Method for preparing active thermosensitive protein in fresh milk through continuous separation

Abstract

The invention discloses a method for preparing active thermosensitive protein in fresh milk through continuous separation, and belongs to the technical field of lactoprotein separation. According to the method, efficient separation of lactoferrin, immunoglobulin G, alpha-lactalbumin and beta-lactoglobulin is realized through the steps of optimization pretreatment, primary membrane separation, continuous chromatography, purification, refining and the like. In the pretreatment stage, casein is removed by adjusting the pH to a casein isoelectric point, and whey protein is stabilized by combining sodium citrate; performing primary membrane separation to remove macromolecular impurities; the continuous ion exchange chromatography realizes synchronous separation of four proteins through gradient elution; and finally, performing ultrafiltration concentration and freeze drying to obtain a finished product. The purity of the four proteins separated by the method reaches 83%-86%, and the recovery rate is 76%-80%. Compared with a traditional method, protein denaturation is reduced through proper temperature control and sodium citrate pretreatment, the production efficiency is improved through continuous chromatography, and the method is suitable for continuously preparing the active milk protein and has high economic value and application prospects.

Description

Technical Field

[0001] This invention relates to the field of milk protein separation technology, and in particular to a method for the continuous separation and preparation of active heat-sensitive proteins in raw milk. Background Technology

[0002] Raw milk is rich in various bioactive milk proteins, including lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin. These proteins, with their unique physiological activities, show great potential for application in the food, pharmaceutical, and cosmetic fields. For example, lactoferrin has antibacterial, antiviral, and immune system-regulating functions; immunoglobulin G provides passive immune protection; and α-lactalbumin and β-lactoglobulin are high-quality protein sources that are easily absorbed and utilized by the human body. However, the efficient and continuous isolation and preparation of high-purity, highly active milk proteins from raw milk has remained a pressing technical challenge for the industry.

[0003] Currently, there are various technical methods for separating milk proteins, but all have obvious limitations. Salting out, as a traditional separation method, is relatively simple to operate and low in cost. However, by adding a large amount of salt to precipitate the protein, it is easy to cause changes in the protein molecular structure, leading to denaturation and loss of some physiological activity. Moreover, the purity of the obtained product is usually low, requiring complex desalting treatment. Subsequent purification steps are cumbersome and it is difficult to accurately separate active heat-sensitive proteins such as lactoferrin and immunoglobulin G.

[0004] Organic solvent precipitation is another common separation method. It utilizes organic solvents to reduce the solubility of proteins in solution, causing the proteins to precipitate. However, the use of organic solvents can not only cause protein denaturation and affect their activity, but also pose a threat to the health of operators due to their volatility and toxicity. Furthermore, organic solvents may remain in the product, limiting their application in food, pharmaceutical, and other fields with high safety requirements. Additionally, they exhibit poor selectivity for separating different types of milk proteins.

[0005] Centrifugation mainly relies on centrifugal force to separate proteins from other components, but its separation efficiency is low. For raw milk with complex components, it is difficult to accurately separate different active milk proteins such as lactoferrin and immunoglobulin G. In addition, this method is usually an intermittent operation with limited processing capacity, which cannot meet the needs of large-scale continuous production.

[0006] Membrane separation has found some application in the field of milk protein separation due to its advantages such as simple operation, no phase change, and low energy consumption. However, single membrane separation technology often fails to achieve ideal separation results. Because active milk proteins such as lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin have different molecular weights, although these differences are not extremely large, a single membrane separation process cannot effectively separate them, resulting in products with low purity. Therefore, it is necessary to combine membrane separation with other techniques to improve the separation efficiency.

[0007] Chromatography can achieve high-purity protein separation, but its traditional intermittent operation mode has significant drawbacks. During the separation process, once the chromatography column reaches adsorption saturation, sample injection needs to be stopped for elution, regeneration, and other operations. This interruption leads to low production efficiency, small throughput, and an inability to meet the requirements of continuous and high-efficiency processing. Moreover, traditional chromatography is not effective for the simultaneous separation of multiple bioactive milk proteins, often only capable of separating one or a few proteins, making it difficult to simultaneously extract multiple high-value bioactive milk proteins such as lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin.

