Improved culture medium containing egg yolk protein and chicken embryo extract and preparation method thereof

Abstract

The invention discloses an improved culture medium containing egg yolk protein and a chicken embryo extract and a preparation method of the improved culture medium. The culture medium is mainly composed of egg yolk protein, a chicken embryo extract and a basic culture medium component according to a specific proportion. The improved culture medium is rich in various nutritional ingredients such as proteins, amino acids, vitamins and cell growth factors, can effectively promote the growth and proliferation of various cell lines and improve the efficiency and quality of cell culture, can be widely applied to the field of in-vitro culture of animal cells, provides a choice with higher quality and better cost effectiveness for cell culture, and has a wide application prospect. The problems that a traditional culture medium is insufficient in nutritional ingredients, poor in cell culture effect and the like are solved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro animal cell culture technology, specifically relating to a modified culture medium containing egg yolk protein and chicken embryo extract and its preparation method. Background Technology

[0002] In vitro culture of animal cells is a crucial technology in the field of bioengineering, widely applied in biopharmaceuticals, vaccine development, cell biology research, and many other areas. Culture medium, as a key substance in in vitro cell culture, provides the necessary nutrients and environmental conditions for cell growth, proliferation, and metabolism.

[0003] Traditional animal cell culture media primarily consist of basal medium supplemented with components such as serum. However, serum has a complex composition, containing various unknown factors, and its quality and batch-to-batch variations can negatively impact the stability and reproducibility of cell culture results. Furthermore, the limited availability and high cost of serum restrict its application in large-scale cell culture.

[0004] To overcome the shortcomings of traditional culture media, researchers are constantly developing novel serum-free or low-serum culture media. These media typically contain known nutrients and growth factors to replace the role of serum. However, existing serum-free or low-serum culture media still fall short in terms of the comprehensiveness and synergistic effect of nutrients, making it difficult to meet the growth needs of various cell lines, and cell culture efficiency and quality need further improvement. Summary of the Invention

[0005] The purpose of this invention is to provide an improved culture medium containing egg yolk protein and chicken embryo extract, and its preparation method. This improved culture medium is rich in various nutrients, which can effectively promote the growth and proliferation of various cell lines, improve the efficiency and quality of cell culture, and is more cost-effective.

[0006] An improved culture medium containing egg yolk protein and chicken embryo extract is composed of the following components in parts by weight: 5-15 parts egg yolk protein, 3-10 parts chicken embryo extract, and 75-92 parts basal culture medium components; wherein the egg yolk protein is obtained by homogenizing egg yolk and subjecting it to a first sonication; wherein the chicken embryo extract is obtained by grinding chicken embryos, subjecting them to a second sonication, freezing them in liquid nitrogen, and melting them in a water bath.

[0007] Optionally, the ultrasonic power of the first ultrasound is 200-400W, and the ultrasound time is 5-15min.

[0008] Optionally, the ultrasonic power of the second ultrasound is 300-500W, and the ultrasound time is 10-20min; the temperature of the water in the water bath melting is 36-38℃.

[0009] Optionally, the basic culture medium is composed of the following components in parts by weight: 1-3 parts glucose, 5-10 parts amino acid mixture, 2-5 parts vitamin mixture, 10-20 parts inorganic salt mixture, and 62-82 parts water.

[0010] Optionally, the amino acid mixture comprises the following components in parts by mass: glycine 0.5-1.5 parts, alanine 0.5-1.5 parts, valine 0.5-1.5 parts, leucine 0.5-1.5 parts, isoleucine 0.5-1.5 parts, phenylalanine 0.3-0.8 parts, tyrosine 0.3-0.8 parts, tryptophan 0.1-0.3 parts, serine 0.5-1.5 parts, threonine 0.5-1.5 parts, cysteine ​​0.3-0.8 parts, methionine 0.3-0.8 parts, aspartic acid 0.5-1.5 parts, glutamic acid 0.5-1.5 parts, asparagine 0.5-1.5 parts, glutamine 0.5-1.5 parts, lysine 0.5-1.5 parts, arginine 0.5-1.5 parts, and histidine 0.3-0.8 parts.

