Method for preparing high-activity bovine colostrum immunoglobulin by multi-stage membrane filtration combined ion exchange resin adsorption

Abstract

The application discloses a method for preparing high-activity bovine colostrum immunoglobulin by using a multi-stage membrane filtration combined ion exchange resin adsorption, and belongs to the technical field of bovine colostrum powder processing. The method for preparing bovine colostrum immunoglobulin is specifically as follows: acidification precipitation, ultrafiltration combined nanofiltration separation, ion exchange resin selective adsorption and other means are cooperatively used for the pretreated bovine colostrum, so that the extraction rate and purity of the immunoglobulin are remarkably improved, the small-molecule growth factors, amino acids and other active components in the bovine colostrum are effectively reserved, the final product is rich in high-activity immunoglobulin, and other important nutritional components in the bovine colostrum are reserved, so that the overall nutritional value and market competitiveness of the product are improved.

Description

Technical Field

[0001] This invention relates to a method for preparing highly active bovine colostrum immunoglobulins by adsorption using a multi-stage membrane filtration combined with ion exchange resin, belonging to the field of bovine colostrum powder processing technology. Background Technology

[0002] Bovine colostrum refers to the milk produced by dairy cows within 7 days after calving, especially the colostrum from the first 3 days. It is rich in various bioactive components such as immunoglobulins (IgG), lactoferrin, lysozyme, growth factors, and amino acids, and has significant physiological functions such as enhancing immunity, fighting infection, and promoting growth and development, earning it the reputation of a "natural treasure trove of nutrients." Among these, immunoglobulins (IgG) are one of the most crucial active components in bovine colostrum, effectively neutralizing pathogens and enhancing the body's immune defense capabilities. Therefore, the extraction and purification of immunoglobulins from bovine colostrum has significant application value.

[0003] Traditional methods for extracting immunoglobulins from bovine colostrum primarily rely on DEAE ion-exchange chromatography. This method selectively adsorbs immunoglobulins through ion-exchange chromatography, followed by elution to separate them. However, this method has several drawbacks, severely limiting the extraction efficiency and product quality of bovine colostrum immunoglobulins. First, the ion-exchange column adsorption process requires large amounts of buffer and eluent, increasing production costs and potentially leading to loss of immunoglobulin activity. Second, the selective adsorption characteristics of the ion-exchange chromatography column cause small-molecule growth factors, amino acids, and other active components in colostrum to be discarded during adsorption, failing to be effectively retained. These small-molecule active components have important physiological functions in bovine colostrum; for example, growth factors promote cell proliferation and tissue repair, and amino acids are the basic building blocks of protein synthesis, crucial for maintaining normal physiological functions. Therefore, traditional methods not only result in low immunoglobulin extraction rates but also waste other valuable active components in bovine colostrum.

[0004] Furthermore, traditional ion chromatography column adsorption methods are complex, with expensive adsorption equipment and columns, and cumbersome operation steps requiring multiple adsorption, elution, and concentration steps. This not only increases production costs but also reduces production efficiency, making large-scale industrial production difficult. In practical applications, the low extraction efficiency and high cost result in expensive bovine colostrum immunoglobulin products produced by traditional methods, limiting their widespread market application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly efficient, simple, and low-cost process for preparing bovine colostrum immunoglobulins that effectively preserves small-molecule growth factors, amino acids, and other active ingredients in colostrum. This invention optimizes the extraction process by employing a synergistic combination of acidification precipitation, ultrafiltration combined with nanofiltration separation, and selective adsorption using ion exchange resins. This significantly improves the extraction rate and purity of immunoglobulins while effectively preserving small-molecule growth factors, amino acids, and other active ingredients in bovine colostrum. This process avoids the destruction and discarding of small-molecule active ingredients during extraction, resulting in a final product rich in highly active immunoglobulins while retaining other important nutrients from bovine colostrum, thereby enhancing the overall nutritional value and market competitiveness of the product.

