A single-cell green algae combined functional factor and its application in cell cultured meat

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a single-cell green algae combined functional factor and its application in cell-cultured meat. The functional factor is obtained by subjecting the raw material to processes such as low-temperature freezing, room temperature thawing, and nano-scale shell adsorption column refining. The extraction process is simple and highly safe, and is suitable for industrial scale-up production. When applied to muscle cell culture, it can be directly dissolved in the basal culture medium, and the compounding method with the culture medium is simple. It has no toxic side effects on muscle cell growth and can completely replace serum for muscle cell culture. When the single-cell green algae combined functional factor mentioned in the present invention is applied to fish muscle cell culture, compared with the group without serum addition, it can provide cells with growth functional factor supplements, promote cell proliferation and differentiation in vitro, and can be applied to the preparation of cell-cultured meat.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a single-cell green algae combined functional factor and an application thereof in cell cultured meat. Background Art

[0002] During the in vitro cell culture process, serum is an ideal cell growth supplement. Serum contains plasma proteins, peptides, fats, carbohydrates, growth factors, hormones and other components, providing nutrients to maintain cell growth. Although serum is an ideal supplement for cell culture, it has disadvantages such as large batch-to-batch quality differences, increased risk of zoonotic diseases, and ethical issues, which have prompted people to continue to look for substitutes for serum. So far, no substance has been found that can widely replace serum and be applied to all cell lines.

[0003] Scholars at home and abroad have discovered a series of serum substitute combinations for myeloma, CHO, BHK, Vero, and lymphocytes, such as various recombinant proteins, galactose, lipids, trace elements, vitamins, amino acids, hormones and other combinations with clear ingredients to replace serum to maintain cell function, but multiple added ingredients and basal culture medium are compounded in different proportions, the additives are redundant, and the optimization conditions are relatively complex. Although the above technology discloses some serum substitutes that can meet certain needs, there are still some defects, such as high cost, low food safety, and low serum replacement degree, which will affect the widespread use of serum substitutes in cell cultured meat. The exploration of serum substitutes is of great significance. This is also one of the research hotspots and focuses in this technical field, and it is also the driving force and starting point for the completion of the present invention.

[0004] Unicellular algae refers to lower plants that reproduce by spores. They are also a source of biological activity that promotes immunity, anti-oxidation and tissue reconstruction. The reproduction of unicellular algae is basically that the unicellular spores or zygotes leave the mother body and germinate into new individuals directly or after a short period of dormancy. Chlorella is a typical unicellular green algae, which is easy to cultivate. It can not only use light energy for autotrophy, but also use organic carbon sources for growth and reproduction under heterotrophic conditions. It also grows and reproduces quickly. It is one of the few organisms in animals and plants that can grow 4 times in 20 hours. This shows that there may be nutrients in Chlorella cells that can promote cell proliferation, and it has the potential to extract serum substitutes. Chlorella cells are rich in proteins, polypeptides, nucleic acids and other components, but due to the presence of Chlorella cell walls, intracellular substances cannot be released, so it is necessary to use certain technical means to extract intracellular active components. The prior art mentions that algae polysaccharides are added as functional ingredients in serum-free culture media for stem cell culture, but it does not specify whether they can be used as serum substitutes, nor does it mention whether other ingredients such as proteins, peptides, nucleic acids, etc. have similar substitution effects. In addition, the culture conditions required for proliferation and differentiation of different cells are different, so serum substitutes developed for different cell types are not universal. At present, most of the serum-free culture media used in muscle cell culture in the prior art have problems such as slow cell culture rate, slow cell growth, limited number of cell culture, slow cell expansion, and difficulty in achieving expanded culture, which limits the production of serum-free cell cultured meat. Summary of the invention

[0005] In order to overcome the above-mentioned problems existing in the prior art, the present invention provides a single-cell green algae combined functional factor, the main components of which are protein functional factors and nucleic acid functional factors. It can be directly dissolved in the basic culture medium to achieve the purpose of replacing serum, provide growth functional factor supplements for cells, promote cell proliferation and differentiation in vitro, and can be used in the preparation of cell cultured meat.

