Preparation method of biological breeding pork rice

Abstract

The invention discloses a preparation method of biological breeding pork rice, and belongs to the technical field of biological breeding meat. The preparation method of the biological breeding pork rice comprises a preparation method of pork breeding cereals, collagen incubation, pig myoblast inoculation, pig myoblast culture and pork rice harvesting. According to the technical scheme, the production process is mature and controllable, and stable product quality can be kept in large-scale production; the production process does not depend on a microcarrier, the production cost is reduced, the production process is simplified, and the whole production process is more efficient and economical; by using biological materials such as fish collagen protein and hyaluronic acid, the nutritional value and biocompatibility of the product are improved, so that the final product has higher market competitiveness; the production process is green, environment-friendly, safe, reliable and resource-saving; the cereal and new protein combined pork rice provides a new possibility for consumers to select in the food market.

Description

Technical Field

[0001] The invention relates to the technical field of bio-cultivated meat, in particular to a preparation method of bio-cultivated pork rice. Background Art

[0002] Meat products can provide high-quality protein for humans. Among them, pork is one of the most consumed meats in the world, and has a long tradition of consumption in Asia and Europe. The huge demand has led to the large scale of pig farming. Pork farming has very significant harm in terms of environmental pollution, resource consumption, and animal welfare. Large-scale farming occupies agricultural land, emits a large amount of greenhouse gases such as methane and ammonia, and produces wastewater and feces that pollute water sources and soil, leading to deforestation and reduction of biodiversity. A large amount of antibiotics are used in farming, which increases bacterial resistance and easily spreads zoonotic diseases such as swine flu. The intensive farming environment is poor and the slaughter process lacks humane care. A large amount of feed and water resources further aggravate resource tensions.

[0003] New protein foods are foods that are developed and produced using modern technology and innovative methods to replace traditional animal proteins. They aim to obtain protein foods without raising or slaughtering animals. Early new protein foods were divided into plant-based protein foods, fermented protein foods, and cell-cultured meat. Plant-based protein foods use plants as raw materials, which can be extracted and purified to obtain plant protein; cell-cultured meat uses animal cells as raw materials, and obtains a large number of animal cells through cell culture, large-scale amplification and other methods, and ultimately obtains animal protein.

[0004] Different types of new protein foods can only provide a single source and type of protein, which does not meet consumers' demand for multiple types of multi-nutrition protein. Cereal food is one of the main sources of energy for humans, mainly by providing a large amount of carbohydrates to provide energy for human activities. The protein content of most cereal foods is about 7.5% (that is, every 100g of cereal can provide 7.5g of protein for the human body), and the average adult needs to consume at least 60g to 90g of protein to meet the body's needs. Cereal food alone cannot fully meet people's daily protein needs.

[0005] Developing sustainable alternatives such as new protein foods can effectively address these issues. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method of biologically cultivated pork rice, which solves the problems of environmental pollution, resource consumption, waste of resources, food safety hazards, animal abuse, single protein, etc.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing biologically cultivated pork rice,

[0008] S1: Preparation method of pork culture grains

[0009] a. Grain softening treatment: Soak the grains in PBS solution;

[0010] b. Sterilization treatment: after uniformly mixing the PBS solution with the softened grains, sterilize the mixture to obtain sterile grains;

[0011] S2: Collagen incubation

[0012] Mixing fish collagen, hyaluronic acid and bovine collagen evenly and preparing a saturated concentration solution, incubating the sterile grains in the saturated concentration solution to obtain incubated grains;

[0013] S3: Porcine myoblast seeding

[0014] The incubated grains are evenly spread on a culture plate, and the porcine myoblast suspension is injected into each well of the culture plate;

[0015] S4: Porcine myoblast culture

[0016] The culture plate containing the porcine myoblast suspension in S3 is placed in an incubator for cell culture, and the culture solution is suspended from the bottom to the top every other day; the porcine myoblast suspension is replaced once every two days;

[0017] S5: Harvest pork rice

[0018] After the culture was completed, the culture plate was rinsed with a PBS solution to remove the residual culture medium, thereby obtaining a pork grain product.

