A protein separation method and related applications

Abstract

The invention discloses a protein separation method and related applications thereof, relating to the technical field of dairy product processing. The method comprises heating a whey solution whose pH is adjusted to 3.5-4.2 by a citric acid solution, microfiltering the heated product, and separating β-lactoglobulin and / or α-lactalbumin from a permeate and a retentate obtained by the microfiltration. The method is based on membrane filtration technology, achieves high-purity separation of β-lactoglobulin and α-lactalbumin, with a purity of more than 70%-90%, is compatible with whey from various sources, has simple equipment, and has the advantages of low separation cost, high efficiency, ease of implementation, high throughput, and the like, thereby providing a path for large-scale industrial production of β-lactoglobulin and α-lactalbumin.

Description

Technical Field

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

[0002] Whey protein accounts for approximately 20% of total milk protein. Whey is a significant byproduct of dairy processing and cheese production, and is produced in high abundance. Two protein fractions, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), account for over 70% of whey protein concentration and exist as a mixed fraction in a ratio of approximately 1:4. Both proteins are important sources of essential amino acids for the human body. Due to their excellent nutritional and absorption properties, they are widely used in sports nutrition and functional foods, either in their natural blend or in separated forms. Because these two fractions have distinct nutritional and physiological properties, which are derived from differences in their secondary and tertiary structures, research into separation techniques has garnered significant attention in recent years.

[0003] α-La contains a high tryptophan content, which can positively impact stress tolerance and improve attention by promoting serotonin synthesis. In the infant food industry, isolating and purifying the α-La fraction can reduce allergenicity and bring its protein composition closer to that of breast milk, as β-Lg is the primary allergen in infant formula (it is virtually absent in breast milk). Notably, a complex formed by partially denatured α-La and oleic acid has been shown to exhibit biological activity in inducing apoptosis in human and mouse tumor cells. Furthermore, the hydrophobic / hydrophilic group distribution of β-Lg is highly homologous to that of the lipocalin family. This structural characteristic enables it to bind and transport small hydrophobic molecules, offering important applications in the food industry (e.g., flavor binding).

[0004] The differences in the molecular properties of α-La and β-Lg result in completely different functional properties. Due to the absence of free thiol groups, isolated α-La exhibits high thermal stability at neutral pH, even under ultra-high temperature heating conditions, making this protein suitable for applications where gelation after heat treatment is undesirable. In contrast, the presence of a free thiol group in the β-Lg structure can play a role in applications that require cross-linking and gelation during heat treatment. The amphiphilic nature of β-Lg gives it excellent emulsification and foaming capabilities.

[0005] To better utilize the various whey proteins, their separation and purification are necessary. Over the past two decades, numerous laboratory-scale separation procedures have been developed for the isolation and purification of 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 these methods can achieve high purity, they suffer from high equipment costs, low separation efficiency, and complex processes, limiting their applicability for large-scale whey protein separation. Alternatively, separation methods based on the selective aggregation of β-Lg at high pH and in the presence of calcium ions exist. However, these methods result in very low α-La yields, necessitating high-frequency diafiltration during membrane filtration to make them suitable for large-scale α-La production.

[0006] At present, the purity of separation of β-Lg and α-La by relying solely on membrane filtration technology is low, usually only reaching 50%~60%.

[0007] Therefore, the market urgently needs 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 proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide a protein separation method and related applications.

[0010] The present invention is achieved in that:

[0011] In a first aspect, an embodiment of the present invention provides a method for separating a protein, comprising the following steps:

[0012] Heating a whey solution whose pH is adjusted to 3.5-4.2 with a citric acid solution, and then microfiltering 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;

[0013] Using ultrafiltration to separate β-lactoglobulin from the first permeate and / or to separate α-lactalbumin from the first retentate;

[0014] The separation of β-lactoglobulin comprises: 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 comprises: adjusting the pH of the first retentate to 6.9-8.0, performing membrane filtration using a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution. The molecular weight cutoffs 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, respectively.

