Preparation and application method of stem cell culture medium taking protein hydrolysate as core raw material

Abstract

Belongs to the field of cell culture media. The stem cell culture medium contains 20-80% of protein hydrolysate on the basis of the weight of the total components in the stem cell culture medium. The protein hydrolysate comprises one or more of a high free amino acid yeast hydrolysate, a high nucleotide yeast hydrolysate, a high glutathione yeast hydrolysate, a high polypeptide type yeast hydrolysate, a rice protein hydrolysate, a high glutamine wheat protein hydrolysate, a pea protein hydrolysate and a soybean protein hydrolysate. The animal stem cell natural culture medium is prepared by taking the full-nutrition protein hydrolysate as a core raw material, so that the nutritional requirements of animal stem cell proliferation can be met under the condition of not additionally adding or adding a small amount of amino acids, vitamins, growth factors and serum, and the cost of the stem cell culture medium is effectively reduced; therefore, the method has a wide application prospect in the field of food such as cell culture meat.

Description

Preparation and application method of stem cell culture medium using protein hydrolysate as core raw material

[0001] Related applications

[0002] This application claims priority to the prior application document with application number 202410668053.4 filed with the State Intellectual Property Office of China on May 27, 2024, and incorporates its entire contents into this document. Technical Field

[0003] The present invention relates to the field of cell culture medium, and in particular to a preparation method of a stem cell culture medium using protein hydrolysate as a core raw material and an application method thereof. Background Art

[0004] Complete protein hydrolysates are natural mixtures rich in protein, peptides of varying molecular weights, trace elements, vitamins, lipids, cholesterol, functional peptides, nucleotides, and amino acids, produced through targeted enzymatic hydrolysis using plant proteins, whole animal cells, and whole microbial cells, either with their own endogenous enzymes or with the addition of exogenous enzymes. These mixtures also include yeast protein hydrolysates and plant protein hydrolysates, providing the various amino acids, nucleotides, vitamins, growth factors, and serum analogs required for animal stem cell growth. Natural animal stem cell culture media prepared using complete protein hydrolysates as the core raw material can meet the nutritional needs of animal stem cell proliferation without the addition of any additional amino acids, vitamins, growth factors, or serum, or with minimal or no addition of serum. Summary of the Invention

[0005] The technical problem solved by the present invention is that the animal stem cell culture medium in the existing technology is generally a non-food grade chemically defined culture medium, which is composed of a combination of nutrients such as energy substances, nitrogen sources, inorganic salts, buffers, growth factors, lipids, vitamins, hormones, transferrin, protein hydrolysates, serum, etc. The raw materials used are generally purified pure raw materials. However, pure raw materials are expensive, have large batch differences, and are difficult to achieve large-scale culture, making them even more difficult to be widely used in food fields such as cell-cultured meat.

[0006] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0007] The method comprises optimizing a certain proportion of one or more of high-free amino acid yeast hydrolysate, high-nucleotide yeast hydrolysate, high-glutathione yeast hydrolysate, yeast hydrolysate containing expressed recombinant stem cell growth factor, high-glutamine wheat protein hydrolysate, pea protein hydrolysate, rice protein hydrolysate, microbial whole-cell protein hydrolysate and plant-derived protein hydrolysate, and mixing the mixture with energy substances, inorganic salts, buffers, growth factors, lipids, vitamins, hormones and transferrin, to obtain a stem cell culture medium with protein hydrolysate as the core raw material and an application method thereof.

[0008] In a first aspect, the present invention provides a stem cell culture medium using protein hydrolysate as a core raw material, characterized in that the stem cell culture medium contains 20-80% protein hydrolysate based on the weight of the total components in the stem cell culture medium;

[0009] The protein hydrolysate comprises one or more of: high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate;

[0010] Wherein, the high-free amino acid yeast hydrolysate has a free amino acid content of ≥50% by weight;

[0011] The high nucleotide yeast hydrolysate has a free nucleotide content of >5% by weight;

[0012] The high-glutathione yeast hydrolysate has a glutathione content greater than 10% by weight;

[0013] The high-polypeptide yeast hydrolysate has a peptide segment with a molecular weight greater than 1000 Daltons accounting for more than 20% of the total peptide segments in the high-polypeptide yeast hydrolysate, based on its weight;

[0014] The rice protein hydrolysate has a total nitrogen content of >12% by weight;

[0015] The high-glutamine wheat protein hydrolysate has a hydrolyzed glutamine content greater than 20% by weight;

[0016] The pea protein hydrolysate has a total nitrogen content greater than 13.5% by weight;

[0017] The soy protein hydrolysate has a carbohydrate content greater than 20% by weight.

[0018] Preferably, based on weight, the free amino acid content of the high-free amino acid yeast hydrolysate is 50%-65%, the free nucleotide content of the high-nucleotide yeast hydrolysate is 5%-20%, the glutathione content of the high-glutathione yeast hydrolysate is 10%-20%, the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the high-polypeptide yeast hydrolysate accounts for 20%-50% of the total peptides in the high-polypeptide yeast hydrolysate, the total nitrogen content of the rice protein hydrolysate is 12%-13%, the hydrolyzed glutamine content of the high-glutamine wheat protein hydrolysate is 20%-35%, the total nitrogen content of the pea protein hydrolysate is 13.5%-14.5%, and the carbohydrate content of the soy protein hydrolysate is 20%-22%.

[0019] Preferably, the components of the stem cell culture medium further include: one or more of: balanced salts, pH regulating solution, antibiotic mixture, serum, vitamin complex, amino acid complex, trace element complex, glucose and growth factors.

[0020] Preferably, the concentrations of the components in the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, protein hydrolyzate 4000-25000 mg / L, balanced salt 2000-6000 mg / L, pH adjusting solution 1500-3500 mg / L, antibiotic mixture 10-120 mg / L, vitamin complex 20-50 mg / L, amino acid complex 100-1000 mg / L, trace element complex 0.5-4.0 mg / L, glucose 4000-6000 mg / L, growth factor 0.15-0.25 mg / L, and serum 1% to 4% by volume;

[0021] More preferably, the concentrations of the components in the stem cell culture medium are as follows: protein hydrolysate 4500-21000 mg / L, balanced salt 2170-5630 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 30-50 mg / L, amino acid complex 200-1000 mg / L, trace element complex 0.5-3.0 mg / L, glucose 4000-6000 mg / L, growth factor 0.20-0.25 mg / L, and serum 1 volume %-3 volume %, based on the total volume of the stem cell culture medium.

[0022] Preferably, the concentrations of the components in the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, protein hydrolysate 4000-25000 mg / L, balanced salt 2000-6000 mg / L, pH adjusting solution 1500-3500 mg / L, vitamin complex 20-50 mg / L, amino acid complex 200-1000 mg / L, trace element complex 0.5-4.0 mg / L, glucose 4000-6000 mg / L, and growth factor 0.15-0.25 mg / L;

[0023] More preferably, the concentrations of the components in the stem cell culture medium are as follows: protein hydrolysate 14000-16000 mg / L, balanced salt 4050-4690 mg / L, pH adjusting solution 2250-3500 mg / L, vitamin complex 40-50 mg / L, amino acid complex 250-500 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L, and growth factor 0.20-0.25 mg / L, based on the total volume of the stem cell culture medium.

[0024] Preferably, the vitamin complex comprises, based on the total weight of the vitamin complex, one or more of 22%-27% choline chloride, 6%-10% folic acid, 40%-50% inositol, 2.5%-7.5% niacinamide, 5%-10% calcium pantothenate, 1%-5% pyridoxal, 1%-5% vitamin B12 and 1%-5% thiamine.

[0025] Preferably, based on the total weight of the amino acid complex, the amino acid complex comprises: 40%-50% asparagine, and / or 20%-30% aspartic acid, and / or 20%-30% serine, and / or 1%-10% cysteine.

[0026] Preferably, based on the total weight of the trace element complex, the trace element complex comprises: 1%-10% Fe(NO3)3·9H2O, and / or 40%-50% FeSO4·7H2O, and / or 40%-50% ZnSO4·7H2O.

[0027] Preferably, the balanced salt includes one or more of calcium chloride, magnesium sulfate, potassium chloride, sodium chloride and sodium phosphate.

[0028] Preferably, the pH regulating solution comprises one or more of sodium bicarbonate buffer and HEPEs, and / or the antibiotic comprises one or more of penicillin and streptomycin, and / or the growth factor comprises growth factor FGF2.

[0029] Preferably, the stem cell culture medium is a stem cell culture medium containing high glutathione yeast hydrolysate; or a stem cell culture medium containing pea protein hydrolysate.

[0030] Preferably, based on the total volume of the stem cell culture medium containing high glutathione yeast hydrolysate, the total volume of the stem cell culture medium containing high glutathione yeast hydrolysate includes 1-3 volume% of serum, 4500-5500 mg / L of high glutathione yeast hydrolysate, 4750-5630 mg / L of balanced salt, 2250-3500 mg / L of pH adjusting solution, 80-120 mg / L of antibiotic mixture, 40-50 mg / L of vitamin complex, 750-1000 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor;

[0031] Alternatively, based on the total volume of the stem cell culture medium containing pea protein hydrolysate, the concentrations of the components in the stem cell culture medium containing pea protein hydrolysate are as follows: 1-3% by volume of serum, 4500-5500 mg / L of pea protein hydrolysate, 4750-5630 mg / L of balanced salt, 2250-350 mg / L of pH adjusting solution, 80-120 mg / L of antibiotic mixture, 40-50 mg / L of vitamin complex, 750-1000 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor.

[0032] Preferably, the balanced salt is calcium chloride 110-130 mg / L, magnesium sulfate 80-100 mg / L, potassium chloride 700-900 mg / L, sodium chloride 3000-3500 mg / L and sodium phosphate 750-1000 mg / L;

[0033] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0034] Preferably, the stem cell culture medium is a stem cell culture medium containing high-polypeptide yeast hydrolysate; or a stem cell culture medium containing soy protein hydrolysate.

[0035] Preferably, based on the total volume of the stem cell culture medium containing high-polypeptide yeast hydrolysate, the total volume of the stem cell culture medium containing high-polypeptide yeast hydrolysate includes 1-2 volume%, serum, 14000-16000 mg / L of high-polypeptide yeast hydrolysate, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH adjusting solution, 80-120 mg / L of antibiotic mixture, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor FGF;

[0036] Alternatively, based on the total volume of the stem cell culture medium containing soy protein hydrolysate, the concentrations of the components in the stem cell culture medium containing soy protein hydrolysate are as follows: serum 1-2%, soy protein hydrolysate 14000-16000 mg / L, balanced salt 4050-4690 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 40-50 mg / L, amino acid complex 250-500 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-5000 mg / L, and growth factor 0.20-0.25 mg / L.

[0037] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0038] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0039] Preferably, the stem cell culture medium is a stem cell culture medium containing high glutamine wheat protein hydrolysate.

[0040] Preferably, based on the total volume of the stem cell culture medium containing high glutamine wheat protein hydrolysate, the concentrations of the components in the stem cell culture medium containing high glutamine wheat protein hydrolysate are: 1-2% serum, 19000-21000 mg / L high glutamine wheat protein hydrolysate, 2170-2760 mg / L balanced salt, 2250-3500 mg / L pH adjusting solution, 80-120 mg / L antibiotic mixture, 30-40 mg / L vitamin complex, 200-400 mg / L amino acid complex, 0.5-2.5 mg / L trace element complex, 4000-6000 mg / L glucose, and 0.20-0.25 mg / L growth factor.

[0041] Preferably, the balanced salt is calcium chloride 70-90 mg / L, magnesium sulfate 50-70 mg / L, potassium chloride 350-500 mg / L, sodium chloride 1400-1600 mg / L and sodium phosphate 300-500 mg / L;

[0042] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0043] Preferably, the stem cell culture medium is a stem cell culture medium containing a composite protein hydrolysate, wherein, based on the total added weight of the composite protein hydrolysate, it contains 5%-60% high-free amino acid yeast hydrolysate, 5%-60% high-nucleotide yeast hydrolysate, 5%-60% high-glutathione yeast hydrolysate, 5%-60% high-polypeptide yeast hydrolysate, 5%-60% rice protein hydrolysate, 5%-60% high-glutamine wheat protein hydrolysate, 5%-60% pea protein hydrolysate and 5%-60% soy protein hydrolysate.

[0044] Preferably, based on the total volume of the stem cell culture medium containing the composite protein hydrolysate, the concentrations of the components in the stem cell culture medium containing the composite protein hydrolysate are: 1-2% by volume of serum, 14000-16000 mg / L of the composite protein hydrolysate according to claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 80-120 mg / L of a mixed solution of antibiotics, 40-50 mg / L of a vitamin complex, 250-500 mg / L of an amino acid complex, 0.5-2.5 mg / L of a trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of a growth factor.

[0045] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0046] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0047] Preferably, the concentrations of the components in the stem cell culture medium containing the composite protein hydrolysate are as follows: based on the total volume of the stem cell culture medium, the composite protein hydrolysate is 14,000-16,000 mg / L, balanced salt is 4,050-4,690 mg / L, pH adjusting solution is 2,250-3,500 mg / L, vitamin complex is 40-50 mg / L, amino acid complex is 250-500 mg / L, trace element complex is 0.5-2.5 mg / L, glucose is 4,000-6,000 mg / L, and growth factor is 0.20-0.25 mg / L.

[0048] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0049] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0050] In a second aspect, the present invention provides a method for screening a stem cell culture medium, comprising the following steps:

[0051] Step 1: Screening protein hydrolysates. Specifically, after animal stem cells are activated, they are transferred to stem cell culture media containing different types of protein hydrolysates at concentrations ranging from 4,500 to 21,000 mg / L for expansion. The growth of animal stem cells in the presence of different types of protein hydrolysates is determined based on stem cell density and the positive rate of stem cell characteristics, thereby selecting the appropriate protein hydrolysate.

[0052] Step 2: Screening the mixing ratio of the composite protein hydrolysate, specifically, mixing two or more different types of protein hydrolysates to obtain the composite protein hydrolysate; and transferring the activated animal stem cells to a stem cell culture medium containing each of the composite protein hydrolysates at a concentration of 4,500-21,000 mg / L to expand the stem cell culture. The growth of the animal stem cells in the composite protein hydrolysates at different mixing ratios is determined based on the stem cell density and the positive rate of stem cell characteristics, so as to screen the suitable mixing ratio of the composite protein hydrolysate. The mixing ratio refers to the mixing ratio of the different types of protein hydrolysates added to prepare the composite protein hydrolysate.

[0053] Step 3: Prepare stem cell culture medium, specifically using the protein hydrolysate selected in step 1 as the core raw material and / or the composite protein hydrolysate selected in step 2 in a suitable mixing ratio to prepare the stem cell culture medium;

[0054] Step 4: Screening stem cell culture media. Specifically, after the animal stem cells are activated, they are transferred to the stem cell culture media prepared in step 3 for expansion culture. The growth of animal stem cells in different stem cell culture media is determined based on the stem cell density and the positive rate of stem cell characteristics to screen out the appropriate stem cell culture media.

[0055] Preferably, the stem cell culture medium or the stem cell culture medium screened by the screening method is used in stem cells, wherein the stem cells include one or more muscle stem cells derived from chicken, cattle, sheep, shrimp and mouse.

[0056] In a third aspect, the present invention provides a chicken muscle stem cell culture medium, which contains, based on the total volume of the chicken muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor.

[0057] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0058] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0059] In a fourth aspect, the present invention provides a bovine muscle stem cell culture medium, comprising, based on the total volume of the bovine muscle stem cell culture medium, 14,000-16,000 mg / L of the composite protein hydrolysate, 4,050-4,690 mg / L of balanced salt, 2,250-3,500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4,000-6,000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor.

[0060] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0061] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0062] In a fifth aspect, the present invention provides a sheep muscle stem cell culture medium, which contains, based on the total volume of the sheep muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor.

[0063] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0064] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0065] In a fifth aspect, the present invention provides a shrimp muscle stem cell culture medium, which contains, based on the total volume of the shrimp muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor.

