Method for improving stability of egg yolk antibody

Abstract

The invention discloses a method for improving the stability of an egg yolk antibody, which comprises the following steps: (1) adding a stabilizing additive into an egg yolk antibody solution to form a mixed solution, and adding a buffer system to adjust the pH value of the solution to 7.0; (2) drying the yolk antibody solution treated in the step (1) to prepare yolk antibody freeze-dried powder; the stabilizing additive in the mixed solution comprises the following raw materials in percentage by mass: 3-8% of trehalose, 3-8% of cane sugar, 2-7% of glycerol, 2-5% of mannitol, 2-7% of glycine and 2-5% of proline; the drying treatment mode is a combination of vacuum freeze drying and spray drying. The egg yolk antibody treated by the method has the advantages that the storage stability at normal temperature is improved by 30-50%, the immunocompetence retention time is prolonged by 2-3 times under the refrigeration condition of 4 DEG C, the application range of the egg yolk antibody is greatly widened, and the egg yolk antibody can be widely applied to the fields of immunodiagnosis, disease prevention and treatment, functional food addition and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the stability of egg yolk antibodies. Background Art

[0002] As a biological product with unique advantages, egg yolk antibodies have shown potential application prospects in many fields such as immunodiagnosis, disease prevention and treatment, and food safety testing. However, their inherent defect of poor stability has seriously restricted their widespread application and market promotion. Egg yolk antibodies are easily negatively affected by various factors such as temperature, humidity, light, pH changes, and repeated freezing and thawing, resulting in reduced or even loss of their activity, which in turn affects their efficacy and use results. Therefore, the development of a universal and effective method to improve the stability of egg yolk antibodies is of urgent practical need and important scientific significance. Summary of the Invention

[0003] To address the poor stability of egg yolk antibodies during storage and application, the present invention provides a method for improving the stability of egg yolk antibodies. This method can increase the shelf life and maintain the biological activity of egg yolk antibodies, thereby expanding the application range and practical value of egg yolk antibodies.

[0004] The technical solutions of the present invention are as follows:

[0005] The object of the present invention is to provide a method for improving the stability of egg yolk antibodies, comprising the following steps:

[0006] (1) adding a stabilizing additive to the egg yolk antibody solution to form a mixed solution, and then adding a buffer system to adjust the pH of the solution to 7.0;

[0007] (2) drying the egg yolk antibody solution treated in step (1) to obtain egg yolk antibody lyophilized powder;

[0008] The mass percentages of the raw materials of the stabilizing additives in the mixed solution are: 3-8% trehalose, 3-8% sucrose, 2-7% glycerol, 2-5% mannitol, 2-7% glycine and 2-5% proline;

[0009] The drying method is a combination of vacuum freeze drying and spray drying.

[0010] In one embodiment of the present invention, the egg yolk antibody solution is prepared by extracting from egg yolk by a two-step salting-out method.

[0011] In one embodiment of the present invention, the egg yolk antibody solution is prepared by:

[0012] (1) Select fresh commercial eggs, separate the yolk and egg white, and obtain the yolk for later use;

[0013] (2) diluting the egg yolk with sterile water in a volume ratio of 1:4-5, salting out and centrifuging in the first step; the ammonium sulfate saturation of the first step salting out is 35%-40%;

[0014] (3) second step of salting out and centrifugation; the ammonium sulfate saturation used in the second step of salting out is 45-50%;

[0015] (4) Remove impurities by ultrafiltration combined with ion exchange chromatography.

[0016] In one embodiment of the present invention, in steps (2) and (3), the salting-out conditions are: 5-7° C., and standing for 5-6 hours.

[0017] In one embodiment of the present invention, in steps (2) and (3), the centrifugal conditions are: a rotation speed of 9000-10000 rpm and a time of 20-25 min.

[0018] In one embodiment of the present invention, in step (4), the ultrafiltration method uses an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa; the ion exchange chromatography uses an anion exchange chromatography column, and linear gradient elution is performed with a phosphate buffer with a concentration of 0.03M-0.05M and a pH of 7.0-7.2 to collect the elution peak containing the egg yolk antibody, thereby effectively removing impurities such as foreign proteins and significantly improving the purity and stability of the egg yolk antibody; the phosphate buffer also contains 0.1M-0.3M NaCl.

[0019] In one embodiment of the present invention, the prepared egg yolk antibody solution has higher purity, fewer impurities, and the solution quality is controllable.

[0020] In one embodiment of the present invention, the buffer system is prepared by mixing phosphate buffer and Tris buffer in a mass ratio of 2:1.

[0021] Phosphate buffer has good buffering capacity and can effectively stabilize the pH value of the solution; Tris buffer has a wide buffering range and low biological toxicity. The combination of the two can better adapt to the stability requirements of egg yolk antibodies under different environmental conditions.