[0015] In a second aspect, embodiments of the present invention provide use of the protein separation method described in the preceding embodiments in the preparation of products containing α-lactalbumin and / or β-lactoglobulin.

[0016] The present invention has the following beneficial effects:

[0017] The present invention establishes a method for separating β-lactoglobulin and / or α-lactalbumin from whey. The method comprises heating a whey solution whose pH is adjusted to 3.5-4.2 by a citric acid solution, and further separating and purifying the heated product by combining microfiltration, ultrafiltration technology and pH adjustment. The steps cooperate with each other, overcome the technical limitation of the existing membrane filtration technology that cannot achieve high-purity separation, achieve high-purity separation of β-lactoglobulin and / or α-lactalbumin, and achieve purity of more than 70%-90%. The method is compatible with whey from various sources, has simple equipment, and has the advantages of low separation cost, high efficiency, easy implementation, high throughput, and the like, thereby providing a path for large-scale industrial production of β-lactoglobulin and α-lactalbumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the process for separating β-lactoglobulin and α-lactalbumin from whey. DETAILED DESCRIPTION

[0020] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0021] At present, the purity of β-Lg and α-La separation by membrane filtration technology is low, usually only reaching 50%~60%. This may be because (1) the molecular weight of β-Lg and α-La is relatively close, β-Lg molecular weight is about 18 kDa, α-La is about 14 kDa, and it is difficult to achieve effective separation of the two by relying solely on membrane filtration technology; (2) at pH close to the isoelectric point (pI≈5.2) or at high concentration, β-Lg is easy to form dimers / octamers. After β-Lg forms dimers, the molecular weight is about 36 kDa, and a 25-30 kDa ultrafiltration membrane can be used to achieve separation from α-La (14 kDa). However, under existing technology, 36 kDa and 14 kDa are still too close. Limited by the membrane production process, the membrane pore size is not perfect in the range of 25-30 kDa. Usually, to achieve complete retention, the membrane pore size should be one-third of the retained product. 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, if the crystallization process is not used under this condition, β-Lg will not form all 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 temporarily bind through hydrophobic interactions or disulfide bonds to form a complex and be retained by the membrane together; (4) The charges of the two are similar, and the separation cannot be enhanced by electrostatic repulsion. Some membrane materials (such as PVDF) are negatively charged and repel negatively charged proteins (such as β-Lg) when pH>pI, but α-La has a similar charge, so the separation effect is limited; (5) The complexity of the whey matrix: whey contains lactose, minerals (Ca²⁺) and lipids, 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.

[0022] Based on research on whey, β-lactoglobulin and α-lactalbumin, and combined with a large number of experimental verifications, this application innovatively established a new separation method. This method involves heating a whey solution whose pH is adjusted to 3.5-4.2 with a citric acid buffer, and microfiltering the heated 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. The various steps work together 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 (containing β-lactoglobulin and / or α-lactalbumin) from various sources, and is suitable for large-scale industrial production.

[0023] Definition of noun

[0024] The terms "first," "second," "third," and "fourth" herein are merely used for distinction and description and are not to be understood as indicating or implying relative importance.

[0025] The term "microfiltration" in this article is a membrane separation technology based on the core principle of physical screening. The pore size range is usually 0.1~1 micron (μm), and separation is achieved through the uniform microporous structure on the membrane surface.

[0026] The term “ultrafiltration” in this article refers to membrane separation technology based on molecular weight cutoff (MWCO), typically in the range of 1,000–1,000,000 Da, through a combination of sieving effects and charge repulsion.

[0027] The term "cross-flow filtration" in this article refers to a solid-liquid separation technology that uses a pump to force the liquid to flow parallel to the membrane surface. The core principle is that the shear force generated by the liquid flow continuously erodes the membrane surface, removing retained particles. This maintains a thin contamination layer and significantly reduces membrane clogging.

[0028] The term "conventional cross-flow filtration" in this article refers to the basic form of cross-flow filtration, which relies on an external pumping system to make the feed flow tangentially along the membrane surface, suppressing fouling through hydrodynamic shear forces, without additional mechanical rotating parts.