[0066] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0067] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0068] In a sixth aspect, the present invention provides a mouse muscle stem cell culture medium, comprising, based on the total volume of the mouse muscle stem cell culture medium, 14,000-16,000 mg / L of the composite protein hydrolysate, 4,050-4,690 mg / L of balanced salt, 2,250-3,500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4,000-6,000 mg / L of glucose, and 0.20-0.25 mg / L of growth factor.

[0069] Preferably, the balancing salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L;

[0070] Preferably, the pH regulating solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

[0071] Beneficial effects of the present invention:

[0072] (1) The present invention uses complete nutritional protein hydrolyzate as the core raw material to prepare a natural culture medium for animal stem cells. It can meet the nutritional needs of animal stem cell proliferation without adding or with a small amount of amino acids, vitamins, growth factors and serum, and can effectively reduce the cost of stem cell culture medium, so that it has broad application prospects in the field of food such as cell cultured meat.

[0073] (2) The present invention uses a complete nutrient source protein hydrolyzate as the core raw material of the animal stem cell culture medium, wherein the stem cell culture medium contains 20%-80% of the protein hydrolyzate based on the weight of the total components in the stem cell culture medium.

[0074] (3) The present invention utilizes all nutrients contained in the complete nutrient source protein hydrolyzate to replace the non-energy substances in the existing chemically defined culture medium, thereby reducing the amount of serum in the culture medium while improving the proliferation effect of animal stem cells. DETAILED DESCRIPTION

[0075] In the existing technology, animal stem cell culture media are generally non-food grade chemically defined culture media, which are composed of a combination of nutrients such as energy substances, nitrogen sources, inorganic salts, buffers, growth factors, lipids, vitamins, hormones, transferrin, protein hydrolysates, serum, etc. The raw materials used are generally purified pure raw materials. However, pure raw materials are expensive, have large differences between batches of raw materials, and are difficult to achieve large-scale cultivation, making them even more difficult to be widely used in food fields such as cell-cultured meat.

[0076] In order to solve the above technical problems, the present invention provides the following specific solutions:

[0077] The present invention provides a method for preparing a stem cell culture medium using protein hydrolysate as a core raw material and its application. By using a nutritionally complete protein hydrolysate as the core raw material to prepare a culture medium for animal stem cells, the medium replaces the non-energy substances in existing chemically defined culture media, reducing the amount of serum in the culture medium while enhancing the proliferation of animal stem cells. This also reduces the cost of preparing the cell culture medium.

[0078] Specifically, the present invention provides a stem cell culture medium with protein hydrolyzate as a core raw material, wherein the stem cell culture medium contains 20-80% protein hydrolyzate based on the weight of the total components in the stem cell culture medium.

[0079] In the present invention, the percentage of the protein hydrolysate contained in the stem cell culture medium refers to the percentage of the weight of the protein hydrolysate or composite protein hydrolysate relative to the weight of the total components in the stem cell culture medium. The weight of the total components in the stem cell culture medium refers to the total weight of the components excluding serum when the stem cell culture medium contains serum; and refers to the total weight of all components in the stem cell culture medium when the stem cell culture medium does not contain serum.

[0080] Wherein, preferably, the serum is fetal bovine serum.

[0081] Preferably, in some specific embodiments, the stem cell culture medium with protein hydrolysate as the core raw material may contain 20%, 22%, 25%, 26%, 27%, 28%, 30%, 35%, 40%, 45%, 50%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75% and 80% of protein hydrolysate, based on the weight of the total components in the stem cell culture medium, or contain a protein hydrolysate content within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0082] The protein hydrolysate includes one or more of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0083] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate and pea protein hydrolysate.

[0084] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate and soy protein hydrolysate.

[0085] Preferably, in some embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0086] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0087] Preferably, in some embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0088] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0089] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0090] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high-nucleotide yeast hydrolysate, high-glutathione yeast hydrolysate, high-polypeptide yeast hydrolysate, rice protein hydrolysate, high-glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0091] Preferably, in some specific embodiments, the protein hydrolysate is a combination of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0092] In some specific embodiments, the high-free amino acid yeast hydrolysate (protein hydrolysate A) has a free amino acid content of ≥50% by weight. It should be noted that any commercially available or homemade high-free amino acid yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that any commercially available or homemade high-free amino acid yeast hydrolysate with a free amino acid content of ≥50% can be used in the present invention, and the free amino acid content is preferably 50%-65%.

[0093] More specifically, the present inventors have found that using a high-free amino acid yeast hydrolyzate containing various amino acids that meet the following conditions for preparing the stem cell culture medium of the present invention can result in a higher density of stem cells.

[0094] Based on the weight of protein hydrolysate A, the free amino acids are 2-3% aspartic acid, 2-3% threonine, 2-3% serine, 6.5-7.4% glutamic acid, 4-5% glycine, 13-15% alanine, 0.5-0.6% cystine, 3-4% valine, 0.5-1% methionine, 2-3% isoleucine, 4-5% leucine, 1-1.5% tyrosine, 2-3% phenylalanine, 2-3% lysine, 1-1.5% histidine, 4-5% arginine and 0.5-1% proline.

[0095] In some embodiments, in order to prepare an ideal stem cell culture medium, the high-free amino acid yeast hydrolyzate may further comprise the following components:

[0096] Based on the weight of the protein hydrolysate A, the protein hydrolysate A has a total nitrogen content of 11-12%, an ammonia nitrogen content of 5-6%, an oxidized glutathione content of 0.5-1% and a reduced glutathione content of 0.1-0.5%.

[0097] Based on the weight of protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate A is 3-5% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 1-3% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 10-15% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 40-45% of the total peptide segments in the protein hydrolysate A, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 35-40% of the total peptide segments in the protein hydrolysate A.

[0098] Based on the weight of the protein hydrolysate A, the free nucleotide content in the protein hydrolysate A is 0.21-0.58%, wherein, based on the weight of the protein hydrolysate A, the free nucleotides include 0.05-0.15% of uracil nucleotide (UMP), 0.1-0.3% of adenine nucleotide (AMP), 0.01-0.03% of guanine nucleotide (GMP) and 0.05-0.1% of cytosine nucleotide (CMP).

[0099] In some specific embodiments, the high-nucleotide yeast hydrolysate (protein hydrolysate B) has a free nucleotide content of >5% by weight. It should be noted that any commercially available high-nucleotide yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that any commercially available or homemade high-nucleotide yeast hydrolysate with a free nucleotide content of >5% can be used in the present invention. The free nucleotide content is preferably 5%-20%, wherein this range does not include the case where it is equal to 5%.

[0100] More specifically, the present inventors have discovered that using a high-nucleotide yeast hydrolyzate containing specific free nucleotide contents that meet the following conditions for preparing the stem cell culture medium of the present invention can result in a higher density of stem cells.

[0101] Based on the weight of protein hydrolysate B, free nucleotides are uracil nucleotide (UMP) 1.3-5%, adenine nucleotide (AMP) 1-5%, inosine nucleotide (IMP) 1.6-5%, guanine nucleotide (GMP) 1-3% and cytosine nucleotide (CMP) 0.1-0.2%.

[0102] In some embodiments, in order to prepare an ideal stem cell culture medium, the high-nucleotide yeast hydrolysate may further comprise the following components:

[0103] Based on the weight of the protein hydrolysate B, the protein hydrolysate B has a total nitrogen content of 11-12%, an ammonia nitrogen content of 4-5%, an oxidized glutathione content of 0.1-0.5% and a reduced glutathione content of 0.1-0.5%.

[0104] Based on the weight of protein hydrolysate B, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate B is 0.01-0.05% of the total peptide segments in the protein hydrolysate B, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 1-2% of the total peptide segments in the protein hydrolysate B, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 15-20% of the total peptide segments in the protein hydrolysate B, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 47-52% of the total peptide segments in the protein hydrolysate B, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 27-32% of the total peptide segments in the protein hydrolysate B.

[0105] The protein hydrolysate B contains 22.71-36% free amino acids based on the weight of the protein hydrolysate B, wherein the free amino acids are 0.5-1% aspartic acid, 1-1.5% threonine, 1-1.5% serine, 6-7% glutamic acid, 0.5-1% glycine, 5-8% alanine, 0.01-0.1% cystine, 1-2% valine, 0.1-0.6% methionine, 1-1.5% isoleucine, 2-3% leucine, 1-2% tyrosine, 1-2% phenylalanine, 1-2% lysine, 0.1-0.5% histidine, 1-1.5% arginine and 0.5-0.8% proline.

[0106] In some specific embodiments, the high-glutathione yeast hydrolysate has a glutathione content of >10% by weight. It should be noted that any commercially available high-glutathione yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that any commercially available or homemade high-glutathione yeast hydrolysate with a glutathione content of >10% can be used in the present invention. The glutathione content is preferably 10%-20%, wherein this range does not include the case where it is equal to 10%.

[0107] More specifically, the present inventors have found that using a high-glutathione yeast hydrolyzate containing glutathione that meets the following conditions for preparing the stem cell culture medium of the present invention can result in a higher density of stem cells.

[0108] Based on the weight of the protein hydrolysate C, the glutathione includes 0.5-5% of oxidized glutathione and 9.5-15% of reduced glutathione.

[0109] In some embodiments, in order to prepare an ideal stem cell culture medium, the high-glutathione yeast hydrolysate may further comprise the following components:

[0110] Based on the weight of the protein hydrolysate C, the total nitrogen content of the protein hydrolysate C is 8-10%, and the ammonia nitrogen content is 2-5%.

[0111] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate C is 3-5% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 10-12% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 20-23% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 23-26% of the total peptide segments in the protein hydrolysate C, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 35-40% of the total peptide segments in the protein hydrolysate C.

[0112] Based on the weight of the protein hydrolysate C, the free nucleotide content in the protein hydrolysate C is 1-2%, wherein, based on the weight of the protein hydrolysate C, the free nucleotides include 0.1-0.3% of uracil nucleotide (UMP), 0.6-1.0% of adenine nucleotide (AMP), 0.1-0.3% of inosine nucleotide (IMP), 0.1-0.2% of guanine nucleotide (GMP) and 0.1-0.2% of cytosine nucleotide (CMP).

[0113] The protein hydrolysate C has a free amino acid content of 8.14-11.01% based on the weight of the protein hydrolysate C, wherein the free amino acids, based on the weight of the protein hydrolysate C, are 0.3-0.4% of aspartic acid, 0.5-0.6% of threonine, 0.3-0.4% of serine, 4-5% of glutamic acid, 0.1-0.6% of glycine, 0.8-1.0% of alanine, 0.6-1.0% of cystine, 0.1-0.3% of valine, 0.01-0.05% of methionine, 0.1-0.5% of isoleucine, 0.1-0.5% of leucine, 0.1-0.5% of tyrosine, 0.01-0.1% of phenylalanine, 0.1-0.5% of lysine, 0.01-1.2% of histidine, 1-1.5% of arginine, and 0.01-0.06% of proline.

[0114] In some specific embodiments, the high-polypeptide yeast hydrolysate (protein hydrolysate D) has a mass percentage of peptides with a molecular weight greater than 1000 Daltons accounting for >20% of the total peptides in the high-polypeptide yeast hydrolysate. It should be noted that any commercially available high-polypeptide yeast hydrolysate can be used in the present invention. Specifically, the inventors have found through research that any commercially available or homemade high-polypeptide yeast hydrolysate can be used in the present invention if the mass percentage of peptides with a molecular weight greater than 1000 Daltons accounting for >20% of the total peptides in the high-polypeptide yeast hydrolysate, preferably 20%-50%, wherein this range does not include the case where the mass percentage is equal to 20%.

[0115] More specifically, the present inventors have discovered that using a high-polypeptide yeast hydrolyzate having the following peptide relative molecular weight distribution for preparing the stem cell culture medium of the present invention can result in a higher density of stem cells.

[0116] The peptide segments with a molecular weight greater than 1000 Daltons account for 3-20% of the total peptide segments in the high-polypeptide yeast hydrolysate; the peptide segments with a molecular weight greater than or equal to 2000 Daltons account for 17-30% of the total peptide segments in the protein hydrolysate D.

[0117] The mass percentage of peptides with a molecular weight greater than 400 and less than or equal to 1000 Daltons in the protein hydrolysate D is 15-20% of the total peptides, the mass percentage of peptides with a molecular weight greater than 180 and less than or equal to 400 Daltons in the protein hydrolysate D is 30-35% of the total peptides, and the mass percentage of peptides with a molecular weight less than or equal to 180 Daltons in the protein hydrolysate D is 33-37%.

[0118] In some embodiments, in order to prepare an ideal stem cell culture medium, the high-polypeptide yeast hydrolysate may further comprise the following components:

[0119] Based on the weight of the protein hydrolysate D, the protein hydrolysate D has a total nitrogen content of 10-15%, an ammonia nitrogen content of 2-7%, an oxidized glutathione content of 0.2-0.6% and a reduced glutathione content of 0.2-0.5%.

[0120] Based on the weight of the protein hydrolysate D, the free nucleotide content in the protein hydrolysate D is 0.05-0.2%, wherein, based on the weight of the protein hydrolysate D, the free nucleotide is 0.05-0.2% of adenine nucleotide (AMP).

[0121] The protein hydrolysate D has a free amino acid content of 17.5-37.6% based on the weight of the protein hydrolysate D, wherein the free amino acids, based on the weight of the protein hydrolysate D, are 1-2% of aspartic acid, 1-2% of threonine, 1-3% of serine, 3-6% of glutamic acid, 1-3% of glycine, 3-6% of alanine, 0.5-1% of cystine, 1-3% of valine, 0.1-0.6% of methionine, 0.5-1.5% of isoleucine, 1-2% of leucine, 0.1-1% of tyrosine, 1-1.5% of phenylalanine, 1-1.5% of lysine, 0.2-0.5% of histidine, 1.5-2% of arginine, and 0.6-1% of proline.

[0122] In some specific embodiments, the rice protein hydrolysate (protein hydrolysate E) has a total nitrogen content of >12% by weight; It should be noted that any commercially available rice protein hydrolysate can be used in the present invention. Specifically, the inventors have found that commercially available or homemade rice protein hydrolysate with a total nitrogen content of >12 can be used in the present invention, preferably 12%-13%, wherein the range does not include 12%. The rice protein hydrolysate is used to prepare the stem cell density obtained by cultivating the stem cell culture medium of the present invention.

[0123] In some embodiments, in order to prepare an ideal stem cell culture medium, the rice protein hydrolyzate may further comprise the following components:

[0124] Based on the weight of the protein hydrolysate E, the ammonia nitrogen content in the protein hydrolysate E is 1-5%.

[0125] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate E is 2-5% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 4-8% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 17-22% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 43-47% of the total peptide segments in the protein hydrolysate E, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 22-27% of the total peptide segments in the protein hydrolysate E.

[0126] The protein hydrolysate E has a free amino acid content of 10.1-23.5% based on the weight of the protein hydrolysate E, wherein the free amino acids, based on the weight of the protein hydrolysate E, are 0.1-0.5% of aspartic acid, 0.5-1% of threonine, 0.5-1% of serine, 1-1.5% of glutamic acid, 0.1-0.5% of glycine, 0.5-1% of alanine, 0.1-0.5% of cystine, 1-1.5% of valine, 0.1-1% of methionine, 0.5-1% of isoleucine, 2-3% of leucine, 0.5-1.5% of tyrosine, 0.5-1.5% of phenylalanine, 0.5-1.5% of lysine, 0.1-1% of histidine, 2-5% of arginine, and 0.1-0.5% of proline.

[0127] In some specific embodiments, the high-glutamine wheat protein hydrolysate (protein hydrolysate F) has a hydrolyzed glutamine content greater than 20% by weight. It should be noted that any commercially available high-glutamine wheat protein hydrolysate can be used in the present invention. Specifically, the inventors have found that any commercially available or homemade high-glutamine wheat protein hydrolysate with a hydrolyzed glutamine content greater than 20% can be used in the present invention. The hydrolyzed glutamine content is preferably between 20% and 35%, with the hydrolyzed glutamine content not including 20%. Using this high-glutamine wheat protein hydrolysate to prepare the stem cell culture medium of the present invention results in a higher density of stem cells.

[0128] In some specific embodiments, in order to prepare an ideal stem cell culture medium, the high glutamine wheat protein hydrolysate may further comprise the following components:

[0129] Based on the weight of the protein hydrolysate F, the protein hydrolysate E has a total nitrogen content of 10-15% and an ammonia nitrogen content of 1-5%.