[0006] To achieve the above object, the present invention is specifically implemented by the following technical scheme: a single-cell green algae combined functional factor, the preparation method of which comprises the following steps:

[0007] (1) Freeze-thaw treatment: Weigh the single-cell green algae raw material, add water, freeze it overnight at -20 to -80°C, thaw it at room temperature, freeze and thaw it repeatedly, and centrifuge to obtain the supernatant; the green algae cell wall is broken by freeze-thaw method to extract the water-soluble active substances in the cell.

[0008] (2) Column loading: Nano-scale shrimp and crab shell powder is loaded into a chromatography column to obtain a special adsorption chromatography column device; the shrimp and crab shells are derived from the shells of aquatic animals such as shrimp and crabs, and the shells are ultra-finely ground to obtain nano-scale shell powder. Chitin is the only alkaline polysaccharide in nature. There are multiple active groups such as amino, hydroxyl, acetylamino, and glycosidic bonds in the molecular structure of chitin. It has many unique functional properties such as good biocompatibility and natural non-toxicity. At the same time, the special sugar ring structure gives the chitin molecule unique interface characteristics. Therefore, the new material based on chitin has unique biological activity and functional properties. Because of the special fibrous structure of chitin, chitin nanofibers can be obtained by mechanical action. Chitin nanofibers can adsorb negatively charged functional molecules such as proteins and nucleic acids, thereby effectively enriching functional factors.

[0009] (3) Refining and purification: The supernatant obtained in step (2) is passed through the adsorption chromatography column prepared in step (3), the effluent is collected, and the effluent is dried.

[0010] Furthermore, in step (1), water is added at a dry basis weight ratio of 1:5 to 1:20, and the mixture is repeatedly frozen and thawed 2 to 5 times.

[0011] Furthermore, step (3) adopts freeze-drying.

[0012] Furthermore, the unicellular green algae is Chlorella vulgaris. Chlorella vulgaris is a typical unicellular green algae, which is easy to culture, can not only utilize light energy for autotrophy, but also utilize organic carbon sources for growth and reproduction under heterotrophic conditions; and has a fast growth and reproduction speed, and is the only one of the very few organisms in plants and animals that can grow four times in 20 hours.

[0013] The unicellular green algae combined functional factors selected by the present invention contain a variety of cell growth factors, the main components of which are protein functional factors and nucleic acid functional factors. Cultivating fish muscle cells as a serum substitute can stimulate and maintain muscle cell proliferation and characteristic gene expression, which is completely different from stem cell proliferation. The unicellular green algae combined functional factors are used as serum substitutes to cultivate fish muscle cells, which can achieve the goal of completely replacing serum and long-term stable expansion of cultured cells. They can be applied to the production process of cell cultured meat, providing new ideas for the development of the cell cultured meat industry.

[0014] like Figure 1As shown in the figure, taking Chlorella as an example, it was found that the combined functional factors of Chlorella contain a variety of growth factors involved in cell cycle regulation and cell metabolism, such as elongation factor, transcription factor, replication factor, DNA replication factor, and kinases that regulate the proliferation of fish muscle cells, such as serine / threonine protein kinase and phosphoglycerate kinase. Serine / threonine protein kinase is mainly involved in regulating the metabolic process of muscle cells, transcription initiation / elongation / termination factors are mainly involved in the protein synthesis process, and replication factors are mainly involved in cell cycle regulation and inducing myoblast proliferation. Fetal bovine serum provides nutrients for cell proliferation, such as hormones, amino acids, proteins, growth factors, trace elements, carbohydrates, etc. Compared with fetal bovine serum, some protein functional factors of single-cell green algae water extract replace active ingredients in FBS, such as growth factors, to promote muscle cell proliferation.

[0015] The above-mentioned single-cell green algae combined functional factors are used to replace serum in cell cultured meat. It can completely replace serum for culturing muscle cells and achieve the same culture effect as 10% FBS.

