Method for separating multiple proteins in milk raw material and application of milk proteins

Abstract

The invention provides a method for separating multiple proteins in a milk raw material and application of milk protein.The method comprises the steps that the temperature of feed liquid is controlled to range from 50 DEG C to 55 DEG C, the influences of salt balance and beta-casein dissociation caused by the temperature are eliminated, and the stability of the milk protein in the feed liquid is kept; and then whey protein and casein are separated through chymosin treatment. Specifically, in the invention, chymosin treatment is carried out in a first reaction solution, so that whey in the first reaction solution can be separated out, which is caused by the fact that a k-CN micelle net-shaped structure is continuously rearranged in a curding process, and the whey distributed in the net-shaped structure is transferred. In the invention, by controlling the pH of a reaction system and the mass ratio of calcium to phosphorus in the chymosin treatment process, the content of whey separated out from the first reaction liquid can be remarkably increased, so that the yield of lactoprotein is further increased.

Description

Technical Field

[0001] This invention belongs to the field of dairy processing technology, and particularly relates to a method for separating multiple proteins from dairy raw materials and the application of milk proteins. Background Technology

[0002] Milk protein, due to its unique biological activity and nutritional characteristics, has become an important source of high-quality protein in the human diet. The core advantages of milk protein lie in its amino acid composition, which is highly compatible with human needs; its excellent digestibility and absorption efficiency; its strategic value for global food security; and its ability to provide precise nutritional support for specific populations. Milk protein is mainly composed of two parts: casein and whey protein. Whey protein, by mass percentage, includes approximately 13-25% α-lactalbumin, 48-58% β-lactoglobulin, 5-15% immunoglobulins, and 0.2-2% lactoferrin. Immunoglobulins are divided into five classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). IgG is the main component of immunoglobulins, accounting for approximately 75-80%.

[0003] Milk protein contains all the essential amino acids and other amino acids required for human growth and development, and the amino acid ratio is highly compatible with human physiological needs. Studies have shown that the proportion of essential amino acids in milk is close to the ideal pattern recommended by the Food and Agriculture Organization of the United Nations. This naturally optimized composition allows it to effectively supplement the amino acid deficiencies of plant proteins (such as lysine deficiency), significantly improving the nutritional value of mixed foods.

[0004] Compared to plant proteins, milk proteins have a bioavailability of over 90%. Their dual-component structure of casein and whey protein (casein provides slow-release nutrition, while whey protein enables rapid absorption), combined with their small-molecule peptide characteristics, can meet the protein metabolism needs of different physiological states, making them especially suitable for groups with weaker digestive functions.

[0005] In developing countries where grains are the staple food, milk protein, as a dietary supplement, can overcome the nutritional limitations of plant protein. Empirical data shows that a daily intake of 10% milk protein can significantly improve the balance of dietary amino acids. This highly efficient nutritional fortification is of great significance in alleviating hidden protein hunger. For example, in some countries, dairy products have formed the basic framework of dietary nutrition.

[0006] For critical stages of life, including the first 1000 days (pregnancy to infancy) and lactation, milk protein's natural milk-derived properties and high biological value make it an ideal nutritional carrier. Clinical studies have confirmed that fortified milk protein formulas can promote infant neural development, enhance pregnant women's resistance to infection, and improve the metabolic health of lactating women by regulating gut microbiota.

[0007] However, the protein content of natural dairy products (such as milk) is relatively limited (about 2.9g / 100g). Multi-component protein extraction technology is needed to achieve efficient enrichment of target proteins, thereby overcoming the limitations of their application scenarios in clinical nutritional support, nutritional food development and other fields. Summary of the Invention

[0008] To achieve efficient enrichment of target milk proteins, this invention provides a method for separating multiple proteins from milk raw materials and the application of milk proteins.

[0009] According to one aspect of the present invention, a method for separating multiple proteins from dairy raw materials is provided, the method comprising the following steps: S1. defatting the dairy raw materials to obtain skim milk, then pasteurizing the skim milk and heating it to 50-55°C, followed by concentration to obtain a first reaction solution; S2. treating the first reaction solution with rennet to obtain a filtrate and a precipitate; wherein the specific operation of the rennet treatment is as follows: adding rennet and an auxiliary agent to the filtrate, and maintaining the pH of the reaction system at 4.5-6.2; the auxiliary agent includes at least one of calcium and phosphorus, wherein the mass content of phosphorus is M. P The mass content of calcium element is M Ca In the additives, M is calculated according to the mass ratio. P / M Ca =0~10; S3. Subsequently, multi-protein separation processing is performed, which includes batch processing and purification processing; the batch processing is: dividing the filtrate into four parts; the purification processing includes casein extraction processing and whey protein extraction processing; wherein, the casein extraction processing is to perform atmospheric pressure spray drying on the precipitate to obtain casein; the whey protein extraction processing is to perform microfiltration and low pressure spray drying on the first part of the filtrate in sequence to obtain whey protein.