[0130] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate F is 1-5% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 1-5% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 10-15% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 30-35% of the total peptide segments in the protein hydrolysate F, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 45-50% of the total peptide segments in the protein hydrolysate F.

[0131] The protein hydrolysate F has a free amino acid content of 8.61-17.55% based on the weight of the protein hydrolysate F, wherein the free amino acids, based on the weight of the protein hydrolysate F, are 0.1-0.5% of aspartic acid, 0.5-1% of threonine, 0.5-1% of serine, 0.5-1% of glutamic acid, 0.1-0.5% of glycine, 0.5-1% of alanine, 0.1-0.5% of cystine, 1-1.5% of valine, 0.1-0.5% of methionine, 1-1.5% of isoleucine, 2-2.5% of leucine, 0.5-1% of tyrosine, 0.5-1% of phenylalanine, 0.1-1% of lysine, 0.1-1% of histidine, 1-1.5% of arginine, and 0.01-0.05% of proline.

[0132] In some specific embodiments, the pea protein hydrolysate (protein hydrolysate G) has a total nitrogen content greater than 13.5% by weight. It should be noted that any commercially available pea protein hydrolysate can be used in the present invention. Specifically, the inventors have found that any commercially available or homemade pea protein hydrolysate with a total nitrogen content greater than 13.5% can be used in the present invention. The total nitrogen content is preferably between 13.5% and 14.5%, with this range excluding 13.5%. Using this pea protein hydrolysate in the preparation of the stem cell culture medium of the present invention results in a higher density of stem cells.

[0133] In some specific embodiments, in order to prepare an ideal stem cell culture medium, the pea protein hydrolyzate may further comprise the following components:

[0134] The ammonia nitrogen content in the protein hydrolysate G is 1-5% based on the weight of the protein hydrolysate G.

[0135] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate G is 1-5% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 5-10% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 25-30% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 37-42% of the total peptide segments in the protein hydrolysate G, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 20-25% of the total peptide segments in the protein hydrolysate G.

[0136] The protein hydrolysate G has a free amino acid content of 10.53-22.32% based on the weight of the protein hydrolysate G, wherein the free amino acids, based on the weight of the protein hydrolysate G, are 0.1-0.5% of aspartic acid, 0.1-1% of threonine, 0.5-1% of serine, 0.5-1% of glutamic acid, 0.1-1% of glycine, 0.5-1% of alanine, 0.01-0.1% of cystine, 0.5-1.5% of valine, 0.01-0.07% of methionine, 0.5-1% of isoleucine, 2-2.5% of leucine, 1-1.5% of tyrosine, 2-2.5% of phenylalanine, 1-1.5% of lysine, 0.2-0.7% of histidine, 1.5-5% of arginine, and 0.01-0.05% of proline.

[0137] In some specific embodiments, the soy protein hydrolysate (protein hydrolysate H) has a carbohydrate content greater than 20% by weight. It should be noted that any commercially available soy protein hydrolysate can be used in the present invention. Specifically, the inventors have found that any commercially available or homemade soy protein hydrolysate with a carbohydrate content greater than 20% can be used in the present invention. The carbohydrate content is preferably 20%-22%, with this range excluding 20%. Using this soy protein hydrolysate in the preparation of the stem cell culture medium of the present invention results in a higher density of stem cells.

[0138] In some specific embodiments, in order to prepare an ideal stem cell culture medium, the soy protein hydrolyzate may further comprise the following components:

[0139] Based on the weight of the protein hydrolysate H, the total nitrogen content of the protein hydrolysate H is 10-15% and the ammonia nitrogen content is 1-5%.

[0140] The mass percentage of peptides with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate H accounts for 2-7% of the total peptides in the protein hydrolysate G, the mass percentage of peptides with a molecular weight greater than 1000 and less than 2000 Daltons accounts for 5-10% of the total peptides in the protein hydrolysate H, the mass percentage of peptides with a molecular weight greater than 400 and less than or equal to 1000 Daltons accounts for 17-22% of the total peptides in the protein hydrolysate H, the mass percentage of peptides with a molecular weight greater than 180 and less than or equal to 400 Daltons accounts for 27-32% of the total peptides in the protein hydrolysate H, and the mass percentage of peptides with a molecular weight less than or equal to 180 Daltons accounts for 35-40% of the total peptides in the protein hydrolysate H.

[0141] Based on the weight of the protein hydrolysate H, the free amino acid content of the protein hydrolysate H is 6.25-13.85%, wherein, based on the weight of the protein hydrolysate H, the free amino acids are 0.1-0.5% of aspartic acid, 0.1-0.5% of threonine, 0.1-0.5% of serine, 0.4-0.7% of glutamic acid, 0.1-0.5% of glycine, 0.1-0.5% of alanine, 0.1-0.5% of cystine, 0.5-1.0% of valine, 0.05-0.15% of methionine, 0.5-1% of isoleucine, 1-1.5% of leucine, 0.5-1.5% of tyrosine, 1-1.5% of phenylalanine, 0.5-1% of lysine, 0.1-0.5% of histidine, 1-1.5% of arginine, and 0.1-0.5% of proline.

[0142] In some specific embodiments, the components in the stem cell culture medium further include one or more substances selected from the group consisting of balanced salts, pH regulating solutions, mixed solutions of antibiotics, serum, vitamin complexes, amino acid complexes, trace element complexes, glucose, and growth factors.

[0143] In a preferred embodiment of the present invention, the concentrations of the components of the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, 3% fetal bovine serum, 5000 mg / L protein hydrolysate, 5000 mg / L balanced salt (120 mg / L calcium chloride, 100 mg / L magnesium sulfate, 750 mg / L potassium chloride, 3280 mg / L sodium chloride, and 750 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPEs), 100 mg / L of a 100× double antibody mixture of penicillin and streptomycin, 45 mg / L vitamin complex, 800 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor FGF2. The protein hydrolysate is protein hydrolysate C: high glutathione yeast hydrolysate, whose glutathione content is 11.2%; or protein hydrolysate G: pea protein hydrolysate, whose total nitrogen content is 13.8%.

[0144] In another preferred embodiment of the present invention, the concentrations of the components of the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, 2% fetal bovine serum, 15,000 mg / L protein hydrolysate, 4,270 mg / L balanced salt (90 mg / L calcium chloride, 80 mg / L magnesium sulfate, 650 mg / L potassium chloride, 2,800 mg / L sodium chloride, and 650 mg / L sodium phosphate), 3,000 mg / L pH adjusting solution (2,000 mg / L sodium bicarbonate buffer, 1,000 mg / L HEPEs), 100 mg / L of a 100× double antibody mixture of penicillin and streptomycin, 40 mg / L vitamin complex, 400 mg / L amino acid complex, 1 mg / L trace element complex, 4,500 mg / L glucose, and 0.23 mg / L growth factor FGF2. The protein hydrolysate is protein hydrolysate D: a high-polypeptide yeast hydrolysate, wherein the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the total peptides in the high-polypeptide yeast hydrolysate is 25.7%; or protein hydrolysate H: a soy protein hydrolysate, wherein the carbohydrate content is 21.4%.

[0145] In another preferred embodiment of the present invention, the concentrations of the stem cell culture medium components are as follows: 2% fetal bovine serum (FBS), 20,000 mg / L protein hydrolysate, 2540 mg / L balanced salts (80 mg / L calcium chloride, 60 mg / L magnesium sulfate, 450 mg / L potassium chloride, 1500 mg / L sodium chloride, and 450 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPES), 100 mg / L penicillin and streptomycin double antibody mixture (100×), 35 mg / L vitamin complex, 300 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor FGF2, based on the total volume of the stem cell culture medium. The protein hydrolysate is protein hydrolysate F: high-glutamine wheat protein hydrolysate, having a hydrolyzed glutamine content of 24.3%.

[0146] In another preferred embodiment of the present invention, the concentrations of the components of the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, 2% fetal bovine serum, 15,000 mg / L complex protein hydrolysate, 4,270 mg / L balanced salt (90 mg / L calcium chloride, 80 mg / L magnesium sulfate, 650 mg / L potassium chloride, 2,800 mg / L sodium chloride, and 650 mg / L sodium phosphate), 3,000 mg / L pH adjusting solution (2,000 mg / L sodium bicarbonate buffer, 1,000 mg / L HEPEs), 100 mg / L of a 100× double antibody mixture of penicillin and streptomycin, 40 mg / L vitamin complex, 400 mg / L amino acid complex, 1 mg / L trace element complex, 4,500 mg / L glucose, and 0.23 mg / L growth factor FGF2. Among them, based on the total added weight of composite protein hydrolysate, the proportion of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate are all 12.5%.

[0147] In another preferred embodiment of the present invention, the concentrations of the components of the stem cell culture medium are as follows: based on the total volume of the stem cell culture medium, 15000 mg / L of complex protein hydrolysate, 4270 mg / L of balanced salts (90 mg / L of calcium chloride, 80 mg / L of magnesium sulfate, 650 mg / L of potassium chloride, 2800 mg / L of sodium chloride, and 650 mg / L of sodium phosphate), 3000 mg / L of pH adjusting solution (2000 mg / L of sodium bicarbonate buffer, 1000 mg / L of HEPEs), 50 mg / L of vitamin complex, 400 mg / L of amino acid complex, 1 mg / L of trace element complex, 4500 mg / L of glucose, and 0.25 mg / L of growth factor FGF2. Among them, based on the total added weight of composite protein hydrolysate, the proportion of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate are all 12.5%.

[0148] In another embodiment of the present invention, a protein hydrolyzate stem cell activation medium is provided, wherein the concentrations of the components in the stem cell activation medium are as follows: based on the total volume of the stem cell activation medium, 1-5% fetal bovine serum, 70-84 mg / L arginine, 55-63 mg / L cystine, 550-584 mg / L glutamine, 36-42 mg / L histidine hydrochloride, 95-105 mg / L isoleucine, 95-105 mg / L leucine, 135-146 mg / L lysine hydrochloride, 25-30 mg / L methionine, 60-66 mg / L phenylalanine, 38-42 mg / L serine, 88-95 mg / L threonine, and 10-15 mg / L tryptophan. 2-16mg / L, valine 90-94mg / L, choline chloride 1-4mg / L, calcium pantothenate 1-4mg / L, folic acid 1-4mg / L, nicotinamide 1-4mg / L, pyridoxine hydrochloride 1-4mg / L, riboflavin 0.1-0.4mg / L, thiamine 1-4mg / L, inositol 3-7.2mg / L, ferric nitrate 0.01-0.1mg / L, magnesium sulfate 90-97mg / L, potassium chloride 400-800mg / L, sodium bicarbonate buffer 2000-3700mg / L, sodium chloride 2700-6000mg / L, glucose 4000-6000mg / L and protein hydrolysate 4500-5500mg / L. The protein hydrolysate comprises one of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0149] In another embodiment of the present invention, a protein hydrolyzate stem cell culture medium is provided, which is used to screen the types of protein hydrolyzates. The concentrations of the components in the protein hydrolyzate stem cell culture medium are as follows: based on the total volume of the protein hydrolyzate stem cell culture medium, 1-5% fetal bovine serum, 70-84 mg / L arginine, 55-63 mg / L cystine, 550-584 mg / L glutamine, 36-42 mg / L histidine hydrochloride, 95-105 mg / L isoleucine, 95-105 mg / L leucine, 135-146 mg / L lysine hydrochloride, 25-30 mg / L methionine, 60-66 mg / L phenylalanine, 38-42 mg / L serine, and threonine. 88-95mg / L, tryptophan 12-16mg / L, valine 90-94mg / L, choline chloride 1-4mg / L, calcium pantothenate 1-4mg / L, folic acid 1-4mg / L, nicotinamide 1-4mg / L, pyridoxine hydrochloride 1-4mg / L, riboflavin 0.1-0.4mg / L, thiamine 1-4mg / L, inositol 3-7.2mg / L, ferric nitrate 0.01-0.1mg / L, magnesium sulfate 90-97mg / L, potassium chloride 400-800mg / L, sodium bicarbonate buffer 2000-3700mg / L, sodium chloride 2700-4000mg / L, glucose 4000-6000mg / L and protein hydrolysate 4500-21000mg / L. The protein hydrolysate includes one or more of high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate.

[0150] In another embodiment of the present invention, a composite protein hydrolyzate stem cell activation medium is provided, wherein the concentrations of the components in the stem cell activation medium are as follows: based on the total volume of the stem cell activation medium, 1-5% fetal bovine serum, 70-84 mg / L arginine, 55-63 mg / L cystine, 550-584 mg / L glutamine, 36-42 mg / L histidine hydrochloride, 95-105 mg / L isoleucine, 95-105 mg / L leucine, 135-146 mg / L lysine hydrochloride, 25-30 mg / L methionine, 60-66 mg / L phenylalanine, 38-42 mg / L serine, 88-95 mg / L threonine, and 12-15 mg / L tryptophan. -16mg / L, valine 90-94mg / L, choline chloride 1-4mg / L, calcium pantothenate 1-4mg / L, folic acid 1-4mg / L, niacinamide 1-4mg / L, pyridoxine hydrochloride 1-4mg / L, riboflavin 0.1-0.4mg / L, thiamine 1-4mg / L, inositol 3-7.2mg / L, ferric nitrate 0.01-0.1mg / L, magnesium sulfate 90-97mg / L, potassium chloride 400-800mg / L, sodium bicarbonate buffer 2000-3700mg / L, sodium chloride 2700-4000mg / L, glucose 4000-6000mg / L, and complex protein hydrolysate 4500-5500mg / L. The composite protein hydrolysate contains, based on the total added weight, one or more of 5%-60% of high-free amino acid yeast hydrolysate, 5%-60% of high-nucleotide yeast hydrolysate, 5%-60% of high-glutathione yeast hydrolysate, 5%-60% of high-polypeptide yeast hydrolysate, 5%-60% of rice protein hydrolysate, 5%-60% of high-glutamine wheat protein hydrolysate, 5%-60% of pea protein hydrolysate and 5%-60% of soy protein hydrolysate.

[0151] In another specific embodiment of the present invention, a composite protein hydrolyzate stem cell culture medium is provided. The composite protein hydrolyzate stem cell culture medium is used to screen the mixing weight ratio of composite protein hydrolyzates. The concentration of each component in the composite protein hydrolyzate stem cell culture medium is as follows: based on the total volume of the composite protein hydrolyzate stem cell culture medium, fetal bovine serum 1-5%, arginine 70-84 mg / L, cystine 55-63 mg / L, glutamine 550-584 mg / L, histidine hydrochloride 36-42 mg / L, isoleucine 95-105 mg / L, leucine 95-105 mg / L, lysine hydrochloride 135-146 mg / L, methionine 25-30 mg / L, phenylalanine 60-66 mg / L, serine 38-42 mg / L. L, threonine 88-95mg / L, tryptophan 12-16mg / L, valine 90-94mg / L, choline chloride 1-4mg / L, calcium pantothenate 1-4mg / L, folic acid 1-4mg / L, nicotinamide 1-4mg / L, pyridoxine hydrochloride 1-4mg / L, riboflavin 0.1-0.4mg / L, thiamine 1-4mg / L, inositol 3-7.2mg / L, ferric nitrate 0.01-0.1mg / L, magnesium sulfate 90-97mg / L, potassium chloride 400-800mg / L, sodium bicarbonate buffer 2000-3700mg / L, sodium chloride 2700-4000mg / L, glucose 4000-6000mg / L and complex protein hydrolysate 4500-21000mg / L. The composite protein hydrolysate comprises, based on the total added weight, one or more substances selected from the group consisting of 5%-60% high-free amino acid yeast hydrolysate, 5%-60% high-nucleotide yeast hydrolysate, 5%-60% high-glutathione yeast hydrolysate, 5%-60% high-polypeptide yeast hydrolysate, 5%-60% rice protein hydrolysate, 5%-60% high-glutamine wheat protein hydrolysate, 5%-60% pea protein hydrolysate and 5%-60% soy protein hydrolysate.

[0152] It should be noted that the amount of each component added to any of the above-mentioned stem cell culture media or stem cell activation media is based on a total volume of 1 L of the stem cell culture media or stem cell activation media, and the stem cell culture media or stem cell activation media is dissolved and fixed to volume with sterilized ultrapure water.

