third filter membrane, and the fourth filter membrane are 3 kDa - 10 kDa, 20 kDa - 50 kDa, and 3 kDa - 10 kDa in sequence.

2. The separation method according to claim 1, wherein The temperature of the heating is 45 - 55 °C; and / or, the heating time is 1 - 4 h.

3. The separation method according to claim 1, wherein The pore size of the first filter membrane is 100 nm - 500 nm; and / or, the operation mode of the microfiltration adopts cross-flow filtration, and the cross-flow filtration is selected from traditional cross-flow filtration or rotary dynamic cross-flow filtration.

4. The separation method according to claim 1, characterized in that, The separation of α-lactalbumin includes: adjusting the pH of the first retentate to 6.9 - 8.0, and then subjecting it to membrane filtration using a third filter membrane to obtain a third permeate and a third retentate; subjecting the third permeate to membrane filtration using the fourth filter membrane to obtain a fourth permeate and a fourth retentate, and the fourth retentate is the α-lactalbumin solution; and / or, the separation method further includes: adjusting the pH of the third retentate to 6.9 - 8.0 to obtain a β-lactoglobulin solution.

5. The separation method according to any one of claims 1 to 4, characterized in that, The preparation of the whey solution includes the following steps: adjusting the pH of the whey protein solution to 4.5 - 5.2 and then performing solid-liquid separation, and then adjusting the pH of the separated clear liquid to 3.5 - 4.2 again.

6. The separation method according to claim 5, characterized in that, The pH regulator used for preparing the whey solution is 0.1 mol / L - 5 mol / L citric acid buffer solution; and / or, the whey protein solution includes at least one of whey permeate separated from fresh milk by membrane, acid whey discharged from cheese production, whey protein powder reconstitution solution, and acidified whey supernatant, and the protein content of the whey protein solution ≤ 15%.

7. The separation method according to claim 1, characterized in that, The structural forms of the first filter membrane, the second filter membrane, the third filter membrane, and the fourth filter membrane are independently selected from: flat membrane, hollow fiber membrane, hollow tube membrane, and spiral wound membrane; and / or, the membrane materials of the first filter membrane, the second filter membrane, the third filter membrane, and the fourth filter membrane are independently selected from: ceramic membrane, composite membrane, and polymer membrane.

8. The separation method according to claim 1, characterized in that The washing filtrate for microfiltration using the first filter membrane includes: the second permeate and / or 0.1 mol / L - 5 mol / L citric acid-sodium citrate buffer solution with a pH of 3.5 - 4.2; and / or, the washing filtration multiple is 1 - 5 times the volume of the raw material liquid; And / or, the washing filtrate obtained by membrane filtration using the second filter membrane includes reverse osmosis water and / or 0.1 - 5 mol / L citric acid - sodium citrate buffer solution with a pH of 3.5 - 4.2, and / or, the washing and filtration multiple is 1 - 3 times the volume of the raw material liquid; And / or, the washing filtrate obtained by membrane filtration using the third filter membrane and / or the fourth filter membrane includes reverse osmosis water, and / or, the washing and filtration multiple is 1 - 3 times the volume of the raw material liquid.

9. The separation method according to any one of claims 1 to 4, characterized in that, The separation method further includes spray - drying the β - lactoglobulin solution and / or the α - lactalbumin solution to obtain β - lactoglobulin powder and / or α - lactalbumin powder; And / or, the spray - drying is electrostatic spray - drying.

10. Use of the method for separating proteins according to any one of claims 1 - 9 in the preparation of a product containing α - lactalbumin and / or β - lactoglobulin.

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