[0016] Furthermore, the cell cultured meat is fish muscle.

[0017] Different cells require different culture conditions for proliferation and differentiation. Embryonic stem cells can proliferate indefinitely, but there is uncertainty in their directed differentiation into muscle cells, and they cannot be directly applied to the production of cell-cultured meat. Cell types that do not have the fate of myogenic cells themselves need to be induced before they can be used as seed cells. The fish muscle cells used in the present invention are mature immortal muscle cells differentiated from fish muscle stem cells, which can express the muscle cell characteristic factor Myf5, and further differentiate into myotubes, and then form cell-cultured meat. The single-cell green algae combined functional factors of the present invention are added to the serum-free culture medium, and the fish muscle cells are cultured in a 96-well plate for 3-7 days, which can make the fish muscle cells achieve the growth effect of adding 10% fetal bovine serum (FBS) culture medium, and do not affect the stability of cell gene expression, and can also achieve expansion culture to culture bottles for continuous subculture to more than 4 generations.

[0018] A culture solution to which the above-mentioned single-cell green algae combination functional factors are added contains 0.5-1.5 mg / mL of the single-cell green algae combination functional factors. If the addition amount of the single-cell green algae combination functional factors is too low, the effect of replacing FBS to promote muscle cell proliferation is not obvious enough; if the addition amount is too high, the cell culture cost is increased, and the cell osmotic pressure is easily increased, resulting in cell wall shrinkage and cell apoptosis.

[0019] Furthermore, the method comprises: 80%-90% basal culture medium, 10% fish muscle extract, 0%-10% serum, 1% penicillin and streptomycin, 1‰ amphotericin B, and lyophilized powder of combined functional factors of unicellular green algae is dissolved in the above culture solution, filtered and sterilized to obtain a culture solution containing the combined functional factors of unicellular green algae; wherein the concentration of the culture solution containing the combined functional factors of unicellular green algae is 0.5-1.5 mg / mL.

[0020] Furthermore, the basic culture medium is L-15 dry powder culture medium aseptically prepared by adding triple distilled water, the fish muscle extract is the supernatant of muscle homogenate above the lateral line of the fish, and the serum dosage is 10%, 5%, 2%, and 0%.

[0021] The culture medium can be used for long-term in vitro expansion of muscle cells. Long-term in vitro expansion of cells requires more fetal bovine serum to maintain cell proliferation, which increases the cost of cell culture. If the amount of serum is reduced, it will be difficult to maintain multiple cell subcultures. In this example, the proliferation activity of the single-cell green algae water extract was first verified in a 96-well plate. The muscle cells were plated in a 96-well plate at a density of 1×10 5 / mL, cultured at 28°C in the absence of CO2 for 24 hours, replaced with culture medium and continued to culture for 3-7 days to achieve the expansion of muscle cells, that is, further transferred cell culture to 24-well plates, 12-well plates, 6-well plates, and T25 culture flasks for continuous subculture and expansion culture to achieve the expansion of fish muscle cells, and continuous subculture can be achieved for more than 4 generations.

[0022] The beneficial effects of the present invention are:

[0023] (1) Based on the CCK8 method, a single-cell green algae combined functional factor that can completely replace serum to promote muscle cell proliferation was screened in a 96-well plate. The main components are protein functional factors and nucleic acid functional factors, which have no adverse effects on muscle cell growth and can meet the needs of long-term stable growth, proliferation and differentiation of muscle cells.

[0024] (2) The combined functional factors of unicellular green algae can be used as serum substitutes to culture muscle cells, which can stimulate muscle cell proliferation and maintain their differentiation potential. It can achieve the goal of completely replacing serum and expanding the culture of cells in a long-term and stable manner, and can be applied to the large-scale production process of cell-cultured meat.

[0025] (3) The single-cell green algae combined functional factor preparation process of the present invention is simple, has a high production yield, and has a good substitution effect, and can be applied to the large-scale production of serum-free cell cultured meat.