[0019] Preferably, the pork culture grain is any one of rice, millet or purple rice.

[0020] Preferably, the grains in step a of S1 are soaked for 2 hours.

[0021] Preferably, in b of S1, the sterilization method is high temperature and high pressure sterilization, which is a sterilization treatment at a pressure of 0.1 MPa and a temperature of 121°C for 20 minutes.

[0022] Preferably, in S1, the PBS solution in b is mixed with the softened cereals in a mass ratio of 1:3.

[0023] Preferably, in S2, the fish collagen, the hyaluronic acid and the bovine collagen are mixed in a mass ratio of 7:2:1.

[0024] Preferably, in S2, the saturated concentration solution is prepared by mixing the fish collagen, the hyaluronic acid and the bovine collagen in proportion and then using PBS to prepare a saturated solution.

[0025] Preferably, in S2, the sterilized grains are incubated in the saturated concentration solution for 120 minutes.

[0026] Preferably, the concentration of the porcine myoblast suspension in S3 is 1×10 7 Pieces / mL.

[0027] Preferably, in S4, the culture conditions of the incubator are a temperature of 37°C, CO 2 The content is 5%, and the culture is for 8 days; the culture medium is DMEM medium containing 10% FBS.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) A certain proportion of hyaluronic acid and bovine collagen are added to the fish collagen solution. After the sterilized grains are incubated in the solution for a period of time, these nutrients can be fully combined. By comparing the cell growth of the final products of the embodiment and the comparative example, it is found that the treatment of the rice grains by the second component solution of the embodiment makes the surface of the cells easier for the cells to adhere.

[0030] (2) The production process of the present invention is mature and controllable, and can maintain stable product quality in large-scale production. In addition, the invention does not rely on microcarriers, which reduces production costs.

[0031] (3) The cell culture process of the present invention does not emit a large amount of greenhouse gases, and does not produce wastewater and waste residue, which is green and environmentally friendly; it does not require the occupation of agricultural land, which saves resources; it does not use a large amount of antibiotics, which is safe and reliable; there is no slaughtering process, which is humanitarian. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a product picture of the pork cultured rice finally obtained in Example 1 of the present invention, which is magnified 10 times;

[0033] Figure 2 This is a product picture of the pork cultured millet finally obtained in Example 1 of the present invention, which is magnified 10 times;

[0034] Figure 3 This is a product picture of the pork cultured purple rice finally obtained in Example 1 of the present invention, magnified 10 times;

[0035] Figure 4 This is a product picture of the pork cultured rice finally obtained in Comparative Example 1 of Example 1 of the present invention, which is magnified 10 times;

[0036] Figure 5 This is a product picture of the pork cultured millet finally obtained in Comparative Example 2 of Example 2 of the present invention, which is magnified 10 times;

[0037] Figure 6This is a product picture of the pork cultured purple rice finally obtained in Comparative Example 3 of Example 3 of the present invention, which is magnified 10 times;

[0038] Figure 7 This is a fluorescence image of cell attachment and proliferation on the cultured pork rice finally obtained in Examples 1, 2, and 3 of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0040] The present invention relates to the food field of new proteins formed by the fusion of animal and plant proteins, and specifically to an innovative protein pork rice and its preparation method and application in the field of new proteins. That is, pork rice is a technical attempt to use rice as a scaffold and modify its surface to allow cells to attach, grow and differentiate, which is an expanded application of cell culture meat preparation technology. The specific preparation method of pork rice is shown in the following three embodiments.

[0041] Example 1

[0042] Pork cultured meat rice

[0043] The present embodiment provides a method for preparing pork cultured rice, wherein the raw materials include a first component: rice, and a second component: fish collagen, hyaluronic acid, and bovine collagen. The mass ratio of fish collagen: hyaluronic acid: bovine collagen is 7:2:1. The present embodiment provides a method for preparing pork cultured rice, comprising the following steps:

[0044] (1) Rice softening treatment: Soak the rice in PBS solution for 2 hours to achieve a softening effect.

