CN116102641A - Method for extracting egg yolk antibody by pasteurization - Google Patents
Method for extracting egg yolk antibody by pasteurization Download PDFInfo
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- CN116102641A CN116102641A CN202310056805.7A CN202310056805A CN116102641A CN 116102641 A CN116102641 A CN 116102641A CN 202310056805 A CN202310056805 A CN 202310056805A CN 116102641 A CN116102641 A CN 116102641A
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- egg yolk
- pasteurization
- yolk antibody
- antibody
- extracting
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- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 65
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 44
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 44
- 235000013345 egg yolk Nutrition 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000009928 pasteurization Methods 0.000 title claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 21
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 9
- 238000001816 cooling Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 8
- 239000000243 solution Substances 0.000 description 19
- 235000013601 eggs Nutrition 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 9
- 239000003223 protective agent Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 210000003278 egg shell Anatomy 0.000 description 4
- 235000014103 egg white Nutrition 0.000 description 4
- 210000000969 egg white Anatomy 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 230000000521 hyperimmunizing effect Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 229940031551 inactivated vaccine Drugs 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 210000002826 placenta Anatomy 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000003837 chick embryo Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
The invention belongs to the technical field of biological products, and particularly relates to an extraction method for pasteurizing in an egg yolk antibody extraction process. A method of extracting egg yolk antibody by pasteurization, the method comprising: adding disodium hydrogen phosphate solution into yolk, adjusting pH to neutrality at a volume ratio of 1:3, stirring uniformly, and performing pasteurization; cooling to room temperature, and adding into extraction system to extract yolk antibody. The invention provides a method for extracting egg yolk antibody by pasteurization, which can ensure sterilization and disinfection, maintain the property of egg yolk, and maintain the stable titer.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to an extraction method for pasteurizing in an egg yolk antibody extraction process.
Background
Egg yolk antibody (IgY): the egg yolk produced by the egg yolk can be used for extracting corresponding antibodies by injecting the egg yolk for preventing and treating corresponding diseases, and the preparation is called egg yolk antibody.
The egg yolk antibody gradually enters the chick embryo blood in the chick embryo hatching process, provides passive immune protection for the chick just coming out of the shell, and plays an important role in chick disease prevention. However, the extraction method is still one of the factors restricting the mass production of IgY.
Currently, in the production of egg yolk antibodies, low-temperature extraction or normal-temperature extraction is generally used to prevent antibody denaturation. But the yolk is rich in nutrition, so that the yolk is easy to be infected by mixed bacteria in the production process, and sufficient nutrition is provided for the production of the mixed bacteria. The low-temperature extraction has longer duration, longer production time and higher production cost, and can not remove microorganisms carried by the production raw materials, and the normal-temperature extraction is extremely easy to infect infectious microbe, leads to production failure and can not effectively sterilize and disinfect egg yolk in the early stage.
Disclosure of Invention
The invention aims to provide a method for pasteurizing yolk and a compound extracting solution together, which ensures that the yolk antibody can be pasteurized, the titer is maintained at a normal level, and the yolk antibody can play a role in killing bacteria and viruses.
The technical scheme adopted by the invention is as follows: a method of extracting egg yolk antibody by pasteurization, the method comprising: adding disodium hydrogen phosphate solution into yolk, adjusting pH to neutrality at a volume ratio of 1:3, stirring uniformly, and performing pasteurization; cooling to room temperature, and adding into extraction system to extract yolk antibody.
Preferably, the final concentration of the disodium hydrogen phosphate solution is 0.04% -0.16%.
Preferably, the pH is 7.5.
The invention provides a method for pasteurizing egg yolk antibody, which can maintain the property of egg yolk unchanged and the potency stable while guaranteeing sterilization and disinfection.
The egg yolk antibody pasteurization extraction method provided by the invention can be used for realizing high production efficiency and high yield on the basis of sterilization and disinfection, and is suitable for large-scale production. The yolk antibody obtained by the method has high titer and high yield, can replace the existing antibody extraction process, and has good popularization value. Detailed description of the preferred embodiments.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated below. Preferred embodiments of the present invention are presented. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Embodiment 1 this example provides a method for extracting egg yolk antibody by pasteurization, comprising the steps of:
(1) Immunizing 150-day-old laying hens by using an adenovirus inactivated vaccine to obtain hyperimmune eggs, immersing the eggs in a 0.1% new Jieer water solution for disinfection for 15 minutes, and taking out the water on the surface of the eggs to be dried. Breaking eggshells, removing egg white, placenta and lacing, and collecting yolk;
(2) Preparing a protective agent: disodium hydrogen phosphate solution was prepared to a final concentration of 0.02%.
(3) The yolk and the protective agent are mixed and stirred uniformly according to the volume ratio of 1:3, the pH value is regulated to 7.5, and then pasteurization is carried out: heating to 60deg.C, and standing for 30min;
(4) And (3) cooling the egg yolk solution sterilized in the step (3) to room temperature, and adding the cooled egg yolk solution into a conventional extraction system to extract the egg yolk antibody.
Embodiment 2 this embodiment provides a method for extracting egg yolk antibody by pasteurization, comprising the steps of:
(1) Immunizing 150-day-old laying hens by using an adenovirus inactivated vaccine to obtain hyperimmune eggs, immersing the eggs in a 0.1% new Jieer water solution for disinfection for 15 minutes, and taking out the water on the surface of the eggs to be dried. Breaking eggshells, removing egg white, placenta and lacing, and collecting yolk;
(2) Preparing a protective agent: disodium hydrogen phosphate solution was prepared to a final concentration of 0.04%.