[0026] (4) When the single-cell green algae combined functional factors of the present invention are applied to fish muscle cell culture, they can be directly dissolved in the basal culture medium to achieve the purpose of replacing serum. The method of compounding with the culture medium is simple, and it has no toxic side effects on the growth of fish muscle cells. It can completely replace serum for fish muscle cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the GO analysis result of protein functional factors in the combined functional factors of Chlorella.

[0028] Figure 2 To compare the proliferation activity of fish muscle cells cultured for 5 days with Chlorella combination functional factors replacing 100% fetal bovine serum (FBS), control group: 10% FBS group, 0% FBS group, experimental group: 1 mg / mL Chlorella combination functional factors + 0% FBS.

[0029] Figure 3 The experimental results of immunofluorescence detection of fish muscle cell characteristic gene Myf5 (20×), A: 0% FBS group, B: 10% FBS group, C: experimental group: 1 mg / mL Chlorella combined functional factor + 0% FBS.

[0030] Figure 4 The growth of the 4th generation of fish muscle cells (10×) was expanded by replacing 50% fetal bovine serum (FBS) with the combined functional factors of Chlorella. The control group included 10% FBS group and 5% FBS group. The experimental group included 1 mg / mL combined functional factors of Chlorella + 5% FBS.

[0031] Figure 5 To compare the proliferation activity of fish muscle cells cultured for 5 days with protease degradation products and nuclease degradation products of Chlorella combined functional factors replacing 100% fetal bovine serum (FBS), control group: 1 mg / mL of Chlorella combined functional factors + 0% FBS, experimental group: 1 mg / mL of Chlorella combined functional factors protease degradation products + 0% FBS; 1 mg / mL of Chlorella combined functional factors nuclease degradation products + 0% FBS. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 making creative work are within the scope of protection of the present invention.

[0033] Embodiment 1:

[0034] A single-cell green algae combined functional factor, the preparation method of which comprises the following steps:

[0035] a) Freeze-thaw treatment: Take 1 kg of Chlorella powder, add water in a mass ratio of 1:15, freeze overnight at -20°C, thaw at room temperature, and repeat freeze-thaw for 3 times.

[0036] b) Centrifugation: Take the algae solution treated in step a, centrifuge for 30 minutes, and collect the supernatant.

[0037] c) Column loading: Load the nano-scale shrimp and crab shell powder into a chromatography column to obtain a dedicated adsorption chromatography column device.

[0038] d) Refining and purification: taking the supernatant obtained in step b, passing it through the adsorption chromatography column prepared in step c, and collecting the effluent.

[0039] e) Drying: taking the supernatant after the treatment in step d, freeze-drying, and obtaining the freeze-dried powder of the Chlorella combination functional factor.

[0040] Experimental results: The yield of the obtained Chlorella combined functional factors was 8.3%. Through GO analysis of the protein functional factors in the Chlorella combined functional factors, it was found that the Chlorella combined functional factors contained a variety of growth factors involved in cell cycle regulation and cell metabolism, such as elongation factor, transcription factor, replication factor, DNA replication factor, and kinases that regulate the proliferation of fish muscle cells, such as serine / threonine protein kinase, phosphoglycerate kinase, etc. ( Figure 1 ).

[0041] Embodiment 2:

[0042] a) Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL of trypsin to form a cell suspension, adjust the cell inoculation density to 1×105 cells / mL with L-15 complete culture medium (79% L-15 basic culture medium + 10% FBS + 10% fish muscle extract + 1% penicillin, streptomycin double antibiotics + 1‰ amphotericin B), plate 100 μL per well on a 96-well plate, and culture in an incubator at 28°C for 24 hours. Control group: 10% FBS group, 2% FBS group, experimental group: 1 mg / mL of Chlorella combined functional factor + 87% L-15 + 2% FBS + 10% fish muscle extract + 1% penicillin, streptomycin double antibiotics + 1‰ amphotericin B. Replace 100 μL of the above culture medium and continue to culture for 3-7 days. Under light-proof conditions, discard the old culture medium, mix CCK8 and L-15 basal culture medium (1:10), add 100 μL to each well, incubate for 2 h, and compare the absorbance values ​​of the cells at 450 nm.