[0045] (2) Sterilization: After softening, mix the PBS solution with the rice in a mass ratio of 1:3, transfer it into an autoclave, and sterilize it at 0.1 MPa and 121°C for 20 minutes to ensure its sterility. This sterilization can also be done by 1 hour ultraviolet sterilization or irradiation sterilization. The irradiation sterilization is specifically 60Co irradiation sterilization for 20 seconds.

[0046] (3) Collagen incubation: fish collagen, hyaluronic acid and bovine collagen were mixed in a mass ratio of 7:2:1 to prepare a saturated concentration solution, and the sterilized rice was incubated in the solution for 120 minutes to fully combine these nutrients. The saturated concentration solution was prepared by adding the mixed powder of fish collagen, hyaluronic acid and bovine collagen to PBS until it could not be dissolved in the liquid.

[0047] (4) Cell inoculation: The incubated rice cereal product was evenly spread on a 24-well culture plate, and the 10% FBS DMEM medium was diluted to a concentration of 1×10 7 The porcine myoblast suspension at 100 / mL was injected into each well to ensure uniform distribution of cells.

[0048] (5) Cell culture: at 37°C, CO 2 Cells are cultured in an incubator with a content of 5%, and the culture medium is replaced from the bottom to the top every other day for 8 days. The frequency of replacing the porcine myoblast suspension is once every two days. The culture medium here is DMEM medium with 10% FBS. DMEM medium can also be replaced with F12 or McCoy's 5A.

[0049] (6) Pork rice harvesting: After the culture is completed, the culture plate is rinsed with PBS solution to remove the residual culture medium to obtain pork rice products.

[0050] Example 2

[0051] Pork cultured meat millet

[0052] The present embodiment provides a method for preparing pork cultured millet, wherein the raw materials include a first component: millet, and a second component: fish collagen, hyaluronic acid, and bovine collagen. The mass ratio of fish collagen: hyaluronic acid: bovine collagen is 7:2:1. The present embodiment provides a method for preparing pork cultured millet, comprising the following steps:

[0053] (1) Millet softening treatment: Soak the millet in PBS solution for 2 hours to achieve a softening effect.

[0054] (2) Sterilization: After the softened millet is mixed with PBS solution and millet in a mass ratio of 1:3, it is transferred into an autoclave and sterilized at 0.1 MPa and 1121°C for 20 minutes to ensure its sterility. This sterilization can also be carried out by 1h UV sterilization or irradiation sterilization. The specific irradiation sterilization is 60Co irradiation sterilization for 20s.

[0055] (3) Collagen incubation: fish collagen, hyaluronic acid and bovine collagen were mixed in a mass ratio of 7:2:1 to prepare a saturated concentration solution, and the sterilized millet was incubated in the solution for 120 minutes to fully combine these nutrients. The saturated concentration solution was prepared by adding the mixed powder of fish collagen, hyaluronic acid and bovine collagen to PBS until it could not be dissolved in the liquid.

[0056] (4) Cell inoculation: The millet cereal products after incubation were evenly spread on a 24-well culture plate, and 10% FBSDMEM medium was diluted to a concentration of 1×10 7 The porcine myoblast suspension at 100 / mL was injected into each well to ensure uniform distribution of cells.

[0057] (5) Cell culture: at 37°C, CO 2 Cells are cultured in an incubator with a content of 5%, and the culture medium is replaced from the bottom to the top every other day for 8 days. The frequency of replacing the porcine myoblast suspension is once every two days. The culture medium here is DMEM medium with 10% FBS. DMEM medium can also be replaced with F12 or McCoy's 5A.

[0058] (6) Pork rice harvesting: After the culture is completed, the culture plate is rinsed with PBS solution to remove the residual culture medium to obtain pork rice products.

