Method for preparing high-activity bovine colostrum immune globulin through combination of multistage membrane filtration and ion exchange resin adsorption

Abstract

The invention discloses a method for preparing high-activity bovine colostrum immune globulin through combination of multistage membrane filtration and ion exchange resin adsorption, and belongs to the technical field of bovine colostrum powder processing. According to the preparation method of the bovine coloctrum immune globulin, specifically, acidizing precipitation, ultrafiltration and nanofiltration separation, ion exchange resin selective adsorption and other means are adopted for pretreated bovine coloctrum for cooperation, so that the extraction rate and purity of the immune globulin are remarkably improved; meanwhile, active ingredients such as micromolecular growth factors and amino acid in the bovine coloctrum are effectively reserved, so that the final product is rich in high-activity immune globulin, other important nutritional ingredients in the bovine coloctrum are reserved, and the overall nutritional value and market competitiveness of the product are improved.

Description

Technical Field

[0001] The invention relates to a method for preparing high-activity bovine colostrum immunoglobulin by combining multi-stage membrane filtration with ion exchange resin adsorption, and belongs to the technical field of bovine colostrum powder processing. Background Art

[0002] Bovine colostrum refers to the milk produced by dairy cows within seven days of calving, particularly the first three days. Rich in a variety of bioactive components, including immunoglobulins (IgG), lactoferrin, lysozyme, growth factors, and amino acids, it boasts significant physiological functions, including enhancing immunity, resisting infection, and promoting growth and development. It is known as a "natural nutritional treasure trove." Immunoglobulins (IgG) are one of the most critical active components in bovine colostrum, effectively neutralizing pathogens and enhancing the body's immune defenses. 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 adsorption technology. This method selectively adsorbs immunoglobulins via ion exchange chromatography, followed by an elution step to separate them. However, this method has numerous drawbacks that severely limit the extraction efficiency and product quality of bovine colostrum immunoglobulins. First, the ion exchange column adsorption process requires large amounts of buffer and eluent, which not only increases production costs but also may result in loss of immunoglobulin activity. Second, the selective adsorption properties of the ion exchange column result in the loss of active components in colostrum, such as small-molecule growth factors and amino acids, during the adsorption process, preventing their effective retention. These small-molecule active components have important physiological functions in bovine colostrum. For example, growth factors promote cell proliferation and tissue repair, while amino acids are the building blocks of protein synthesis and are 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, the traditional ion chromatography column adsorption method is complex, with expensive adsorption equipment and columns. The operation is cumbersome, 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 practice, bovine colostrum immunoglobulin products produced by traditional methods are expensive due to low extraction efficiency and high costs, limiting their widespread market adoption. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a highly efficient, simple, low-cost process for preparing bovine colostrum immunoglobulins that effectively retains small-molecule growth factors, amino acids, and other active ingredients in the colostrum. By optimizing the extraction process and employing a synergistic combination of acidification precipitation, ultrafiltration combined with nanofiltration separation, and selective adsorption with ion exchange resins, the present invention not only significantly improves the extraction rate and purity of the immunoglobulins, but also effectively retains the small-molecule growth factors, amino acids, and other active ingredients in the colostrum. This process avoids the destruction and discarding of small-molecule active ingredients during the extraction process, resulting in a final product that is not only rich in highly active immunoglobulins but also retains other important nutrients in the colostrum, thereby enhancing the product's overall nutritional value and market competitiveness.

[0006] In order to achieve the above objectives, the technical solutions provided are as follows:

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

[0008] (1) Raw material pretreatment: The colostrum is filtered and centrifuged to remove impurities and fat to obtain defatted colostrum;

[0009] (2) Acidification precipitation: Adjust the pH value of defatted bovine colostrum to 4-5, let it settle, 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 an ultrafiltration membrane with a molecular weight cutoff of 10 to 50 kDa to obtain permeate 1 and retentate 1; the permeate 1 is then further concentrated and separated by a nanofiltration membrane with a molecular weight cutoff of 500 to 1500 Da. Concentration is stopped after the concentration ratio reaches 15 to 20 times to obtain retentate 2 and permeate 2;

[0012] (5) Selective adsorption with ion exchange resin: The retentate 1 obtained in step (4) is adsorbed on an ion exchange resin and eluted after adsorption. The specific elution process is as follows: first, a 0.3-0.5% NaCl solution with a mass fraction of 1-3 times the volume of the resin is used for elution to remove impurities to remove impurity proteins with weak adsorption properties on the surface, and then a mixed solution with a mass fraction of 3-5% NaCl and 0.01-0.05% sodium acetate is used for elution, and the eluate is collected;

[0013] (6) The permeate 2 collected in step (4) and the eluate 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 colostrum in step (1) is sourced from healthy and disease-free bovine colostrum within 24 hours after calving.

[0015] In one embodiment, the standing 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 strongly acidic cation exchange resin.