[0043] b) Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL of trypsin to form a cell suspension, and adjust the cell inoculation density to 1×10 5 / mL, 100μL per well was plated on a 96-well plate and cultured in an incubator at 28℃ for 24h. Control group: 10% FBS group, 0% FBS group, experimental group: 1mg / mL of Chlorella combined functional factor + 89% L-15 + 10% fish muscle extract + 1% penicillin, streptomycin double antibody + 1‰ amphotericin B. Replace 100μL of the above culture medium and continue to culture for 3-7d. Under light-proof conditions, discard the old culture medium, mix CCK8 and L-15 basic culture medium (1:10) and add 100μL to each well, incubate for 2h, and compare the absorbance values ​​of the cells at 450nm.

[0044] Experimental results: It was found that the condition for adding the substance to low serum culture medium is 0.5-1.5 mg / ml, and when replacing 100% FBS ( Figure 2 ) experiment, the experimental group was able to achieve a cell absorbance equivalent to that of 10% FBS, indicating that the combined functional factors of Chlorella can achieve the same growth effect as 10% FBS and can completely replace serum in the production of cell-cultured meat.

[0045] Embodiment 3:

[0046] Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL of trypsin to form a cell suspension, and adjust the cell suspension density to 2×10 5 500 μL per well was inoculated in a 24-well plate and cultured at 28°C for 24 h.

[0047] Control group: 10% FBS group, 0% FBS group, experimental group: 1 mg / mL Chlorella combined functional factor + 89% L-15 + 10% fish muscle extract + 1% penicillin, streptomycin double antibody + 1‰ amphotericin B. Replace 500 μL of the above culture medium, culture at 28°C for 3 days, and detect the expression of the characteristic gene Myf5 of fish muscle cells in the nucleus by immunofluorescence method.

[0048] Immunofluorescence detection was performed as follows:

[0049] (1) Cell fixation: Add 200 μL of cell fixation solution to each well, incubate at room temperature for 15 min, then discard and wash twice with 200 μL of PBS.

[0050] (2) Cell permeabilization: Add 150 μL of cell permeabilization agent to each well, incubate at room temperature for 15 min, then discard and wash twice with 200 μL of PBS.

[0051] (3) Cell blocking: Add 200 μL of cell blocking solution (10% goat serum) to each well, incubate at room temperature for 60 min, then discard and wash twice with 200 μL of PBS.

[0052] (4) Primary antibody incubation: Add 150 μL of primary antibody to each well and incubate at 4°C overnight.

[0053] (5) Secondary antibody incubation: Equilibrate the 24-well plate at room temperature for 30 min, discard the primary antibody, wash twice with PBS, add 150 μL of secondary antibody, and incubate for 1 h in the dark.

[0054] (6) DAPI staining: discard the secondary antibody, wash twice with PBS, add 150 μL of DAPI in dark room, incubate for 5 min in dark room. Discard DAPI, wash twice with PBS, and take pictures under a fluorescence microscope.

[0055] Experimental results: During the proliferation of fish muscle cells, immunofluorescence results showed that the muscle stem cell characteristic factor Pax 7 was not expressed in fish muscle cells, but the muscle cell characteristic gene Myf5 could be expressed normally in the cell nucleus. At the same time, the addition of Chlorella combined functional factors did not affect the stability of fish muscle cell gene expression ( Figure 3 ).

[0056] Embodiment 4:

[0057] Control group: 10% FBS group, 5% FBS group; Experimental group: 1 mg / mL Chlorella combined functional factor + 84% L-15 + 5% FBS + 10% fish muscle extract + 1% penicillin and streptomycin double antibiotics + 1‰ amphotericin B.