[0153] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0154] Unless otherwise specified, the various reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The experimental materials and instrument information used in the present invention are shown in the following table:

[0155] Table 1 Experimental materials / instruments and manufacturers

[0156] The culture medium components involved in the embodiment are as follows:

[0157] Control culture medium (CK): 1 L of control culture medium (CK) was prepared according to the ratio of 90% by volume of DMEM + 10% by volume of fetal bovine serum.

[0158] The other components of the culture medium involved in the embodiment are as follows:

[0159] The pH regulating solution includes sodium bicarbonate buffer and HEPEs, wherein the sodium bicarbonate buffer is prepared by dissolving sodium bicarbonate in sterilized ultrapure water to prepare a sodium bicarbonate buffer with a concentration of 7.5%.

[0160] The antibiotic mixture is preferably a 100× double antibiotic mixture of penicillin and streptomycin, wherein the mixing ratio of the 100× double antibiotic mixture of penicillin and streptomycin is: 10g penicillin + 10g streptomycin dissolved in 1L of sterilized ultrapure water to prepare a 10g / L double antibiotic mixture. When preparing the culture medium, 10ml of the double antibiotic mixture is added to 1L of culture medium to make a working concentration of 100mg / L.

[0161] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0162] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0163] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0164] The protein hydrolysates ah and AH involved in the examples are both products sold by Angel Yeast Co., Ltd. To study the use of stem cell culture media, the present inventors tested the contents of various amino acids or polypeptides, carbohydrates, nucleotides, and total nitrogen in them. The specific parameters are as follows:

[0165] Protein hydrolysate a: ordinary yeast hydrolysate, with a free amino acid content of 25%;

[0166] Protein hydrolysate b: nucleotide-containing yeast hydrolysate, with a free nucleotide content of 0.5%;

[0167] Protein hydrolysate c: glutathione-containing yeast hydrolysate, with a glutathione content of 1%;

[0168] Protein hydrolysate d: a polypeptide-containing yeast hydrolysate, wherein the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the total peptides in the high-polypeptide yeast hydrolysate is 15%;

[0169] Protein hydrolysate e: conventional rice protein hydrolysate, with a total nitrogen content of 11%;

[0170] Protein hydrolysate f: ordinary high glutamine wheat protein hydrolysate, its hydrolyzed glutamine content is 10%;

[0171] Protein hydrolysate g: conventional pea protein hydrolysate, with a total nitrogen content of 11%;

[0172] Protein hydrolysate h: Soy protein hydrolysate, its carbohydrate content is 10%.

[0173] Protein hydrolysate A: high free amino acid yeast hydrolysate, its free amino acid content is 57.1%.

[0174] Protein hydrolysate B: high nucleotide yeast hydrolysate, with a free nucleotide content of 6.5%;

[0175] Protein hydrolysate C: high glutathione yeast hydrolysate, with a glutathione content of 11.2%;

[0176] Protein hydrolysate D: a high-polypeptide yeast hydrolysate, wherein the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the high-polypeptide yeast hydrolysate is 25.7% of the total peptides in the high-polypeptide yeast hydrolysate;

[0177] Protein hydrolysate E: rice protein hydrolysate, with a total nitrogen content of 12.5%;

[0178] Protein hydrolysate F: high glutamine wheat protein hydrolysate, with a hydrolyzed glutamine content of 24.3%;

[0179] Protein hydrolysate G: pea protein hydrolysate, with a total nitrogen content of 13.8;

[0180] Protein hydrolysate H: Soy protein hydrolysate, its carbohydrate content is 21.4%.

[0181] It should be noted that the inventors have discovered through extensive experiments that although protein hydrolysate AH contains other untested components, these untested components have been shown to have no effect on the experimental results of culturing stem cells according to the present invention. In other words, the components in protein hydrolysate AH that have an effect on stem cells are specifically as follows:

[0182] (1) Protein hydrolysate A is a high-free amino acid yeast hydrolysate (Angel yeast extract CM05 (powder) for cell culture). The free amino acid content of the protein hydrolysate A is 57.1% by weight, wherein the free amino acids are 2.77% aspartic acid, 2.47% threonine, 2.78% serine, 6.92% glutamic acid, 4.64% glycine, 13.98% alanine, 0.56% cystine, 3.44% valine, 0.96% methionine, 2.12% isoleucine, 4.13% leucine, 1.37% tyrosine, 2.04% phenylalanine, 2.79% lysine, 1.01% histidine, 4.17% arginine and 0.95% proline.

[0183] Based on the weight of the protein hydrolysate A, the protein hydrolysate A has a total nitrogen content of 11.6%, an ammonia nitrogen content of 5.8%, an oxidized glutathione content of 0.7%, and a reduced glutathione content of 0.2%.

[0184] Based on the weight of protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate A is 3.17% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 2.03% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 13.37% of the total peptide segments in the protein hydrolysate A, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 42.61% of the total peptide segments in the protein hydrolysate A, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 38.83% of the total peptide segments in the protein hydrolysate A.

[0185] Based on the weight of the protein hydrolysate A, the free nucleotide content in the protein hydrolysate A is 0.41%, wherein, based on the weight of the protein hydrolysate A, the free nucleotides are 0.10% of uracil nucleotide (UMP), 0.20% of adenine nucleotide (AMP), 0.00% of inosine nucleotide (IMP), 0.02% of guanine nucleotide (GMP) and 0.09% of cytosine nucleotide (CMP).

[0186] (2) Protein hydrolysate B is a high-nucleotide yeast hydrolysate (model: Angel Yeast Extract Powder FM502). The free nucleotide content of the protein hydrolysate B is 6.5% based on the weight of the protein hydrolysate B. Among them, the free nucleotides are 1.39% of uracil nucleotide (UMP), 1.52% of adenine nucleotide (AMP), 2.12% of inosine nucleotide (IMP), 1.36% of guanine nucleotide (GMP) and 0.11% of cytosine nucleotide (CMP).

[0187] Based on the weight of protein hydrolysate B, the protein hydrolysate B has a total nitrogen content of 11.14%, an ammonia nitrogen content of 4.17%, an oxidized glutathione content of 0.1%, and a reduced glutathione content of 0.1%.

[0188] Based on the weight of protein hydrolysate B, the mass percentage of peptides with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate B is 0.04% of the total peptides in the protein hydrolysate B, the mass percentage of peptides with a molecular weight greater than 1000 and less than 2000 Daltons is 1.76% of the total peptides in the protein hydrolysate B, the mass percentage of peptides with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 18.06% of the total peptides in the protein hydrolysate B, the mass percentage of peptides with a molecular weight greater than 180 and less than or equal to 400 Daltons is 50.51% of the total peptides in the protein hydrolysate B, and the mass percentage of peptides with a molecular weight less than or equal to 180 Daltons is 29.27% ​​of the total peptides in the protein hydrolysate B.

[0189] Based on the weight of the protein hydrolysate B, the free amino acid content of the protein hydrolysate B is 29.44%, wherein, based on the weight of the protein hydrolysate B, the free amino acids are 0.97% of aspartic acid, 1.07% of threonine, 1.09% of serine, 6.77% of glutamic acid, 0.75% of glycine, 5.92% of alanine, 0.09% of cystine, 1.71% of valine, 0.48% of methionine, 1.25% of isoleucine, 2.55% of leucine, 1.40% of tyrosine, 1.67% of phenylalanine, 1.45% of lysine, 0.39% of histidine, 1.31% of arginine and 0.54% of proline.

[0190] (3) Protein hydrolysate C is a high-glutathione yeast hydrolysate (Angel yeast extract CM07 (powder) for cell culture), and its glutathione content is 11.2%, wherein, based on the weight of protein hydrolysate C, the glutathione includes 0.7% oxidized glutathione and 10.5% reduced glutathione.

[0191] Based on the weight of the protein hydrolysate C, the total nitrogen content of the protein hydrolysate C is 9.2% and the ammonia nitrogen content is 3.0%.

[0192] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate C is 4.58% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 11.39% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 21.24% of the total peptide segments in the protein hydrolysate C, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 25.04% of the total peptide segments in the protein hydrolysate C, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 37.76% of the total peptide segments in the protein hydrolysate C.

[0193] Based on the weight of the protein hydrolysate C, the free nucleotide content in the protein hydrolysate C is 1.4%, wherein, based on the weight of the protein hydrolysate C, the free nucleotides include 0.20% of uracil nucleotide (UMP), 0.70% of adenine nucleotide (AMP), 0.20% of inosine nucleotide (IMP), 0.20% of guanine nucleotide (GMP) and 0.10% of cytosine nucleotide (CMP).

[0194] The protein hydrolysate C has a free amino acid content of 9.96%, based on the weight of the protein hydrolysate C. The free amino acids, based on the weight of the protein hydrolysate C, are aspartic acid 0.38%, threonine 0.54%, serine 0.31%, glutamic acid 4.22%, glycine 0.28%, alanine 0.96%, cystine 0.62%, valine 0.18%, methionine 0.04%, isoleucine 0.14%, leucine 0.23%, tyrosine 0.31%, phenylalanine 0.07%, lysine 0.24%, histidine 0.09%, arginine 1.31%, and proline 0.04%.

[0195] (4) Protein hydrolysate D is a high-polypeptide yeast hydrolysate (Angel Yeast Peptone CM08 (powder)), and the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the total peptides in the high-polypeptide yeast hydrolysate is 25.7%, wherein the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the total peptides in the high-polypeptide yeast hydrolysate includes: the mass percentage of peptides with a molecular weight greater than or equal to 2000 Daltons in the total peptides in the protein hydrolysate D is 4.5%, and the mass percentage of peptides with a molecular weight greater than 1000 and less than 2000 Daltons in the total peptides in the protein hydrolysate D is 21.2%.

[0196] The mass percentage of peptides with a molecular weight greater than 400 and less than or equal to 1000 Daltons in the protein hydrolysate D is 17.28% of the total peptides in the protein hydrolysate D, the mass percentage of peptides with a molecular weight greater than 180 and less than or equal to 400 Daltons in the protein hydrolysate D is 31.83% of the total peptides in the protein hydrolysate D, and the mass percentage of peptides with a molecular weight less than or equal to 180 Daltons in the protein hydrolysate D is 34.75%.

[0197] Based on the weight of the protein hydrolysate D, the protein hydrolysate D has a total nitrogen content of 11.3%, an ammonia nitrogen content of 5%, an oxidized glutathione content of 0.45%, and a reduced glutathione content of 0.39%.

[0198] Based on the weight of the protein hydrolysate D, the free nucleotide content in the protein hydrolysate D is 0.1%, wherein, based on the weight of the protein hydrolysate D, the free nucleotides include 0.00% of uracil nucleotide (UMP), 0.10% of adenine nucleotide (AMP), 0.00% of inosine nucleotide (IMP), 0.00% of guanine nucleotide (GMP) and 0.00% of cytosine nucleotide (CMP).

[0199] Based on the weight of the protein hydrolysate D, the free amino acid content of the protein hydrolysate D is 26.21%, wherein, based on the weight of the protein hydrolysate D, the free amino acids are 1.06% of aspartic acid, 1.19% of threonine, 1.13% of serine, 4.73% of glutamic acid, 1.27% of glycine, 5.41% of alanine, 0.79% of cystine, 1.71% of valine, 0.50% of methionine, 1.11% of isoleucine, 1.81% of leucine, 0.48% of tyrosine, 1.16% of phenylalanine, 1.18% of lysine, 0.30% of histidine, 1.62% of arginine, and 0.76% of proline.

[0200] (5) Protein hydrolysate E is rice protein hydrolysate (Angel Rice Peptone FP230 (powder)), and its total nitrogen content is 12.5%.

[0201] Based on the weight of the protein hydrolysate E, the ammonia nitrogen content in the protein hydrolysate E is 2.1%.

[0202] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate E is 2.55% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 6.55% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 20.85% of the total peptide segments in the protein hydrolysate E, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 45.29% of the total peptide segments in the protein hydrolysate E, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 24.75% of the total peptide segments in the protein hydrolysate E.

[0203] Based on the weight of the protein hydrolysate E, the free amino acid content of the protein hydrolysate E is 15.42%, wherein, based on the weight of the protein hydrolysate E, the free amino acids are 0.38% of aspartic acid, 0.73% of threonine, 0.6% of serine, 1.11% of glutamic acid, 0.31% of glycine, 0.84% ​​of alanine, 0.25% of cystine, 1.27% of valine, 0.4% of methionine, 0.93% of isoleucine, 2.23% of leucine, 1.05% of tyrosine, 1.38% of phenylalanine, 0.92% of lysine, 0.44% of histidine, 2.48% of arginine and 0.1% of proline.

[0204] (6) Protein hydrolysate F is a high-glutamine wheat protein hydrolysate (Angel Wheat Protein Hydrolysate PU020 (powder)), and its hydrolyzed glutamine content is 24.3%.

[0205] Based on the weight of the protein hydrolysate F, the protein hydrolysate E has a total nitrogen content of 11.72% and an ammonia nitrogen content of 2.08%.

[0206] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate F is 1.72% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 3.85% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 14.08% of the total peptide segments in the protein hydrolysate F, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 33.62% of the total peptide segments in the protein hydrolysate F, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 46.73% of the total peptide segments in the protein hydrolysate F.

[0207] The protein hydrolysate F has a free amino acid content of 12.08% based on the weight of the protein hydrolysate F, wherein the free amino acids, based on the weight of the protein hydrolysate F, are 0.11% of aspartic acid, 0.70% of threonine, 0.74% of serine, 1.08% of glutamic acid, 0.17% of glycine, 0.63% of alanine, 0.18% of cystine, 1.27% of valine, 0.41% of methionine, 1.07% of isoleucine, 2.16% of leucine, 0.68% of tyrosine, 0.86% of phenylalanine, 0.49% of lysine, 0.43% of histidine, 1.07% of arginine, and 0.03% of proline.

[0208] (7) Protein hydrolysate G is pea protein hydrolysate (Angel Pea Peptone FP210 (powder)), and its total nitrogen content is 13.8%.

[0209] Based on the weight of the protein hydrolysate G, the ammonia nitrogen content in the protein hydrolysate G is 2.08%.

[0210] The mass percentage of peptide segments with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate G is 2.08% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 1000 and less than 2000 Daltons is 6.92% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 400 and less than or equal to 1000 Daltons is 27.12% of the total peptide segments in the protein hydrolysate G, the mass percentage of peptide segments with a molecular weight greater than 180 and less than or equal to 400 Daltons is 40.90% of the total peptide segments in the protein hydrolysate G, and the mass percentage of peptide segments with a molecular weight less than or equal to 180 Daltons is 22.98% of the total peptide segments in the protein hydrolysate G.

[0211] The protein hydrolysate G has a free amino acid content of 14.55% based on the weight of the protein hydrolysate G, wherein the free amino acids, based on the weight of the protein hydrolysate G, are 0.15% of aspartic acid, 0.68% of threonine, 0.71% of serine, 0.81% of glutamic acid, 0.28% of glycine, 0.66% of alanine, 0.07% of cystine, 0.9% of valine, 0.05% of methionine, 0.63% of isoleucine, 2.17% of leucine, 1.34% of tyrosine, 2.27% of phenylalanine, 1.22% of lysine, 0.42% of histidine, 2.15% of arginine, and 0.04% of proline.

[0212] (8) Protein hydrolysate H is a soy protein hydrolysate (Angel soy protein hydrolysate PU048 (powder)), and its carbohydrate content is 21.4%.

[0213] Based on the weight of the protein hydrolysate H, the total nitrogen content of the protein hydrolysate H is 10.65% and the ammonia nitrogen content is 1.91%.

[0214] The mass percentage of peptides with a molecular weight greater than or equal to 2000 Daltons in the protein hydrolysate H accounts for 5.40% of the total peptides in the protein hydrolysate G, the mass percentage of peptides with a molecular weight greater than 1000 and less than 2000 Daltons accounts for 7.36% of the total peptides in the protein hydrolysate H, the mass percentage of peptides with a molecular weight greater than 400 and less than or equal to 1000 Daltons accounts for 20.29% of the total peptides in the protein hydrolysate H, the mass percentage of peptides with a molecular weight greater than 180 and less than or equal to 400 Daltons accounts for 30.02% of the total peptides in the protein hydrolysate H, and the mass percentage of peptides with a molecular weight less than or equal to 180 Daltons accounts for 36.92% of the total peptides in the protein hydrolysate H.