[0010] The method provided by this invention first eliminates the effects of temperature-induced salt balance and β-casein dissociation by controlling the temperature of the feed solution to 50℃~55℃, thus maintaining the stability of the milk proteins in the feed solution. Then, whey protein and casein are separated by rennet treatment. Specifically, in this invention, rennet treatment in the first reaction solution allows whey to precipitate out. This is because the k-CN micelle network structure continuously rearranges during the coagulation process, causing the whey distributed within the network structure to transfer. The applicant has found that in the prior art, since rennet treatment is generally used in cheese preparation, people are more concerned with the quality and flavor of the cheese formed from milk raw materials using the rennet method, rather than the whey content precipitated during the formation process. Through actual research, the applicant has found that the relative whey precipitation rate varies significantly with changes in calcium, phosphorus, and pH, especially the mass ratio between calcium and phosphorus. Therefore, in this invention, by controlling the pH, calcium, and phosphorus mass ratio of the reaction system during rennet treatment, the whey content precipitated in the first reaction solution can be significantly increased, thereby further increasing the yield of milk protein.

[0011] Preferably, the dairy raw materials include at least one of raw milk and dairy products.

[0012] Preferably, the raw milk includes any one of cow's milk, sheep's milk, camel's milk, horse's milk, and donkey's milk.

[0013] Preferably, the dairy products include any one of whole milk powder, skim milk powder, whey powder, and whey protein powder.

[0014] Preferably, in S1, the ceramic membrane used for microfiltration has a diameter of 0.1 μm.

[0015] Preferably, in the casein extraction process, the inlet air temperature of the atmospheric pressure spray drying process is 150℃~180℃, the outlet air temperature is 70℃~90℃, and the flow rate is 9mL / min~15mL / min.

[0016] Preferably, in the whey protein extraction process, the ceramic membrane used for microfiltration has a diameter of 0.05 μm to 0.1 μm.

[0017] Preferably, in the whey protein extraction process, the inlet air temperature of the low-pressure spray drying process is 60℃~85℃, the outlet air temperature is 45℃~60℃, the vacuum degree is 0.02MPa~0.4MPa, and the flow rate is 5mL / min~8mL / min. Generally, the permeate is mostly water, but it contains half of the original milk proteins. During the separation of casein and whey protein, heat treatment alters the protein structure in whey protein, leading to changes in whey protein composition. Atmospheric pressure spray drying can be carried out at temperatures of 150-200℃, while freeze drying is carried out at temperatures of -20℃ to -80℃. Existing powdering processes commonly use spray drying. Although it performs worse in terms of solubility, emulsification, and thermal stability compared to powder obtained from freeze-dried coal, the extremely high cost of freeze drying makes it difficult to promote on an industrial scale. Therefore, in this application, whey protein is first enriched by pretreatment of permeate A and then subjected to low-pressure spray drying, which not only protects the structure of whey protein but also reduces the production cost of whey protein.

[0018] Preferably, the rennet includes at least one of animal rennet and plant rennet; in the rennet treatment, condition (a) and / or condition (b) are satisfied; (a) the rennet includes animal rennet, the pH of the reaction system is 5.3-6.0, M P / M Ca =0~3; (b). Rennet includes plant rennet, the pH of the reaction system is 4.5~5.5, M P / M Ca =0~2.

[0019] Based on their source, rennets can be divided into four main categories: animal rennets, plant rennets, microbial rennets, and genetically engineered rennets. Different rennets result in significant differences in curdling effects. Animal rennets generally refer to abomasum enzymes extracted from the fourth stomach (abomasum) of polygastric ruminants (calves, lambs, etc.). The curd structure formed using abomasum enzymes is compact and can maintain its activity for a long time at low temperatures (below 20°C) or under frozen conditions, making it a commonly used industrial rennet. Many plants in nature contain proteases; therefore, plant rennets are low-cost, environmentally friendly, and suitable for the vegetarian market. However, plant rennets may present various problems, such as over-hydrolyzing proteins to produce bitter peptides, affecting flavor and curd; or they may cause loose curds and difficulty in whey removal. The most representative microbial rennets are those from *Mucor miehei* and *Mucor pusillus*, but cheese produced using them has a low yield, less than ideal flavor after maturation, and is prone to over-fermentation, resulting in poor cheese texture. Genetically engineered rennet, produced through gene editing, enhances the enzyme's thermal stability, pH tolerance, and catalytic efficiency. It boasts advantages such as short production cycles, high yields, lack of geographical limitations, low manufacturing costs, and high enzyme purity. However, the resulting products must undergo safety assessments for genetically modified foods, and some markets are resistant to genetically modified products. Furthermore, a longer research and development period is required to improve the product's flavor.