[0029] The term "Rotating Dynamic Cross-Flow Filtration" in this article refers to an improvement on traditional cross-flow filtration technology that actively generates high shear forces or turbulence on the membrane surface through rotating components (such as rotors, cyclones or rotating membranes themselves), significantly enhancing the anti-fouling ability.

[0030] It should be noted that, in the absence of conflict, the features in the embodiments or implementations of the present invention may be combined with each other.

[0031] Technical Solution

[0032] An embodiment of the present invention provides a method for separating a protein, comprising the following steps:

[0033] Heating a whey solution whose pH is adjusted to 3.5-4.2 by a citric acid solution, and then microfiltering 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;

[0034] Using ultrafiltration to separate β-lactoglobulin from the first permeate and / or to separate α-lactalbumin from the first retentate;

[0035] The separation of β-lactoglobulin comprises: 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 comprises: adjusting the pH of the first retentate to 6.9-8.0, performing membrane filtration using a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution. The molecular weight cutoffs 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, respectively.

[0036] Some embodiments further provide a method for separating a protein, comprising:

[0037] S1. Prepare a whey solution whose pH is adjusted to 3.5-4.2 with a citric acid solution.

[0038] Specifically, in some embodiments, the preparation of the whey solution comprises the following steps: adjusting the pH of the whey protein solution to 4.5-5.2, performing solid-liquid separation, and then adjusting the pH of the separated clear liquid to 3.5-4.2 again.

[0039] Using citric acid solution as a pH regulator to adjust the pH of the whey solution to a limited range can achieve a better separation effect of β-lactoglobulin and α-lactalbumin.

[0040] In some embodiments, the pH adjuster used to prepare the whey solution is 0.1 mol / L~5 mol / L citric acid buffer.

[0041] In some embodiments, the concentration of the citric acid buffer is 0.1-5 mol / L, specifically any one or a 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.

[0042] In some embodiments, the pH is adjusted to 4.5-5.2, and the pH can be specifically adjusted to any one of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 and 5.2, or a range between any two of them.

[0043] In some embodiments, the pH of the whey solution is adjusted to a 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.

[0044] In some embodiments, the solid-liquid separation method includes centrifugation, and the centrifugal speed is 1000~14000 rpm, specifically any one of 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000 and 14000 rpm or a range between any two of them.

[0045] In some embodiments, the whey protein solution includes at least one of whey permeate from fresh milk membrane separation, acid whey discharged from cheese production, and a whey protein powder reconstituted solution, and the protein content of the whey protein solution is ≤15% (w / w).

[0046] In some embodiments, the acid whey discharged from cheese production refers to acid whey (Acid Whey) which is a by-product separated after rennet or lactic acid fermentation during cheese production.

[0047] In some embodiments, the whey permeate obtained by membrane separation of fresh milk refers to the liquid component that passes through the membrane pores and enters the filtration side when fresh milk passes through membrane separation technology (such as ultrafiltration and microfiltration).

[0048] In some embodiments, the fresh milk includes at least one of cow's milk, goat's milk, camel's milk, horse's milk, donkey's milk and yak's milk.

[0049] In some embodiments, the whey protein powder reconstituted solution is obtained by dissolving whey protein powder in pure water.

[0050] In some embodiments, the protein content of the whey protein solution is 5% to 10%, specifically any one or a range between any two of 5%, 6%, 7%, 8%, 9% and 10%.

[0051] S2. Heating the whey solution whose pH is adjusted to 3.5-4.2 by citric acid solution, and then microfiltering the heated product using a first filter membrane to obtain a first permeate and a first retentate.

[0052] Specifically, in some embodiments, the temperature of the heated whey solution is 45-55°C, specifically any one of 45, 46, 48, 50, 52, 54 or 55°C or a range between any two of them.

[0053] In some embodiments, the whey solution is heated for 1 to 4 hours, specifically any one of 1, 1.5, 2, 2.5, 3, 3.5 and 4 hours, or a range between any two of these.