[0011] Optionally, the vitamin mixture comprises the following components in parts by weight: 0.1-0.3 parts vitamin B1, 0.1-0.3 parts vitamin B2, 0.1-0.3 parts vitamin B6, 0.01-0.05 parts vitamin B12, 0.1-0.3 parts niacin, 0.1-0.3 parts calcium pantothenate, 0.05-0.15 parts folic acid, 0.01-0.05 parts biotin, 0.5-1.5 parts vitamin C, 0.01-0.05 parts vitamin D, and 0.05-0.15 parts vitamin E.

[0012] Optionally, the inorganic salt mixture comprises the following components in parts by mass: 5-10 parts sodium chloride, 0.5-2 parts potassium chloride, 0.1-0.5 parts calcium chloride, 0.1-0.5 parts magnesium sulfate, 0.5-2 parts sodium dihydrogen phosphate, 1-3 parts sodium bicarbonate, 0.001-0.005 parts copper sulfate, 0.005-0.015 parts zinc sulfate, 0.01-0.05 parts ferrous sulfate, and 0.001-0.005 parts manganese chloride.

[0013] A method for preparing the above-mentioned modified culture medium containing egg yolk protein and chicken embryo extract includes the following steps:

[0014] The egg yolk protein, the chicken embryo extract, and the basic culture medium components are stirred and mixed at 36-38°C for 15-30 minutes, and then sterilized to obtain the modified culture medium.

[0015] Optionally, the method for preparing the egg yolk protein includes:

[0016] (1) Obtain fresh eggs, clean them, and separate the yolks and whites in a sterile environment;

[0017] (2) Place the egg yolk in a sterile container, add an appropriate amount of sterile PBS buffer, and homogenize it using a homogenizer to obtain egg yolk homogenate.

[0018] (3) Place the egg yolk homogenate in an ultrasonic homogenizer and perform the first ultrasonic treatment. The ultrasonic power of the first ultrasonic treatment is 200-400W, the ultrasonic time is 5-15min, the first ultrasonic treatment is a first pulse ultrasonic treatment, and the first pulse ultrasonic treatment is ultrasonic for 3-5s, followed by a pause of 2-3s.

[0019] (4) After the first ultrasound, the mixture is centrifuged at 3-5℃ and 8000-10000r / min for 15-25min, and the supernatant is taken as egg yolk protein.

[0020] Optionally, the preparation method of the chicken embryo extract includes:

[0021] (1) Select healthy chicken embryos that have developed for 9-12 days and remove the embryonic eyes, beak, air sacs, membranous sacs, allantoic membranes, etc. under sterile conditions;

[0022] (2) Rinse the treated chicken embryos 3-5 times with sterile PBS buffer;

[0023] (3) Place the rinsed chicken embryos in a sterile mortar, add an appropriate amount of sterile PBS buffer, and grind until homogenized.

[0024] (4) Transfer the ground homogenate to a sterile centrifuge tube and place it in an ultrasonic homogenizer for a second ultrasonic treatment. The ultrasonic power of the second ultrasonic treatment is 300-500W and the ultrasonic time is 10-20min. The second ultrasonic treatment is a second pulse ultrasonic treatment, which lasts for 5-8s and is paused for 3-5s.

[0025] (5) After the second ultrasound, the mixture is placed in liquid nitrogen to freeze for 10-15 minutes, then taken out and thawed in a water bath at 36-38℃. The freezing and thawing process is repeated 3-5 times.

[0026] (6) Centrifuge the mixture at 3-5℃ and 10000-12000r / min for 20-30min, and take the supernatant, which is the chicken embryo extract.

