Novel pet food protein substitute and preparation method thereof

Abstract

The invention belongs to the field of cell application, and particularly relates to a novel pet food protein substitute and a preparation method thereof. The novel pet food protein substitute comprises the following components: 10-20% of cell culture meat, 25-35% of pea meal, 5-15% of corn flour, 5-15% of wheat flour, 3-8% of kelp powder, 10-20% of vegetable fat, 3-8% of a rosemary extract, 3-8% of a yeast extract and 0.05-0.12% of NaCl. According to the method, the suspension type CHO cells are taken as seeds, the cell culture meat is obtained by culturing the suspension type CHO cells with a culture medium without serum and animal protein addition through a Fed-batch process, and compared with animal protein from a traditional culture source, the cell culture meat is free of hormones and drug residues and is easier to digest and absorb by intestinal tracts.

Description

Technical Field

[0001] The present invention belongs to the field of cell application, and in particular, relates to a novel pet food protein substitute and a preparation method thereof. Background Art

[0002] In 2021, the national output of pet feed was 1.13 million tons, a year-on-year increase of 17.3%. Among them, protein, an important nutrient in pet food, mainly comes from animal husbandry. Therefore, to a certain extent, there is competition between pets and humans for meat. Secondly, it was found that 34% of pet dogs were allergic to beef, 18% of pet cats were allergic to beef, and 17% of pet cats were allergic to fish. In addition, the animal protein provided by livestock meat may have residues of chemical agents such as pesticides, antibiotics, hormones and growth promoters during the breeding process, and there are also raw material statements in the market that have replaced the concept. The actual raw material used is meat meal, but it is falsely marked as dehydrated meat on the label. Therefore, some pet owners consider using plant protein to replace meat protein produced by animal husbandry, but plants contain too much indigestible plant protein, which can easily cause abdominal pain or even diarrhea in pets. Long-term consumption will seriously affect the digestive function of pets.

[0003] In view of the above problems faced by pet breeding, the production of cell cultured meat can solve these contradictions. Cell cultured meat refers to the use of cell culture engineering and tissue engineering technologies to culture animal muscle tissue in vitro as edible material.

[0004] For example, CN117866883A discloses a method for producing cell cultured meat using animal highly active muscle and fat precursor cells, the method comprising the following steps: S1, screening to obtain highly active vole muscle precursor cells, and performing preliminary amplification;

[0005] S2, screen and obtain highly active vole fat precursor cells, and perform preliminary expansion; S3, use the immersion removal method to obtain a thin layer of decellularized plant extracellular matrix; S4, use the plant extracellular matrix as a cell growth scaffold for culture to obtain an animal cell meat culture layer; S5, obtain a three-dimensional structural tissue that differentiates into mature muscle fiber cells, that is, cell cultured meat. This method has higher efficiency and lower cost, can separate more cells, and can enhance the nutrition and taste of cell meat. However, the above-mentioned cultured muscle cells are expensive and time-consuming to culture.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a new pet food protein substitute and a preparation method thereof.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A novel pet food protein substitute, wherein the novel pet food protein substitute comprises the following components:

[0010]

[0011] As a preferred embodiment, the novel pet food protein substitute comprises the following components:

[0012]

[0013] In the present invention, the percentages are percentages by mass.

[0014] Furthermore, the cell cultured meat is obtained by using suspended CHO cells as seeds and culturing them in a bioreactor through a Fed-batch process using serum-free culture medium.

[0015] The present invention also provides a method for preparing the novel pet food protein substitute, comprising the following steps:

[0016] S1. Obtaining cell cultured meat

[0017] Suspension CHO cells are used as seeds and cultured in a bioreactor using serum-free medium through the Fed-batch process to obtain cell-cultured meat;

[0018] S2. Preparation of pet food protein substitutes

[0019] The cell cultured meat obtained in step S1 is used as an animal protein source, pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added, and dried to obtain the new pet food protein substitute.

[0020] Furthermore, step S1 specifically includes the following steps:

[0021] S11, inoculating CHO cells suspended in a serum-free medium in a shake flask into a 700 mL bioreactor at 30 w / ml, adding serum-free medium to set the working volume to 300 mL, and obtaining a serum-free medium inoculated with CHO cells;

[0022] S12. Stir the serum-free culture medium inoculated with CHO cells at a stirring speed of 150r / min±10 under the conditions of temperature 37°C, dissolved oxygen concentration 40%±5%, and pH 6.8-7.2, and cultivate the CHO cells inoculated in the serum-free culture medium to obtain cell-cultured meat.