[0054] In some embodiments, the pore size of the first filter membrane is any one of 100, 150, 200, 250, 300, 350, 400, 450 and 500 nm, or a range between any two of the ranges.

[0055] In some embodiments, the structure of the first filter membrane is selected from the group consisting of: a flat sheet membrane, a hollow fiber membrane, a hollow tubular membrane, and a spiral membrane.

[0056] In some embodiments, the membrane material of the first filter membrane is selected from the group consisting of ceramic membranes, composite membranes, and polymer membranes. Ceramic membranes (e.g., alumina, zirconia, and titania) are inorganic membranes; composite membranes include organic-inorganic composite membranes and purely inorganic composite membranes (e.g., ceramic-metal composite membranes); and polymer membranes are organic membranes, such as polyethersulfone (PES), polysulfone (PS), polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), regenerated cellulose (RC), and polyamide (PA).

[0057] In some embodiments, the first filter membrane is selected from: a microfiltration membrane (eg, ceramic), a hollow fiber membrane, or a hollow tubular membrane.

[0058] In some embodiments, the microfiltration operation mode adopts cross-flow filtration, and the cross-flow filtration is selected from traditional cross-flow filtration or rotary dynamic cross-flow filtration.

[0059] In some embodiments, the diafiltration liquid for microfiltration using the first filter membrane includes: the second permeate and / or 0.1-5 mol / L citric acid-sodium citrate buffer with a pH of 3.5-4.2; and / or the diafiltration multiple is 1-5 times the volume of the raw liquid, specifically any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 times, or a range between any two of them.

[0060] S3. The separation of β-lactoglobulin comprises: 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.

[0061] Specifically, in some embodiments, the molecular weight cut-off of the second filter membrane is any one or a range between any two of 3, 4, 5, 6, 7, 8, 9 and 10 kDa.

[0062] In some embodiments, the structure and membrane raw material of the second filter membrane are the same as the structure and membrane raw material of the first filter membrane described in the above embodiment, and are not repeated here.

[0063] In some embodiments, the second filter membrane is a spiral wound membrane (polymer).

[0064] In some embodiments, the pH is adjusted to 6.9-8.0, specifically any one 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, or a range between any two thereof.

[0065] In some embodiments, the diafiltration liquid for membrane filtration using the second filter membrane includes reverse osmosis water (RO water) and / or 0.1-5 mol / L citric acid-sodium citrate buffer with a pH of 3.5-4.2, and / or the diafiltration multiple is 1-3 times the volume of the raw liquid, specifically any one of 1, 1.5, 2, 2.5 and 3 times, or a range between any two of them.

[0066] In some embodiments, the pH of the citric acid-sodium citrate buffer can be any one of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 and 4.2, or a range between any two thereof, and the concentration can be any one of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mol / L, or a range between any two thereof.

[0067] S4, the separation of the α-lactalbumin comprises: 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.

[0068] Specifically, in some embodiments, the molecular weight cut-off of the third filter membrane is any one or a range between any two of 20, 25, 30, 35, 40, 45 and 50 kDa.

[0069] In some embodiments, the molecular weight cut-off of the fourth filter membrane is any one or a range between any two of 3, 4, 5, 6, 7, 8, 9 and 10 kDa.

[0070] In some embodiments, the structure and membrane raw materials of the third filter membrane and / or the fourth filter membrane are the same as the structure and membrane raw materials of the first filter membrane described in the above embodiment, and are not repeated here.

[0071] In some embodiments, the third filter membrane and / or the fourth filter membrane is a spiral wound membrane (polymer).

[0072] In some embodiments, 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 to obtain a third permeate and a third retentate; the third permeate is an α-lactalbumin solution.

[0073] In some embodiments, the separation of α-lactalbumin includes: adjusting the pH of the first retentate to 6.9-8.0, and filtering the third permeate using the fourth filter membrane to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution.

[0074] In some embodiments, the separation of α-lactalbumin includes: adjusting the pH of the first retentate to 6.9-8.0, filtering the first retentate through a third filter membrane to obtain a third permeate and a third retentate; and filtering the third permeate through a fourth filter membrane to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution. Using both the third and fourth filter membranes results in higher separation purity.