[0020] In this application, the pH and M in the reaction systems of animal chymotrypsin and plant chymotrypsin are optimized. P / M Ca It can improve the separation speed and content of whey protein and casein.

[0021] Preferably, the animal chymoses include at least one of calf rennet, lamb chymoses, and camel chymoses; the plant chymoses include at least one of aspartic protease, cysteine ​​protease, and serine protease.

[0022] Preferably, the pH is maintained at 5.8–6.6 during rennet treatment. Soluble Ca 2+ It dissolves easily, but it causes a significant decrease in pH, releasing free Ca. 2+ Increased concentration often reduces the stability of dairy products. While offering advantages in increasing nutritional value, adding calcium to dairy products in actual processing is challenging due to the complexity of salt balance and its impact on casein micelle structure and product stability. This application addresses this issue by adjusting the pH during rennet treatment to maintain the salt balance of the system and improve the solubility of subsequently isolated lactoferrin, based on production needs.

[0023] Preferably, the amount of rennet added in the rennet treatment is 0.005%~0.007%, the temperature is 38℃~46℃, and the reaction time is 40~60 minutes.

[0024] Since whey protein includes various milk proteins such as α-lactalbumin, β-lactoglobulin, immunoglobulins, and lactoferrin, this invention can further separate and purify the whey protein contained in the permeate through diverse operations to obtain higher-value lactoferrin. Therefore, the method provided by this invention can improve the utilization rate of milk raw materials, the extraction efficiency, and achieve diversified production.

[0025] Preferably, before microfiltration, the pH of the second filtrate is adjusted to 4.5–5.0, the temperature to 30–40°C, and the calcium ion concentration to 0.05 mol / L–0.1 mol / L. The presence of calcium ions promotes the thermal denaturation of α-lactalbumin and β-lactoglobulin, as well as their interaction with κ-casein. This causes the "hair" portion at the C-terminus of κ-casein on the micelle surface to stiffen, thereby reducing the electrostatic repulsion and steric hindrance of the micelles. Calcium bridges are formed between casein molecules, leading to flocculation of β-lactoglobulin. This further reduces the subsequent steps for extracting lactoferrin. Furthermore, since lactoferrin has the strongest heat resistance among whey proteins, the flocculation of α-lactalbumin and β-lactoglobulin can be promoted by appropriately increasing the temperature during lactoferrin extraction.

[0026] The isoelectric point (pI) of casein is approximately 4.6–4.8, that of α-lactalbumin is approximately 4.2–4.8, that of β-lactoglobulin is approximately 5.1–5.3, and that of lactoferrin is approximately 8–9. When the solution pH is close to their pI, the surface charge of the proteins is neutralized, resulting in the lowest solubility and making them prone to flocculation. Therefore, adjusting the pH to the range of 4.5–5.0 can simultaneously reduce the solubility of both proteins and promote aggregation.

[0027] In the extraction of lactoferrin, ion exchange chromatography (IEC) typically uses a salt-containing buffer solution, such as NaCl or citrate, for elution. Desalting effectively removes these residual salts, preventing lactoferrin aggregation, functional inactivation, or decreased stability due to high salt concentrations in subsequent processing or applications. Desalting also optimizes protein solubility and function; the reduced ionic strength of the solution decreases after desalting, minimizing non-specific precipitation of lactoferrin due to salting out, while restoring its native conformation and ensuring the integrity of its antibacterial and iron-binding functions.

[0028] Preferably, in the extraction of lactoferrin, the low-temperature drying process includes at least one of freeze-drying and low-temperature spray drying.

[0029] In raw milk, lactoferrin readily forms complexes with α-lactoglobulin, β-lactoglobulin, casein micelles, and immunoglobulins. Because immunoglobulins and lactoferrin are present in relatively low amounts in cow's milk, their purification is challenging. Lactoferrin generally exists in two forms: one is reversibly bound and immobilized with casein colloids; the other is free lactoferrin existing in whey. Lactoferrin has a high isoelectric point and, under natural conditions in raw milk (pH 6.6–6.7), carries a positive charge, while casein micelles carry a negative charge. The two bind electrostatically due to their opposite charges.