[0023] Furthermore, during the culture process of step S12, the feeding mode control was performed, specifically: on the 5th day of culture, 300 mL of serum-free medium was added; on the 6th day of culture, 2 mL of 2.5 M glucose stock solution was supplemented to a final concentration of 22.83 mM; on the 7th day of culture, 6.206 mL of 2.5 M glucose stock solution was supplemented to a final concentration of 33.3 mM, and 4% 008.2 + 0.4% 009 was added; the entire culture process lasted for 9 days, and the viable cell density on the 8th day of culture reached 1.3×10 7 cells / ml.

[0024] In the present invention, the glucose storage solution is a glucose aqueous solution, which is configured to a concentration of 2.5M and stored for later use.

[0025] 008.2 is a feed medium.

[0026] 009 is a feed medium.

[0027] Furthermore, during the culture process of step S12, the cell density and cell viability are monitored every day during the entire culture cycle.

[0028] Furthermore, the serum-free culture medium in step S11 and step S12 is CD 04+4mM L-Glu.

[0029] In the present invention, CD 04 is CHO CD 04 medium.

[0030] Further, in step S2, the drying is performed by one of the following four processes:

[0031] 1) Steam for 10-30 minutes and then dry at 45-68°C for 15-20 hours;

[0032] 2) Steam for 10-30 minutes and then freeze-dry for 6-10 hours;

[0033] 3) Drying at 45℃-68℃ for 15-20h;

[0034] 4) Freeze-dry for 6h-10.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] The present invention uses suspended CHO cells as seeds and cultured the cell-cultured meat through the Fed-batch process in a serum-free, animal protein-free culture medium. Compared with animal protein from traditional breeding sources, the cell-cultured meat is hormone-free, drug-free, and easier to digest and absorb in the intestines. DETAILED DESCRIPTION

[0037] The following are specific embodiments of the present invention, and the embodiments are intended to further describe the present invention rather than to limit the present invention.

[0038] Example 1

[0039] Components:

[0040]

[0041] Wherein, the vegetable oil is soybean oil.

[0042] Preparation method:

[0043] S1. Obtaining cell cultured meat

[0044] S11, CHO cells suspended and cultured in a 250 ml shake flask with a serum-free medium (CD 04 + 4 mM L-Glu) were inoculated into a 700 ml stirred tank bioreactor at 30 w / ml, and serum-free medium (CD 04 + 4 mM L-Glu) was added to set the working volume to 300 ml to obtain a serum-free medium inoculated with CHO cells;

[0045] S12. Stir the serum-free medium inoculated with CHO cells at a temperature of 37°C, a dissolved oxygen concentration of 40%, and a pH of 6.8 to 7.2 at a stirring speed of 150 r / min, and culture the CHO cells inoculated in the serum-free medium to obtain cell culture meat; during the culture process, take samples every day to determine the cell density and cell viability. And control the feeding method, specifically: on the 5th day of culture, add 300mL of serum-free medium (CD 04+4mM L-Glu); on the 6th day of culture, supplement 2mL of 2.5M glucose stock solution to a final concentration of approximately 22.83mM; on the 7th day of culture, supplement 6.206mL of 2.5M glucose stock solution to a final concentration of approximately 33.3mM, and add 4% 008.2+0.4% 009. The entire culture process lasts for 9 days, and the viable cell density on the 8th day of culture reaches 1.3×10 7 cells / ml.

[0046] S2. Preparation of pet food protein substitutes

[0047] The cell cultured meat obtained in step S1 is used as an animal protein source, and pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added. After steaming for 10 minutes, the mixture is dried at 45° C. for 20 hours to obtain the new pet food protein substitute.

[0048] Example 2

[0049] Components:

[0050]

[0051] Wherein, the vegetable oil is peanut oil.

[0052] Preparation method:

[0053] S1. Obtaining cell cultured meat

[0054] The specific process is the same as that of Example 1, except that in step S12, the dissolved oxygen concentration is 35% and the stirring speed is 140 r / min.