[0008] Furthermore, existing technologies lack sufficient measures to protect the activity of active milk proteins during the separation process. Many separation methods suffer from improper temperature control during operation; high temperatures can easily lead to protein denaturation and inactivation, reducing the physiological activity and application value of the product. Simultaneously, a lack of continuity in the separation steps, with excessively long residence times in intermediate stages, can also affect the retention of protein activity. In conclusion, developing a separation and preparation method that enables continuous operation, simultaneously separates multiple active milk proteins such as lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin, and ensures high purity and high activity of the products, is of great practical significance for promoting the continuous production and application of active milk proteins. Summary of the Invention

[0009] The purpose of this invention is to provide a method for the continuous separation and preparation of active heat-sensitive proteins in raw milk. This method can achieve continuous production, and can simultaneously separate multiple heat-sensitive active milk proteins with high purity and good activity.

[0010] To achieve the above objectives, the present invention provides a method for the continuous separation and preparation of active heat-sensitive proteins in raw milk, comprising the following steps: S1. Pretreatment: The raw milk is defat-free by centrifugation at 2-6℃, at a speed of 3000-4000 r / min, for 20-30 min. The defat-free milk is collected. The pH of the defat-free milk is adjusted to the isoelectric point of casein, and the casein precipitate and supernatant are separated.

[0011] Add 0.05-0.1% sodium citrate to the supernatant to adjust the pH to 6.5-7.0, and let it stand at 2-6℃ for 30-60 minutes.

[0012] The S1 process effectively removes fat and casein, and the addition of sodium citrate stabilizes the structure of milk proteins such as lactoferrin and immunoglobulin G, preventing their denaturation.

[0013] S2. Primary Membrane Separation: The pretreated supernatant is passed into a primary ceramic membrane separation system at 20-25℃. The membrane pore size is 0.2-0.5 μm, and the operating pressure is 0.1-0.15 MPa. The permeate is collected. Primary membrane separation removes macromolecular impurities and microorganisms from milk. The ceramic membrane has good chemical stability and mechanical strength, maintaining high flux at lower pressures, reducing energy consumption. Its precise pore size setting effectively retains macromolecular impurities while allowing small molecules and active milk proteins to permeate, laying the foundation for subsequent continuous chromatography separation.

[0014] S3. Continuous Chromatographic Separation: The permeate from S2 is passed into a continuous ion exchange chromatography system. The chromatography column is packed with a weak anion exchange resin, and gradient elution is used. The eluent is 0.05-0.3 mol / L phosphate buffer, pH 6.0-7.5, flow rate 1-2 column volumes / h, and temperature 16-20℃. Fractions from different elution stages are continuously collected to obtain lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin, respectively. The continuous chromatography system enables uninterrupted separation, improving production efficiency, and gradient elution achieves effective separation of different proteins.

[0015] Continuous chromatography systems enable uninterrupted separation, improving production efficiency, while gradient elution allows for the effective separation of different proteins. Weak anion exchange resins exhibit differentiated adsorption capacities for active milk proteins with varying isoelectric points. As the concentration and pH of the phosphate buffer solution change gradient, lactoferrin, immunoglobulins, and whey proteins desorb from the resin sequentially according to their own characteristics. By precisely controlling the elution conditions and collection time, accurate separation of various active milk proteins can be achieved, effectively avoiding the problems of low protein separation efficiency and easy contamination in traditional batch chromatography methods.

[0016] S4. Purification and refining: The components collected in S3 are concentrated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane is 10-50 kDa, the operating pressure is 0.1-0.15 MPa, and the temperature is 2-6℃. After concentration, the active milk protein product is obtained by freeze drying.

[0017] Preferably, in S3, the continuous ion exchange chromatography system includes at least three chromatography columns connected in series, and adopts a continuous operation mode of cocurrent injection and countercurrent elution.

[0018] Preferably, in S3, the specific procedure for gradient elution is as follows: 0-30 min, 0.05 mol / L phosphate buffer; 30-50 min, 0.1 mol / L phosphate buffer; 50-80 min, 0.2 mol / L phosphate buffer; 80-120 min, 0.3 mol / L phosphate buffer, corresponding to the collection of lactoferrin, immunoglobulin G, α-lactalbumin and β-lactoglobulin.

[0019] Preferably, in S4, the freeze-drying conditions are: vacuum degree 0.01-0.02MPa, temperature -40 to -50℃, and drying time 24-36h.

[0020] Therefore, the method for continuous separation and preparation of active heat-sensitive proteins in raw milk according to the present invention has the following beneficial effects: (1) The present invention uses a continuous ion exchange chromatography system, combined with optimized pretreatment and membrane separation technology, to realize the continuous separation and preparation of lactoferrin, immunoglobulin G, α-lactalbumin and β-lactoglobulin, which improves production efficiency and is suitable for continuous preparation.

[0021] (2) Low temperature operation is used throughout the process, which effectively avoids denaturation and inactivation of active milk proteins.