[0030] This application reduces the interaction between lactoferrin and casein micelles by treating the first reaction solution with rennet, thereby increasing the release of lactoferrin. Subsequently, lactoferrin is enriched by ultrafiltration and ion-exchange chromatography, achieving the separation and purification of lactoferrin.

[0031] Preferably, in the process of extracting lactoferrin, the ceramic membrane used for microfiltration is 0.45 μm.

[0032] Preferably, in the process of extracting lactoferrin, the pore size of the ultrafiltration membrane is 50kDa~100kDa, and the operating pressure is 0.2MPa~0.27MPa.

[0033] Preferably, in the extraction of lactoferrin, the adsorption gel in the ion exchange chromatography process includes any one of CM-sephadex C-25, SP-Sepharose FF, and CM-sepharose FF.

[0034] Preferably, the purification process further includes immunoglobulin extraction, which involves ultrafiltration, ion-exchange chromatography, and freeze-drying of the third filtrate to obtain immunoglobulins. Ultrafiltration can separate proteins of similar size using electrostatic charge differences, and is an effective method for protein concentration and fractionation without causing structural damage. The proteins in the pretreated permeate C have molecular weights mainly distributed between 245 kDa and 135 kDa, and 20 kDa and 11 kDa, with trace amounts present between 70 kDa and 40 kDa. The main proteins present are Ig, β-Lg, and α-La.

[0035] Preferably, step S1 further includes the following step: mixing skim milk with a high-temperature protectant before pasteurization.

[0036] Preferably, the thermoprotectant includes at least one of sucrose, glucose, fructose, maltose, glutamic acid, lactose, and glycerol. Among these, sugars (such as maltose and sucrose) can form hydrogen bonds with proteins through hydroxyl groups, replacing water molecules to maintain the native conformation; polyols (such as glycerol) can increase solution viscosity, reduce molecular motion rate, and delay the denaturation process; ionic thermoprotectants (such as glutamic acid) can regulate the ionic strength of the solution and stabilize the surface charge distribution of the protein.

[0037] Preferably, the purification process further includes: an immunoglobulin G extraction process, wherein the immunoglobulin G extraction process involves pre-extraction, ultrafiltration, and freeze-drying of the fourth part of the filtrate to obtain immunoglobulin G; wherein the ultrafiltration membrane has a pore size of 100 kDa, an ultrafiltration pressure of 0.06 MPa to 0.1 MPa, and an ultrafiltration temperature of 25°C to 30°C.

[0038] Pasteurization requires high temperatures to inactivate pathogens or contaminating proteins, but IgG itself is temperature-sensitive. All food processing involves heat sterilization; temperatures too low are ineffective. Under heat treatment conditions below 65°C, the IgG molecular structure of dairy raw materials maintains high stability; however, when the temperature exceeds this threshold, its denaturation rate exhibits a non-linear increasing trend. After heating at 65°C for 30 minutes, IgG activity is reduced by nearly 30%; as the temperature continues to rise, IgG denatures rapidly; at 75°C, activity decreases to over 80% after 6 minutes; and at 85°C, irreversible denaturation occurs after 2.5 minutes, essentially resulting in complete inactivation. Traditional food industry processes involving high-temperature sterilization (such as ultra-high temperature sterilization and dry heat treatment) (e.g., infant formula and sterilized dairy product production) easily lead to a significant loss of IgG biological activity, significantly limiting its application in functional foods. This application, however, protects IgG by introducing a thermoprotectant before pasteurization, ensuring that IgG maintains its good structure even after sterilization.

[0039] On the other hand, IgG has a molecular weight of approximately 106 kDa, and theoretically, it can be retained in pores with a diameter smaller than 100 kDa. However, in practical applications, the applicant found that the purity of IgG decreases as the pore size decreases. This may be because smaller pore sizes retain more impurity proteins, leading to a decrease in purity. Conversely, smaller pore sizes significantly improve IgG recovery, possibly due to the enhanced physical retention capacity of the membrane pores for the target molecule. In ultrafiltration, since protein molecules are flexible, the retention effect depends on multiple factors such as membrane pore size, ultrafiltration temperature, and ultrafiltration pressure. Therefore, this application aims to achieve a balance between purity and recovery rate by optimizing the ultrafiltration process.

[0040] In a second aspect, the present invention provides a composition comprising at least one of whey protein obtained by the above method, casein obtained by the above method, lactoferrin obtained by the above method, immunoglobulin obtained by the above method, and immunoglobulin G obtained by the above method.

[0041] A third aspect of the invention provides the use of the above-described composition in the preparation of pharmaceuticals, dietary supplements, health products, general foods, or pet foods.