[0006] To achieve the above objectives, the following technical solution is provided:

[0007] This invention provides a method for preparing highly active bovine colostrum immunoglobulins by adsorption using a multi-stage membrane filtration combined with ion exchange resin, the method comprising the following steps:

[0008] (1) Raw material pretreatment: Bovine colostrum is filtered and centrifuged to remove impurities and fat, resulting in defatted bovine colostrum;

[0009] (2) Acidification precipitation: Adjust the pH of the skimmed bovine colostrum to 4-5, let it stand to precipitate, separate and remove the precipitate, and collect the supernatant;

[0010] (3) Ceramic microfiltration: The supernatant collected in step (2) is subjected to ceramic membrane microfiltration to remove residual casein micelles and fat droplets, and the permeate is collected;

[0011] (4) Ultrafiltration combined with nanofiltration separation: The permeate collected in step (3) is concentrated by ultrafiltration membrane with a molecular weight cutoff of 10-50 kDa to obtain permeate 1 and retentate 1; then permeate 1 is further concentrated and separated by nanofiltration membrane with a molecular weight cutoff of 500-1500 Da. After the concentration factor reaches 15-20 times, the concentration is stopped to obtain retentate 2 and permeate 2.

[0012] (5) Selective adsorption by ion exchange resin: The retentate 1 obtained in step (4) is adsorbed by ion exchange resin and then eluted. The specific elution process is as follows: First, use 1 to 3 times the volume of resin with a mass fraction of 0.3 to 0.5% NaCl solution to remove impurities and proteins with weak adsorption characteristics on the surface. Then, use a mixed solution of 3 to 5% NaCl and 0.01 to 0.05% sodium acetate to elute and collect the eluent.

[0013] (6) The permeate 2 collected in step (4) and the eluent collected in step (6) are mixed and then freeze-dried to obtain bovine colostrum immunoglobulin rich in highly active ingredients.

[0014] In one embodiment, the bovine colostrum in step (1) is sourced from healthy, disease-free bovine colostrum within 24 hours after calving.

[0015] In one embodiment, the settling time in step (2) is 1 to 2 hours.

[0016] In one embodiment, the pore size of the ceramic membrane in step (3) is 20-50 nm.

[0017] In one embodiment, the ion exchange resin in step (5) is a strong acid cation exchange resin.

[0018] In one embodiment, the strong acid cation exchange resin includes either Mitsubishi DIAION SK1B strong acid cation exchange resin or DuPont AmberLite IR-120 strong acid cation exchange resin.

[0019] In one embodiment, the adsorption conditions in step (5) are as follows: the concentration of the buffer salt in the adsorption process is controlled at 0.02-0.1 mol / L, the pH value is 5.0-6.0, and the time is 20-60 min.

[0020] In one embodiment, the elution temperature in step (5) is 25-30°C.

[0021] The present invention also provides a highly active bovine colostrum immunoglobulin obtained by the method described above.

[0022] In one embodiment, the highly active bovine colostrum immunoglobulin contains ≥40% immunoglobulin, retains ≥90% of its immune activity, and preserves active ingredients such as small molecule growth factors and amino acids from bovine colostrum.

[0023] In one embodiment, the highly active bovine colostrum immunoglobulin contains immunoglobulin content greater than 0.4 g / g, lactoferrin content ≥ 3.2 mg / g, epidermal growth factor (EGF) content ≥ 1.5 μg / g, insulin-like growth factor (IGF-1) content ≥ 4.0 μg / g, transforming growth factor α content ≥ 1.2 μg / g, taurine content ≥ 0.25 μg / g, and folic acid content ≥ 0.25 μg / g.

[0024] In one embodiment, the highly active bovine colostrum immunoglobulin can be used as an immune-enhancing health product for the elderly and children, and also as a high-quality protein supplement for postoperative patients.

[0025] The present invention also provides the application of the above-described highly active bovine colostrum immunoglobulin in the preparation of drugs for enhancing immunity and preventing and adjuvant treatment of infectious diseases.