[0022] (3) By combining multi-stage membrane separation with gradient elution chromatography, the simultaneous separation of multiple active milk proteins such as lactoferrin, immunoglobulins, and whey protein was achieved, and the purity and recovery rate were both at a high level.

[0023] (4) Adding sodium citrate to the pretreatment step can not only remove some impurities, but also stabilize the structure of milk protein, further improving the activity and recovery rate of protein.

[0024] (5) The low-temperature operation throughout the process of this invention can inhibit the growth of microorganisms, and the primary membrane separation can directly retain microorganisms in the milk; on the other hand, by combining the low-temperature process with membrane separation, the protein activity is preserved to the maximum extent while ensuring product safety.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0031] Example 1 A method for continuous separation and preparation of active heat-sensitive proteins in raw milk, the process is as follows: Figure 1 As shown, the specific steps include the following: S1. Pretreatment: 10L of raw milk is centrifuged at 4℃ for 20 minutes at a speed of 3000r / min. The skimmed milk is then collected.

[0032] Add hydrochloric acid to the skim milk to adjust the pH to 4.6, centrifuge at 8000 r / min for 20 min at 2℃, and collect the casein precipitate and supernatant separately; add 0.05% (w / v) sodium citrate to the supernatant, adjust the pH to 6.5 with 1 mol / L NaOH, and let stand at 2℃ for 30 min.

[0033] S2. Primary membrane separation: The pretreated supernatant is passed into a primary ceramic membrane separation system at 20°C. The membrane material is zirconia, the membrane pore size is 0.1μm, the membrane channel diameter is 4mm, the operating pressure is 0.1MPa, and the permeate is collected.

[0034] S3. Continuous Chromatography Separation: The permeate from the primary membrane separation is passed into a continuous ion exchange chromatography system. This system consists of three chromatography columns connected in series, filled with weak anion exchange resin, and operates in a continuous mode of co-current injection and counter-current elution. The eluent is phosphate buffer, and the gradient elution program is as follows: 0-30 min, 0.05 mol / L phosphate buffer; 30-50 min, 0.1 mol / L phosphate buffer; 50-80 min, 0.2 mol / L phosphate buffer; 80-120 min, 0.3 mol / L phosphate buffer; flow rate 1 column volume / h, temperature 10℃; the eluent fractions from 30-50 min, 50-80 min, 80-100 min, and 100-120 min are collected respectively to obtain lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin.

[0035] S4. Purification and Refining: The components collected in S4 are concentrated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane is 10 kDa, the operating pressure is 0.1 MPa, and the temperature is 4℃. After being concentrated to 1 / 10 of the original volume, the product is freeze-dried for 24 hours under vacuum of 0.01 MPa and temperature of -40℃ to obtain the active milk protein product.

[0036] Example 2 A method for continuous separation and preparation of active heat-sensitive proteins in raw milk includes the following steps: S1. Pretreatment: 10L of raw milk is centrifuged at 6℃ for 15 minutes at a speed of 4000r / min. The skimmed milk is then collected.

[0037] Add hydrochloric acid to skim milk to adjust the pH to 4.8, centrifuge at 10000 r / min for 30 min at 4℃, and collect casein precipitate and supernatant separately; add 0.1% (w / v) sodium citrate to supernatant, adjust the pH to 7.0 with 1 mol / L NaOH, and let stand at 4℃ for 60 min.

[0038] S2. Primary membrane separation: The pretreated supernatant is passed into a primary ceramic membrane separation system at 25°C. The membrane material is zirconia, the membrane pore size is 0.2μm, the membrane channel diameter is 6mm, the operating pressure is 0.15MPa, and the permeate is collected.

[0039] S3. Continuous Chromatographic Separation: The permeate from the primary membrane separation is passed into a continuous ion exchange chromatography system. This system consists of four chromatography columns connected in series, filled with weak anion exchange resin, and operates in a continuous mode of co-current injection and counter-current elution. The eluent is phosphate buffer, and the gradient elution program is as follows: 0-30 min, 0.05 mol / L, pH 6.0; 30-50 min, 0.1 mol / L, pH 6.5; 50-80 min, 0.2 mol / L, pH 7.0; 80-120 min, 0.3 mol / L, pH 7.5; flow rate 2 column volumes / h, temperature 20℃. The eluent fractions from 30-50 min, 50-80 min, 80-100 min, and 100-120 min are collected respectively to obtain lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin.