[0075] In some embodiments, the separation method further comprises adjusting the pH of the third retentate to 6.9-8.0 to obtain a β-lactoglobulin solution. The third retentate contains β-lactoglobulin, and the third retentate can be mixed with the second retentate and then pH-adjusted to obtain a β-lactoglobulin solution. Alternatively, the third retentate can be pH-adjusted to obtain a β-lactoglobulin solution, which can then be mixed with the β-lactoglobulin solution obtained from the second retentate to obtain a final β-lactoglobulin solution.

[0076] In some embodiments, the pH is adjusted to 6.9-8.0, specifically any one 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, or a range between any two thereof.

[0077] In some embodiments, the pH is adjusted to 6.9-8.0 using a pH adjuster comprising 1-5 mol / L sodium hydroxide solution.

[0078] In some embodiments, the concentration of the sodium hydroxide solution may be any one of 1, 2, 3, 4, and 5 mol / L, or a range between any two thereof.

[0079] In some embodiments, the diafiltration liquid for membrane filtration using the third filter membrane and / or the fourth filter membrane includes reverse osmosis water (RO water), and / or the diafiltration multiple is 1 to 3 times the volume of the raw liquid, specifically any one of 1, 1.5, 2, 2.5 and 3 times, or a range between any two of them.

[0080] S5. Spray-drying the β-lactoglobulin solution and / or the α-lactalbumin solution to obtain β-lactoglobulin powder and / or α-lactalbumin powder.

[0081] Specifically, in some embodiments, the spray drying is electrostatic spray drying.

[0082] 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 98% or more.

[0083] In some embodiments, the purity of β-lactoglobulin in the β-lactoglobulin liquid and / or β-lactoglobulin powder can reach 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or 98% or more.

[0084] In addition, an embodiment of the present invention further provides the use of the protein separation method described in any of the above embodiments in the preparation of a product containing α-lactalbumin and / or β-lactoglobulin.

[0085] In some embodiments, the product containing α-lactalbumin and / or β-lactoglobulin includes any one or more of milk powder, fresh milk, yogurt, cheese, whey protein powder, nutritional supplements, and purified protein products.

[0086] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0087] Materials in the examples:

[0088] The preparation method of 1 mol / L citric acid buffer is as follows: dissolve 210.14 g of citric acid monohydrate in 1 L of distilled water.

[0089] The preparation method of 1 mol / L citric acid-sodium citrate buffer at pH 4.2 is as follows: 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 adjust the volume to obtain a 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 adjust the volume to obtain a 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 the 1 mol / L citric acid-sodium citrate buffer at pH 4.2 is obtained.

[0090] The preparation method of 4 mol / L sodium hydroxide solution is as follows: dissolve 160 g of sodium hydroxide in 1 L of water.

[0091] Example 1

[0092] This embodiment provides a method for producing α-lactalbumin and β-lactoglobulin, comprising the following steps.

[0093] A. Preparation of working whey: The whey permeate from fresh bovine milk membrane separation is concentrated by ultrafiltration to a protein content of 9% (w / w);

[0094] B. pH adjustment and centrifugation: Adjust the pH of the working whey to 4.6 using 1 mol / L citric acid buffer (pH adjuster), then centrifuge at 10,000 rpm in a tubular centrifuge and collect the supernatant.

[0095] 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 adjuster), the supernatant was allowed to stand at 50°C (heating temperature) for 2 hours. Conventional cross-flow filtration was then performed using a 100 nm ceramic microfiltration membrane (first filter membrane). After microfiltration of the raw liquid to a minimum volume, the raw liquid was diafiltered using 1 mol / L citric acid-sodium citrate buffer (washing solution) at pH 4.2 at a diafiltration rate of twice the raw liquid volume to obtain a first permeate and a first retentate.