[0027] Beneficial effects

[0028] The improved culture medium of this invention is rich in various nutrients such as proteins, amino acids, vitamins, and cell growth factors, which can provide comprehensive and sufficient nutritional support for cell growth and effectively promote the growth and proliferation of various cell lines. Detailed Implementation

[0029] One embodiment of the present invention provides a modified culture medium containing egg yolk protein and chicken embryo extract. This modified culture medium comprises the following components in parts by weight: 5-15 parts egg yolk protein, 3-10 parts chicken embryo extract, and 75-92 parts basal culture medium components. The egg yolk protein is obtained by homogenizing egg yolk and then sonicating it. The chicken embryo extract is obtained by grinding chicken embryos, followed by sonication, liquid nitrogen freezing, and water bath thawing. This component combination provides rich and balanced nutrition for cell growth, effectively promoting the growth and proliferation of various cell lines, improving cell culture efficiency and quality, while maintaining relatively low cost, thus exhibiting good practicality and economy.

[0030] In some embodiments, the ultrasonic power of the first ultrasound is 200-400W, and the ultrasound time is 5-15 minutes. The first ultrasound can fully extract the protein and other nutrients in the egg yolk, which is beneficial for them to exert their nutritional role in the culture medium and promote cell growth.

[0031] In some embodiments, the ultrasonic power of the second ultrasound is 300-500W, and the ultrasound time is 10-20 minutes; the water temperature in the water bath melting is 36-38℃. The second ultrasound can effectively break chicken embryo cells, releasing the nutrients and growth factors therein, and can better promote cell growth and proliferation.

[0032] In some embodiments, the basal culture medium comprises the following components in parts by weight: 1-3 parts glucose, 5-10 parts amino acid mixture, 2-5 parts vitamin mixture, 10-20 parts inorganic salt mixture, and 62-82 parts water. This formulation of the basal culture medium covers the essential nutrients required for cell growth. Glucose provides energy, while amino acids, vitamins, and inorganic salts meet the needs of cell metabolism and synthesis. The components work synergistically to lay a good foundation for cell growth.

[0033] In some embodiments, the amino acid mixture comprises the following components in parts by weight: glycine 0.5-1.5 parts, alanine 0.5-1.5 parts, valine 0.5-1.5 parts, leucine 0.5-1.5 parts, isoleucine 0.5-1.5 parts, phenylalanine 0.3-0.8 parts, tyrosine 0.3-0.8 parts, tryptophan 0.1-0.3 parts, serine 0.5-1.5 parts, threonine 0.5-1.5 parts, cysteine ​​0.3-0.8 parts, methionine 0.3-0.8 parts, aspartic acid 0.5-1.5 parts, glutamic acid 0.5-1.5 parts, asparagine 0.5-1.5 parts, glutamine 0.5-1.5 parts, lysine 0.5-1.5 parts, arginine 0.5-1.5 parts, and histidine 0.3-0.8 parts. This amino acid mixture contains a variety of amino acids essential for cell growth in an appropriate ratio, which can meet the needs of cells to synthesize biological macromolecules such as proteins, promote cell growth and proliferation, and improve the effect of cell culture.

[0034] In some embodiments, the vitamin mixture comprises the following components in parts by weight: 0.1-0.3 parts vitamin B1, 0.1-0.3 parts vitamin B2, 0.1-0.3 parts vitamin B6, 0.01-0.05 parts vitamin B12, 0.1-0.3 parts niacin, 0.1-0.3 parts calcium pantothenate, 0.05-0.15 parts folic acid, 0.01-0.05 parts biotin, 0.5-1.5 parts vitamin C, 0.01-0.05 parts vitamin D, and 0.05-0.15 parts vitamin E. The rational combination of multiple vitamins participates in various cellular metabolic processes, such as energy metabolism and substance synthesis, maintaining normal cellular physiological functions, enhancing cellular vitality and stress resistance, and promoting cell growth and proliferation in in vitro culture environments.