[0026] The present invention also provides a formulation comprising the above-described highly active bovine colostrum immunoglobulin, wherein the formulation is in the form of an oral liquid, capsule, tablet or powder, for enhancing immunity and preventing and adjuvant treatment of infectious diseases.

[0027] Beneficial effects:

[0028] The present invention provides a highly efficient, simple, and low-cost method for preparing highly active bovine colostrum immunoglobulins. It employs a combination of acidification precipitation, ultrafiltration combined with nanofiltration separation, and selective adsorption with ion exchange resin. This process avoids the destruction and discarding of small molecule active ingredients during extraction, resulting in a final product that is not only rich in highly active immunoglobulins but also retains other important nutrients from bovine colostrum, thereby enhancing the overall nutritional value and market competitiveness of the product.

[0029] Compared with traditional processes, this method not only significantly improves the extraction rate and purity of immunoglobulins, but also effectively preserves active ingredients such as small molecule growth factors and amino acids in bovine colostrum. Attached Figure Description

[0030] Figure 1 This is a graph showing the immunoglobulin content analysis in bovine colostrum immunoglobulins obtained in the embodiments and comparative examples of the present invention;

[0031] Figure 2 This is a graph showing the analysis of lactoferrin content in bovine colostrum immunoglobulins obtained in the embodiments and comparative examples of the present invention;

[0032] Figure 3 This is a graph showing the yield of active proteins in bovine colostrum immunoglobulins obtained in Example 1 and Comparative Example 7 of the present invention.

[0033] Figure 4 This is a schematic diagram of the process of the present invention. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0035] Example 1

[0036] A method for preparing highly active bovine colostrum immunoglobulins by adsorption using a multi-stage membrane filtration combined with ion exchange resin includes the following steps:

[0037] (1) Raw material pretreatment: Select healthy, disease-free bovine colostrum within 24 hours after calving as raw material, and remove impurities and fats by filtration and centrifugation to obtain defatted bovine colostrum;

[0038] (2) Acidification precipitation: Adjust the pH of the skimmed bovine colostrum to 4.5, let it stand for 1 hour to precipitate, separate and remove the precipitate, and collect the supernatant;

[0039] (3) Ceramic microfiltration: The supernatant collected in step (2) is subjected to ceramic membrane microfiltration to remove residual casein micelles and fat droplets. The ceramic membrane used has a pore size of 30 nm to obtain the permeate.

[0040] (4) Ultrafiltration combined with nanofiltration separation: The permeate collected in step (3) is concentrated by ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain permeate 1 and retentate 1; then permeate 1 is further concentrated and separated by nanofiltration membrane with a molecular weight cutoff of 1000 Da. After the concentration factor reaches 15-20 times, the concentration is stopped to obtain retentate 2 and permeate 2.

[0041] (5) Selective adsorption by ion exchange resin: The retentate 1 obtained in step (4) was adsorbed by ion exchange resin. The adsorption pH was 5.0. In this pH range, bovine colostrum immunoglobulins and lactoferrin are both positively charged and can be selectively adsorbed on the ion exchange resin. The concentration of the sample buffer salt was 0.05 mol / L and the adsorption time was 40 min. The cation exchange resin used was Mitsubishi DIAION SK1B strong acid cation exchange resin.

[0042] (6) Ion exchange resin elution: The ion exchange resin adsorbed in step (5) is eluted: The specific elution process is to first use a 0.5% NaCl solution with a mass fraction of 3 times the resin volume to remove impurity proteins with weak adsorption characteristics on the surface, and then use a mixed solution of 4% NaCl and 0.05% sodium acetate to elute. The eluent is collected and the temperature is maintained at 25℃ during the elution process.

[0043] (7) Mix the permeate 2 collected in step (4) and the eluent collected in step (6), and then freeze-dry to obtain bovine colostrum immunoglobulin rich in highly active ingredients.