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

[0019] In one embodiment, the adsorption conditions in step (5) are as follows: the salt concentration of the adsorption process control buffer is 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 above method.

[0022] In one embodiment, the highly active bovine colostrum immunoglobulin has an immunoglobulin content of ≥40%, an immune activity retention rate of ≥90%, and retains active ingredients such as small molecule growth factors and amino acids in the bovine colostrum.

[0023] In one embodiment, the highly active bovine colostrum immunoglobulin has an immunoglobulin content greater than 0.4 g / g, a lactoferrin content ≥3.2 mg / g, an epidermal growth factor EGF content ≥1.5 μg / g, an insulin-like growth factor content IGF-1 ≥4.0 μg / g, a transforming growth factor α content 1.2 ≥μg / g, a taurine content ≥0.25 μg / g, and a 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 can also be used as a high-quality protein supplement food for postoperative patients.

[0025] The present invention also provides the use of the highly active bovine colostrum immunoglobulin in the preparation of medicines for enhancing immunity, preventing and assisting in the treatment of infectious diseases.

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

[0027] Beneficial effects:

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

[0029] Compared with traditional processes, it not only significantly improves the extraction rate and purity of immunoglobulins, but also effectively retains active ingredients such as small molecule growth factors and amino acids in bovine colostrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an analysis chart of immunoglobulin content in bovine colostrum immunoglobulins obtained in the examples of the present invention and the comparative examples;

[0031] Figure 2 This is an analysis chart of lactoferrin content in bovine colostrum immunoglobulins obtained in the examples of the present invention and the comparative examples;

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

[0033] Figure 4 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The following specific embodiments further describe the present invention.

[0035] Example 1

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

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

[0038] (2) Acidification precipitation: Adjust the pH value of defatted bovine colostrum to 4.5, let it settle for 1 hour, 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, and a permeate is obtained;

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

[0041] (5) Selective adsorption with ion exchange resin: The retentate 1 obtained in step (4) was subjected to ion exchange resin adsorption, and the adsorption pH value was 5.0. Within this pH range, bovine colostrum immunoglobulin and lactoferrin were both positively charged and could be selectively adsorbed on the ion exchange resin. The salt concentration of the sample buffer during the adsorption process was 0.05 mol / L, and the adsorption time was 40 min. The cation exchange resin used was Mitsubishi DIAION SK1B strongly acidic cation exchange resin.

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

[0043] (7) The permeate 2 collected in step (4) and the eluate collected in step (6) are mixed and then freeze-dried 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 model in the adjustment step (5) is DuPont AmberLite IR-120 strongly acidic cation exchange resin; other parameters and conditions are the same as those in Example 1.

[0046] Example 3

[0047] The only difference from Example 1 is that the elution conditions in step (6) are adjusted to: elution with a mass fraction of 0.3% NaCl solution of 3 times the volume of the resin to remove impurity proteins with weak adsorption properties on the surface, and then elution with a mixed solution of 5% NaCl and 0.05% sodium acetate, the eluate is collected, and the temperature of the elution process is maintained at 30°C; 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), the permeate obtained in step (3) is directly subjected to nanofiltration, and the retentate after nanofiltration is used in subsequent processes such as step (5); other parameters and conditions are the same as those 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 concentrated by the ultrafiltration membrane and the eluate 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 those 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 of step (4) are directly mixed. Other parameters and conditions are the same as those of Example 1.

[0056] Comparative Example 5

[0057] The only difference from Example 1 is that in step (6), instead of using a 0.5% by mass NaCl solution for elution and impurity removal, a mixed solution of 4% by mass NaCl and 0.05% by mass sodium acetate is directly used for elution. Other parameters and conditions are the same as those in Example 1.

[0058] Comparative Example 6

[0059] The only difference from Example 1 is that in step (7), the eluate 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 use of sodium acetate solution is omitted during the elution process in step (6), and the other parameters and conditions are the same as those in Example 1.

[0062] Result Analysis

[0063] The performance of the bovine colostrum immunoglobulins obtained in the examples and comparative examples was analyzed, and the results are shown in Table 1 and Figures 1 to 3 As shown:

[0064] Table 1 Components of the products of each embodiment and comparative example

[0065]

[0066]

[0067] From the table and Figures 1-2 It can be seen that each embodiment can meet the requirements of immunoglobulin content greater than 0.4g / g and lactoferrin content greater than 3.2mg / g. After adjusting steps (4)-(6) in Comparative Examples 1-5, the contents of immunoglobulin and lactoferrin are greatly affected. Among them, Comparative Examples 1, 2 and 4 have the greatest impact on the contents of immunoglobulin and lactoferrin. This shows that the use of an ultrafiltration and nanofiltration two-stage membrane filtration system 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] In comparative example 3, a nanofiltration membrane with a higher molecular weight cutoff was used in step (4), which reduced the immunoglobulin and lactoferrin contents to a certain extent and did not 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 pass through the membrane, increasing the impurity protein content, and thus causing the target protein content to decrease.