[0035] In some embodiments, the inorganic salt mixture comprises the following components in parts by weight: 5-10 parts sodium chloride, 0.5-2 parts potassium chloride, 0.1-0.5 parts calcium chloride, 0.1-0.5 parts magnesium sulfate, 0.5-2 parts sodium dihydrogen phosphate, 1-3 parts sodium bicarbonate, 0.001-0.005 parts copper sulfate, 0.005-0.015 parts zinc sulfate, 0.01-0.05 parts ferrous sulfate, and 0.001-0.005 parts manganese chloride. These inorganic salts maintain the osmotic pressure and pH stability of the culture medium, provide the necessary ionic environment for cells, participate in cellular physiological activities such as enzyme activation and signal transduction, ensure normal cell growth and metabolism, and improve the stability of cell culture.

[0036] Another embodiment of the present invention provides a method for preparing the above-mentioned modified culture medium containing egg yolk protein and chicken embryo extract, comprising the following steps: stirring and mixing egg yolk protein, chicken embryo extract and basic culture medium components at 36-38°C for 15-30 minutes, followed by sterilization to obtain the modified culture medium. This preparation method is simple and easy to implement, and is suitable for widespread application.

[0037] Optionally, the method for preparing egg yolk protein includes:

[0038] (1) Obtain fresh eggs, clean them, and separate the yolks and whites in a sterile environment;

[0039] (2) Place the egg yolk in a sterile container, add an appropriate amount of sterile PBS buffer, and homogenize it using a homogenizer to obtain egg yolk homogenate.

[0040] (3) Place the egg yolk homogenate in an ultrasonic homogenizer and perform the first ultrasonic treatment. The ultrasonic power of the first ultrasonic treatment is 200-400W and the ultrasonic time is 5-15min. The first ultrasonic treatment is a first pulse ultrasonic treatment, which lasts for 3-5 seconds and is paused for 2-3 seconds.

[0041] (4) After the first ultrasound, the mixture is centrifuged at 3-5℃ and 8000-10000r / min for 15-25min, and the supernatant is taken as egg yolk protein.

[0042] This preparation method can fully extract nutrients such as protein from egg yolks, and the operation is carried out in a sterile environment, which ensures the purity and safety of egg yolk protein, and is conducive to its nutritional role in the culture medium and promotes cell growth.

[0043] Optionally, the preparation method of chicken embryo extract includes:

[0044] (1) Select healthy chicken embryos that have developed for 9-12 days and remove the embryonic eyes, beak, air sacs, membranous sacs, allantoic membranes, etc. under sterile conditions;

[0045] (2) Rinse the treated chicken embryos 3-5 times with sterile PBS buffer;

[0046] (3) Place the rinsed chicken embryos in a sterile mortar, add an appropriate amount of sterile PBS buffer, and grind until homogenized.

[0047] (4) Transfer the ground homogenate to a sterile centrifuge tube and place it in an ultrasonic homogenizer for a second ultrasonic treatment. The ultrasonic power of the second ultrasonic treatment is 300-500W, the ultrasonic time is 10-20min, the second ultrasonic treatment is a second pulse ultrasonic treatment, the second pulse ultrasonic treatment is ultrasonic for 5-8s, and pause for 3-5s.

[0048] (5) After the second ultrasound, the mixture is placed in liquid nitrogen to freeze for 10-15 minutes, then taken out and thawed in a water bath at 36-38℃. The freezing and thawing process is repeated 3-5 times.

[0049] (6) Centrifuge the mixture at 3-5℃ and 10000-12000r / min for 20-30min, and take the supernatant, which is the chicken embryo extract.

[0050] This preparation method can effectively break chicken embryo cells, releasing their nutrients and growth factors. Through multiple rinsing and centrifugation steps, the purity of the chicken embryo extract is improved, and its biological activity is preserved, which can better promote cell growth and proliferation.

[0051] Example 1

[0052] In this embodiment:

[0053] 1. Methods for preparing egg yolk protein include:

[0054] (1) Obtain fresh eggs, clean them, and separate the yolks and whites in a sterile environment;

[0055] (2) Place the egg yolk in a sterile container, add an appropriate amount of sterile PBS buffer, and homogenize it using a homogenizer to obtain egg yolk homogenate.