[0022] The buffer system maintains the stability of the solution's pH, effectively resisting external factors (such as temperature changes and microbial metabolites) that interfere with the stability of the egg yolk antibody, ensuring that the antibody remains active for a long time. A high or low pH value will affect the stability of the egg yolk antibody, ultimately leading to a decrease in its activity and a reduction in its activity retention rate during storage.

[0023] In one embodiment of the present invention, the drying process is performed by spray drying followed by vacuum freeze drying, with a total drying time of 5-18 hours.

[0024] In one embodiment of the present invention, the drying process is to first spray into droplets of appropriate size, then rapidly freeze using freezing technology, and finally perform sublimation drying. The total drying time is 5-18 hours.

[0025] In one embodiment of the present invention, the vacuum freeze drying temperature is liquid nitrogen -196°C, the vacuum degree is 1-100 Pa, the sublimation temperature is -40°C to -20°C; and the spray pressure is 0.2-0.8 MPa.

[0026] The pre-freezing temperature for vacuum freeze drying is below -196°C, and the liquid nitrogen flow rate can be 5-10 L / min. The vacuum degree of the vacuum drying chamber reaches 1-100 Pa, the sublimation temperature is between -40°C and -20°C, and the drying time is 5-18 hours. During the spraying process, the spray pressure is precisely controlled to 0.2-0.8 MPa to ensure uniform atomization and the formation of appropriate droplet size, which is conducive to subsequent rapid freezing. The freezing process uses multi-stage freezing technology, initially cooling with cold air at -20°C to -10°C, and then entering the liquid nitrogen low-temperature environment (-196°C) for deep freezing. This not only reduces the damage to the antibody structure caused by ice crystal growth, but also improves freezing efficiency. During vacuum sublimation drying, the vacuum degree is strictly controlled between 1-100 Pa and the temperature is maintained between -40°C and -20°C to ensure smooth water sublimation and avoid temperature and pressure fluctuations that may cause antibody denaturation.

[0027] The spray freeze-drying method combines vacuum freeze drying with spray drying. First, the egg yolk antibody solution is sprayed through the spray dryer's nozzle. The spray pressure is generally set between 0.2 and 0.8 MPa, which ensures uniform atomization of the egg yolk solution. The distance between the nozzle and the freezing zone is adjusted, typically between 10 and 30 cm, to ensure smooth entry of the atomized droplets into the freezing zone and prevent them from coalescing or sticking during flight. Within the freezing zone, the atomized droplets are rapidly frozen using liquid nitrogen. The liquid nitrogen flow rate is adjusted based on the spray velocity and the number of droplets to ensure rapid freezing of each droplet. Generally, a liquid nitrogen flow rate of 5 to 10 L / min is recommended, depending on the size of the equipment and the spray volume. The frozen ice particles are transferred to a vacuum drying chamber, and the vacuum pump is activated to achieve a vacuum level of 1 to 100 Pa. During the drying process, the temperature is maintained between -40°C and -20°C. The drying progress is monitored by monitoring the pressure and temperature sensors on the vacuum drying chamber, as well as the amount of water vapor collected. The drying process may take anywhere from a few hours to more than ten hours, depending on the number and size of the ice particles.

[0028] In one embodiment of the present invention, the prepared lyophilized egg yolk antibody powder is packaged using a multi-layer composite packaging material with high barrier properties, such as a composite material of aluminum foil and polyethylene or polypropylene. This packaging material has excellent barrier properties to oxygen, water vapor, and light, effectively preventing environmental factors from eroding the egg yolk antibody and ensuring its stability during storage. The dried egg yolk antibody powder is sealed in a packaging bag or bottle made of this material, providing a good protective barrier and extending its shelf life and activity.

[0029] The beneficial technical effects of the present invention are:

[0030] The present invention utilizes the steric hindrance effect and hydrogen bonding of polysaccharide molecules to reduce the interaction between antibody molecules and reduce the risk of aggregation. Trehalose can replace water molecules and bind to antibodies during the drying process of egg yolk antibodies, forming a stable glassy structure and effectively preventing antibody denaturation; sucrose enhances its stability through hydrogen bonding with antibody molecules. Glycerol can lower the freezing point of the solution, reduce the damage of ice crystals to the antibody structure, and at the same time has moisturizing properties to prevent excessive water loss of antibodies during the drying process; mannitol can increase the osmotic pressure of the solution, inhibit microbial growth, and further protect the antibodies. Glycine can adjust the ionic strength of the solution and reduce the impact of electrostatic effects on antibodies; proline can increase the stability of proteins and prevent them from aggregation and denaturation.