[0055] S2. Preparation of pet food protein substitutes

[0056] The cell cultured meat obtained in step S1 is used as an animal protein source, pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added, and the mixture is steamed for 10 minutes and freeze-dried for 6 hours to obtain the novel pet food protein substitute.

[0057] Example 3

[0058] Components:

[0059]

[0060] Wherein, the vegetable oil is rapeseed oil.

[0061] Preparation method:

[0062] S1. Obtaining cell cultured meat

[0063] The specific process is the same as that of Example 1, except that in step S12, the dissolved oxygen concentration is 45% and the stirring speed is 160 r / min.

[0064] S2. Preparation of pet food protein substitutes

[0065] The cell cultured meat obtained in step S1 is used as an animal protein source, pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added, and the mixture is dried at 45° C. for 20 hours to obtain the new pet food protein substitute.

[0066] Example 4

[0067] Components:

[0068]

[0069] Wherein, the vegetable oil is sesame oil.

[0070] Preparation method:

[0071] S1. Obtaining cell cultured meat

[0072] The same as Example 1, except that in step S12, the dissolved oxygen concentration is 42% and the stirring speed is 146 r / min.

[0073] S2. Preparation of pet food protein substitutes

[0074] The cell cultured meat obtained in step S1 is used as an animal protein source, pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added, and freeze-dried for 6 hours to obtain the new pet food protein substitute.

[0075] Example 5

[0076] Components:

[0077] Same as Example 1.

[0078] Preparation method:

[0079] S1. Obtaining cell cultured meat

[0080] Same as Example 1.

[0081] S2. Preparation of pet food protein substitutes

[0082] The specific process is the same as that of Example 1, except that the drying step is steaming for 30 minutes and then drying at 68° C. for 15 hours.

[0083] Example 6

[0084] Components:

[0085] Same as Example 2.

[0086] Preparation method:

[0087] S1. Obtaining cell cultured meat

[0088] Same as Example 2.

[0089] S2. Preparation of pet food protein substitutes

[0090] The specific process is the same as that of Example 1, except that the drying step is steaming for 30 minutes and then freeze-drying for 10 hours.

[0091] Example 7

[0092] Components:

[0093] Same as Example 3.

[0094] Preparation method:

[0095] S1. Obtaining cell cultured meat

[0096] Same as Example 3.

[0097] S2. Preparation of pet food protein substitutes

[0098] The specific process is the same as that of Example 1, except that the drying is performed at 68° C. for 15 h.

[0099] Example 8

[0100] Components:

[0101] Same as Example 4.

[0102] Preparation method:

[0103] S1. Obtaining cell cultured meat

[0104] Same as Example 4.

[0105] S2. Preparation of pet food protein substitutes

[0106] The specific process is the same as in Example 1, except that the drying is freeze-drying for 10 hours.

[0107] Experimental Example 1: Determination of seeding density of CHO cells in suspension culture

[0108] This test example investigated the seeding density of CHO cells in suspension culture.

[0109] In this experiment, 6 groups of cell starting culture densities were set up: 30w / mL, 40w / mL, 50w / mL, 100w / mL, 200w / mL, and 200w / mL. The initial inoculation density of 30w / ml was screened out to ensure high cell viability while maintaining the cell doubling time at a shorter time, which is most suitable for the culture system of the present invention.

[0110] 30w / ml concentration——125ml shake bottle——2%GlutaMAX+CD04

[0111]

[0112] 40w / ml concentration——125ml shake bottle——2%GlutaMAX+CD04

[0113]

[0114] 50w / ml concentration——125ml shake bottle——2%GlutaMAX+CD04

[0115]

[0116]

[0117] 100w / ml concentration——125ml shake bottle——2%GlutaMAX+CD04

[0118]

[0119] 150w / ml concentration - 125ml shake bottle - 2% GlutaMAX + CD04 - second batch

[0120] 200w / ml concentration——125ml shake bottle——2%GlutaMAX+CD04

[0121]

[0122] It can be seen from the above results that the initial inoculation density of 30w / ml can ensure a high cell viability while maintaining the cell doubling time at a relatively short time, which is most suitable for the culture system of the present invention.