[0056] (3) Place the egg yolk homogenate in an ultrasonic homogenizer and perform the first ultrasonic treatment. The ultrasonic power of the first ultrasonic treatment is 300W and the ultrasonic time is 10min. The first ultrasonic treatment is a first pulse ultrasonic treatment, which lasts for 4s and pauses for 2s.

[0057] (4) After the first ultrasound, the mixture is centrifuged at 4℃ and 9000r / min for 20min, and the supernatant is taken as egg yolk protein.

[0058] 2. The preparation methods of chicken embryo extract include:

[0059] (1) Select healthy chicken embryos that have developed for 9-12 days and remove the embryonic eyes, beak, air sacs, membranous sacs, allantoic membranes, etc. under sterile conditions;

[0060] (2) Rinse the treated chicken embryos four times with sterile PBS buffer;

[0061] (3) Place the rinsed chicken embryos in a sterile mortar, add an appropriate amount of sterile PBS buffer, and grind until homogenized.

[0062] (4) Transfer the ground homogenate to a sterile centrifuge tube and place it in an ultrasonic homogenizer for a second ultrasonic treatment. The ultrasonic power of the second ultrasonic treatment is 400W and the ultrasonic time is 12min. The second ultrasonic treatment is a second pulse ultrasonic treatment, which consists of 8 seconds of ultrasonic treatment followed by a 4-second pause.

[0063] (5) After the second ultrasound, the mixture was placed in liquid nitrogen and frozen for 12 minutes, then taken out and thawed in a 37°C water bath. The freezing and thawing were repeated 4 times.

[0064] (6) Centrifuge the mixture at 4℃ and 11000r / min for 25min, and take the supernatant, which is the chicken embryo extract.

[0065] 3. The mass fractions of the modified culture medium are: 8 parts egg yolk protein, 6 parts chicken embryo extract, and 86 parts basal culture medium components.

[0066] 4. The basic culture medium consists of the following components in parts by weight: 2 parts glucose, 7 parts amino acid mixture, 3 parts vitamin mixture, 15 parts inorganic salt mixture, and 73 parts water.

[0067] 5. The amino acid mixture consists of the following components in parts by mass: 1 part glycine, 1 part alanine, 1 part valine, 1 part leucine, 1 part isoleucine, 0.5 part phenylalanine, 0.5 part tyrosine, 0.2 part tryptophan, 1 part serine, 1 part threonine, 0.5 part cysteine, 0.5 part methionine, 1 part aspartic acid, 1 part glutamic acid, 1 part asparagine, 1 part glutamine, 1 part lysine, 1 part arginine, and 0.5 part histidine.

[0068] 6. The vitamin mixture consists of the following components in parts by weight: Vitamin B1 0.3 parts, Vitamin B2 0.3 parts, Vitamin B6 0.3 parts, Vitamin B12 0.05 parts, Niacin 0.3 parts, Calcium Pantothenate 0.3 parts, Folic Acid 0.15 parts, Biotin 0.05 parts, Vitamin C 1.5 parts, Vitamin D 0.05 parts, and Vitamin E 0.15 parts.

[0069] 7. The inorganic salt mixture is composed of the following components in parts by mass: 10 parts sodium chloride, 2 parts potassium chloride, 0.5 parts calcium chloride, 0.5 parts magnesium sulfate, 2 parts sodium dihydrogen phosphate, 3 parts sodium bicarbonate, 0.005 parts copper sulfate, 0.015 parts zinc sulfate, 0.05 parts ferrous sulfate, and 0.005 parts manganese chloride.

[0070] 8. The method for preparing the modified culture medium is as follows:

[0071] Egg yolk protein and chicken embryo extract were added to the basal culture medium and stirred at 300 rpm for 20 minutes at 37°C to ensure complete dissolution. The mixture was then sterilized by filtration through a 0.22 μm filter membrane to obtain the modified culture medium.