[0042] A fourth aspect of the present invention provides a method for preparing a composition, wherein the raw materials for preparing the composition include lactoferrin obtained by the method described above, and the preparation method comprises the following steps: Step 1. Dividing the lactoferrin into two parts, and premixing the first part of lactoferrin with calcium lactate to obtain premix I; Step 2. Dividing glucose into two parts, and mixing premix I with whey protein, the second part of lactoferrin, and the first part of glucose to obtain premix II; Step 3. Mixing premix II with the second part of glucose to obtain the composition.

[0043] Generally, whey protein has good solubility, while lactoferrin has poor solubility. When these two are used as raw materials to prepare a composition, incomplete dissolution of lactoferrin may occur, affecting the homogeneity of the mixed solution and impacting the product's taste and appearance. For example, in the preparation of a mixed beverage of lactoferrin and whey protein, lactoferrin may form aggregates. In this application, calcium lactate is first introduced to compound with a portion of the lactoferrin, which improves the solubility of this portion. Furthermore, the introduction of glucose can form hydrogen bonds with another portion of the lactoferrin and whey protein, thereby increasing the hydrophilicity between protein molecules and improving the solubility of the remaining portion of lactoferrin. The composition obtained through the above preparation process significantly improves the solubility of lactoferrin. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the process flow provided by the present invention. Detailed Implementation

[0045] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0046] To more clearly understand the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in further detail. In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered exemplary in nature and not restrictive.

[0047] Example 1 This embodiment performs multi-protein separation on dairy raw materials according to the following steps, wherein the process flow diagram of this embodiment is as follows: Figure 1 As shown.

[0048] S1. Take approximately 500 kg of fresh raw milk, preheat it to 63°C, and then defatt it using a disc centrifuge to obtain approximately 460 kg of skimmed milk. After pasteurization (72°C, 15s), cool it to 20°C using circulating water and store it in a cold storage at 4°C. Heat the skimmed milk to 50°C and maintain it for 10 minutes, then perform microfiltration using a 0.10 μm ceramic membrane. Specifically: during the microfiltration separation process, control the feed temperature at 50~55°C, start the pipeline circulation system, keep the valves unchanged, and perform 20 minutes of pre-filtration to allow the membrane surface mass transfer process to reach dynamic equilibrium. Adjust the pipeline valves to the filtration condition, set the membrane inlet pressure to 120 kPa, reduce the outlet pressure to 40 kPa, and connect the permeate side pipeline to the atmosphere (pressure 0 kPa) to form an 80... kPa transmembrane pressure difference; continuous filtration under constant pressure difference, real-time monitoring of liquid volume change, microfiltration is stopped when the liquid is concentrated to 3 times, and the first reaction solution is obtained; S2. Treat the first reaction solution with rennet to obtain a filtrate and a precipitate; the specific procedure for rennet treatment is as follows: place the filtrate in a constant temperature incubator at 42℃ for 30 minutes, and after the temperature stabilizes, add 0.005% calf rennet and 0.03% CaCl2, react at 42℃ for 40 minutes, and maintain the pH of the reaction system at 4.8. Calculate M according to the mass ratio. P / M Ca =0; S3. Subsequently, multi-protein separation processing is performed, which includes batch processing and purification processing. Batch processing involves dividing the filtrate into four parts. Purification processing includes extraction of casein, extraction of whey protein, extraction of lactoferrin, extraction of immunoglobulins, and extraction of immunoglobulin G. The casein extraction process involved spray drying the precipitate under normal pressure to obtain casein; the inlet air temperature of the spray drying process was 180℃, the outlet air temperature was 80℃, and the flow rate was 12 mL / min.

[0049] The whey protein extraction process involved sequentially microfiltration and low-pressure spray drying of the first filtrate to obtain whey protein. The microfiltration process followed step S1, except that the ceramic membrane used in the microfiltration process was 0.05 μm. The low-pressure spray drying process had an inlet air temperature of 80℃, an outlet air temperature of 55℃, a vacuum degree of 0.03 MPa, and a flow rate of 8.5 mL / min.

[0050] The extraction of lactoferrin involves sequentially performing microfiltration, ultrafiltration, ion exchange chromatography, desalting, and drying on the filtrate described in Part II to obtain lactoferrin.

[0051] The ultrafiltration process involves controlling the temperature of the third permeate to 36°C, adjusting the pH to 6.8, fixing the operating pressure to 0.20 MPa, and performing ultrafiltration using a 100 kDa polysulfone spiral wound membrane.