[0123] Test Example 2: Test of nutritional components of cell cultured meat

[0124] This test example tested the nutritional components of the cell cultured meat prepared in this application, and the results are shown in Table 1 below:

[0125] Table 1. Test results of nutritional components of cell cultured meat

[0126] Cell cultured meat Protein content Fat Content Example 1 8.8158％ 0.7725％

[0127] The present invention selects Chinese hamster ovary cells (CHO) as cell seeds, cultivates CHO cells in a laboratory environment, and tests the nutritional components of CHO cell cultured meat, showing that the protein content of CHO cell cultured meat is: 8.8158%, and the fat content is: 0.7725%. Therefore, CHO cell cultured meat can completely replace the meat produced by traditional breeding, and the cell cultured meat produced in the laboratory can reduce the energy consumption of traditional animal husbandry and reduce the emission of greenhouse gases such as carbon dioxide into the atmosphere by animals. At the same time, it will also save water resources, reduce the number of breeding and slaughtering animals, and increase animal welfare. In addition, the culture medium used to cultivate CHO cells is serum-free and free of animal-derived proteins. Therefore, the cell cultured meat cultivated by the present invention can also be used as food for pets of strict vegetarians. It can be seen that the pet cell meat provided by the method of the present invention has the advantages of sufficient nutrition, customizability, and high quality.

[0128] Test Example 3: Animal Tasting Test

[0129] The new pet food protein substitute prepared by the present invention was tasted by Teddy Bear (twice, each time providing about 25g of pet food), and the dog finished eating it within about 1 minute and 30 seconds, with good feedback.

Claims

1. A new pet food protein substitute, characterized in that: The novel pet food protein substitute comprises the following components:

2. A novel pet food protein substitute according to claim 1, characterized in that: The novel pet food protein substitute comprises the following components:

3. The novel pet food protein substitute according to claim 1 or 2, characterized in that: The cell cultured meat is obtained by using suspended CHO cells as seeds and culturing them in a bioreactor using a serum-free culture medium through a Fed-batch process.

4. A method for preparing a novel pet food protein substitute according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: S1. Obtaining cell cultured meat Suspension CHO cells are used as seeds and cultured in a bioreactor using serum-free medium through the Fed-batch process to obtain cell-cultured meat; S2. Preparation of pet food protein substitutes The cell cultured meat obtained in step S1 is used as an animal protein source, pea flour, corn flour, wheat flour, kelp powder, vegetable oil, rosemary extract, yeast extract and NaCl are added, and dried to obtain the new pet food protein substitute.

5. The preparation method according to claim 4, characterized in that: Step S1 specifically includes the following steps: S11, inoculating CHO cells suspended in a serum-free medium in a shake flask into a 700 mL bioreactor at 30 w / ml, adding serum-free medium to set the working volume to 300 mL, and obtaining a serum-free medium inoculated with CHO cells; S12. Stir the serum-free culture medium inoculated with CHO cells at a stirring speed of 150r / min±10 under the conditions of temperature 37°C, dissolved oxygen concentration 40%±5%, and pH 6.8-7.2, and cultivate the CHO cells inoculated in the serum-free culture medium to obtain cell-cultured meat.

6. The preparation method according to claim 5, characterized in that: During the cultivation process of step S12, the feeding mode control was performed, specifically: on the 5th day of cultivation, 300 mL of serum-free medium was added; on the 6th day of cultivation, 2 mL of 2.5 M glucose stock solution was supplemented to a final concentration of 22.83 mM; on the 7th day of cultivation, 6.206 mL of 2.5 M glucose stock solution was supplemented to a final concentration of 33.3 mM, and 4% 008.2 + 0.4% 009 was added; the entire cultivation process lasted for 9 days, and the viable cell density reached 1.3×10 on the 8th day of cultivation. 7 cells / ml.

7. The preparation method according to claim 6, characterized in that: During the culture process of step S12, the cell density and cell viability are also monitored every day during the entire culture cycle.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The serum-free culture medium described in step S11 and step S12 is CD 04+4mM L-Glu.

9. The preparation method according to claim 4, characterized in that: In step S2, the drying is carried out by one of the following four processes: 1) Steam for 10-30 minutes and then dry at 45-68°C for 15-20 hours; 2) Steam for 10-30 minutes and then freeze-dry for 6-10 hours; 3) Drying at 45℃-68℃ for 15-20h; 4) Freeze-dry for 6-10 hours.

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