[0072] Example 2

[0073] Compared with Example 1, the difference is that the mass fractions of the modified culture medium are: 5 parts egg yolk protein, 10 parts chicken embryo extract, and 92 parts basal culture medium components.

[0074] Example 3

[0075] Compared with Example 1, the difference is that the mass fractions of the modified culture medium are: 15 parts egg yolk protein, 3 parts chicken embryo extract, and 75 parts basal culture medium components.

[0076] Comparative Example 1

[0077] In this comparative example, the culture medium was a traditional serum-containing medium with a serum content of 10%, and the basal medium was commercially available DMEM medium.

[0078] Comparative Example 2

[0079] Compared with Example 1, the difference is that the mass fractions of the modified culture medium are: 3 parts egg yolk protein, 15 parts chicken embryo extract, and 70 parts basal culture medium components.

[0080] Comparative Example 3

[0081] Compared with Example 1, the difference is that the mass fractions of the modified culture medium are: 18 parts egg yolk protein, 1 part chicken embryo extract, and 98 parts basal culture medium components.

[0082] Comparative Example 4

[0083] The difference from Example 1 is that egg yolk protein was replaced with an equal mass of chicken embryo extract.

[0084] Comparative Example 5

[0085] The difference from Example 1 is that chicken embryo extract was replaced with an equal mass of egg yolk protein.

[0086] Test case

[0087] Cell proliferation rate assay: The MTT assay was used. HeLa cells, CHO cells, and Vero cells in logarithmic growth phase were seeded into 96-well plates, 100 μL per well, at a cell density of 5 × 10⁶ cells / well. 3Cells / mL. Culture media from both the experimental and comparative examples were added, with 5 replicates per group. All wells were incubated at 37°C with 5% CO2 for 72 h. After incubation, 20 μL MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. The supernatant was then discarded, and 150 μL LDMSO was added to each well, shaken for 10 min to fully dissolve the crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. Cell proliferation rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, where the control group was comparative example 1. The test results are shown in the table below.

[0088]

[0089] As can be seen from the table above, the improved culture medium of the present invention can effectively promote the proliferation of various cell types.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0091] For those skilled in the art, based on the ideas of this invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A modified medium containing egg yolk protein and chicken embryo extract, characterized in that, The egg yolk protein is obtained by homogenizing and first ultrasonic treatment of egg yolk; the chicken embryo extract is obtained by grinding, second ultrasonic treatment, liquid nitrogen freezing and water bath melting of chicken embryo.

2. The modified medium containing egg yolk protein and chick embryo extract according to claim 1, wherein, The first ultrasonic treatment is performed at a power of 200-400 W for 5-15 min.

3. The modified medium containing egg yolk protein and chick embryo extract according to claim 1, wherein, The second ultrasonic treatment is performed at a power of 300-500 W for 10-20 min; the water in the water bath melting is at a temperature of 36-38℃.

4. The modified medium containing egg yolk protein and chick embryo extract according to claim 1, wherein, The base medium component is composed of the following components in parts by mass: glucose 1-3 parts, amino acid mixture 5-10 parts, vitamin mixture 2-5 parts, inorganic salt mixture 10-20 parts, and water 62-82 parts.

5. The modified medium containing egg yolk protein and chick embryo extract according to claim 4, wherein, The amino acid mixture is composed of the following components in parts by mass: glycine 0.5-1.5 parts, alanine 0.5-1.5 parts, valine 0.5-1.5 parts, leucine 0.5-1.5 parts, isoleucine 0.5-1.5 parts, phenylalanine 0.3-0.8 parts, tyrosine 0.3-0.8 parts, tryptophan 0.1-0.3 parts, serine 0.5-1.5 parts, threonine 0.5-1.5 parts, cysteine 0.3-0.8 parts, methionine 0.3-0.8 parts, aspartic acid 0.5-1.5 parts, glutamic acid 0.5-1.5 parts, asparagine 0.5-1.5 parts, glutamine 0.5-1.5 parts, lysine 0.5-1.5 parts, arginine 0.5-1.5 parts, and histidine 0.3-0.8 parts.