[0096] D. Separation of β-lactoglobulin: The first permeate was ultrafiltered using a 10 kDa ultrafiltration membrane (secondary membrane, polyethersulfone (PES) spiral membrane). After ultrafiltration to the minimum volume of the raw liquid, RO water was used for filtration at a rate of 1.5 times the volume of the raw liquid to obtain a second permeate and a second retentate. The pH of the second retentate was adjusted to 7.0 using 4 mol / L sodium hydroxide solution to obtain the β-lactoglobulin solution.

[0097] E. Separation of α-lactalbumin: The first retentate was adjusted to pH 7.0 using a 4 mol / L sodium hydroxide solution, and then ultrafiltered using a 30 kDa ultrafiltration membrane (third filter membrane, polyethersulfone spiral wound membrane). After ultrafiltration of the raw liquid to a minimum volume, the raw liquid was diafiltered using RO water (washing filtrate) at a diafiltration rate of 2 times the volume of the raw liquid to obtain a third permeate and a third retentate. The third permeate was ultrafiltered using a 10 kDa ultrafiltration membrane (fourth filter membrane, polyethersulfone spiral wound membrane). After ultrafiltration of the raw liquid to a minimum volume, the raw liquid was diafiltered using RO water (washing filtrate) at a diafiltration rate of 1.5 times the volume of the raw liquid to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution.

[0098] F. Spray drying: The α-lactalbumin solution and the β-lactoglobulin solution were subjected to electrostatic spray drying (60°C) to prepare the α-lactalbumin powder and the β-lactoglobulin powder.

[0099] Please refer to the flow chart Figure 1 .

[0100] Example 2

[0101] This embodiment provides a method for producing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that step B is omitted, that is, the working whey prepared in step A is directly subjected to step C.

[0102] Example 3

[0103] This embodiment provides a method for producing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the step of filtering with the third filter membrane in step E is omitted, that is, the first retentate adjusted to pH 7.0 is directly ultrafiltered with the fourth filter membrane.

[0104] Example 4

[0105] This embodiment provides a method for producing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that this embodiment further comprises: mixing the third retentate in step E and the second retentate in step D, and then adjusting the pH to 7.0 using a 4 mol / L sodium hydroxide solution to obtain a β-lactoglobulin solution.

[0106] Example 5

[0107] This embodiment provides a method for separating α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the pore size of the first filter membrane is 500 nm.

[0108] Example 6

[0109] This example provides a method for separating α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the molecular weight cutoff of the third filter membrane in step E is set to 50 kDa.

[0110] Example 7

[0111] This example provides a method for separating α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the molecular weight cutoff of the fourth filter membrane in step E is set to 5 kDa.

[0112] Comparative Example 1

[0113] This example provides a method for producing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that:

[0114] In step C, microfiltration was not performed, a 30 kDa ultrafiltration membrane was used as the first filter membrane, and RO water was used as the filtration solution. In step D, the first retentate was diafiltered using a second filter membrane, and the pH of the second retentate was not adjusted. In step E, membrane filtration using a third filter membrane was omitted, and the first permeate was membrane filtered using a fourth filter membrane.

[0115] Steps C, D and E of Comparative Example 1 are specifically as follows.

[0116] 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 adjuster), ultrafiltration is performed using a 30 kDa ultrafiltration membrane (first filter membrane, polyethersulfone spiral membrane) at approximately 35°C (30-40°C). After ultrafiltration to a minimum volume, the raw liquid is diafiltered using RO water (washing liquid) at a diafiltration rate of 1.5 times the volume of the raw liquid to obtain a first permeate and a first retentate.

[0117] Step D: Ultrafiltration of the first retentate using a 10 kDa ultrafiltration membrane (second filter membrane, polyethersulfone spiral membrane) to ultrafilter the raw liquid to a minimum volume, and then diafiltration of the raw liquid using RO water (washing liquid) at a diafiltration rate of 1.5 times the volume of the raw liquid to obtain a second permeate and a second retentate, wherein the second retentate is a β-lactoglobulin solution;

[0118] Step E: The first permeate is ultrafiltered using a 10 kDa ultrafiltration membrane (fourth filter membrane, polyethersulfone spiral membrane). After the raw liquid is ultrafiltered to a minimum volume, RO water (washing liquid) is used to wash the raw liquid at a washing ratio of 1.5 times the volume of the raw liquid to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin liquid.