[0215] Based on the weight of the protein hydrolysate H, the free amino acid content of the protein hydrolysate H is 9.02%, wherein, based on the weight of the protein hydrolysate H, the free amino acids are 0.12% of aspartic acid, 0.42% of threonine, 0.31% of serine, 0.58% of glutamic acid, 0.11% of glycine, 0.29% of alanine, 0.14% of cystine, 0.60% of valine, 0.10% of methionine, 0.62% of isoleucine, 1.41% of leucine, 0.97% of tyrosine, 1.21% of phenylalanine, 0.63% of lysine, 0.25% of histidine, 1.15% of arginine and 0.13% of proline.

[0216] The methods for determining the free amino acid content, total nitrogen content, ammonia nitrogen content, peptide relative molecular weight distribution, free nucleotide content, hydrolyzed glutamine content, carbohydrate content, oxidized glutathione content, and reduced glutathione content mentioned in the above-mentioned protein hydrolysates AH are specifically as follows:

[0217] (1) Determination of free amino acid content

[0218] Weigh 0.5g to 1g of the sample (accurate to 0.001g) and place it in a 50mL volumetric flask. Add 20mL of sulfosalicylic acid and sonicate until fully dissolved. Then dilute to 50mL and mix thoroughly. After standing for 1h, accurately pipette 1mL of the supernatant into a 25mL volumetric flask and add 0.02mol / L hydrochloric acid solution or sodium citrate buffer to the mark. After mixing, filter through a 0.22μm microporous filter into a sample injection bottle for testing. According to the instructions of the amino acid automatic analyzer, the concentration of the mixed amino acid standard solution is within the optimal detection range of the instrument and is measured as an external standard on the instrument.

[0219] The amino acid content of the sample solution is calculated according to formula (1):

[0220] Where:

[0221] C i : The content of amino acid i in the sample solution, in nmol / mL;

[0222] A i : Peak area of ​​amino acid i in the sample determination solution;

[0223] A s : The peak area of ​​amino acid s in the amino acid standard working solution;

[0224] C s : The content of amino acid s in the amino acid standard working solution, in nmol / mL.

[0225] The content of each amino acid in the sample is calculated according to formula (2):

[0226] Where:

[0227] X i : The content of amino acid i in the sample, in %;

[0228] C i : The content of amino acid i in the sample solution, in nmol / mL;

[0229] F: dilution factor;

[0230] V: volume of the sample, in mL;

[0231] M: molar mass of amino acid i, in g / mol;

[0232] m: sample weight, in g.

[0233] (2) Determination of total nitrogen: Use the Kjeldahl method 6.4 in the national standard GB / T 23530-2009: take a sample (equivalent to 30440 mg of total nitrogen), add 20 mL of concentrated sulfuric acid to digest it in the presence of 5 g of mixed catalyst a (potassium sulfate and sewage copper sulfate mixed in a ratio of 97:3) and 2.5 g of catalyst b (selenium powder and potassium sulfate mixed in a ratio of 0.1:100); then distill it and absorb the product ammonia with boric acid; then titrate it with 0.1 mol / L hydrochloric acid, read the data, and calculate the total nitrogen content.

[0234] (3) Determination of Ammonia Nitrogen (Amino Acid Nitrogen) Content: The amino acid nitrogen detection method of 6.5 in the national standard GB / T 23530-2009 was used: 5 g of sample was diluted and titrated with 0.5 mol / L sodium hydroxide solution to a pH of 8.2, and the pH was maintained for 1 min. 10 mL of 36% formaldehyde solution was slowly added to react with the non-dissociated amino groups in the neutral amino acids to generate monomethylol and dimethylol derivatives. This reaction was completely quantitative. The hydrogen ions released were titrated with the sodium hydroxide solution described above, and the amino acid nitrogen content was calculated based on the amount of alkali solution consumed.

[0235] (4) Determination of peptide relative molecular weight distribution

[0236] Determination of peptide relative molecular mass distribution: Modify the standard sample based on the determination method of peptide relative molecular mass distribution in Appendix A of GB / T 22492-2008 soybean peptide powder. The specific operation steps are as follows:

[0237] Weigh 10 mg of the sample into a 10 mL volumetric flask, add a small amount of mobile phase, and ultrasonically vibrate to fully dissolve and mix the sample. Dilute to the scale with mobile phase, pass through a 0.45 μm organic membrane, and wait for injection analysis. Weigh 5 mg of each standard (cytochrome C, bacitracin, tyrosine-tyrosine-arginine, tyrosine-tyrosine-tyrosine) into a 10 mL volumetric flask, dissolve and adjust to volume with mobile phase, filter and inject with a 0.45 μm organic phase membrane, and obtain a chromatogram of the standard. Plot the logarithm of the relative molecular mass against the retention time to obtain a calibration curve and its equation. X = A / A 总 ×100%

[0238] Where:

[0239] X—mass fraction of a peptide segment with a certain relative molecular mass in the sample, %

[0240] A—peak area of ​​a peptide segment with a certain relative molecular mass;

[0241] A 总 —The sum of the peak areas of peptides of each relative molecular mass

[0242] (5) Determination of free nucleotide content

[0243] Determined according to Appendix H of GB / T 20886.2-2021.

[0244] (6) Determination of hydrolyzed glutamine content: Determine according to the method in GB 5009.124-2016.

[0245] (7) Determination of carbohydrate content:

[0246] Carbohydrate content = 100% - (fat content + total nitrogen content + free nucleotide content + moisture content + ash content).

[0247] The determination methods of total nitrogen content and free nucleotide content are as shown above.

[0248] Determination of fat content: According to the near infrared method specified in GB / T24870-2010.

[0249] Determination of moisture content: Determine according to Appendix A of GB / T 20886.2-2021.

[0250] Determination of ash content: Determine according to Appendix G of GB / T 20886.2-2021.

[0251] (8) Determination of oxidized glutathione content and reduced glutathione content: Determination shall be carried out according to the method in Appendix A of GB / T 35882-2018.

[0252] Example 1: Screening of protein hydrolysate types

[0253] (1) Preparation of culture medium:

[0254] (1-1) The amount of each component added to the protein hydrolyzate stem cell activation medium is based on a total volume of 1 L of the stem cell activation medium. The concentrations of each component in the stem cell activation medium are as follows:

[0255] Based on the total volume of protein hydrolyzate stem cell activation medium, fetal bovine serum 5%, arginine 84mg / L, cystine 63mg / L, glutamine 584mg / L, histidine hydrochloride 42mg / L, isoleucine 105mg / L, leucine 105mg / L, lysine hydrochloride 146mg / L, methionine 30mg / L, phenylalanine 66mg / L, serine 42mg / L, threonine 95mg / L, tryptophan 16mg / L, valine 94mg / L, choline chloride 4mg / L, calcium pantothenate 4mg / L, folic acid 4mg / L, niacinamide 4mg / L, salt The medium contains 4 mg / L pyridoxine, 0.4 mg / L riboflavin, 4 mg / L thiamine, 7.2 mg / L inositol, 0.1 mg / L ferric nitrate, 97 mg / L magnesium sulfate, 400 mg / L potassium chloride, 3700 mg / L sodium bicarbonate buffer, 6000 mg / L sodium chloride, 4500 mg / L glucose, and 5000 mg / L protein hydrolyzate (A, B, C, D, E, F, G, H, a, b, c, d, e, f, g, or h), which are named stem cell activation medium A, B, C, D, E, F, G, H, a, b, c, d, e, f, g, or h, respectively.

[0256] For the protein hydrolyzate stem cell activation medium, the total weight of all components except fetal bovine serum is taken as the total weight of the protein hydrolyzate stem cell activation medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the protein hydrolyzate stem cell activation medium is calculated to be 23.6%. The specific calculation method is as follows:

[0257] Protein Hydrolysate / (Protein Hydrolysate + Arginine + Cystine + Glutamine + Histidine HCl + Isoleucine + Leucine + Lysine HCl + Methionine + Phenylalanine + Serine + Threonine + Tryptophan + Valine + Choline Chloride + Calcium Pantothenate + Folic Acid + Nicotinamide + Pyridoxine Hydrochloride + Riboflavin + Thiamine + Inositol + Ferric Nitrate + Magnesium Sulfate + Potassium Chloride + Sodium Bicarbonate Buffer + Sodium Chloride + Glucose)

[0258] =5000 / (5000+84+63+584+42+105+105+146+30+66+42+95+16+94+4+4+4+4+4+0.4+4+7.2+0.1+97+400+3700+6000+4500)=23.6%.

[0259] (1-2) The amount of each component added to the protein hydrolyzate stem cell culture medium of the embodiment was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to the desired volume, based on a total volume of 1 L of the protein hydrolyzate stem cell culture medium of the embodiment. The specific concentrations of the components in the protein hydrolyzate stem cell culture medium of the embodiment are as follows:

[0260] Based on the total volume of the protein hydrolyzate stem cell culture medium of the embodiment, the following ingredients are included: 5% fetal bovine serum, 84 mg / L arginine, 63 mg / L cystine, 584 mg / L glutamine, 42 mg / L histidine hydrochloride, 105 mg / L isoleucine, 105 mg / L leucine, 146 mg / L lysine hydrochloride, 30 mg / L methionine, 66 mg / L phenylalanine, 42 mg / L serine, 95 mg / L threonine, 16 mg / L tryptophan, 94 mg / L valine, 4 mg / L choline chloride, 4 mg / L calcium pantothenate, 4 mg / L folic acid, 4 mg / L nicotinamide, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, and 4 mg / L thiamine. g / L, inositol 7.2 mg / L, ferric nitrate 0.1 mg / L, magnesium sulfate 97 mg / L, potassium chloride 400 mg / L, sodium bicarbonate buffer 3700 mg / L, sodium chloride 6000 mg / L, glucose 4500 mg / L and 5000 mg / L, 15000 mg / L and 20000 mg / L of protein hydrolysate (A, B, C, D, E, F, G or H), respectively, the protein hydrolyzate stem cell culture medium of the embodiment is set to A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3, E1, E2, E3, F1, F2, F3, G1, G2, G3, H1, H2 or H3. Among them, A1 represents the stem cell culture medium added with 5000 mg / L protein hydrolysate A, A2 represents the stem cell culture medium added with 15000 mg / L protein hydrolysate A, and A3 represents the stem cell culture medium added with 20000 mg / L protein hydrolysate A. Similarly, protein hydrolysate stem cell culture media added with B1, B2, B3, C1, C2, C3, D1, D2, D3, E1, E2, E3, F1, F2, F3, G1, G2, G3, H1, H2 and H3 are obtained. Among them, protein hydrolysate A is a high-free amino acid yeast hydrolysate with a free amino acid content of 57.1%; protein hydrolysate B is a high-nucleotide yeast hydrolysate with a free nucleotide content of 6.5%; protein hydrolysate C is a high-glutathione yeast hydrolysate with a glutathione content of 11.2%; protein hydrolysate D is a high-polypeptide yeast hydrolysate, and the mass percentage of peptides with a molecular weight greater than 1000 Daltons in the high-polypeptide yeast hydrolysate is 25.7% of the total peptides in the high-polypeptide yeast hydrolysate; protein hydrolysate E is a rice protein hydrolysate with a total nitrogen content of 12.5%; protein hydrolysate F is a high-glutamine wheat protein hydrolysate with a hydrolyzed glutamine content of 24.3%; protein hydrolysate G is a pea protein hydrolysate with a total nitrogen content of 13.8; and protein hydrolysate H is a soy protein hydrolysate with a carbohydrate content of 21.4%.

[0261] For the protein hydrolyzate stem cell culture medium (referred to as "stem cell culture medium"), the total weight of each component except fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the stem cell culture medium is calculated. The specific calculation method is as follows:

[0262] Weight ratio of protein hydrolysate to stem cell culture medium = 100% * protein hydrolysate / (protein hydrolysate + arginine + cystine + glutamine + histidine hydrochloride + isoleucine + leucine + lysine hydrochloride + methionine + phenylalanine + serine + threonine + tryptophan + valine + choline chloride + calcium pantothenate + folic acid + niacinamide + pyridoxine hydrochloride + riboflavin + thiamine + inositol + ferric nitrate + magnesium sulfate + potassium chloride + sodium bicarbonate buffer + sodium chloride + glucose)

[0263] Specifically, (a) when the added weight of the protein hydrolysate is 5000 mg, the weight ratio of the protein hydrolysate to the stem cell culture medium is calculated to be 23.6%;

[0264] (b) When the added weight of the protein hydrolysate is 15,000 mg, the weight ratio of the protein hydrolysate to the stem cell culture medium is calculated to be 48.1%;

[0265] (c) When the added weight of the protein hydrolysate was 20,000 mg, the weight ratio of the protein hydrolysate to the stem cell culture medium was calculated to be 55.2%.

[0266] (1-3) The amount of each component added to the protein hydrolyzate stem cell culture medium of the comparative example was based on a total volume of 1 L of the protein hydrolyzate stem cell culture medium of the comparative example. The concentrations of each component in the protein hydrolyzate stem cell culture medium of the comparative example were as follows:

[0267] Based on the total volume of the protein hydrolyzate stem cell culture medium in the comparative example, the following ingredients are included: 5% fetal bovine serum, 84 mg / L arginine, 63 mg / L cystine, 584 mg / L glutamine, 42 mg / L histidine hydrochloride, 105 mg / L isoleucine, 105 mg / L leucine, 146 mg / L lysine hydrochloride, 30 mg / L methionine, 66 mg / L phenylalanine, 42 mg / L serine, 95 mg / L threonine, 16 mg / L tryptophan, 94 mg / L valine, 4 mg / L choline chloride, 4 mg / L calcium pantothenate, 4 mg / L folic acid, 4 mg / L nicotinamide, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, and 4 mg thiamine. / L, inositol 7.2 mg / L, ferric nitrate 0.1 mg / L, magnesium sulfate 97 mg / L, potassium chloride 400 mg / L, sodium bicarbonate buffer 3700 mg / L, sodium chloride 6000 mg / L, glucose 4500 mg / L, and 5000 mg / L, 15000 mg / L and 20000 mg / L of protein hydrolysate (a, b, c, d, e, f, g or h), respectively, and the protein hydrolyzate stem cell culture medium of the comparative example is set to a1, a2, a3, b1, b2, b3, c1, c2, c3, d1, d2, d3, e1, e2, e3, f1, f2, f3, g1, g2, g3, h1, h2 or h3. Among them, a1 represents the stem cell culture medium added with 5000 mg / L protein hydrolysate a, a2 represents the stem cell culture medium added with 15000 mg / L protein hydrolysate a, and a3 represents the stem cell culture medium added with 20000 mg / L protein hydrolysate a. Similarly, protein hydrolysate stem cell culture media added with b1, b2, b3, c1, c2, c3, d1, d2, d3, e1, e2, e3, f1, f2, f3, g1, g2, g3, h1, h2 and h3 are obtained. Among them, protein hydrolysate a is ordinary yeast hydrolysate with a free amino acid content of 25%; protein hydrolysate b is a nucleotide-containing yeast hydrolysate with a free nucleotide content of 0.5%; protein hydrolysate c is a glutathione-containing yeast hydrolysate with a glutathione content of 1%; protein hydrolysate d is a polypeptide-containing yeast hydrolysate, and the mass percentage of peptide segments with a molecular weight greater than 1000 Daltons in the high-polypeptide yeast hydrolysate is 15%; protein hydrolysate e is a conventional rice protein hydrolysate with a total nitrogen content of 11%; protein hydrolysate f is an ordinary high-glutamine wheat protein hydrolysate with a hydrolyzed glutamine content of 10%; protein hydrolysate g is a conventional pea protein hydrolysate with a total nitrogen content of 11%; and protein hydrolysate h is a soy protein hydrolysate with a carbohydrate content of 10%.