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

[0070] Comparative Example 6 is different from Example 1 in that the eluate obtained in step (6) is used as the final product. The obtained product is characterized in that the content of immunoglobulin and lactoferrin can meet the product requirements, but 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 the bovine colostrum obtained is extremely low, among which transforming growth factor α, taurine, and folic acid are not detected. This shows 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 the bovine colostrum, such as epidermal growth factor EGF, insulin-like growth factor IGF-1, transforming growth factor α, taurine, and folic acid, thereby giving the product multiple nutritional functions.

[0071] Comparative Example 7 significantly reduced the elution efficiency of immunoglobulin and lactoferrin compared with Example 1 (e.g. Figure 3 ), in a common salt ion neutral environment, the adsorption of lactoferrin and cationic resin is strong, resulting in some lactoferrin being difficult to elute at a set salt concentration. However, in the present invention, sodium acetate solution is used to adjust the alkalinity of the eluent, thereby achieving the purpose of improving the elution efficiency by regulating the dual effects of the solute's own charge and the electrostatic shielding of salt ions, and ultimately effectively increasing the yields of immunoglobulins and lactoferrin.

[0072] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing highly active bovine colostrum immunoglobulin by multi-stage membrane filtration combined with ion exchange resin adsorption, characterized in that: The method comprises the following steps: (1) Raw material pretreatment: The colostrum is filtered and centrifuged to remove impurities and fat to obtain defatted colostrum; (2) Acidification precipitation: Adjust the pH value of defatted bovine colostrum to 4-5, let it settle, 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; (4) Ultrafiltration combined with nanofiltration separation: The permeate collected in step (3) is concentrated by an ultrafiltration membrane with a molecular weight cutoff of 10 to 50 kDa to obtain permeate 1 and retentate 1; the permeate 1 is then further concentrated and separated by a nanofiltration membrane with a molecular weight cutoff of 500 to 1500 Da. Concentration is stopped after the concentration ratio reaches 15 to 20 times to obtain retentate 2 and permeate 2; (5) Selective adsorption with ion exchange resin: The retentate 1 obtained in step (4) is adsorbed on an ion exchange resin and eluted after adsorption. The specific elution process is as follows: first, a 0.3-0.5% NaCl solution with a mass fraction of 1-3 times the volume of the resin is used for elution to remove impurities to remove impurity proteins with weak adsorption properties on the surface, and then a mixed solution with a mass fraction of 3-5% NaCl and 0.01-0.05% sodium acetate is used for elution, and the eluate is collected; (6) The permeate 2 collected in step (4) and the eluate collected in step (6) are mixed and then freeze-dried to obtain bovine colostrum immunoglobulin rich in highly active ingredients.

2. The method according to claim 1, characterized in that The pore size of the ceramic membrane in step (3) is 20-50 nm.

3. The method according to claim 1, characterized in that The ion exchange resin in step (5) is a strongly acidic cation exchange resin.

4. The method according to claim 3, characterized in that The strong acid cation exchange resin includes any one of Mitsubishi DIAION SK1B strong acid cation exchange resin and DuPont AmberLite IR-120 strong acid cation exchange resin.

5. The method according to claim 1, wherein The adsorption conditions of step (5) are as follows: the salt concentration of the adsorption process control buffer is 0.02-0.1 mol / L, the pH value is 5.0-6.0, and the time is 20-60 min.

6. Highly active bovine colostrum immunoglobulin obtained by the method according to any one of claims 1 to 5.

7. The highly active bovine colostrum immunoglobulin according to claim 6, characterized in that The immunoglobulin content in the highly active bovine colostrum immunoglobulin is ≥40%, the immune activity retention rate is ≥90%, and the small molecule growth factors and amino acid active components in the bovine colostrum are retained.

8. The highly active bovine colostrum immunoglobulin according to claim 6, characterized in that The highly active bovine colostrum immunoglobulin has an immunoglobulin content greater than 0.4 g / g, a lactoferrin content ≥3.2 mg / g, an epidermal growth factor (EGF) content ≥1.5 μg / g, an insulin-like growth factor (IGF-1) content ≥4.0 μg / g, a transforming growth factor α content 1.2 ≥μg / g, a taurine content ≥0.25 μg / g, and a folic acid content ≥0.25 μg / g.

9. Use of the highly active bovine colostrum immunoglobulin according to any one of claims 6 to 8 in the preparation of drugs for enhancing immunity, preventing and assisting in the treatment of infectious diseases.

10. A preparation comprising the highly active bovine colostrum immunoglobulin according to any one of claims 6 to 8, characterized in that: The preparation is in the form of oral liquid, capsule, tablet or powder, and is used for enhancing immunity, preventing and assisting in the treatment of infectious diseases.

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