[0059] Example 3

[0060] Pork cultured meat purple rice

[0061] The present embodiment provides a method for preparing purple rice cultivated with pork, wherein the raw materials include a first component: purple rice, and a second component: fish collagen, hyaluronic acid, and bovine collagen. The mass ratio of fish collagen: hyaluronic acid: bovine collagen is 7:2:1. The present embodiment provides a method for preparing purple rice cultivated with pork, comprising the following steps:

[0062] (1) Purple rice softening treatment: Soak the purple rice in PBS solution for 2 hours to achieve a softening effect.

[0063] (2) Sterilization: After softening, mix the purple rice with PBS solution and millet in a mass ratio of 1:3, transfer it into an autoclave, and sterilize it at 0.1 MPa and 121°C for 20 minutes to ensure its sterility. This sterilization can also be done by 1h UV sterilization or irradiation sterilization. Irradiation sterilization is specifically 60Co irradiation sterilization for 20s.

[0064] (3) Collagen incubation: fish collagen, hyaluronic acid and bovine collagen were mixed in a mass ratio of 7:2:1 and prepared into a saturated concentration solution, and the sterilized purple rice was incubated in the solution for 120 minutes to fully combine these nutrients. The saturated concentration solution was prepared by adding fish collagen, hyaluronic acid and bovine collagen mixed powder into PBS until it could not be dissolved in the liquid.

[0065] (4) Cell inoculation: The purple rice cereal products after incubation were evenly spread on a 24-well culture plate, and 10% FBSDMEM medium was diluted to a concentration of 1×10 7 The porcine myoblast suspension at 100 / mL was injected into each well to ensure uniform distribution of cells.

[0066] (5) Cell culture: at 37°C, CO 2 Cells are cultured in an incubator with a content of 5%, and the culture medium is replaced from the bottom to the top every other day for 8 days. The frequency of replacing the porcine myoblast suspension is once every two days. The culture medium here is DMEM medium with 10% FBS. DMEM medium can also be replaced with F12 or McCoy's 5A.

[0067] (6) Pork rice harvesting: After the culture is completed, the culture plate is rinsed with PBS solution to remove the residual culture medium to obtain the pork purple rice product.

[0068] Comparative Example 1

[0069] This comparative example provides a pork bio-cultured meat rice, which is different from Example 1 only in that the second component uses only fish collagen. Fish collagen is added to PBS until it cannot be dissolved in the liquid to prepare a saturated fish collagen solution.

[0070] The only difference between the above method for preparing pork bio-cultured rice and Example 1 is that the sterilized rice is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0071] Comparative Example 2

[0072] This comparative example provides a pork bio-cultured meat millet, which is different from Example 2 only in that the second component uses only fish collagen. Fish collagen is added to PBS until it cannot be dissolved in the liquid to prepare a saturated fish collagen solution.

[0073] The preparation method of the above pork bio-cultured meat millet is different from that of Example 2 only in that the sterilized millet is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0074] Comparative Example 3

[0075] This comparative example provides a pork bio-cultured purple rice, which is different from Example 2 only in that the second component uses only fish collagen. Fish collagen is added to PBS until it cannot be dissolved in the liquid to prepare a saturated fish collagen solution.

[0076] The preparation method of the above pork bio-cultured purple rice is different from that of Example 2 only in that the second component is replaced with a fish collagen solution. The sterilized purple rice is incubated in a saturated fish collagen solution for 120 minutes to fully combine these nutrients.

[0077] Performance testing and result discussion

[0078] Verification of the attachment efficiency of pork cultured rice cells

[0079] Immunofluorescence labeling of cells that adhered and proliferated on the cultured pork meat was performed to verify the cell proliferation efficiency. PBST: PBS solution containing 0.5% Triton X-100 (volume percentage).

[0080] Specific operations:

[0081] 1) Cultivate pork rice, discard the culture medium, and wash twice with PBS.

[0082] 2) Add 4% paraformaldehyde and fix at room temperature for 15 minutes. You can also add anhydrous methanol and fix at room temperature for 5-10 minutes, or at -20℃ for 2 minutes.

[0083] 3) Wash with PBS three times, 5 minutes each time.