[0058] Generation 1: Take 1 bottle of fish muscle cells with a fusion rate of 90%, wash the cell monolayer twice with PE, digest the cells with trypsin, and pipette into a cell suspension with L-15 complete culture medium. After counting, adjust the cell suspension with L-15 complete culture medium to 1×10 5 / mL, aliquot the cell suspension, collect the cells by centrifugation, resuspend the cells with the above culture medium, inoculate them in 24-well plates for culture, change the medium half every 1-2 days, and take photos continuously to observe the cell adhesion and growth conditions.

[0059] Generation 2: Wash the cell monolayer twice with PE, digest the cells with trypsin, prepare culture medium to resuspend the cells, pipette into cell suspension, observe whether the cell monolayer is completely digested under an inverted microscope, and inoculate all of them into 12-well plates for culture. Add the remaining fresh culture medium, change the medium half every 1-2 days, and take pictures continuously to observe the cell adhesion and growth density.

[0060] Generation 3: Wash the cell monolayer twice with PE, digest the cells with trypsin, prepare culture medium to resuspend the cells, pipette into cell suspension, observe whether the cell monolayer is completely digested under an inverted microscope, and inoculate all of them into 6-well plates for culture. Add the remaining fresh culture medium, change the medium half every 1-2 days, and take pictures continuously to observe the cell adhesion and growth density.

[0061] Generation 4: Wash the cell monolayer twice with PE, digest the cells with trypsin, prepare culture medium to resuspend the cells, pipette into cell suspension, observe whether the cell monolayer is completely digested under an inverted microscope, and inoculate all of them into T25 culture flasks for culture, add the remaining fresh culture medium, and take photos continuously to observe the cell adhesion and growth density.

[0062] Generation 5: Wash the cell monolayer twice with PE, digest the cells with trypsin, terminate the digestion, pipette into a cell suspension, count the cells, and compare the cell numbers in the control and experimental groups.

[0063] Experimental results: During the process of continuous cell passage and expansion, when 50% FBS was replaced, at the 4th generation, it was found that the number of cells in the Chlorella combination functional factor + 5% FBS experimental group was higher than that in the 5% FBS group, further verifying the proliferation effect of the Chlorella combination functional factor. At the same time, the number of cells in the Chlorella combination functional factor + 5% FBS experimental group was also higher than that in the 10% FBS group, which can achieve the effect of replacing 50% FBS in the process of expansion culture ( Figure 4 ).

[0064] Embodiment 5:

[0065] (1) Proteolysis: The powder of Chlorella combination functional factors was dissolved in 1×PBS to prepare a solution with a concentration of 1 mg / mL, and trypsin was added to react in a water bath at 37°C for 2 h to perform protein enzymolysis. After the reaction was completed, the enzyme was inactivated in a boiling water bath for 10 min, and the supernatant was collected after centrifugation to obtain the protease degradation product of Chlorella combination functional factors, which was lyophilized.

[0066] (2) Nuclease hydrolysis: Dissolve the Chlorella combination functional factor powder in 1×PBS to prepare a solution with a concentration of 1 mg / mL, add nuclease, and react in a water bath at 37°C for 2 hours to perform nuclease hydrolysis. After the reaction is completed, inactivate the enzyme in a boiling water bath for 10 minutes, centrifuge, and take the supernatant to obtain the Chlorella combination functional factor nuclease degradation product powder, which is freeze-dried.

[0067] (3) Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL of trypsin to form a cell suspension, and adjust the cell seeding density to 1×10 5 / mL, 100μL per well was plated on a 96-well plate and cultured in an incubator at 28℃ for 24h. Control group: 1mg / mL of Chlorella combined functional factor + 0% FBS culture medium, experimental group: 1mg / mL Chlorella combined functional factor protease degradation product + 0% FBS culture medium; 1mg / mL Chlorella combined functional factor nuclease degradation product + 0% FBS culture medium. Replace 100μL of the above culture medium and continue to culture for 3-7d. Under light-proof conditions, discard the old culture medium, mix CCK8 and L-15 basic culture medium (1:10) and add 100μL to each well, incubate for 2h, and compare the absorbance values ​​of the cells at 450nm.

