A kind of blue algae water extract and its application as substitute for serum

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a blue algae water extract and its use as a substitute for serum. The water extract is obtained by processes such as raw material extraction, low-temperature freezing, room temperature thawing, and purification by nano-scale shell adsorption columns of blue algae. 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. The blue algae water extract mentioned in the present invention is applied to the muscle cell culture process to replace serum, which can meet the effect of long-term continuous subculture of cells in culture bottles, and can be compounded into a culture fluid and applied to a bioreactor to expand the culture of cell meat.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a blue algae water extract and an application thereof as a substitute for serum. 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 continuously search 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 developed a series of serum substitute combinations for myeloma, CHO, BHK, Vero, and lymphocytes. For example, various recombinant proteins, galactose, lipids, trace elements, vitamins, amino acids, hormones and other clear combinations of ingredients replace serum to maintain cell function, but multiple additives and basal culture media are compounded in different proportions, additives are redundant, and 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 application of serum substitutes in cell cultured meat. In order to further solve the above problems and reduce the shortcomings of animal-derived additives, plant-derived serum substitutes have received more and more attention.

[0004] Blue algae are the most nutritious and comprehensive organisms in nature. They are rich in high-quality protein, with a protein content of up to 60%-70%, and the protein is mainly water-soluble. Therefore, blue algae water extracts may play a positive role in cell proliferation and differentiation, and thus have the potential to replace serum. The prior art only 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 algae proteins can have similar substitution effects. Moreover, the culture conditions required for the proliferation and differentiation of different cells are different, and serum substitutes developed for different cell types are not universal. At present, most of the serum-free culture media used in the prior art for muscle cell culture have problems such as slow subculture rate, slow cell growth, limited number of subcultures, 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 blue algae water extract, which can be used as a serum substitute to culture fish muscle cells, and can achieve the goal of completely replacing serum and expanding the cultured cells stably for a long time, and can be applied to the large-scale production process of cell-cultured meat.

[0006] To achieve the above object, the present invention is specifically implemented by the following technical scheme: a blue algae water extract, the preparation method of which comprises the following steps:

[0007] (1) Add water to the blue algae powder and mix well, and let it stand;

[0008] (2) Freeze-thaw extraction: The algae solution obtained in step (1) is subjected to freeze-thaw treatment, and the supernatant is obtained by centrifugation; the cell walls of the cyanobacteria are ruptured by freeze-thaw treatment to extract the water-soluble active substances.

[0009] (3) 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.

[0010] (4) Refining and purification: Take the supernatant obtained in step (2), pass it through the adsorption chromatography column prepared in step (3), collect the effluent, and dry it.

[0011] Furthermore, the cyanobacteria is Spirulina or Synechococcus.

[0012] Furthermore, in the step (1), the liquid-to-solid ratio of spirulina powder to water is 1:5 to 1:20, and the mixture is allowed to stand overnight.

[0013] Furthermore, the step (2) is to freeze at -20 to -80°C overnight and then thaw at room temperature, repeat freezing and thawing 2 to 5 times and then centrifuge to collect the supernatant.

[0014] Furthermore, step (4) adopts freeze-drying.

[0015] like Figure 1As shown, taking Spirulina as an example, the main component of its water extract is protein, and the protein content is 61%. Through GO analysis of protein functional factors in water extract, it is found that the water extract of the present invention contains a variety of functional factors involved in cell division and cycle regulation, such as elongation factor, trigger factor, cell division protein FtsZ, etc., and contains kinases that regulate cell signaling processes, such as serine / threonine protein kinase, histidine kinase, etc. Serine / threonine protein kinase is mainly involved in regulating muscle cell metabolism, transcription initiation / elongation / termination factors, etc. are mainly involved in protein synthesis, and growth factors can induce myoblast proliferation and maintain muscle cell differentiation potential. 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 cyanobacteria water extract can replace active ingredients in FBS, such as growth factors, to promote muscle cell proliferation.

[0016] The above-mentioned blue algae water extract is used to replace serum in cell cultured meat and can completely replace serum for culturing muscle cells.