[0119] Comparative Example 2

[0120] This embodiment provides a method for separating α-lactalbumin and β-lactoglobulin, which is substantially the same as that of embodiment 1, except that the pore size of the first filter membrane is 1000 nm.

[0121] After microfiltration begins, α-lactalbumin particles will quickly clog the microfiltration membrane and the process cannot proceed.

[0122] Comparative Example 3

[0123] This example provides a method for producing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the acidic pH regulator in step C is replaced with 1 mol / L hydrochloric acid.

[0124] Comparative Example 4

[0125] This example provides a method for preparing α-lactalbumin and β-lactoglobulin, which is substantially the same as that of Example 1, except that the heating temperature in step C is set to 65° C. After heating, the proteins denature and flocculate.

[0126] Verification Example 1

[0127] 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 were obtained, and at least one of the following was tested: total solids, moisture, ash, protein, α-lactalbumin content, β-lactoglobulin content, total casein, immunoglobulin IgG, bovine serum albumin, and casein glycomacropeptide. The total solids and moisture content were tested according to the first method of GB 5009.3-2016, the ash content was tested according to the first method of GB 5009.4-2016, and the protein content was tested according to the first method of GB 5009.5-2016. The α-lactalbumin and β-lactoglobulin contents were tested using high performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS / MS) under the following testing conditions:

[0128] Chromatographic column: Acquity UPLC BEH 300 C18 column (particle size 1.7 μm, 2.1 × 100 mm)

[0129] Chromatographic conditions: Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile in water. The injection volume was 5 μL, and the column temperature was 40°C. The LC elution gradient program was as follows: initial 5% mobile phase B, held for 1.2 minutes, followed by a linear gradient increase to 60% mobile phase B over 1.8 minutes, then to 80% mobile phase B over 1 minute, and then to 100% mobile phase B over 0.2 minutes. Equilibration at 100% mobile phase B was continued for 1.4 minutes, and then returned to 5% mobile phase B over 2.2 minutes. The total run time for each injection was 8 minutes.

[0130] Ion source: electrospray ionization (ESI);

[0131] The mass spectrometry conditions were as follows: capillary voltage 3.50 kV, cone voltage 30 V, source temperature 150°C, desolvation temperature 350°C, cone gas flow rate 50 L / h nitrogen, desolvation gas flow rate 900 L / h nitrogen, and argon collision gas pressure of 3 × 10 -3 mbar;

[0132] The purity of α-La and β-Lg was calculated according to the following formula:

[0133] ;

[0134] ;

[0135] Here, x represents the purity of α-La and y represents the purity of β-Lg.

[0136] Table 1 Partial test results of Example 1

[0137]

[0138] Table 2 Partial test results of Example 1

[0139]

[0140] Table 3 Test results of Example 2

[0141]

[0142] Table 4 Test results of Example 3

[0143]

[0144] Table 5 Test results of Example 4

[0145]

[0146] Table 6 Test results of Example 5

[0147]

[0148] Table 7 Test results of Example 6

[0149]

[0150] Table 8 Test results of Example 7

[0151]

[0152] Table 9 Test results of Comparative Example 1

[0153]

[0154] Table 10 Test results of Comparative Example 3

[0155]

[0156] From the above results, we can see that:

[0157] When step B is omitted (Example 2), the purity of α-lactalbumin and β-lactoglobulin is lower than that of Example 1, but the purity of α-lactalbumin is still high;

[0158] By omitting the ultrafiltration step using the third filter membrane in step E (Example 3), the purity of α-lactalbumin is lower than that of Example 1, but the purity of β-lactoglobulin is still high;

[0159] After recycling the third retentate to prepare a β-lactoglobulin solution (Example 4), the purity of the β-lactoglobulin is lower than that of Example 1, but the utilization rate of the raw materials can be improved, the waste generated in the production process can be reduced, and resources and production costs can be saved;

[0160] Replacing the pore size of the first filter membrane with 500 nm (Example 5) will cause α-lactalbumin to partially pass through the microfiltration membrane, reducing the purity of β-lactoglobulin. At the same time, α-lactalbumin particles will clog the microfiltration membrane pores in the middle and late stages of microfiltration, reducing the separation efficiency.