[0268] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using the stem cell activation medium A, B, C, D, E, F, G, H, a, b, c, d, e, f, g, or h in step (1-1), as well as the control medium (CK). 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0269] (3) Proliferation and culture of stem cells: The activated C2C12 mouse myoblasts were digested with trypsin and centrifuged to collect the cells. The cells were inoculated into the corresponding example culture medium (for example, the C2C12 mouse myoblasts activated by using stem cell activation medium A in step (2) were inoculated into protein hydrolyzate A1 stem cell culture medium, protein hydrolyzate A2 stem cell culture medium, and protein hydrolyzate A3 stem cell culture medium for expansion culture), the corresponding comparative example protein hydrolyzate stem cell culture medium, and the control medium (CK) in a 6 cm cell culture dish, and the inoculation cell density was 4×10 4 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0270] (4) Detection of stem cell growth and status:

[0271] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and the density of viable cells after expansion was detected using a cell counter.

[0272] To determine the stemness-positive rate, cells were collected after expansion and culture, and immunofluorescence staining with a PAX7 antibody was performed to detect PAX7 protein expression. The cells were then observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 2.

[0273] Table 2. Effects of different types of protein hydrolysate stem cell culture media on stem cell growth and status detection

[0274] As shown in Table 2, the results indicate that protein hydrolysates A, B, C, D, E, F, G, or H used in the protein hydrolysate stem cell culture medium of the Examples can all be used as core raw materials for stem cell culture medium. Furthermore, by comparing the viable cell density and stemness positive rate after expansion, the protein hydrolysates A, B, C, D, E, F, G, or H used in the comparative culture medium were inferior to the protein hydrolysates A, B, C, D, E, F, G, or H used in the culture medium of the Examples in terms of both cell viability and stemness positive rate. Therefore, it can be concluded that culture media prepared with protein hydrolysates A, B, C, D, E, F, G, or H at appropriate concentrations can meet the requirements for stem cell expansion and maintenance of stem cell characteristics. Therefore, the protein hydrolyzates A, B, C, D, E, F, G, or H used in the culture medium of the examples can all be used as core raw materials for developing stem cell culture media. Furthermore, when the optimal solution is determined based on the criteria that the viable cell density and stemness positive rate after expansion are both no less than 80% of the control group CK, the culture media of the examples A1, A2, B2, C1, C2, D2, E1, E2, F2, F3, G1, H2, and H3 are optimal solutions. In contrast, when the concentration of protein hydrolyzate A, B, C, D, E, F, G, or H used in the protein hydrolyzate stem cell culture medium of the examples is 15,000 mg / L, the viable cell density and stem cell positive rate of the cultured stem cells after expansion are higher. Therefore, in the following Example 2, a composite protein hydrolyzate stem cell culture medium is prepared at a concentration of 15,000 mg / L to screen the mixing weight of the composite protein hydrolyzate.

[0275] Example 2: Screening of the mixing weight ratio of the composite protein hydrolysate

[0276] (1) Preparation of culture medium:

[0277] (1-1) The amount of each component added to the composite protein hydrolyzate stem cell activation medium is based on a total volume of 1 L of stem cell activation medium. The components are dissolved and fixed to volume with sterilized ultrapure water. The specific concentrations of the components in the stem cell activation medium are as follows:

[0278] The concentrations of the components in the composite protein hydrolyzate stem cell activation medium are as follows: based on the total volume of the stem cell activation medium, 5% fetal bovine serum, 84 mg / L arginine, 63 mg / L cystine, 584 mg / L glutamine, 42 mg / L histidine hydrochloride, 105 mg / L isoleucine, 105 mg / L leucine, 146 mg / L lysine hydrochloride, 30 mg / L methionine, 66 mg / L phenylalanine, 42 mg / L serine, 95 mg / L threonine, 16 mg / L tryptophan, 94 mg / L valine, 4 mg / L choline chloride, and 4 mg / L calcium pantothenate. , folic acid 4 mg / L, nicotinamide 4 mg / L, pyridoxine hydrochloride 4 mg / L, riboflavin 0.4 mg / L, thiamine 4 mg / L, inositol 7.2 mg / L, ferric nitrate 0.1 mg / L, magnesium sulfate 97 mg / L, potassium chloride 400 mg / L, sodium bicarbonate buffer 3700 mg / L, sodium chloride 6000 mg / L, glucose 4500 mg / L and 5000 mg / L composite protein hydrolysate, wherein the composite protein hydrolysate is obtained by mixing protein hydrolysates AH in a weight ratio, and the weight ratio of the protein hydrolysates AH is specifically shown in Table 3.

[0279] For the composite protein hydrolyzate stem cell activation medium, the total weight of each component except fetal bovine serum is taken as the total weight of the composite protein hydrolyzate stem cell activation medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the composite protein hydrolyzate stem cell activation medium is calculated to be 23.6%. The specific calculation method is as follows:

[0280] Complex protein hydrolysate / (complex protein hydrolysate + arginine + cystine + glutamine + histidine hydrochloride + isoleucine + leucine + lysine hydrochloride + methionine + phenylalanine + serine + threonine + tryptophan + valine + choline chloride + calcium pantothenate + folic acid + niacinamide + pyridoxine hydrochloride + riboflavin + thiamine + inositol + ferric nitrate + magnesium sulfate + potassium chloride + sodium bicarbonate buffer + sodium chloride + glucose).

[0281] (1-2) The amount of each component added to the composite protein hydrolyzate stem cell culture medium of the embodiment was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to the total volume of 1 L. The specific concentrations of the components in the composite protein hydrolyzate stem cell culture medium of the embodiment are as follows:

[0282] Based on the total volume of the composite protein hydrolysate stem cell culture medium, the following ingredients are included: 5% fetal bovine serum, 84mg / L arginine, 63mg / L cystine, 584mg / L glutamine, 42mg / L histidine hydrochloride, 105mg / L isoleucine, 105mg / L leucine, 146mg / L lysine hydrochloride, 30mg / L methionine, 66mg / L phenylalanine, 42mg / L serine, 95mg / L threonine, 16mg / L tryptophan, 94mg / L valine, 4mg / L choline chloride, 4mg / L calcium pantothenate, 4mg / L folic acid, 4mg / L nicotinic acid. The invention relates to a 5-well plate containing 4 mg / L glutamine, 4 mg / L pyridoxine hydrochloride, 0.4 mg / L riboflavin, 4 mg / L thiamine, 7.2 mg / L inositol, 0.1 mg / L ferric nitrate, 97 mg / L magnesium sulfate, 400 mg / L potassium chloride, 3700 mg / L sodium bicarbonate buffer, 6000 mg / L sodium chloride, 4500 mg / L glucose, and 15000 mg / L composite protein hydrolysate, wherein the composite protein hydrolysate is obtained by mixing protein hydrolysates A and H in a mixing weight ratio, and the specific mixing weight ratio of protein hydrolysates A and H is shown in Table 3.

[0283] For the composite protein hydrolyzate stem cell culture medium (referred to as "stem cell culture medium"), the total weight of each component except fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the stem cell culture medium is calculated as 48.1%. The specific calculation method is as follows:

[0284] Compound protein hydrolysate / (compound protein hydrolysate + arginine + cystine + glutamine + histidine hydrochloride + isoleucine + leucine + lysine hydrochloride + methionine + phenylalanine + serine + threonine + tryptophan + valine + choline chloride + calcium pantothenate + folic acid + niacinamide + pyridoxine hydrochloride + riboflavin + thiamine + inositol + ferric nitrate + magnesium sulfate + potassium chloride + sodium bicarbonate buffer + sodium chloride + glucose)

[0285] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using the culture medium in step (1-1) and the control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0286] (3) Stem cell expansion and culture: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into a 6 cm cell culture dish containing the corresponding composite protein hydrolysate stem cell culture medium at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0287] (4) Detection of stem cell growth and status:

[0288] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0289] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 3.

[0290] Table 3. Effects of compound protein hydrolysate stem cell culture medium on stem cell growth and status detection

[0291] As shown in Table 3, the results showed that the stem cells cultured in the composite protein hydrolysate stem cell medium (Group 7) in which protein hydrolysates A and H were mixed at a ratio of 12.50% based on the total weight of the composite protein hydrolysate had significantly higher viable cell density and stemness positive rate after expansion than those of the other groups.

[0292] Example 3: Low-concentration single protein hydrolysate stem cell culture medium

[0293] (1) Preparation of culture medium:

[0294] The amount of each component added to the low-concentration single protein hydrolysate stem cell culture medium is based on the total volume of 1L of the low-concentration single protein hydrolysate stem cell culture medium. It is dissolved and fixed to volume with sterilized ultrapure water. The specific concentrations of each component in the low-concentration single protein hydrolysate stem cell culture medium are as follows:

[0295] The total volume of the low-concentration single protein hydrolysate stem cell culture medium is composed of 3% fetal bovine serum, 5000 mg / L protein hydrolysate, 5000 mg / L balanced salt (120 mg / L calcium chloride, 100 mg / L magnesium sulfate, 750 mg / L potassium chloride, 3280 mg / L sodium chloride, and 750 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPEs), 100 mg / L penicillin and streptomycin double antibody mixture (100×), 45 mg / L vitamin complex, 800 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor FGF2. The culture media using protein hydrolysates A and H as raw materials are named culture media A-1, B-1, C-1, D-1, E-1, F-1, G-1, and H-1, respectively.

[0296] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0297] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0298] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0299] For the low-concentration single protein hydrolyzate stem cell culture medium (referred to as the "stem cell culture medium"), the total weight of all components except fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the stem cell culture medium is calculated as 27.1%. The specific calculation method is as follows:

[0300] Protein hydrolysate / (protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0301] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using culture media A-1, B-1, C-1, D-1, E-1, F-1, G-1, H-1, and control culture medium (CK) at a concentration of 4 × 10 4The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0302] (3) Proliferation and culture of stem cells: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing culture media A-1, B-1, C-1, D-1, E-1, F-1, G-1, and H-1 and control culture medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0303] (4) Detection of stem cell growth and status:

[0304] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0305] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x magnification lens. A green color under the microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 4.

[0306] Table 4. Stem cell growth and status detection in low concentration single protein hydrolysate stem cell culture medium

[0307] As shown in Table 4, the results indicate that low-concentration single protein hydrolysate stem cell culture media A-1 to H-1 are all capable of maintaining the growth of stem cells, and low-concentration single protein hydrolysate stem cell culture media A-1, C-1, E-1, and G-1 are superior culture media. Among them, low-concentration single protein hydrolysate stem cell culture media C-1 and G-1 more significantly promoted the overall expansion of stem cells and effectively maintained the characteristics of stem cells.

[0308] Example 4: Medium-concentration single protein hydrolysate stem cell culture medium

[0309] (1) Preparation of culture medium:

[0310] The amount of each component added to the medium-concentration single protein hydrolysate stem cell culture medium is based on a total volume of 1L of the medium-concentration single protein hydrolysate stem cell culture medium. Dissolve and adjust to volume with sterilized ultrapure water. The specific concentrations of each component in the medium-concentration single protein hydrolysate stem cell culture medium are as follows:

[0311] Based on the total volume of the medium-concentration single protein hydrolysate stem cell culture medium, the composition includes 2% fetal bovine serum, 15,000 mg / L protein hydrolysate, 4270 mg / L balanced salt (90 mg / L calcium chloride, 80 mg / L magnesium sulfate, 650 mg / L potassium chloride, 2800 mg / L sodium chloride, and 650 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPEs), 100 mg / L of a double antibody mixture of penicillin and streptomycin (100×), 40 mg / L vitamin complex, 400 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor FGF2. The culture media using protein hydrolysates A and H as raw materials are named culture media A-2, B-2, C-2, D-2, E-2, F-2, G-2, and H-2, respectively.

[0312] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0313] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0314] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0315] For the medium-concentration single protein hydrolyzate stem cell culture medium (hereinafter referred to as the "stem cell culture medium"), the total weight of all components except fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolyzate relative to the total weight of the stem cell culture medium is calculated as 54.9%. The specific calculation method is as follows:

[0316] Protein hydrolysate / (protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0317] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using culture media A-2, B-2, C-2, D-2, E-2, F-2, G-2, H-2, and control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0318] (3) Stem cell expansion culture: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing culture medium A-2, B-2, C-2, D-2, E-2, F-2, G-2, H-2 and control culture medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0319] (4) Stem cell growth and status detection:

[0320] Detection of stem cell expansion: C2C12 mouse myoblasts were collected after expansion and culture, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0321] Stem cell positivity testing: After expansion and culture, cells were collected, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the fluorescence microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stem cell positivity rate, or stemness positivity rate, and thus to assess the maintenance of stem cell characteristics in the expanded C2C12 mouse myoblasts. The results are shown in Table 5.

[0322] Table 5. Stem cell growth and status detection in medium concentration single protein hydrolysate stem cell culture medium

[0323] As shown in Table 5, the results indicate that medium-concentration single protein hydrolyzate stem cell culture media A-2 to H-2 are all capable of maintaining the growth of stem cells and are all superior culture media. Among them, medium-concentration single protein hydrolyzate stem cell culture media D-2 and H-2 more significantly promoted the overall expansion of stem cells and effectively maintained the characteristics of stem cells.

[0324] Example 5: High-concentration single protein hydrolysate stem cell culture medium

[0325] (1) Preparation of culture medium:

[0326] The amount of each component added to the high-concentration single protein hydrolysate stem cell culture medium is based on a total volume of 1L of the high-concentration single protein hydrolysate stem cell culture medium. Dissolve and adjust to volume with sterilized ultrapure water. The specific concentrations of each component in the high-concentration single protein hydrolysate stem cell culture medium are as follows:

[0327] Based on the total volume of high-concentration single protein hydrolysate stem cell culture medium, fetal bovine serum 2%, protein hydrolysate 20,000 mg / L, balanced salt 2540 mg / L (calcium chloride 80 mg / L, magnesium sulfate 60 mg / L, potassium chloride 450 mg / L, sodium chloride 1500 mg / L and sodium phosphate 450 mg / L), pH adjustment solution 3000 mg / L (sodium bicarbonate buffer 2000 mg / L, HEPEs 1000 mg / L), penicillin and streptomycin The double antibody mixture (100×) is 100 mg / L, vitamin complex is 35 mg / L, amino acid complex is 300 mg / L, trace element complex is 1 mg / L, glucose is 4500 mg / L and growth factor FGF2 is 0.23 mg / L. Among them, the culture media with protein hydrolysate (A, B, C, D, E, F, G or H) as raw materials are named culture media A-3, B-3, C-3, D-3, E-3, F-3, G-3, and H-3, respectively.

[0328] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0329] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0330] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0331] For the high-concentration single protein hydrolysate stem cell culture medium (hereinafter referred to as "stem cell culture medium"), the total weight of all components except fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolysate relative to the total weight of the stem cell culture medium is calculated as 65.6%. The specific calculation method is as follows:

[0332] Protein hydrolysate / (protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0333] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using culture media A-3, B-3, C-3, D-3, E-3, F-3, G-3, H-3, and control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0334] (3) Proliferation and culture of stem cells: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing culture medium A-3, B-3, C-3, D-3, E-3, F-3, G-3, H-3 and control culture medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0335] (4) Detection of stem cell growth and status:

[0336] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0337] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 6.

[0338] Table 6 Stem cell growth and status detection in high concentration single protein hydrolysate stem cell culture medium

[0339] As shown in Table 6, the results indicate that high-concentration single protein hydrolyzate stem cell culture media A-3-H-3 are all capable of maintaining the growth of stem cells. High-concentration single protein hydrolyzate stem cell culture media D-3, F-3, and H-3 are superior culture media. Among them, high-concentration single protein hydrolyzate stem cell culture media F-3 more significantly promotes the overall expansion of stem cells and effectively maintains stem cell characteristics.

[0340] Example 6: Low-concentration composite protein hydrolysate stem cell culture medium

[0341] (1) Preparation of culture medium:

[0342] (1-1) The amount of each component added to the low-concentration composite protein hydrolyzate stem cell culture medium (Mix 1 stem cell culture medium) of the embodiment was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the low-concentration composite protein hydrolyzate stem cell culture medium (Mix 1 stem cell culture medium). The specific concentrations of the components in the low-concentration composite protein hydrolyzate stem cell culture medium (Mix 1 stem cell culture medium) of the embodiment are as follows:

[0343] Based on the total volume of the low-concentration composite protein hydrolysate stem cell culture medium (Mix 1 stem cell culture medium) of the embodiment, fetal bovine serum (fetal bovine serum) is 2%, composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%) 5000 mg / L, balanced salt 5000 mg / L (calcium chloride 120 mg / L, magnesium sulfate 100 mg / L, potassium chloride 750 mg / L, sodium chloride 3280 mg / L, and sodium phosphate 750 mg / L), pH adjusting solution 3000 mg / L (sodium bicarbonate buffer 2000 mg / L and HEPEs 1000 mg / L), penicillin and streptomycin double antibody mixture (100×) 100 mg / L, vitamin complex 45 mg / L, amino acid complex 800 mg / L, trace element complex 1 mg / L, glucose 4500 mg / L, and growth factor FGF2 0.23 mg / L.