[0044] Example 2

[0045] The only difference from Example 1 is that the ion exchange resin in step (5) is DuPont AmberLiteIR-120 strong acid cation exchange resin; all other parameters and conditions are the same as in Example 1.

[0046] Example 3

[0047] The only difference from Example 1 is that the elution conditions in step (6) are adjusted as follows: elution is performed using a 0.3% NaCl solution with a mass fraction of 3 times the resin volume to remove impurity proteins with weak adsorption properties on the surface, followed by elution using a mixed solution of 5% NaCl and 0.05% sodium acetate, and the eluent is collected. The elution process temperature is maintained at 30°C. All other parameters and conditions are the same as in Example 1.

[0048] Comparative Example 1

[0049] The only difference from Example 1 is that the ultrafiltration membrane concentration process is omitted in step (4), and the permeate obtained in step (3) is directly subjected to nanofiltration filtration, and the retentate after nanofiltration is used for subsequent processes such as step (5); other parameters and conditions are the same as in Example 1.

[0050] Comparative Example 2

[0051] The only difference from Example 1 is that the nanofiltration membrane separation process is omitted in step (4), and the permeate after ultrafiltration membrane concentration and the eluent collected in step (6) are mixed in step (7); other parameters and conditions are the same as in Example 1.

[0052] Comparative Example 3

[0053] The only difference from Example 1 is that the molecular weight cutoff of the nanofiltration membrane in step (4) is adjusted to 2000 Da; other parameters and conditions are the same as in Example 1.

[0054] Comparative Example 4

[0055] The only difference from Example 1 is that steps (5) and (6) are deleted, and the ultrafiltration concentrate and nanofiltration permeate 3 from step (4) are directly mixed. All other parameters and conditions are the same as in Example 1.

[0056] Comparative Example 5

[0057] The only difference from Example 1 is that step (6) did not use a 0.5% NaCl solution for elution and impurity removal, but instead used a mixed solution of 4% NaCl and 0.05% sodium acetate. All other parameters and conditions were the same as in Example 1.

[0058] Comparative Example 6

[0059] The only difference from Example 1 is that in step (7), the eluent obtained in step (6) is directly freeze-dried to obtain bovine colostrum immunoglobulin.

[0060] Comparative Example 7

[0061] The only difference from Example 1 is that the sodium acetate solution was omitted in the elution process of step (6), while the other parameters and conditions are the same as in Example 1.

[0062] Results Analysis

[0063] Performance analysis was performed on the bovine colostrum immunoglobulins obtained in the examples and comparative examples. The results are shown in Table 1 and... Figures 1-3 As shown:

[0064] Table 1. Product components of each embodiment and comparative example.

[0065]

[0066]

[0067] From the table and Figures 1-2 It can be seen that all embodiments can meet the requirements of immunoglobulin content greater than 0.4 g / g and lactoferrin content greater than 3.2 mg / g. After adjusting steps (4)-(6), comparative examples 1-5 significantly affected the content of immunoglobulin and lactoferrin. Among them, comparative examples 1, 2, and 4 had the greatest impact on the content of immunoglobulin and lactoferrin. This indicates that using a two-stage membrane filtration system of ultrafiltration and nanofiltration to separate and reconstitute the components in bovine colostrum is an effective means to increase the content of immunoglobulin and lactoferrin in bovine colostrum products.

[0068] Comparative Example 3 used a nanofiltration membrane with a higher molecular weight cutoff in step (4), which reduced the content of immunoglobulins and lactoferrin to a certain extent and failed to meet the target product requirements. This may be because the molecular weight cutoff of the nanofiltration membrane used was too high, causing some lactoglobulin molecules to permeate through the membrane, increasing the content of impurity proteins, thereby leading to a decrease in the content of the target protein.