[0052] The ion exchange chromatography process was as follows: First, 40 ml of CM-sepharose FF gel was taken, 20% ethanol was removed, and the surface liquid was dried. The gel was mixed with PB buffer at a ratio of 3:1 to form a gel mixture. Then, the chromatography column and filter membrane were wetted with PB buffer, ensuring that there were no air bubbles and that the liquid level was higher than the filter membrane. The gel mixture was injected into the column in one go along the glass rod, and allowed to settle while keeping the liquid level covering the gel. A peristaltic pump was connected, and the column was equilibrated with PB buffer at a flow rate of 1.5 mL / min to three times the column volume to ensure column bed stability. The flow rate was reduced to 1 mL / min until the conductivity and pH of the eluent remained constant. Immediately before loading the sample, the sample was prepared with equilibration buffer to keep the sample pH stable. The loading rate was 0.6 mL / min. After loading the sample, impurities were washed away with PB buffer containing 0.3 mol / L NaCl. After the baseline was leveled, lactoferrin was eluted with PB buffer containing 0.8 mol / L NaCl.

[0053] The immunoglobulin extraction process involved ultrafiltration, ion exchange chromatography, and freeze-drying of the third filtrate to obtain immunoglobulins. The specific steps of ultrafiltration treatment are as follows: temperature is controlled at 40℃, pH is adjusted to 6.8, operating pressure is fixed at 0.30MP, and ultrafiltration treatment is performed using a 100kDa polysulfone spiral wound membrane to concentrate the pretreated permeate C by seven times. The ultrafiltration concentrate was then purified by ion exchange chromatography. The ion exchange chromatography process in this embodiment refers to the ion exchange chromatography process provided in Example 2, except that the resin used in this embodiment is DEAE-Sepharose FF, and impurities were washed with 10 mmol / L phosphate buffer (pH 6.8). After the baseline was leveled, immunoglobulins were eluted with 0.03 mol / L phosphate buffer (pH 7.2).

[0054] The process for extracting immunoglobulin G involved pre-extraction treatment, ultrafiltration treatment, and freeze-drying of the fourth part of the filtrate to obtain immunoglobulin G. The pre-extraction treatment is a low-temperature ethanol method. The specific steps are as follows: adjust the pH of the mixture to 6.7, then add 20% volume of -20℃ ethanol, then add NaCl to adjust the ionic strength to 0.06 mol / kg, then let the reaction system stand at 4℃ for 2 hours and then centrifuge. The ultrafiltration process is as follows: Take the above centrifuged precipitate, dissolve it with sterile PBS, dilute it by 2 times to obtain a feed solution; then control the temperature of the feed solution at 30℃, and use a MIF503 hollow fiber membrane module (molecular weight cutoff of 100kDa) to concentrate the feed solution by ultrafiltration at 0.1MPa.

[0055] Example 2 This embodiment refers to the method provided in Embodiment 1 for the separation of multiple proteins from milk raw materials. The difference from Embodiment 1 is that a high-temperature protectant is added in S1 in this embodiment. Apart from the above differences, the operation steps for the separation of multiple proteins from milk raw materials in this embodiment are strictly consistent with those in Embodiment 1.

[0056] Specifically, in this embodiment, the steps for separating multiple proteins from the milk raw material are as follows: S1. Take approximately 500 kg of fresh raw milk, preheat it to 63°C, and then defatt it using a disc centrifuge to obtain approximately 460 kg of skimmed milk. Then, add 10 wt% of a skimmed milk hyperthermia agent, which includes 20 wt% glutamic acid, 45 wt% sucrose, and 35 wt% lactose. After pasteurization (72°C, 15 s), cool it to 20°C using circulating water and store it in a cold storage at 4°C. Heat the skimmed milk to 50°C and maintain it for 10 minutes. Then, perform microfiltration using a 0.10 μm ceramic membrane. Specifically, during the microfiltration separation process, control the feed liquid temperature at 50~55°C, start the pipeline circulation system, keep the valve status unchanged, and perform 20 minutes of pre-filtration to allow the mass transfer process on the membrane surface to reach dynamic equilibrium. Adjust the pipeline valves to the filtration condition and set the membrane inlet pressure to 120. kPa, the outlet pressure drops to 40 kPa, the permeate side pipeline is connected to the atmosphere (pressure is 0 kPa), forming an 80 kPa transmembrane pressure difference; filtration continues under constant pressure difference, the change in feed volume is monitored in real time, and microfiltration is stopped when the feed is concentrated to 3 times to obtain the first reaction solution. Steps S2 and S3 are performed strictly in accordance with the steps provided in Example 1.