[0344] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0345] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0346] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0347] For the low-concentration composite protein hydrolysate stem cell culture medium (hereinafter referred to as "stem cell culture medium"), the total weight of all components excluding fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolysate relative to the total weight of the stem cell culture medium is calculated as 27.1%. The specific calculation method is as follows:

[0348] Complex protein hydrolysate / (complex protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0349] (1-2) The amount of each component added to the comparative example low-concentration composite protein hydrolyzate stem cell culture medium (mix 1 stem cell culture medium) was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the comparative example low-concentration composite protein hydrolyzate stem cell culture medium (mix 1 stem cell culture medium). The specific concentrations of the components in the comparative example low-concentration composite protein hydrolyzate stem cell culture medium (mix 1 stem cell culture medium) are as follows:

[0350] Based on the total volume of the low-concentration composite protein hydrolysate stem cell culture medium (mix 1 stem cell culture medium) of the comparative example, the following ingredients are included: 2% fetal bovine serum, 5000 mg / L composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%), 5000 mg / L balanced salt (120 mg / L calcium chloride, 100 mg / L magnesium sulfate, 750 mg / L potassium chloride, 3280 mg / L sodium chloride, and 750 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer and 1000 mg / L HEPEs), 100 mg / L of a double antibody mixture of penicillin and streptomycin (100×), 45 mg / L vitamin complex, 800 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor FGF2.

[0351] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0352] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0353] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0354] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using Mix 1 stem cell culture medium, Mix 1 stem cell culture medium, and control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0355] (3) Stem cell expansion and culture: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing Mix 1 stem cell culture medium, Mix 1 stem cell culture medium, and control medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0356] (4) Detection of stem cell growth and status:

[0357] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0358] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the fluorescence microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 7.

[0359] Table 7. Stem cell growth and status detection in low concentration composite protein hydrolysate stem cell culture medium

[0360] As shown in Table 7, the results showed that, in comparison, Mix 1 stem cell culture medium was able to promote the overall expansion of stem cells and maintain stem cell characteristics, while Mix 1 stem cell culture medium was less able to promote the overall expansion of stem cells and maintain stem cell characteristics.

[0361] Example 7: Medium-concentration composite protein hydrolysate stem cell culture medium

[0362] (1) Preparation of culture medium:

[0363] (1-1) The amount of each component added to the medium-concentration composite protein hydrolyzate stem cell culture medium (Mix 2 stem cell culture medium) of the embodiment was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the medium-concentration composite protein hydrolyzate stem cell culture medium (Mix 2 stem cell culture medium). The specific concentrations of the components in the medium-concentration composite protein hydrolyzate stem cell culture medium (Mix 2 stem cell culture medium) of the embodiment are as follows:

[0364] Based on the total volume of the medium-concentration composite protein hydrolysate stem cell culture medium (Mix 2 stem cell culture medium) in the embodiment, fetal bovine serum (fetal bovine serum) is 2%, composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%) is 15000 mg / L, balanced salt is 4270 mg / L (calcium chloride 90 mg / L, magnesium sulfate 80 mg / L, potassium chloride 650 mg / L, sodium chloride 2800 mg / L and sodium phosphate 650 mg / L), pH adjustment solution is 3000 mg / L (sodium bicarbonate buffer 2000 mg / L, HEPEs 1000 mg / L), penicillin and streptomycin double antibody mixture (100×) is 100 mg / L, vitamin complex is 40 mg / L, amino acid complex is 400 mg / L, trace element complex is 1 mg / L, glucose is 4500 mg / L and growth factor FGF2 is 0.23 mg / L.

[0365] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0366] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0367] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0368] For the medium-concentration composite protein hydrolysate stem cell culture medium (hereinafter referred to as "stem cell culture medium"), the total weight of all components excluding fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolysate relative to the total weight of the stem cell culture medium is calculated as 54.9%. The specific calculation method is as follows:

[0369] Complex protein hydrolysate / (complex protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0370] (1-2) The amount of each component added to the comparative example medium-concentration composite protein hydrolyzate stem cell culture medium (mix 2 stem cell culture medium) was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the comparative example medium-concentration composite protein hydrolyzate stem cell culture medium (mix 2 stem cell culture medium). The specific concentrations of the components in the comparative example medium-concentration composite protein hydrolyzate stem cell culture medium (mix 2 stem cell culture medium) are as follows:

[0371] Based on the total volume of the medium-concentration composite protein hydrolysate stem cell culture medium (mix 2 stem cell culture medium) of the comparative example, the following ingredients are included: 2% fetal bovine serum, 15,000 mg / L composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%), 4,270 mg / L balanced salt (90 mg / L calcium chloride, 80 mg / L magnesium sulfate, 650 mg / L potassium chloride, 2,800 mg / L sodium chloride, and 650 mg / L sodium phosphate), 3,000 mg / L pH adjusting solution (2,000 mg / L sodium bicarbonate buffer, 1,000 mg / L HEPEs), 100 mg / L of a double antibody mixture of penicillin and streptomycin (100×), 40 mg / L vitamin complex, 400 mg / L amino acid complex, 1 mg / L trace element complex, 4,500 mg / L glucose, and 0.23 mg / L growth factor FGF2.

[0372] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0373] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0374] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0375] (2) Activation of stem cells: C2C12 mouse myoblasts were activated using Mix 2 stem cell culture medium, Mix 2 stem cell culture medium, and control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0376] (3) Stem cell expansion and culture: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing Mix 2 stem cell culture medium, Mix 2 stem cell culture medium, and control medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0377] (4) Detection of stem cell growth and status:

[0378] Detection of stem cell expansion: C2C12 mouse myoblasts after expansion and culture were collected, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0379] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 8.

[0380] Table 8. Stem cell growth and status detection in medium concentration composite protein hydrolysate stem cell culture medium

[0381] As shown in Table 8, the results showed that, in comparison, Mix 2 stem cell medium significantly promoted the overall expansion of stem cells and effectively maintained stem cell characteristics, while Mix 2 stem cell medium had poorer ability to promote the overall expansion of stem cells and maintain stem cell characteristics.

[0382] Example 8: High-concentration composite protein hydrolysate stem cell culture medium

[0383] (1) Preparation of culture medium:

[0384] (1-1) The amount of each component added to the high-concentration composite protein hydrolyzate stem cell culture medium (Mix 3 stem cell culture medium) of the embodiment was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the high-concentration composite protein hydrolyzate stem cell culture medium (Mix 3 stem cell culture medium). The specific concentrations of the components in the high-concentration composite protein hydrolyzate stem cell culture medium (Mix 3 stem cell culture medium) of the embodiment are as follows:

[0385] Based on the total volume of the high-concentration composite protein hydrolysate stem cell culture medium (Mix 3 stem cell culture medium) of the embodiment, the following ingredients are included: 2% fetal bovine serum, 20,000 mg / L composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%), 2540 mg / L balanced salt (80 mg / L calcium chloride, 60 mg / L magnesium sulfate, 450 mg / L potassium chloride, 1500 mg / L sodium chloride, and 450 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPEs), 100 mg / L of a double antibody mixture of penicillin and streptomycin (100×), 35 mg / L vitamin complex, 300 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor (FGF2).

[0386] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0387] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0388] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0389] For the high-concentration composite protein hydrolysate stem cell culture medium (hereinafter referred to as "stem cell culture medium"), the total weight of all components excluding fetal bovine serum is taken as the total weight of the stem cell culture medium, and the percentage of the weight of the added protein hydrolysate relative to the total weight of the stem cell culture medium is calculated as 65.6%. The specific calculation method is as follows:

[0390] Complex protein hydrolysate / (complex protein hydrolysate + balanced salt + pH regulating solution + double antibiotic mixture of penicillin and streptomycin (100×) + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0391] (1-2) The amount of each component added to the comparative example high-concentration composite protein hydrolyzate stem cell culture medium (mix 3 stem cell culture medium) was determined by dissolving the components with sterilized ultrapure water and adjusting the volume to a constant volume, based on a total volume of 1 L of the comparative example high-concentration composite protein hydrolyzate stem cell culture medium (mix 3 stem cell culture medium). The specific concentrations of the components in the comparative example high-concentration composite protein hydrolyzate stem cell culture medium (mix 3 stem cell culture medium) are as follows:

[0392] Based on the total volume of the high-concentration composite protein hydrolysate stem cell culture medium (mix 3 stem cell culture medium) in the comparative example, the following ingredients are included: 2% fetal bovine serum, 20,000 mg / L composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%), 2540 mg / L balanced salt (80 mg / L calcium chloride, 60 mg / L magnesium sulfate, 450 mg / L potassium chloride, 1500 mg / L sodium chloride, and 450 mg / L sodium phosphate), 3000 mg / L pH adjusting solution (2000 mg / L sodium bicarbonate buffer, 1000 mg / L HEPEs), 100 mg / L of a double antibody mixture of penicillin and streptomycin (100×), 35 mg / L vitamin complex, 300 mg / L amino acid complex, 1 mg / L trace element complex, 4500 mg / L glucose, and 0.23 mg / L growth factor (FGF2).

[0393] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0394] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0395] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0396] (2) Activation of cells: C2C12 mouse myoblasts were activated using Mix 3 stem cell culture medium, Mix 3 stem cell culture medium, and control culture medium (CK) at a concentration of 4 × 10 4The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0397] (3) Cell expansion: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into 6 cm cell culture dishes containing Mix 3 stem cell culture medium, Mix 3 stem cell culture medium, and control medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0398] (4) Detection of stem cell growth and status:

[0399] Detection of stem cell expansion: C2C12 mouse myoblasts were collected after expansion and culture, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0400] To determine the stemness-positive rate, cells were collected after expansion and culture, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope at a 40x objective and a 10x eyepiece. A green color under the fluorescence microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stemness-positive rate, or stemness-positive rate, and thus to assess the maintenance of stemness characteristics in C2C12 mouse myoblasts after expansion and culture. The results are shown in Table 9.

[0401] Table 9. Stem cell growth and status detection in high concentration composite protein hydrolysate stem cell culture medium

[0402] As shown in Table 9, the results indicate that, by comparison, Mix 3 stem cell culture medium significantly promoted overall stem cell expansion but was not effective in maintaining stem cell characteristics. Mix 3 stem cell culture medium was also less effective in promoting overall stem cell expansion and maintaining stem cell characteristics.

[0403] Therefore, according to Tables 7, 8 and 9, the results show that, by comparison, the culture medium using AH protein hydrolysate compound is better than the culture medium using ah protein hydrolysate compound; among them, Mix 2 stem cell culture medium is more suitable and applicable to the culture of stem cells than Mix 1 stem cell culture medium and Mix 3 stem cell culture medium.

[0404] Example 9: Food-grade stem cell culture medium containing a serum-free, antibiotic-free mixture of medium-concentration composite protein hydrolysates

[0405] (1) Preparation of culture medium:

[0406] The amount of each component added to the food-grade stem cell culture medium (Mix 4 stem cell culture medium) containing a serum-free and antibiotic-free mixture of medium-concentration compound protein hydrolysates (Mix 4 stem cell culture medium) is based on a total volume of 1 L of the food-grade stem cell culture medium (Mix 4 stem cell culture medium) containing a serum-free and antibiotic-free mixture of medium-concentration compound protein hydrolysates (Mix 4 stem cell culture medium). The concentrations of each component in the food-grade stem cell culture medium (Mix 4 stem cell culture medium) containing a serum-free and antibiotic-free mixture of medium-concentration compound protein hydrolysates (Mix 4 stem cell culture medium) are as follows:

[0407] Based on the total volume of a serum-free, antibiotic-free food-grade stem cell culture medium (Mix 4 stem cell culture medium) containing a medium-concentration composite protein hydrolysate mixture, the following ingredients are included: 15,000 mg / L composite protein hydrolysate (based on the total weight of the composite protein hydrolysate, the proportion of protein hydrolysates A and H is 12.5%), 4,270 mg / L balanced salt (90 mg / L calcium chloride, 80 mg / L magnesium sulfate, 650 mg / L potassium chloride, 2,800 mg / L sodium chloride, and 650 mg / L sodium phosphate), 3,000 mg / L pH adjusting solution (2,000 mg / L sodium bicarbonate buffer, 1,000 mg / L HEPEs), 50 mg / L vitamin complex, 400 mg / L amino acid complex, 1 mg / L trace element complex, 4,500 mg / L glucose, and 0.25 mg / L growth factor FGF2.

[0408] The vitamin complex comprises, based on the total weight of the vitamin complex, 25.2% choline chloride, 8.1% folic acid, 45.1% inositol, 5.4% niacinamide, 8.1% calcium pantothenate, 2.7% pyridoxal, 2.7% vitamin B12 and 2.7% thiamine.

[0409] The amino acid complex comprises, based on the total weight of the amino acid complex, 42% asparagine, 25.5% aspartic acid, 25.5% serine and 7% cysteine.

[0410] The trace element complex comprises: based on the total weight of the trace element complex, 7.7% Fe(NO3)3·9H2O, 46.2% FeSO4·7H2O and 46.1% ZnSO4·7H2O.

[0411] For the serum-free, antibiotic-free food-grade stem cell culture medium containing a medium-concentration composite protein hydrolysate (hereinafter referred to as the "stem cell culture medium"), the total weight of the components excluding fetal bovine serum is taken as the total weight of the stem cell culture medium. The percentage of the weight of the added protein hydrolysate relative to the total weight of the stem cell culture medium is calculated as 55.1%. The specific calculation method is as follows:

[0412] Complex protein hydrolysate / (complex protein hydrolysate + balanced salt + pH regulating solution + vitamin complex + amino acid complex + trace element complex + glucose + growth factor FGF2).

[0413] (2) Activation of cells: C2C12 mouse myoblasts were activated using Mix 4 stem cell culture medium and control culture medium (CK) at a concentration of 4 × 10 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0414] (3) Cell expansion: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into a 6 cm cell culture dish containing Mix 4 stem cell culture medium and control medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0415] (4) Stem cell growth and status detection:

[0416] Detection of stem cell expansion: C2C12 mouse myoblasts were collected after expansion and culture, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0417] Stem cell positivity testing: After expansion and culture, cells were collected, incubated and stained with a PAX7 antibody, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope with a 40x objective lens and a 10x eyepiece. A green color under the fluorescence microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stem cell positivity rate, or stemness positivity rate, and thus to assess the maintenance of stem cell characteristics in C2C12 mouse myoblasts after expansion and culture. This is shown in Table 10.

[0418] Table 10. Stem cell growth and status detection in food-grade stem cell culture medium containing a serum-free and antibiotic-free mixture of medium-concentration complex protein hydrolysates

[0419] As shown in Table 10, the results demonstrate that Mix 4 Stem Cell Medium is capable of maintaining stem cell growth and is a superior medium, significantly promoting overall stem cell expansion and effectively maintaining stem cell characteristics. Furthermore, the cost of Mix 4 Stem Cell Medium is significantly lower than that of the control medium (CK), effectively reducing the cost of stem cell culture media.

[0420] Application example: Application of food-grade stem cell culture medium containing a serum-free and antibiotic-free mixture of medium-concentration complex protein hydrolysates in different muscle stem cells

[0421] (1) Cell activation: Porcine muscle stem cells, chicken muscle stem cells, bovine muscle stem cells, and shrimp muscle stem cells were activated using the Mix 4 stem cell culture medium obtained in Example 9 and the control culture medium (CK), respectively. 4 The cells were seeded at a density of 1.5 mL / well in a 6-well plate. Fresh culture medium was replaced in time according to the cell growth. Cells were passaged when the culture dish density reached 90%.