[0069] In Comparative Example 5, elution with a 0.3-0.5% NaCl solution was not used in step (6). Instead, a mixed solution of 3-5% NaCl and 0.01%-0.05% sodium acetate was used directly for elution. This failed to effectively remove the weakly adsorbed impurity proteins on the surface of the cation exchange resin, resulting in a slightly lower content of the target protein in the prepared bovine colostrum immunoglobulin powder than the target value.

[0070] Comparative Example 6 differs from Example 1 in that the eluent obtained in step (6) is used as the final product. The product obtained is characterized by the fact that the content of immunoglobulins and lactoferrin can meet the product requirements. However, the content of small molecules such as epidermal growth factor (EGF), insulin-like growth factor (IGF-1), transforming growth factor α, taurine, and folic acid in bovine colostrum is extremely low. Among them, transforming growth factor α, taurine, and folic acid were not detected. This indicates that the technical solution of the present invention can not only effectively enrich the macromolecular immune active proteins in bovine colostrum to meet the product requirements, but also effectively retain the small molecules with growth-promoting effects in bovine colostrum, such as epidermal growth factor (EGF), insulin-like growth factor (IGF-1), transforming growth factor α, taurine, and folic acid, thereby endowing the product with multiple nutritional functions.

[0071] Compared with Example 1, Comparative Example 7 significantly reduced the elution efficiency of immunoglobulins and lactoferrin (e.g., Figure 3 In a normal salt ion neutral environment, lactoferrin and cationic resin have strong adsorption effects, making it difficult for some lactoferrin to be eluted at the set salt concentration. However, this invention uses sodium acetate solution to adjust the alkalinity of the eluent, thereby improving the elution efficiency by adjusting the charge of the solute itself and the electrostatic shielding effect of salt ions, ultimately effectively increasing the yield of immunoglobulins and lactoferrin.

[0072] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing highly active bovine colostrum immunoglobulins by adsorption using a multi-stage membrane filtration combined with ion exchange resin, characterized in that, The method includes the following steps: (1) Raw material pretreatment: Bovine colostrum is filtered and centrifuged to remove impurities, resulting in defatted bovine colostrum; (2) Acidification precipitation: Adjust the pH of the skimmed bovine colostrum to 4-5, let it stand to precipitate, separate and remove the precipitate, and collect the supernatant; (3) Ceramic microfiltration: The supernatant collected in step (2) is subjected to ceramic membrane microfiltration to remove residual casein micelles and fat droplets, and the permeate is collected; The ceramic membrane has a pore size of 30 nm; (4) Ultrafiltration combined with nanofiltration separation: The permeate collected in step (3) is concentrated by ultrafiltration membrane. The molecular weight cutoff of the ultrafiltration membrane is 10~50kDa, to obtain permeate 1 and retentate 1. Then, permeate 1 is further concentrated and separated by nanofiltration membrane. The molecular weight cutoff of the nanofiltration membrane is 500-1500 Da. After the concentration factor reaches 15-20 times, the concentration is stopped, to obtain retentate 2 and permeate 2. (5) Selective adsorption by ion exchange resin: The retentate 1 obtained in step (4) is adsorbed by ion exchange resin and then eluted. The specific elution process is as follows: First, use 1 to 3 times the volume of resin with a mass fraction of 0.3 to 0.5% NaCl solution to remove impurities and proteins with weak adsorption characteristics on the surface. Then, use a mixed solution of 3 to 5% NaCl and 0.01 to 0.05% sodium acetate to elute and collect the eluent. The ion exchange resin is either Mitsubishi DIAION SK1B strong acid cation exchange resin or DuPont AmberLite IR-120 strong acid cation exchange resin. (6) Mix the permeate 2 collected in step (4) and the eluent collected in step (5), and then freeze-dry to obtain bovine colostrum immunoglobulin rich in highly active ingredients.

2. The method according to claim 1, characterized in that, The adsorption conditions in step (5) are as follows: the concentration of the buffer salt is controlled at 0.02~0.1mol / L, the pH value is 5.0-6.0, and the time is 20~60min.

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