[0057] Example 3 This embodiment describes the preparation of a composition using the whey protein and lactoferrin obtained in Examples 1-2, and the testing and observation of the solubility of the prepared composition. Specifically, the formulation of the composition provided in this embodiment is shown in Table 1. The treatment groups 1A-3A and the control group 1A prepared the composition according to the following method, while the control group 2A directly prepared the composition by mixing the raw materials.

[0058] Step 1. Divide lactoferrin into two parts. Premix the first part of lactoferrin with calcium lactate to obtain premix I. Step 2. Divide the glucose into two parts. Mix premix I with whey protein, lactoferrin (part 2), and glucose (part 1) to obtain premix II. Step 3. Mix premix II with the second part of glucose to obtain the composition.

[0059] Table 1. Formulations of the compositions provided in this test example

[0060] Test Example 1 (1) Test methods and test objects The reconstituteability of the above-prepared composition was tested as follows: At 25°C, 100g of water was mixed with 5g of the composition, and the precipitation and stratification of the solution were observed. "O" indicates no precipitation or stratification, "X" indicates precipitation or stratification, and "XX" indicates both precipitation and stratification. Precipitation and stratification are two different issues. Specifically, precipitation refers to the presence of solid matter at the bottom of the system; stratification refers to the natural separation of substances in a mixed system due to differences in density, solubility, or phase state. For example, when iodine solution (density 3.34 g / mL) is mixed with carbon tetrachloride (density 1.47 g / mL), the iodine layer settles at the bottom, while the carbon tetrachloride floats on top.

[0061] (2) Test results and analysis The test results in this example are shown in Table 2. The reconstitution properties of treatment groups 1A, 2A, and 3A are all "0", while control group 1A is "X" and control group 2A is "XX", indicating that the solubility of lactoferrin prepared by the method of this invention is significantly improved when mixed with whey protein. The dissolution rates of treatment groups 2A and 3A are both "fast", while the dissolution rate of control group 1A is "slow" and control group 2A is "insoluble", further confirming this point.

[0062] On the other hand, after the introduction of the thermoprotectant, the dissolution rate of treatment group 3A was also "fast", indicating that the thermoprotectant can further improve the solubility of lactoferrin and whey protein.

[0063] While conventional commercial lactoferrin and whey protein were used in control groups 1A and 2A, their solubility was still improved after using the composition preparation method provided by the present invention.

[0064] The test results show that the lactoferrin and whey protein prepared by the method provided in this invention significantly improve their solubility after mixing; furthermore, introducing a high-temperature protectant further improves their solubility. Moreover, data from comparative groups 1 and 2 indicate that the composition prepared by the method provided in this invention improves the solubility of both lactoferrin and whey protein, even when using commercially available lactoferrin and whey protein.

[0065] Table 2. Test results of this embodiment

[0066] Example 4 In this embodiment, each treatment group and the control group prepared lactoferrin and whey protein according to the formula and method provided in Example 1, and the pH, type of rennet, and M of the rennet treatment in S2 were used. P / M CaThe variables mentioned above are shown in Table 3. Apart from the differences described above, the procedures for preparing lactoferrin and whey protein in each treatment group and control group of this embodiment are strictly consistent with those in Example 1. Furthermore, the procedures for preparing the compositions in each treatment group and control group of this embodiment are strictly consistent with those in treatment group 1A of Example 3.

[0067] Table 3. Variables involved in each treatment group in Example 2

[0068] Test Example 2 (1) Test methods and test objects The compositions obtained from each treatment group in Example 4 were tested according to the method provided in Test Example 1.

[0069] (2) Test results and analysis The test results for this test case are shown in Table 4.

[0070] M in the data P / M Ca Groups with non-zero values ​​(such as treatments 2B~5B and 7B~10B) all introduced Ca and phosphorus. In contrast, the groups without introduced elements (M) P / M Ca =0, as in treatment groups 1B and 6B), the dissolution rate showed diverse changes after the introduction of Ca and phosphorus, indicating that the introduction of Ca and phosphorus does not directly determine the dissolution rate; it needs to be combined with pH regulation. This may be because after the introduction of calcium and phosphorus, pH regulation can effectively adjust the ionic strength and protein charge state of the system, thereby improving the solubility of lactoferrin. Furthermore, rennet from different sources has different effects on pH and M... P / M Ca The responses differed. Calf abomasal enzymes responded at higher pH (5.6) and M... P / M Ca Higher pH levels resulted in better efficacy, while aspartic protease showed better efficacy at lower pH (4.8) and M. P / M Ca It performs exceptionally well at lower levels.