[0017] Furthermore, the cell cultured meat is fish muscle.

[0018] 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 cyanobacteria water extract of the present invention is added to a serum-free culture medium, and the fish muscle cells are cultured in a 96-well plate for 3-7 days. The fish muscle cells can achieve the growth effect of adding 10% fetal bovine serum (FBS) culture medium, and the stability of fish muscle cell gene expression is not affected. It can also achieve expansion culture to culture bottles for continuous subculture to more than 4 generations.

[0019] A culture solution containing the above-mentioned cyanobacteria water extract contains 0.5-1.5 mg / mL of cyanobacteria water extract. If the amount of cyanobacteria water extract added is too low, the effect of replacing FBS to promote muscle cell proliferation is not obvious enough; if the amount added is too high, the cost of cell culture will be increased, and the cell osmotic pressure will be easily increased, resulting in cell wall shrinkage and cell apoptosis.

[0020] Further, it comprises: 80%-90% basal culture medium, 10% fish muscle extract, 0%-10% serum, 1% penicillin, streptomycin, 1‰ amphotericin B, lyophilized powder of cyanobacteria water extract is dissolved in the above culture solution, filtered and sterilized, and a culture solution containing cyanobacteria water extract is obtained; wherein the concentration of the culture solution containing cyanobacteria water extract is 0.5-1.5 mg / mL. Further, the amount of serum used is 10%, 5%, 2%, 0%.

[0021] The culture medium can be used for long-term in vitro expansion and culture of muscle cells. Long-term in vitro expansion and culture 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 is difficult to maintain multiple cell subcultures. In this example, the proliferation activity of the blue algae water extract was first verified in a 96-well plate.

[0022] Furthermore, the cells were transferred and cultured in 24-well plates, 12-well plates, 6-well plates, and T25 culture flasks for continuous subculture and expansion culture to achieve the expansion culture of fish muscle cells, and continuous subculture for more than 4 generations was achieved.

[0023] Further, the following steps are included:

[0024] (1) collecting cells by centrifugation at 800-1200 rpm for 5-10 min, collecting 0.1-1 mL of conditioned medium and mixing it with 0.1-1 mL of the low serum culture medium described in claim 3, resuspending the cells in the mixed culture medium, and then inoculating them in a 24-well plate for culture;

[0025] (2) Collect the cell supernatant from the 24-well plate, prepare 0.1-1 mL of conditioned medium and 1-2 mL of culture medium; after cell digestion, blow the cell suspension and inoculate it into a 12-well plate for culture;

[0026] (3) Collect the cell supernatant from the 12-well plate, prepare 0.1-1 mL of conditioned medium and 1-2 mL of culture medium; after cell digestion, blow the cell suspension and inoculate it into a 6-well plate for culture;

[0027] (4) Collect the cell supernatant from the 6-well plate and prepare 0.1-1 mL of conditioned medium and 3-5 mL of culture medium; after cell digestion, blow the cell suspension and inoculate it into a T25 culture flask for culture.

[0028] The beneficial effects of the present invention are:

[0029] (1) The cyanobacteria water extract selected by the present invention can be used as a serum substitute to stably replace serum to culture fish muscle cells, and can stimulate and maintain muscle cell proliferation and characteristic gene expression, which is completely different from stem cell proliferation.

[0030] (2) Cyanobacteria water extract can be used as a serum substitute to culture fish muscle cells, which can achieve the goal of completely replacing serum and expanding the culture of cells in a long-term and stable manner. It can be applied to the large-scale production process of cell-cultured meat.

[0031] (3) The blue algae water extract of the present invention has a simple preparation process, a high production yield, and a good substitution effect, and can be applied to the large-scale production of serum-free cell cultured meat.

[0032] (4) When the cyanobacteria water extract of the present invention is used for fish muscle cell culture, it can be directly dissolved in the basal culture medium, and the method of compounding with the culture medium is simple. It has no toxic side effects on the growth of fish muscle cells and can completely replace serum for fish muscle cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of GO analysis of the functional factors of proteins in Spirulina water extract.