6. The modified medium containing egg yolk protein and chick embryo extract according to claim 4, wherein, The vitamin mixture is composed of the following components in parts by mass: vitamin B1 0.1-0.3 parts, vitamin B2 0.1-0.3 parts, vitamin B6 0.1-0.3 parts, vitamin B12 0.01-0.05 parts, nicotinic acid 0.1-0.3 parts, calcium pantothenate 0.1-0.3 parts, folic acid 0.05-0.15 parts, biotin 0.01-0.05 parts, vitamin C 0.5-1.5 parts, vitamin D 0.01-0.05 parts, and vitamin E 0.05-0.15 parts.

7. The modified medium containing egg yolk protein and chick embryo extract according to claim 4, wherein, The inorganic salt mixture is composed of the following components in parts by mass: sodium chloride 5-10 parts, potassium chloride 0.5-2 parts, calcium chloride 0.1-0.5 parts, magnesium sulfate 0.1-0.5 parts, sodium dihydrogen phosphate 0.5-2 parts, sodium bicarbonate 1-3 parts, copper sulfate 0.001-0.005 parts, zinc sulfate 0.005-0.015 parts, ferrous sulfate 0.01-0.05 parts, and manganese chloride 0.001-0.005 parts.

8. A method for preparing the modified medium of claim 1-7 containing egg yolk protein and chicken embryo extract, characterized by, The method comprises the following steps: The egg yolk protein, the chicken embryo extract and the base medium component are stirred and mixed at 36-38℃ for 15-30 min, and then sterilized to obtain the improved medium.

9. The production method according to claim 8, characterized by: The preparation method of the egg yolk protein comprises: (1) obtaining fresh eggs, cleaning them, and separating the egg yolk and egg white in a sterile environment; (2) Put the egg yolk into a sterile container, add an appropriate amount of sterile PBS buffer, and homogenize it with a homogenizer to obtain egg yolk homogenate; (3) Put the egg yolk homogenate into an ultrasonic cell disruptor and perform the first ultrasonic treatment, wherein the ultrasonic power is 200-400 W, the ultrasonic time is 5-15 min, the first ultrasonic treatment is a first pulsed ultrasonic treatment, and the first pulsed ultrasonic treatment is ultrasonic treatment for 3-5 s followed by a pause of 2-3 s; (4) After the first ultrasonic treatment, centrifuge the mixture at 3-5℃ and 8000-10000 r / min for 15-25 min, and take the supernatant, which is the egg yolk protein.

10. The production method according to any one of claims 8 to 9, characterized by: The preparation method of the chicken embryo extract comprises the following steps: (1) Select healthy chicken embryos developed for 9-12 days, remove the embryo eyes, beaks, air sacs, membrane sacs, and allantoic membranes in a sterile environment; (2) Rinse the treated chicken embryos with sterile PBS buffer for 3-5 times; (3) Put the rinsed chicken embryos into a sterile mortar, add an appropriate amount of sterile PBS buffer, and grind them into a homogenate state; (4) Transfer the ground homogenate into a sterile centrifuge tube, and perform second ultrasonic treatment in an ultrasonic cell disruptor, wherein the ultrasonic power is 300-500 W, the ultrasonic time is 10-20 min, the second ultrasonic treatment is a second pulsed ultrasonic treatment, and the second pulsed ultrasonic treatment is ultrasonic treatment for 5-8 s followed by a pause of 3-5 s; (5) After the second ultrasonic treatment, freeze the mixture in liquid nitrogen for 10-15 min, then take it out and melt it in a 36-38℃ water bath, and repeat the freezing and melting for 3-5 times; (6) Centrifuge the mixture at 3-5℃ and 10000-12000 r / min for 20-30 min, and take the supernatant, which is the chicken embryo extract.

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