[0071] Therefore, the test results above show that in rennet treatment, the salt balance of the system can be maintained by introducing Ca and phosphorus elements and adjusting the pH during rennet treatment, and the synergistic effect of calcium and phosphorus with the pH of the enzymatic hydrolysis system can be utilized to improve the solubility rate of lactoferrin in the subsequent separation.

[0072] Table 4. Test results of this embodiment

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for separating multiple proteins from dairy raw materials, characterized in that, The method includes the following steps: S1. The milk raw material is defatted to obtain defatted milk, which is then pasteurized and heated to 50°C~55°C, followed by concentration to obtain the first reaction solution; S2 treats the first reaction solution with rennet to obtain filtrate and precipitate; The specific operation of the rennet treatment is as follows: rennet and an auxiliary agent are added to the filtrate, and the pH of the reaction system is maintained at 4.5-6.2; the auxiliary agent includes at least one of calcium and phosphorus, and the mass content of phosphorus is M. P The mass content of the calcium element is M. Ca In the aforementioned additives, M is calculated according to the mass ratio. P / M Ca =0~10; S3. Subsequently, a multi-protein separation process is performed, which includes batch processing and purification. The batch processing involves dividing the filtrate into four parts. The purification process includes casein extraction and whey protein extraction; wherein, the casein extraction process involves spray drying the precipitate at normal pressure to obtain casein; and the whey protein extraction process involves microfiltration and low-pressure spray drying of the filtrate in the first part to obtain whey protein.

2. The method as described in claim 1, characterized in that: The rennet includes at least one of animal rennet and plant rennet; in the rennet treatment, condition (a) and / or condition (b) are satisfied. (a) The rennet includes animal rennet, and the pH of the reaction system is 5.3–6.0, M P / M Ca =0~3; (b) The rennet includes plant-based rennet, and the pH of the reaction system is 4.5–5.5, M P / M Ca =0~2.

3. The method as described in claim 2, characterized in that: The animal chymotrypsin includes at least one of calf abomasal enzyme, lamb chymotrypsin, and camel chymotrypsin; the plant chymotrypsin includes at least one of aspartic protease, cysteine ​​protease, and serine protease.

4. The method as described in claim 1, characterized in that: The purification process also includes lactoferrin extraction; the lactoferrin extraction process involves sequentially performing microfiltration, ultrafiltration, ion exchange chromatography, desalting, and drying on the filtrate from the second part to obtain lactoferrin.

5. The method as described in claim 4, characterized in that: Before performing the microfiltration treatment, the pH of the filtrate in the second part is adjusted to 4.5~5.0, the temperature to 30~40℃, and the concentration of calcium ions to 0.05~0.1mol / L.

6. The method as described in claim 1, characterized in that, The purification process also includes immunoglobulin extraction, which involves ultrafiltration, ion exchange chromatography, and freeze-drying of the filtrate from the third part to obtain immunoglobulins.

7. The method as described in claim 1, characterized in that, The S1 process further includes the following steps: mixing skim milk with a high-temperature protectant and then pasteurizing it; the high-temperature protectant includes at least one of sucrose, glucose, fructose, maltose, glutamic acid, lactose, and glycerol.

8. The method as described in claim 1, characterized in that, The purification process also includes the extraction of immunoglobulin G, which involves pre-extracting the filtrate from Part IV, followed by ultrafiltration and freeze-drying to obtain immunoglobulin G. The ultrafiltration membrane used in the ultrafiltration process has a molecular weight of 100 kDa, an ultrafiltration pressure of 0.06 MPa to 0.1 MPa, and an ultrafiltration temperature of 25°C to 30°C.

9. A composition, characterized in that, The composition comprises at least one of the following: whey protein prepared by the method of any one of claims 1 to 8; casein prepared by the method of any one of claims 1 to 8; lactoferrin prepared by the method of claim 4 or 5; immunoglobulin prepared by the method of claim 6; and immunoglobulin G prepared by the method of claim 8.

10. The use of the composition of claim 9 in the preparation of pharmaceuticals, dietary supplements, health products, general foods or pet foods.

11. A method for preparing a composition, characterized in that, The raw materials for preparing the composition include the lactoferrin obtained by the method described in claim 4 or 5, wherein the preparation method includes the following steps: Step 1. Divide the lactoferrin into two parts, and premix the first part of the lactoferrin with calcium lactate to obtain premix I; Step 2. Divide the glucose into two parts, and mix the premix I with whey protein, the second part of lactoferrin, and the first part of glucose to obtain premix II; Step 3. Mix the premix II with the second portion of glucose to obtain the composition.

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