[0034] Figure 2 To compare the proliferation activity of fish muscle cells cultured for 5 days with Spirulina water extract replacing 100% FBS, the control group included 10% FBS group and 0% FBS group, and the experimental group included 1 mg / mL Spirulina water extract + 0% FBS.

[0035] 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 Spirulina water extract + 0% FBS.

[0036] Figure 4 The growth of fish muscle cells at the 4th generation (10×) was expanded by using Spirulina water extract instead of 50% FBS. Control group: 10% FBS group, 5% FBS group; experimental group: 1 mg / mL Spirulina water extract + 5% FBS.

[0037] Figure 5 To compare the proliferation activity of fish muscle cells cultured for 5 days with protease degradation products of Spirulina water extract replacing 100% fetal bovine serum (FBS), control group: 1 mg / mL Spirulina water extract + 0% FBS, experimental group: 1 mg / mL Spirulina water extract protease degradation products + 0% FBS. DETAILED DESCRIPTION

[0038] 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.

[0039] Embodiment 1:

[0040] A blue algae water extract, the preparation method of which comprises the following steps:

[0041] a) Raw material extraction: Take 1 kg of spirulina powder, add water in a mass ratio of 1:10, mix well, and let stand overnight.

[0042] b) Freeze-thaw extraction: Take the algae solution obtained in step a, freeze it at -20°C overnight, thaw it at room temperature, repeat the freeze-thaw treatment 3 times, centrifuge it for 30 minutes, and collect the supernatant.

[0043] c) Column loading: Load the nano-scale shrimp and crab shell powder into a chromatography column to obtain a dedicated adsorption chromatography column device.

[0044] 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.

[0045] e) Freeze drying: freeze-dry the effluent obtained in step d to obtain freeze-dried powder of water extract.

[0046] Experimental results: The main component of Spirulina water extract is protein, with a protein content of 61%. Through GO analysis of protein functional factors in Spirulina water extract, it was found that Spirulina water extract contains a variety of functional factors involved in cell division and cycle regulation, such as elongation factor, trigger factor, cell division protein FtsZ, etc., and contains kinases that regulate cell signaling processes, such as serine / threonine protein kinases, histidine kinases, etc. ( Figure 1 ).

[0047] Embodiment 2:

[0048] (1) Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL trypsin to form a cell suspension, and adjust the cell inoculation density to 1×10 with L-15 complete culture medium (79% L-15 basal culture medium + 10% FBS + 10% muscle extract + 1% penicillin and streptomycin + 1‰ amphotericin B). 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, 2% FBS group, experimental group: 1mg / mL Spirulina water extract + 87% L-15 + 2% FBS + 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 per well, incubate for 2h, and compare the absorbance values ​​of the cells at 450nm.

[0049] (2) Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL 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: 10% FBS group, 0% FBS group, experimental group: 1mg / mL Spirulina water extract + 89% L-15 + 0% FBS + 10% 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 per well, incubate for 2h, and compare the absorbance values ​​of the cells at 450nm.

[0050] Experimental results: In the experiment of replacing 100% FBS (group (2) above), the water extract of Spirulina can achieve a cell absorbance equivalent to that of 10% FBS, indicating that the water extract of Spirulina can achieve a similar growth effect as 10% FBS and can replace serum in the production of cell-cultured meat.

[0051] Embodiment 3:

[0052] Take fish muscle cells with a fusion rate of 80-90%, digest the cell monolayer with 300 μL 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.

[0053] Control group: 10% FBS group, 0% FBS group, experimental group: 1 mg / mL Spirulina water extract + 89% L-15 + 5% FBS + 10% 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.

[0054] The process of immunofluorescence detection is as follows:

[0055] (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.

[0056] (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.

[0057] (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.

[0058] (4) Primary antibody incubation: Add 150 μL of primary antibody to each well and incubate at 4°C overnight.

[0059] (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.

[0060] (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.

[0061] 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 Spirulina water extract did not affect the stability of gene expression in fish muscle cells ( Figure 3 ).