[0068] Experimental results: Figure 5 As shown, Figure 5 As shown in the figure, after the Chlorella combined functional factors were treated with proteases and nucleases, the cell proliferation activity was significantly reduced, indicating that the main active ingredients in the substitute were protein functional factors and nucleic acid functional factors. The Chlorella combined functional factors contain a variety of growth factors involved in cell cycle regulation and cell metabolism, such as elongation factors, transcription factors, replication factors, and DNA replication factors, and contain kinases that regulate the proliferation of fish muscle cells, such as serine / threonine protein kinases and phosphoglycerate kinases. Serine / threonine protein kinases are mainly involved in regulating muscle cell metabolism, transcription initiation / elongation / termination factors are mainly involved in protein synthesis, and replication factors are mainly involved in cell cycle regulation and inducing myoblast proliferation. Therefore, some protein functional factors in the unicellular green algae water extract can replace active ingredients such as growth factors in FBS to promote muscle cell proliferation.

[0069] In summary, it can be seen that the present invention screened and obtained a single-cell green algae combined functional factor that can completely replace serum to promote the proliferation of fish muscle cells in a 96-well plate according to the CCK8 method, and explored that the condition for adding the substance to the low-serum culture medium is 0.5-1.5 mg / mL, and the cells can achieve an effect equivalent to 10% fetal bovine serum (FBS) after culturing the cells for a period of time; the single-cell green algae combined functional factor culture medium is applied to the in vitro expanded culture of fish muscle cells, and continuous subculture can be achieved to more than 4 generations, and the final harvested cell number is higher than that of the 10% fetal bovine serum (FBS) group; at the same time, the single-cell green algae combined functional factor does not affect the stable expression of the characteristic genes of muscle cells in the process of promoting the proliferation of fish muscle cells, and has no adverse effect on the growth of fish muscle cells. It is an effective and very promising serum substitute and culture medium supplement, which can be applied to the production process of cell cultured meat.

[0070] It should be noted that, although the specific embodiments of the present invention have been described, those skilled in the art may make various modifications and improvements to the present invention based on the basic concept of the invention. As long as they do not deviate from the basic concept of the present invention, they should all be within the scope of protection of the present invention.

Claims

1. A method for preparing a single-cell green algae combined functional factor comprising the following steps: (1) Freeze-thaw treatment: weigh a single-cell green algae raw material, add water according to a dry basis weight ratio of 1:5 to 1:20, freeze overnight at -20 to -80°C, thaw at room temperature, repeat freeze-thaw 2 to 5 times, and centrifuge to obtain the supernatant; the single-cell green algae is Chlorella vulgaris; (2) column loading: loading the nano-scale shrimp and crab shell powder into a chromatography column to obtain a dedicated adsorption chromatography column device; (3) Refining and purification: The supernatant obtained in step (2) is passed through the adsorption chromatography column prepared in step (3), the effluent is collected, and the effluent is dried.

2. The use according to claim 1, characterized in that: Step (3) adopts freeze drying.

3. The use according to claim 1, characterized in that: The cell cultured meat is fish muscle.

4. A culture solution to which the combined functional factors of the unicellular green algae according to claim 1 are added, characterized in that: Contains 0.5-1.5mg / mL of combined functional factors of single-cell green algae.

5. The culture solution according to claim 4, characterized in that: The invention comprises the following components: 80%-90% basic culture medium, 10% fish muscle extract, 0%-10% serum, 1% penicillin and streptomycin double antibiotics, 1‰ amphotericin B, and lyophilized powder of single-cell green algae combined functional factors is dissolved in the culture solution, filtered and sterilized to obtain the culture solution containing the single-cell green algae combined functional factors.

6. Use of the culture solution of the combined functional factors of single-cell green algae according to claim 4 or 5 in continuous passage and expanded culture of muscle cells in vitro.

7. The use according to claim 6, characterized in that: Muscle cells were plated in 96-well plates at a density of 1 × 10 5 / mL, culture at 28℃ in the absence of CO2 for 24 hours, replace with culture medium and continue to culture for 3-7 days to achieve the expansion of muscle cell culture.

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