[0040] S4. Purification and refining: The components collected in S3 are concentrated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane is 30kDa, the operating pressure is 0.15MPa, and the temperature is 8℃. After being concentrated to 1 / 10 of the original volume, the product is freeze-dried for 36 hours under vacuum of 0.02MPa and temperature of -50℃ to obtain the active milk protein product.

[0041] Comparative Example 1 The following steps are taken to separate active milk proteins from raw milk using a traditional batch chromatography method: S1. Pretreatment: 10L of raw milk was defatted by centrifugation at 4℃, 3000r / min, for 20min, and the defatted milk was collected. Hydrochloric acid was added to the defatted milk to adjust the pH to 4.6, and after standing for 30min, casein and supernatant were separated by centrifugation.

[0042] S2. Ion exchange chromatography: A single-column intermittent ion exchange chromatography system was used, filled with strong anion exchange resin. The sample loading volume was 10% of the column volume. The eluent was 0.5 mol / L NaCl solution, pH 7.0, and the flow rate was 1 column volume / h. The elution peak was collected.

[0043] S3. Ultrafiltration Concentration: The collected eluent is concentrated by ultrafiltration. The ultrafiltration membrane has a molecular weight cutoff of 10 kDa, the operating pressure is 0.2 MPa, and the temperature is 25℃.

[0044] S4. Freeze-drying: The concentrate is freeze-dried at a vacuum of 0.05 MPa and a temperature of -30°C for 48 hours to obtain the mixed whey protein product.

[0045] Comparative Example 2 A method for continuous separation and preparation of active heat-sensitive proteins in raw milk includes the following steps: S1. Pretreatment: 10L of raw milk was defatted by centrifugation at 4℃, 3000r / min, for 30min, and the defatted milk was collected. Hydrochloric acid was added to the defatted milk to adjust the pH to 4.6, and the mixture was centrifuged at 2℃, 8000r / min for 20min. The casein precipitate and supernatant were collected separately.

[0046] S2. Primary membrane separation: The pretreated supernatant is passed into a primary ceramic membrane separation system at 25°C. The membrane material is zirconia, the membrane pore size is 0.1μm, the membrane channel diameter is 4mm, the operating pressure is 0.1MPa, and the permeate is collected.

[0047] S3. Continuous Chromatography Separation: The permeate from the primary membrane separation is passed into a continuous ion exchange chromatography system. This system consists of three chromatography columns connected in series, filled with weak anion exchange resin, and operates in a continuous mode of co-current injection and counter-current elution. The eluent is phosphate buffer, and the gradient elution program is as follows: 0-30 min, 0.05 mol / L phosphate buffer; 30-50 min, 0.1 mol / L phosphate buffer; 50-80 min, 0.2 mol / L phosphate buffer; 80-120 min, 0.3 mol / L phosphate buffer; flow rate 1 column volume / h; temperature 20℃. The elution fractions from 30-50 min, 50-80 min, 80-100 min, and 100-120 min are collected respectively to obtain lactoferrin, immunoglobulin G, α-lactalbumin, and β-lactoglobulin.

[0048] S4. Purification and refining: The components collected in S3 are concentrated by ultrafiltration. The molecular weight cutoff of the ultrafiltration membrane is 10 kDa, the operating pressure is 0.1 MPa, and the temperature is 4℃. After being concentrated to 1 / 10 of the original volume, the mixture is freeze-dried for 24 hours under a vacuum of 0.01 MPa and a temperature of -40℃ to obtain the finished milk protein product.

[0049] Comparative Example 3 A method for continuous separation and preparation of active heat-sensitive proteins in raw milk includes the following steps: S1. Pretreatment: 10L of raw milk was centrifuged at 4℃ to remove fat, and the skimmed milk was collected. Hydrochloric acid was added to the skimmed milk to adjust the pH to 4.6, and the mixture was centrifuged at 8000r / min for 20min at 2℃. The casein precipitate and supernatant were collected separately. 0.05% (w / v) sodium citrate was added to the supernatant to adjust the pH to 6.5.

[0050] S2. Membrane separation: The pretreated supernatant is passed into an ultrafiltration membrane system with a membrane molecular weight cutoff of 30 kDa and an operating pressure of 0.2 MPa. The retentate is then collected.

[0051] S3. Freeze-drying: The retentate is freeze-dried at a vacuum of 0.01 MPa and a temperature of -40°C for 24 hours to obtain the mixed whey protein product.

[0052] Test Example 1 The active milk protein products prepared in Examples 1, 2, 1, 2 and 3 were subjected to performance tests. The performance tests mainly measured the purity, recovery rate and processing time of the active milk protein.