[0062] Embodiment 4:

[0063] Control group: 10% FBS group, 5% FBS group; Experimental group: 1 mg / mL Spirulina water extract + 84% L-15 + 5% FBS + 10% muscle extract + 1% penicillin and streptomycin + 1‰ amphotericin B.

[0064] 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 halfway every 1-2 days, and take photos continuously to observe the cell adhesion and growth conditions.

[0065] 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, perform half-culture every 1-2 days, and take photos continuously to observe the cell adhesion and growth density.

[0066] 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, perform half-culture every 1-2 days, and take photos continuously to observe the cell adhesion and growth density.

[0067] 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.

[0068] 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.

[0069] Experimental results: Figure 4 As shown in the data, during the continuous passage and expansion of fish muscle cells, 50% FBS was replaced. When it was passed to the 4th generation, the number of cells in the experimental group (Spirulina water extract + 5% FBS) was higher than those in the 5% FBS group and the 10% FBS group, which further demonstrated that Spirulina water extract can promote cell proliferation and can achieve the effect of replacing serum in the process of expansion culture.

[0070] Embodiment 5:

[0071] (1) Proteolysis: The Spirulina water extract powder was dissolved in 1×PBS to prepare a solution with a concentration of 1 mg / mL, and trypsin was added. The solution was reacted 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. The supernatant was collected after centrifugation to obtain the protease degradation product of Spirulina water extract, which was lyophilized.

[0072] (2) 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 Spirulina water extract + 0% FBS culture medium, experimental group: 1mg / mL of Spirulina water extract protease 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.

[0073] Experimental results: Figure 5 As shown in the figure, after the Spirulina water extract was treated with protease, the cell proliferation activity was significantly reduced, indicating that the main active ingredients in the substitute are protein functional factors. Among them, serine / threonine protein kinase DST1 is mainly involved in regulating cell metabolism and transferase activity. Epidermal growth factor and platelet activating factor induce myoblast proliferation and differentiation, and transcription initiation / elongation / termination factors participate in protein synthesis.

[0074] In summary, it can be seen that the present invention screened and obtained a cyanobacteria water extract that can replace FBS to promote the proliferation of fish muscle cells according to the CCK8 method, and explored that the condition for adding the substance to the basal culture medium is 0.5-1.5 mg / mL, and the fish muscle cells are cultured in a 96-well plate for 3-7 days to reach the cell proliferation rate of the 10% FBS group; the cyanobacteria water extract culture medium is applied to the in vitro expansion 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% FBS group; at the same time, the cyanobacteria water extract does not affect the stable expression of the cell characteristic gene MyoD in the process of promoting the proliferation of fish muscle cells, and can achieve the effect of complete serum replacement, and is used to make cell cultured meat.

[0075] 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. Application of cyanobacteria water extract to replace serum in cell cultured meat; the preparation method of cyanobacteria water extract comprises the following steps: (1) Mix the blue algae powder with water and let it stand; (2) Freeze-thaw extraction: Take the algae solution obtained in step (1), freeze-thaw it, and centrifuge it to obtain the supernatant: (3) Column loading: loading the nano-scale shrimp and crab shell powder into a chromatography column to obtain a dedicated adsorption chromatography column device; (4) Refining and purification: taking the supernatant obtained by the treatment in step (2), passing it through the adsorption chromatography column prepared in step (3) to collect the effluent, and then drying the effluent; The blue algae is Spirulina; In the step (1), the ratio of cyanobacteria powder to water is 1:5-1:20, and the mixture is allowed to stand overnight; and in the step (2), the mixture is frozen at -20--80°C overnight and then thawed at room temperature, and the mixture is repeatedly frozen and thawed 2-5 times and then centrifuged to collect the supernatant.

2. The use according to claim 1, characterized in that : Step (4) adopts freeze drying.

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

4. A culture solution containing the cyanobacteria water extract of claim 1, characterized in that :The culture solution contains 0.5~1.5mg / ml of cyanobacteria water extract.

5. Use of the culture solution of the cyanobacteria water extract according to claim 4 in continuous passage and expanded culture of muscle cells in vitro.

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