[0031] The egg yolk antibodies treated by the method of the present invention have storage stability improved by 30%-50% at room temperature, and the immune activity retention time is extended by 2-3 times under refrigerated conditions at 4°C, which greatly broadens the application range of egg yolk antibodies and can be widely used in the fields of immune diagnosis, disease prevention and treatment, and functional food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the drying process of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0034] In the following embodiments of the present invention, the preparation method of the egg yolk antibody solution is as follows:

[0035] (1) Select fresh commercial eggs, gently separate the yolk and egg white, and obtain the yolk for later use;

[0036] (2) The egg yolk was diluted with sterile water in a volume ratio of 1:4, and the first step was salting out and centrifugation; the ammonium sulfate saturation of the first step was 35%; the salting out conditions were: 5°C, standing for 6 hours; the centrifugation conditions were: speed of 9000 rpm, centrifugation for 25 minutes;

[0037] (3) Second step: salting out and centrifugation; the second step: salting out ammonium sulfate saturation is 45%; salting out conditions are: 5°C, standing for 6 hours; centrifugation conditions are: speed of 9000 rpm, centrifugation for 25 minutes;

[0038] (4) Impurities were removed by ultrafiltration combined with ion exchange chromatography. The ultrafiltration method used an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa. The ion exchange chromatography used an anion exchange column and linear gradient elution was performed with phosphate buffer (0.04 M, pH 7.0) containing 0.2 M NaCl. The elution peak containing the egg yolk antibody was collected, thereby effectively removing impurities such as foreign proteins and significantly improving the purity and stability of the egg yolk antibody.

[0039] In the following embodiments of the present invention, the spray freeze drying treatment method is spray drying first and then vacuum freeze drying. The vacuum freeze drying temperature is liquid nitrogen -196°C, the vacuum degree is 100 Pa, the sublimation temperature is -40°C; and the spray pressure is 0.8 MPa.

[0040] Example 1

[0041] A stabilizing additive was added to the egg yolk antibody solution to form a mixed solution containing 5wt% trehalose, 5wt% sucrose, 4wt% glycerol, 3wt% mannitol, 4wt% glycine and 3wt% proline. The system was adjusted with a mixed buffer (phosphate buffer and Tris buffer mixed in a mass ratio of 2:1, pH 7.0), and then spray freeze-dried to obtain a dry egg yolk antibody powder.

[0042] Example 2

[0043] The same as Example 1, except that the mixed solution contains 3 wt% trehalose, 3 wt% sucrose, 2 wt% glycerol, 2 wt% mannitol, 2 wt% glycine and 2 wt% proline.

[0044] Example 3

[0045] The same as Example 1, except that the mixed solution contains 8 wt% trehalose, 8 wt% sucrose, 7 wt% glycerol, 5 wt% mannitol, 7 wt% glycine and 5 wt% proline.

[0046] Comparative Example 1

[0047] The same as Example 1, except that the mixed solution contains 5 wt% trehalose, 4 wt% glycerol, 3 wt% mannitol, 4 wt% glycine and 3 wt% proline.

[0048] Comparative Example 2

[0049] The same as Example 1, except that the mixed solution contains 5 wt% sucrose, 4 wt% glycerol, 3 wt% mannitol, 4 wt% glycine and 3 wt% proline.

[0050] Comparative Example 3

[0051] The same as Example 1, except that the mixed solution contains 5 wt% trehalose, 5 wt% sucrose, 4 wt% glycerol, 4 wt% glycine and 3 wt% proline.

[0052] Comparative Example 4

[0053] The same as Example 1, except that the mixed solution contains 5 wt% trehalose, 5 wt% sucrose, 3 wt% mannitol, 4 wt% glycine and 3 wt% proline.

[0054] Comparative Example 5

[0055] The same as Example 1, except that the mixed solution contains 5 wt% trehalose, 5 wt% sucrose, 4 wt% glycerol, 3 wt% mannitol, and 4 wt% glycine.

[0056] Comparative Example 6

[0057] The same as Example 1, except that the mixed solution contains 5 wt% trehalose, 5 wt% sucrose, 4 wt% glycerol, 3 wt% mannitol and 3 wt% proline.

[0058] Test example:

[0059] 1. The egg yolk antibody powders prepared in the Examples and Comparative Examples were stored at 4°C, 25°C, and 37°C, respectively, and their activity was regularly tested using an enzyme-linked immunosorbent assay (ELISA). The results showed that after nine months of storage at 4°C, the egg yolk antibody activity retention rate was still as high as 92%; after six months of storage at 25°C, the activity retention rate was 80%; and after three months of storage at 37°C, the activity retention rate was 65%, significantly superior to the stability of egg yolk antibodies not treated by the method of the present invention.