[0053] The testing method is as follows: (1) The purity of the protein was determined by high performance liquid chromatography (HPLC), and the results are shown in Table 1 below (n=3, mean ± standard deviation): Table 1. Comparison of protein purity (%)

[0054] As shown in Table 1, the purity of the four proteins in Examples 1 and 2 was significantly higher than that in Comparative Examples 1, 2, and 3. This indicates that the present invention can effectively improve protein purity through optimized pretreatment, continuous chromatography, and ultrafiltration steps. Comparative Example 1, due to the use of a traditional batch method, had low separation efficiency; Comparative Example 2 lacked sodium citrate pretreatment, resulting in poor protein separation; and Comparative Example 3, using only a single membrane separation, could not achieve effective protein separation.

[0055] (2) The recovery rate was calculated by weighing and protein content determination. The results are shown in Table 2 below: Table 2 Comparison of protein recovery rates (%)

[0056] The recovery rates of Examples 1 and 2 were significantly higher than those of the comparative examples. This is attributed to the continuous operation and optimized process parameters of the examples, which reduced protein loss during the separation process. The lower recovery rates of Comparative Examples 1 and 3 were mainly due to significant protein loss caused by imperfect process steps.

[0057] (3) Processing time record From the start of the pretreatment of raw milk to the final freeze-dried product, the continuous operation time of the entire process (excluding the initial equipment debugging time) was recorded, and the average value was taken from three parallel measurements.

[0058] The processing times for 10L of raw milk in Examples 1 and 2 were 2.5h and 2.0h, respectively, significantly shorter than the 8.0h of Comparative Example 1. This is because the continuous chromatography system does not require shutdown for regeneration and can operate continuously, greatly improving production efficiency. Comparative Example 2 had the same processing time as Example 1, indicating that sodium citrate pretreatment does not significantly increase processing time.

[0059] In summary, the method of the present invention outperforms existing technologies in terms of purity, recovery rate, and production efficiency of the four active milk proteins, and has significant technical advantages.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for continuous separation for producing active heat-sensitive proteins in raw milk, characterized by, The method comprises the following steps: S1, pretreatment: centrifugal separation of raw milk at 2-6℃, rotation speed of 3000-4000r / min, centrifugal time of 20-30min, and collection of skim milk; adjusting the pH of skim milk to the isoelectric point of casein, separating casein precipitate and supernatant; adding 0.05-0.1% sodium citrate to the supernatant, adjusting the pH to 6.5-7.0, and standing at 2-6℃ for 30-60min; S2, primary membrane separation: passing the pretreated supernatant into a primary ceramic membrane separation system at 20-25℃, membrane pore size of 0.2-0.5μm, operating pressure of 0.1-0.15MPa, and collection of permeate; S3, continuous chromatography separation: passing the permeate in S2 into a continuous ion exchange chromatography system, the chromatography column being filled with weak anion exchange resin, gradient elution being adopted, eluent being 0.05-0.3mol / L phosphate buffer, pH 6.0-7.5, flow rate being 1-2 column volumes / h, temperature being 10-20℃, and continuously collecting components at different elution time periods to obtain lactoferrin, immunoglobulin G, α-lactalbumin and β-lactoglobulin, respectively; S4, purification and refining: ultrafiltration concentration of each component collected in S3, ultrafiltration membrane molecular weight cut-off being 10-50kDa, operating pressure being 0.1-0.15MPa, temperature being 2-6℃, and obtaining active heat-sensitive milk protein finished product by freeze-drying after concentration.

2. A method of continuous separation for the preparation of active heat sensitive proteins in raw milk as claimed in claim 1, wherein: In S3, the continuous ion exchange chromatography system comprises at least 3 chromatography columns connected in series, and adopts a continuous operation mode of forward flow sampling and reverse flow elution.

3. A method of continuous separation for preparing active heat sensitive proteins in raw milk as claimed in claim 1, wherein, In S3, the specific procedure of gradient elution is as follows: 0-30min, 0.05mol / L phosphate buffer; 30-50min, 0.1mol / L phosphate buffer; 50-80min, 0.2mol / L phosphate buffer; and 80-120min, 0.3mol / L phosphate buffer, corresponding to collection of lactoferrin, immunoglobulin G, α-lactalbumin and β-lactoglobulin.

4. A method of continuous separation for preparing active heat sensitive proteins in raw milk as claimed in claim 1, wherein, In S4, the freeze-drying conditions are as follows: vacuum degree of 0.01-0.02MPa, temperature of -40 to -50℃, and drying time of 24-36h.

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