[0161] After the microfiltration membrane in step C was replaced with a 30 kDa ultrafiltration membrane (Comparative Example 1), the purity of α-lactalbumin and β-lactoglobulin was significantly reduced compared with that in Example 1.

[0162] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for separating protein, characterized in that: It includes the following steps: Heating a whey solution whose pH is adjusted to 3.5-4.2 by a citric acid solution, and then microfiltering 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; Using ultrafiltration to separate β-lactoglobulin from the first permeate and / or to separate α-lactalbumin from the first retentate; The separation of β-lactoglobulin comprises: filtering the first permeate through 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 comprises: adjusting the pH of the first retentate to 6.9-8.0, filtering through a third filter membrane and / or a fourth filter membrane to obtain an α-lactalbumin solution; the molecular weight cutoffs 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, respectively; The heating temperature is 45-55°C; the heating time is 1-4 hours; The pore size of the first filter membrane is 100 nm to 500 nm.

2. The separation method according to claim 1, characterized in that 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.

3. 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, performing membrane filtration using a third filter membrane to obtain a third permeate and a third retentate; and performing membrane filtration on the third permeate using the fourth filter membrane to obtain a fourth permeate and a fourth retentate, wherein the fourth retentate is an α-lactalbumin solution.

4. The separation method according to claim 3, characterized in that 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 comprises the following steps: adjusting the pH of the whey protein solution to 4.5-5.2, 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 in preparing the whey solution is 0.1 mol / L~5 mol / L citric acid buffer.

7. The separation method according to claim 5, characterized in that The whey protein solution comprises at least one of whey permeate separated from fresh milk membrane, acid whey discharged from cheese production, whey protein powder reconstituted solution and acidified whey supernatant, and the protein content of the whey protein solution is ≤15%.

8. The separation method according to claim 1, characterized in that The structures of the first filter membrane, the second filter membrane, the third filter membrane and the fourth filter membrane are independently selected from the group consisting of: flat membrane, hollow fiber membrane, hollow tubular membrane and spiral membrane.

9. The separation method according to claim 1, characterized in that 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.

10. The separation method according to claim 1, characterized in that The filtrate obtained by microfiltration using the first filter membrane includes: the second permeate and / or a 0.1 mol / L to 5 mol / L citric acid-sodium citrate buffer solution with a pH of 3.5 to 4.

2.

11. The separation method according to claim 10, characterized in that The filtration multiple of microfiltration using the first filter membrane is 1 to 5 times the volume of the raw material liquid.

12. The separation method according to claim 1, characterized in that The washing filtrate for membrane filtration using the second filter membrane includes reverse osmosis water and / or 0.1-5 mol / L citric acid-sodium citrate buffer with a pH of 3.5-4.

2.

13. The separation method according to claim 12, characterized in that The filtration multiple of membrane filtration using the second filter membrane is 1 to 3 times the volume of the raw material liquid.

14. The separation method according to claim 1, characterized in that The washing filtrate obtained by membrane filtration using the third filter membrane and / or the fourth filter membrane includes reverse osmosis water.

15. The separation method according to claim 14, characterized in that The diafiltration multiple of membrane filtration using the third filter membrane and / or the fourth filter membrane is 1 to 3 times the volume of the raw material liquid.

16. The separation method according to any one of claims 1 to 4, characterized in that The separation method further comprises spray-drying the β-lactoglobulin liquid and / or the α-lactalbumin liquid to obtain β-lactoglobulin powder and / or α-lactalbumin powder.

17. The separation method according to claim 16, characterized in that The spray drying is electrostatic spray drying.

18. Use of the protein separation method according to any one of claims 1 to 17 in the preparation of a product containing α-lactalbumin and / or β-lactoglobulin.

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