(3) Mixing yolk and protective agent in the volume ratio of 1:3, stirring well, regulating pH to 7.5, and pasteurizing: heating to 60deg.C, and standing for 30min;
(4) And (3) cooling the egg yolk solution sterilized in the step (3) to room temperature, and adding the cooled egg yolk solution into a conventional extraction system to extract the egg yolk antibody.
Embodiment 3 this embodiment provides a method for extracting egg yolk antibody by pasteurization, comprising the steps of:
(1) Immunizing 150-day-old laying hens by using an adenovirus inactivated vaccine to obtain hyperimmune eggs, immersing the eggs in a 0.1% new Jieer water solution for disinfection for 15 minutes, and taking out the water on the surface of the eggs to be dried. Breaking eggshells, removing egg white, placenta and lacing, and collecting yolk;
(2) Preparing a protective agent: disodium hydrogen phosphate solution was prepared to a final concentration of 0.08%.
(3) Mixing yolk and protective agent in the volume ratio of 1:3, stirring well, regulating pH to 7.5, and pasteurizing: heating to 60deg.C, and standing for 30min;
(4) And (3) cooling the egg yolk solution sterilized in the step (3) to room temperature, and adding the cooled egg yolk solution into a conventional extraction system to extract the egg yolk antibody.
Embodiment 4 this embodiment provides a method for extracting egg yolk antibody by pasteurization, comprising the steps of:
(1) Immunizing 150-day-old laying hens by using an adenovirus inactivated vaccine to obtain hyperimmune eggs, immersing the eggs in a 0.1% new Jieer water solution for disinfection for 15 minutes, and taking out the water on the surface of the eggs to be dried. Breaking eggshells, removing egg white, placenta and lacing, and collecting yolk;
(2) Preparing a protective agent: disodium hydrogen phosphate solution was prepared at a final concentration of 0.16%.
(3) Mixing yolk and protective agent in the volume ratio of 1:3, stirring well, regulating pH to 7.5, and pasteurizing: heating to 60deg.C, and standing for 30min;
(4) And (3) cooling the egg yolk solution sterilized in the step (3) to room temperature, and adding the cooled egg yolk solution into a conventional extraction system to extract the egg yolk antibody.
EXAMPLE 5 bacterial detection of the yolk antibody extracted in example 2 above was performed 48 hours after extraction
Inoculating the extracted yolk antibody on common LB agar medium, culturing in a constant temperature incubator at 37deg.C for 36 hr, observingNo bacteria growth. The normal temperature extraction method was used as a control group. The results are shown in Table 1, and when the dilution ratio is more than 10 -6 The colony count is obviously less than that of the normal temperature extraction.
TABLE 1 egg yolk antibody sterilizing effect extracted in example 2
Example 6 titers of the yolk antibodies extracted in examples 1-4 were determined using the FADV-I kit, as follows:
sample preparation: each sample was diluted 1:100, using a 2-step procedure recommended.
The operation steps are as follows: according to the procedure in the kit instructions.
Results: to ensure that the assay is effective, the average absorbance of the negative control should be less than 0.30, the average absorbance of the positive control should be greater than or equal to 0.30, and the difference between the average absorbance of the positive and negative controls should be greater than 0.15.
The result judging method comprises the following steps:
the P value of the sample is greater than or equal to 0.5, which indicates that the FADV-I antibody is positive
Calculation of S/P value (average of sample to be tested-average of negative control)/(average of positive control-average of negative control) =s/P
2. Calculation of antibody titres
Lg potency = 1.1 x lgs/p+3.361
Anti-log = potency
3. Determination criteria: see table 2.
TABLE 2FADV-I kit result criterion
TABLE 3 determination of the titer of yolk antibodies extracted in examples 1 to 4 of the present invention
Through detection, the titer of the yolk antibody extracted by the method provided by the invention is detected to be qualified and is equal to the titer extracted at normal temperature. The method provided by the invention can maintain the property of the egg yolk antibody unchanged and maintain the stable titer while ensuring sterilization and disinfection.
Claims (3)
1. A method for extracting egg yolk antibody by pasteurization, which is characterized in that: the method comprises the following steps: adding disodium hydrogen phosphate solution into yolk, adjusting pH to neutrality at a volume ratio of 1:3, stirring uniformly, and performing pasteurization; cooling to room temperature, and adding into extraction system to extract yolk antibody.
2. The method for extracting egg yolk antibody by pasteurization according to claim 1, wherein: the final concentration of the disodium hydrogen phosphate solution is 0.04% -0.16%.
3. The method for extracting egg yolk antibody by pasteurization according to claim 1 or 2, wherein: the pH was 7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310056805.7A CN116102641A (en) | 2023-01-13 | 2023-01-13 | Method for extracting egg yolk antibody by pasteurization |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310056805.7A CN116102641A (en) | 2023-01-13 | 2023-01-13 | Method for extracting egg yolk antibody by pasteurization |
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| Publication Number | Publication Date |
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| CN116102641A true CN116102641A (en) | 2023-05-12 |
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| CN202310056805.7A Pending CN116102641A (en) | 2023-01-13 | 2023-01-13 | Method for extracting egg yolk antibody by pasteurization |
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| Country | Link |
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| CN (1) | CN116102641A (en) |
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- 2023-01-13 CN CN202310056805.7A patent/CN116102641A/en active Pending
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