[0084] 4) Permeabilize with PBST solution at room temperature for 10 minutes.

[0085] 5) Wash with PBS three times, 5 minutes each time.

[0086] 6) Add the final concentration of DAPI / Hoechst and incubate at room temperature for 5 minutes.

[0087] 7) Wash with PBS three times, 5 minutes each time.

[0088] 8) Add 500 μl PBS and take pictures under a fluorescence microscope.

[0089] 9) Observe the growth and proliferation morphology of cells on the scaffold under a fluorescence microscope to verify the efficiency of scaffold cell culture.

[0090] from Figures 1 to 6 ,as well as Figure 7It can be concluded that the final fluorescence results show that a large number of cells attach and proliferate after the treated rice, millet, and purple rice cells in Example 1, Example 2, and Example 3 are inoculated, and a layer of cells is attached to the surface of the rice, indicating that the surface treatment of grains in the invention is effective and has a beneficial effect on cell attachment and growth. The difference between Examples 1, 2, and 3 and Comparative Examples 1, 2, and 3 mainly lies in the second component. Fish collagen is a mild type I collagen, and type I collagen is the main component of the extracellular matrix. In order to better simulate the extracellular matrix and promote the adherent growth of cells, a certain proportion of hyaluronic acid and bovine collagen are added to the fish collagen solution. By comparing the cell growth of the final finished products of the embodiments and the comparative examples, it is found that the treatment of the rice grains by the second component solution of the embodiment makes the surface of the cells easier for cells to attach.

[0091] Pork rice combined with cereals and novel proteins provides new possibilities for consumers to choose in the food market. In addition, porcine myoblasts can be replaced with other types of porcine cells to prepare pork rice products with different types and nutritional properties.

[0092] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing biologically cultivated pork rice, characterized in that: S1: Preparation method of pork culture grains a. Grain softening treatment: Soak the grains in PBS solution; b. Sterilization treatment: after the PBS solution and the softened grains are evenly mixed, sterilization is performed to obtain sterile grains; S2: Collagen incubation Mixing fish collagen, hyaluronic acid and bovine collagen evenly and preparing a saturated concentration solution, incubating the sterile grains in the saturated concentration solution to obtain incubated grains; S3: Porcine myoblast seeding Evenly spreading the incubated grains on a culture plate, and injecting the porcine myoblast suspension into each well of the culture plate; S4: Porcine myoblast culture The culture plate containing the porcine myoblast suspension in S3 is placed in an incubator for cell culture, and the culture solution is suspended from the bottom to the top every other day; the porcine myoblast suspension is replaced once every two days; S5: Harvest pork rice After the culture was completed, the culture plate was rinsed with a PBS solution to remove the residual culture medium, thereby obtaining a pork grain product.

2. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: The pork culture grain is any one of rice, millet or purple rice.

3. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: The grains in S1a were soaked for 2 hours.

4. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: In b of S1, the sterilization method is high temperature and high pressure sterilization, which is a sterilization treatment at a pressure of 0.1 MPa and a temperature of 121°C for 20 minutes.

5. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: In S1, the PBS solution in b is mixed with the softened cereals at a mass ratio of 1:

3.

6. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: In S2, the fish collagen, the hyaluronic acid and the bovine collagen are mixed in a mass ratio of 7:2:

1.

7. The method for preparing biologically cultivated pork rice according to claim 6, characterized in that: In S2, the saturated concentration solution is prepared by mixing the fish collagen, the hyaluronic acid and the bovine collagen in proportion and then using PBS to prepare a saturated solution.

8. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: In S2, the incubation time of the sterilized grains in the saturated concentration solution is 120 minutes.

9. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: The concentration of the porcine myoblast suspension in S3 is 1×10 7 Pieces / mL.

10. The method for preparing biologically cultivated pork rice according to claim 1, characterized in that: In S4, the culture conditions of the incubator are a temperature of 37° C., a CO 2 content of 5%, and a culture period of 8 days; the culture medium is a DMEM medium containing 10% FBS.

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