[0422] (2) Cell expansion: The activated cells were digested with trypsin and centrifuged, and the cells were collected and inoculated into a 6 cm cell culture dish containing Mix 4 stem cell culture medium and control medium (CK) at a cell density of 3 × 10 5 / mL, and culture continuously for 6 days, observing daily and replacing fresh medium as needed. After 6 days, the cells were digested and seeded into T25 culture flasks and cultured continuously until day 10, observing daily and replacing fresh medium as needed. After 10 days, the cells were digested and seeded into T75 culture flasks and cultured continuously until day 15, observing daily and replacing fresh medium as needed. After 15 days, the cells were digested and seeded into T175 culture flasks and cultured until day 24.

[0423] (3) Stem cell growth and status detection:

[0424] Detection of stem cell expansion: C2C12 mouse myoblasts were collected after expansion and culture, stained with 0.2% trypan blue dye, and placed on a cell counter to detect the density of viable cells after expansion.

[0425] Stem cell positivity testing: After expansion and culture, cells were collected, incubated and stained with PAX7 antibodies, and PAX7 antibody expression was detected by immunofluorescence staining. The cells were observed under a fluorescence microscope with a 40x objective lens and a 10x eyepiece. A green color under the fluorescence microscope indicated that PAX7 was positively expressed in the cells. This was used to determine the stem cell positivity rate, or stemness positivity rate, and thus to assess the maintenance of stem cell characteristics in C2C12 mouse myoblasts after expansion and culture. This is shown in Table 11.

[0426] Table 11. Effects of food-grade stem cell culture medium containing a serum-free, antibiotic-free mixture of medium-concentration complex protein hydrolysates on the growth and status of different muscle stem cells.

[0427] As shown in Table 11, the results demonstrate that Mix 4 stem cell culture medium, when applied to porcine, chicken, bovine, and shrimp muscle stem cells, is equally effective in maintaining stem cell growth and performing as well as the control medium (CK). Specifically, Mix 4 significantly promoted the overall expansion of porcine and bovine muscle stem cells and effectively maintained their stem cell properties.

[0428] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any form. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A stem cell culture medium with protein hydrolysate as the core raw material, characterized in that: The stem cell culture medium contains 20-80% protein hydrolysate based on the weight of the total components in the stem cell culture medium; The protein hydrolysate includes one or more of: high free amino acid yeast hydrolysate, high nucleotide yeast hydrolysate, high glutathione yeast hydrolysate, high polypeptide yeast hydrolysate, rice protein hydrolysate, high glutamine wheat protein hydrolysate, pea protein hydrolysate and soy protein hydrolysate; Wherein, the high free amino acid yeast hydrolysate has a free amino acid content of ≥50% by weight; The high-nucleotide yeast hydrolysate has a free nucleotide content of >5% by weight; The high-glutathione yeast hydrolysate has a glutathione content of >10% by weight; The high-polypeptide yeast hydrolysate, based on its weight, has a peptide segment with a molecular weight greater than 1000 Daltons accounting for more than 20% of the total peptide segments in the high-polypeptide yeast hydrolysate; The rice protein hydrolysate has a total nitrogen content of >12% by weight; The high-glutamine wheat protein hydrolysate has a hydrolyzed glutamine content of >20% by weight; The pea protein hydrolysate has a total nitrogen content of >13.5% by weight; The soy protein hydrolysate has a carbohydrate content of more than 20% by weight.

2. The stem cell culture medium according to claim 1, characterized in that By weight, the free amino acid content of the high free amino acid yeast hydrolysate is 50%-65%, the free nucleotide content of the high nucleotide yeast hydrolysate is 5%-20%, the glutathione content of the high glutathione yeast hydrolysate is 10%-20%, the mass percentage of the peptide segments with a molecular weight greater than 1000 Daltons in the high polypeptide yeast hydrolysate accounts for 20%-50% of the total peptide segments in the high polypeptide yeast hydrolysate, the total nitrogen content of the rice protein hydrolysate is 12%-13%, the hydrolyzed glutamine content of the high glutamine wheat protein hydrolysate is 20%-35%, the total nitrogen of the pea protein hydrolysate is 13.5%-14.5% and the carbohydrate content of the soy protein hydrolysate is 20%-22%.

2. The stem cell culture medium according to claim 1, characterized in that the components in the stem cell culture medium also include: one or more of balanced salts, pH regulating solution, antibiotic mixture, serum, vitamin complex, amino acid complex, trace element complex, glucose and growth factor.

3. The stem cell culture medium according to claim 1 or 2, characterized in that The concentration of each component in the stem cell culture medium is: based on the total volume of the stem cell culture medium, protein hydrolyzate 4000-25000 mg / L, balanced salt 2000-6000 mg / L, pH adjusting solution 1500-3500 mg / L, antibiotic mixture 10-120 mg / L, vitamin complex 20-50 mg / L, amino acid complex 100-1000 mg / L, trace element complex 0.5-4.0 mg / L, glucose 4000-6000 mg / L, growth factor 0.15-0.25 mg / L and serum 1 volume %-4 volume %; Preferably, the concentration of each component in the stem cell culture medium is: based on the total volume of the stem cell culture medium, protein hydrolyzate 4500-21000 mg / L, balanced salt 2170-5630 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 30-50 mg / L, amino acid complex 200-1000 mg / L, trace element complex 0.5-3.0 mg / L, glucose 4000-6000 mg / L, growth factor 0.20-0.25 mg / L and serum 1 volume %-3 volume %.

4. The stem cell culture medium according to claim 1 or 2, characterized in that The concentration of each component in the stem cell culture medium is: based on the total volume of the stem cell culture medium, protein hydrolyzate 4000-25000 mg / L, balanced salt 2000-6000 mg / L, pH adjusting solution 1500-3500 mg / L, vitamin complex 20-50 mg / L, amino acid complex 200-1000 mg / L, trace element complex 0.5-4.0 mg / L, glucose 4000-6000 mg / L and growth factor 0.15-0.25 mg / L; Preferably, the concentration of each component in the stem cell culture medium is: based on the total volume of the stem cell culture medium, protein hydrolyzate 14000-16000 mg / L, balanced salt 4050-4690 mg / L, pH adjusting solution 2250-3500 mg / L, vitamin complex 40-50 mg / L, amino acid complex 250-500 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L and growth factor 0.20-0.25 mg / L.

5. The stem cell culture medium according to any one of claims 1 to 4, characterized in that The vitamin complex comprises, based on the total weight of the vitamin complex, one or more of 22%-27% choline chloride, 6%-10% folic acid, 40%-50% inositol, 2.5%-7.5% nicotinamide, 5%-10% calcium pantothenate, 1%-5% pyridoxal, 1%-5% vitamin 12 and 1%-5% thiamine.

6. The stem cell culture medium according to any one of claims 1 to 5, characterized in that Based on the total weight of the amino acid complex, the amino acid complex comprises: 40%-50% asparagine, and / or 20%-30% aspartic acid, and / or 20%-30% serine, and / or 1%-10% cysteine.

7. The stem cell culture medium according to any one of claims 1 to 6, characterized in that Based on the total weight of the trace element complex, the trace element complex comprises: 1%-10% Fe(NO3)3·9H2O, and / or 40%-50% FeSO4·7H2O, and / or 40%-50% ZnSO4·7H2O.

8. The stem cell culture medium according to any one of claims 1 to 7, characterized in that The balanced salt includes one or more of calcium chloride, magnesium sulfate, potassium chloride, sodium chloride and sodium phosphate.

9. The stem cell culture medium according to any one of claims 1 to 8, characterized in that The pH regulating solution includes one or more of sodium bicarbonate buffer and HEPEs, and / or the antibiotic includes one or more of penicillin and streptomycin, and / or the growth factor includes growth factor FGF2.

10. The stem cell culture medium according to any one of claims 1 to 9, characterized in that The stem cell culture medium is a stem cell culture medium containing high glutathione yeast hydrolysate; or a stem cell culture medium containing pea protein hydrolysate.

11. The stem cell culture medium according to claim 10, characterized in that Based on the total volume of the stem cell culture medium containing high glutathione yeast hydrolysate, the concentrations of the components in the stem cell culture medium containing high glutathione yeast hydrolysate are: serum 1-3 volume%, high glutathione yeast hydrolysate 4500-5500 mg / L, balanced salt 4750-5630 mg / L, pH adjusting solution 2250-3500 mg / Lmg / L, antibiotic mixture 80-120 mg / L, vitamin complex 40-50 mg / L, amino acid complex 750-1000 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L and growth factor 0.20-0.25 mg / L; Or based on the total volume of the stem cell culture medium containing pea protein hydrolysate, the concentrations of the components in the stem cell culture medium containing pea protein hydrolysate are: serum 1-3% by volume, pea protein hydrolysate 4500-5500 mg / L, balanced salt 4750-5630 mg / L, pH adjusting solution 2250-350 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 40-50 mg / L, amino acid complex 750-1000 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L and growth factor 0.20-0.25 mg / L, Preferably, the balanced salt is 110-130 mg / L calcium chloride, 80-100 mg / L magnesium sulfate, 700-900 mg / L potassium chloride, 3000-3500 mg / L sodium chloride and 750-1000 mg / L sodium phosphate; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

12. The stem cell culture medium according to any one of claims 1 to 9, characterized in that The stem cell culture medium is a stem cell culture medium containing high-polypeptide yeast hydrolysate; or a stem cell culture medium containing soy protein hydrolysate.

13. The stem cell culture medium according to claim 12, characterized in that Based on the total volume of the stem cell culture medium containing high-polypeptide yeast hydrolysate, the concentrations of the components in the stem cell culture medium containing high-polypeptide yeast hydrolysate are: serum 1-2 volume%, high-polypeptide yeast hydrolysate 14000-16000 mg / L, balanced salt 4050-4690 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 40-50 mg / L, amino acid complex 250-500 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L and growth factor FGF 0.20-0.25 mg / L; Or based on the total volume of the stem cell culture medium containing soy protein hydrolysate, the concentrations of the components in the stem cell culture medium containing soy protein hydrolysate are: serum 1-2%, soy protein hydrolysate 14000-16000 mg / L, balanced salt 4050-4690 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 40-50 mg / L, amino acid complex 250-500 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-5000 mg / L and growth factor 0.20-0.25 mg / L, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution comprises 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

14. The stem cell culture medium according to any one of claims 1 to 9, characterized in that The stem cell culture medium is a stem cell culture medium containing high-glutamine wheat protein hydrolyzate.

15. The stem cell culture medium according to claim 14, characterized in that Based on the total volume of the stem cell culture medium containing high glutamine wheat protein hydrolysate, the concentrations of the components in the stem cell culture medium containing high glutamine wheat protein hydrolysate are: serum 1-2%, high glutamine wheat protein hydrolysate 19000-21000 mg / L, balanced salt 2170-2760 mg / L, pH adjusting solution 2250-3500 mg / L, antibiotic mixture 80-120 mg / L, vitamin complex 30-40 mg / L, amino acid complex 200-400 mg / L, trace element complex 0.5-2.5 mg / L, glucose 4000-6000 mg / L and growth factor 0.20-0.25 mg / L, Preferably, the balanced salt is calcium chloride 70-90 mg / L, magnesium sulfate 50-70 mg / L, potassium chloride 350-500 mg / L, sodium chloride 1400-1600 mg / L and sodium phosphate 300-500 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

16. The stem cell culture medium according to any one of claims 1 to 9, characterized in that The stem cell culture medium is a stem cell culture medium containing a composite protein hydrolysate, wherein, based on the total added weight of the composite protein hydrolysate, the composite protein hydrolysate contains 5%-60% high free amino acid yeast hydrolysate, 5%-60% high nucleotide yeast hydrolysate, 5%-60% high glutathione yeast hydrolysate, 5%-60% high polypeptide yeast hydrolysate, 5%-60% rice protein hydrolysate, 5%-60% high glutamine wheat protein hydrolysate, 5%-60% pea protein hydrolysate and 5%-60% soy protein hydrolysate.

17. The stem cell culture medium according to claim 16, characterized in that Based on the total volume of the stem cell culture medium containing the composite protein hydrolysate, the concentrations of the components in the stem cell culture medium containing the composite protein hydrolysate are: 1-2% by volume of serum, 14000-16000 mg / L of the composite protein hydrolysate, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 80-120 mg / L of mixed solution of antibiotics, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

18. The stem cell culture medium according to claim 17, characterized in that The concentrations of the components in the stem cell culture medium containing the composite protein hydrolysate are as follows: based on the total volume of the stem cell culture medium, the composite protein hydrolysate is 14000-16000 mg / L, the balanced salt is 4050-4690 mg / L, the pH adjusting solution is 2250-3500 mg / L, the vitamin complex is 40-50 mg / L, the amino acid complex is 250-500 mg / L, the trace element complex is 0.5-2.5 mg / L, the glucose is 4000-6000 mg / L and the growth factor is 0.20-0.25 mg / L. Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

19. The method for screening a stem cell culture medium according to any one of claims 1 to 18, characterized in that: The following steps are involved: Step 1: Screening the types of protein hydrolysates. Specifically, after animal stem cells are activated, they are transferred to stem cell culture media containing different types of protein hydrolysates at a concentration of 4500-21000 mg / L for expansion culture. The growth of animal stem cells under different types of protein hydrolysates is determined based on the stem cell density and the positive rate of stem cell characteristics, so as to screen out the suitable types of protein hydrolysates. Step 2: Screening the mixing ratio of the composite protein hydrolysate, specifically, mixing two or more different types of protein hydrolysates to obtain the composite protein hydrolysate; and after the animal stem cells are activated, respectively transferred to the stem cell culture medium containing each of the composite protein hydrolysates at a concentration of 4500-21000 mg / L to expand the stem cell culture, and judging the growth of the animal stem cells under the composite protein hydrolysates of different mixing ratios according to the stem cell density and the positive rate of stem cell characteristics, so as to screen out the mixing ratio suitable for the composite protein hydrolysate; wherein the mixing ratio refers to: the mixing ratio of different types of protein hydrolysates added to prepare the composite protein hydrolysate; Step 3: preparing stem cell culture medium, specifically, using the protein hydrolysate selected in step 1 as the core raw material and / or preparing the stem cell culture medium according to the suitable mixing ratio of the composite protein hydrolysate selected in step 2; Step 4: Screening stem cell culture media. Specifically, after the animal stem cells are activated, they are transferred to the stem cell culture media prepared in step 3 for expansion culture. The growth of animal stem cells under different stem cell culture media is determined based on the stem cell density and the positive rate of stem cell characteristics, so as to screen out suitable stem cell culture media.

20. Use of the stem cell culture medium according to any one of claims 1 to 19 or the stem cell culture medium screened by the screening method according to claim 19 in stem cells, wherein: The stem cells include one or more muscle stem cells derived from chicken, cattle, sheep, shrimp and mouse.

21. A chicken muscle stem cell culture medium, characterized in that: The chicken muscle stem cell culture medium contains, based on the total volume of the culture medium, 14000-16000 mg / L of the composite protein hydrolysate of claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor. Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

22. A bovine muscle stem cell culture medium, characterized in that: The invention contains, based on the total volume of the bovine muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate of claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

23. A sheep muscle stem cell culture medium, characterized in that: The invention contains, based on the total volume of the sheep muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate of claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

24. A shrimp muscle stem cell culture medium, characterized in that The invention contains, based on the total volume of the shrimp muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate of claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.

25. A culture medium for mouse muscle stem cells, characterized in that: The invention contains, based on the total volume of the mouse muscle stem cell culture medium, 14000-16000 mg / L of the composite protein hydrolysate of claim 16, 4050-4690 mg / L of balanced salt, 2250-3500 mg / L of pH regulating solution, 40-50 mg / L of vitamin complex, 250-500 mg / L of amino acid complex, 0.5-2.5 mg / L of trace element complex, 4000-6000 mg / L of glucose and 0.20-0.25 mg / L of growth factor, Preferably, the balanced salt is calcium chloride 80-100 mg / L, magnesium sulfate 70-90 mg / L, potassium chloride 600-800 mg / L, sodium chloride 2700-2900 mg / L and sodium phosphate 600-800 mg / L; Preferably, the pH adjusting solution is 1500-2500 mg / L sodium bicarbonate buffer and 750-1000 mg / L HEPEs.