[0060] To further highlight the advantages of the method of the present invention, the activity retention rate of the egg yolk antibody was compared with that of the egg yolk antibody that was not treated by the method of the present invention at the same temperature. After storage at 4°C for 9 months, the activity retention rate of the untreated egg yolk antibody was only 60%; after storage at 25°C for 6 months, the activity retention rate was 40%; after storage at 37°C for 3 months, the activity retention rate was 20%. At the same time, based on Example 1, a comparative experiment was set up to reduce one additive, and the activity retention rate of the egg yolk antibody after adding only a single additive and the specific raw material ratio of the present invention at different storage temperatures was compared as follows: Specific experimental data are shown in Table 1 below:

[0061] Table 1

[0062] Storage temperature Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 4℃ 92％ 83％ 80％ 87％ 85％ 84％ 82％ 25℃ 80％ 70％ 65％ 75％ 70％ 72％ 68％ 37℃ 65％ 55％ 50％ 60％ 55％ 56％ 52％

[0063]

[0064] It can be seen that the stability of egg yolk antibodies treated by the method of the present invention is significantly better than that of untreated egg yolk antibodies and egg yolk antibodies with one protective agent reduced, which fully proves the effectiveness of the method of the present invention in improving the stability of egg yolk antibodies.

[0065] 2. The obtained dry powder was placed in a simulated gastrointestinal environment (incubated in simulated gastric fluid (components include: hydrochloric acid, sodium chloride, pepsin and water) at pH 2.0 for 2 hours, and then in simulated intestinal fluid (components include: phosphate buffer, trypsin, pancreatic lipase, bile salts, sodium chloride and water) at pH 7.5 for 4 hours. The antibody integrity was detected by Western blot and the activity was detected by ELISA.

[0066] The results showed that the treated egg yolk antibodies remained largely intact in the simulated gastrointestinal environment, with an activity retention rate of 68%. However, the untreated egg yolk antibodies were severely degraded under the same conditions, with almost no detectable activity. A comparative test was also conducted using only a single additive of the same type. The specific results are shown in Table 2 below:

[0067] Table 2

[0068] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Activity retention rate 68％ 53％ 50％ 58％ 55％ 54％ 52％

[0069] This fully demonstrates that the method of the present invention significantly improves the stability of egg yolk antibodies in complex environments, and further illustrates that the effect will be significantly worse if the combination method is not limited by the present invention.

[0070] 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 improving the stability of egg yolk antibodies, characterized in that: The method comprises the following steps: (1) adding a stabilizing additive to the egg yolk antibody solution to form a mixed solution, and then adding a buffer system to adjust the pH of the solution to 7.0; (2) drying the egg yolk antibody solution treated in step (1) to obtain egg yolk antibody lyophilized powder; The mass percentages of the raw materials of the stabilizing additives in the mixed solution are: 3-8% trehalose, 3-8% sucrose, 2-7% glycerol, 2-5% mannitol, 2-7% glycine and 2-5% proline; The drying method is a combination of vacuum freeze drying and spray drying.

2. The method according to claim 1, characterized in that The egg yolk antibody solution is extracted from the egg yolk by a two-step salting-out method.

3. The method according to claim 1, characterized in that The preparation method of egg yolk antibody solution is as follows: (1) Select fresh commercial eggs, separate the yolk and egg white, and obtain the yolk for later use; (2) diluting the egg yolk with sterile water in a volume ratio of 1:4-5, salting out and centrifuging in the first step; the ammonium sulfate saturation of the first step salting out is 35-40%; (3) second step of salting out and centrifugation; the ammonium sulfate saturation used in the second step of salting out is 45-50%; (4) Remove impurities by ultrafiltration combined with ion exchange chromatography.

4. The method according to claim 3, characterized in that In steps (2) and (3), the salting-out conditions are: 5-7° C. and standing for 5-6 h.

5. The method according to claim 3, characterized in that In steps (2) and (3), the centrifugal conditions are: a rotation speed of 9000-10000 rpm and a time of 20-25 minutes.

6. The method according to claim 3, characterized in that In step (4), the ultrafiltration method uses an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa; the ion exchange chromatography uses an anion exchange chromatography column, and linear gradient elution is performed with a phosphate buffer with a concentration of 0.03M-0.05M and a pH of 7.0-7.2; the phosphate buffer also contains 0.1M-0.3M NaCl.

7. The method according to claim 1, characterized in that The buffer system was prepared by mixing phosphate buffer and Tris buffer in a mass ratio of 2:

1.

8. The method according to claim 1, characterized in that The drying method is spray drying followed by vacuum freeze drying, with a total drying time of 5-18 hours.

9. The method according to claim 8, characterized in that The temperature of vacuum freeze drying is liquid nitrogen -196°C, the vacuum degree is 1-100 Pa, the sublimation temperature is -40°C to -20°C; and the spray pressure is 0.2-0.8 MPa.

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