CN115125195A - Single-cell green alga combined functional factor and application thereof in cell culture meat - Google Patents
Single-cell green alga combined functional factor and application thereof in cell culture meat Download PDFInfo
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Abstract
The invention relates to the technical field of bioengineering, in particular to a single-cell green alga combined functional factor and application thereof in cell culture meat. The functional factors are obtained by processing the raw materials through low-temperature freezing, room-temperature thawing, nanometer carapace adsorption column refining and the like, the extraction process is simple, the safety is high, and the method is suitable for industrial amplification production. When the compound preparation is applied to muscle cell culture, the compound preparation can be directly dissolved in a basic culture medium, has a simple compounding mode with the culture medium, has no toxic or side effect on the growth of muscle cells, and can completely replace serum to be used for the muscle cell culture. When the single-cell green alga combined functional factor provided by the invention is applied to fish muscle cell culture, compared with the case of not adding a serogroup, the single-cell green alga combined functional factor can provide growth functional factor supplement for cells, promotes in-vitro proliferation and differentiation of the cells, and can be applied to preparation of cell culture meat.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a single-cell green alga combined functional factor and application thereof in cell culture meat.
Background
Serum is an ideal cell growth supplement during in vitro cell culture. Serum contains plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, etc. and provides nutrients for maintaining cell growth. Although serum is an ideal supplement for cell culture, it has the disadvantages of large quality difference between batches, easy increase of the risk of zoonosis, ethical problems, and the like, and the serum is continuously searched for a substitute. To date, no substance has been found that can broadly replace serum and be applied to all cell lines.
Researchers at home and abroad find a series of serum substitute combinations for myeloma, CHO, BHK, Vero and lymphocytes, for example, the serum substitute combinations with definite components such as various recombinant proteins, galactose, lipid, trace elements, vitamins, amino acids, hormones and the like are used for maintaining cell functions, but various additive components and basic culture media are compounded in different proportions, the additives are complicated, and the optimization conditions are complex. Although the above-mentioned techniques disclose some serum substitutes which can meet certain requirements, there are some disadvantages, such as high cost, low food safety, low serum substitution degree, etc., which may affect the wide application of serum substitutes to cell culture meat. The research on the serum substitute is of great significance, which is one of the research hotspots and the emphasis in the technical field, and is the impetus and the starting point for the completion of the invention.
Unicellular algae refer to lower plants that reproduce as spores, and are a source of biological activity that promotes immunity, oxidation resistance, and tissue remodeling. The reproduction of unicellular algae is basically the germination of new individuals from spores or zygotes of single cells leaving the mother, either directly or after a short period of dormancy. The chlorella is typical unicellular green algae, is easy to culture, can not only utilize light energy for autotrophy, but also can utilize organic carbon source for growth and propagation under heterotrophic conditions; and the growth and propagation speed is high, and the growth and propagation speed is less than that of organisms which can grow 4 times in 20 hours in animals and plants, so that the chlorella probably has nutrient components capable of promoting cell proliferation in cells, and has the potential of extracting serum substitutes. Chlorella intracellular contains abundant proteins, polypeptides, nucleic acids and other components, but intracellular substances cannot be released due to the existence of chlorella cell walls, so that extraction of intracellular active components by certain technical means is required. The prior art mentions that algal polysaccharides are added as functional components in serum-free culture media for stem cell culture, but does not indicate whether the algal polysaccharides can be used as serum substitutes, nor does it mention whether other components such as proteins, polypeptides, nucleic acids and the like have similar substitution effects, and the culture conditions required by different cell proliferation and differentiation are different, so that the serum substitutes developed for different cell types have no universality. At present, most of serum-free culture media applied to muscle cell culture in the prior art have the problems of slow passage rate, slow cell growth, limited passage times, slow cell amplification speed, difficulty in realizing expanded culture and the like, and limit the production of serum-free cell culture meat.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a single-cell chlorella combined functional factor, which mainly comprises a protein functional factor and a nucleic acid functional factor, is directly dissolved in a basic culture medium to achieve the purpose of replacing serum, can provide growth functional factor supplement for cells and promote the in-vitro proliferation and differentiation of the cells, and can be applied to the preparation of cell culture meat.
In order to achieve the purpose, the invention is realized by the following technical scheme, and the preparation method of the single-cell green alga combined functional factor comprises the following steps:
(1) freeze-thaw treatment: weighing a single-cell green alga raw material, adding water, freezing overnight at the temperature of-20 to-80 ℃, unfreezing at room temperature, repeatedly freezing and thawing, and centrifuging to obtain a supernatant; the cell wall of green algae is broken by freeze thawing to extract water soluble active substance in cells.
(2) Column assembling: loading the nano-scale shrimp and crab shell powder into a chromatographic column to obtain a special adsorption chromatographic column device; the shrimp and crab shells are derived from shells of aquatic animals such as shrimps, crabs and the like, and the shells are subjected to superfine grinding to obtain nano-scale shell powder. Chitin is the only basic polysaccharide in nature, and the molecular structure of chitin has a plurality of active groups such as amino, hydroxyl, acetamido, glycosidic bond and the like, and has a plurality of unique functional properties such as good biocompatibility, nature, no toxicity and the like, and meanwhile, the special sugar ring structure endows chitin molecules with unique interface characteristics. Therefore, the novel chitin-based material has unique biological activity and functional characteristics. Because of the special fibrous structure of chitin, chitin nano-fibers can be obtained through mechanical action. The chitin nanofiber can adsorb negatively charged functional molecules such as protein, nucleic acid and the like, so that functional factors are effectively enriched.
(3) Refining and purifying: and (3) taking the supernatant obtained by the treatment in the step (2), flowing through the adsorption chromatography column prepared in the step (3), collecting effluent liquid, and drying the effluent liquid.
Further, water is added in the step (1) according to the mass ratio of the dry basis to the dry basis of 1: 5-1: 20, and freeze thawing is carried out repeatedly for 2-5 times.
Further, step (3) adopts freeze drying.
Further, the single-cell green algae is chlorella. The chlorella is typical unicellular green algae, is easy to culture, can not only utilize light energy for autotrophy, but also can utilize organic carbon source for growth and propagation under heterotrophic conditions; and the growth and reproduction speed is high, and the growth and reproduction speed is very few and only one organism which can grow 4 times in 20 hours in animals and plants.
The single-cell chlorella combined functional factor selected by the invention contains various cell growth factors, and the main components are protein functional factors and nucleic acid functional factors. The fish muscle cells cultured as the serum substitute can stimulate and maintain muscle cell proliferation and characteristic gene expression, and are completely different from stem cell proliferation. The single-cell green alga combined functional factor is used as a serum substitute for culturing fish muscle cells, can realize the aims of completely replacing the serum and stably expanding and culturing the cells for a long time, can be applied to the preparation process of cell culture meat, and provides a new idea for the development of the cell culture meat industry.
As shown in fig. 1, it was found that chlorella combined functional factors include various growth factors involved in cell cycle regulation and cell metabolism, such as elongation factor (elongation factor), transcription factor (transcription factor), replication factor (replication factor), DNA replication factor (DNA replication factor), and kinases involved in the proliferation of fish muscle cells, such as serine/threonine protein kinase, phosphoglycerate kinase, etc. Serine/threonine protein kinases are mainly involved in regulating muscle cell metabolic processes, transcription initiation/elongation/termination factors and the like are mainly involved in protein synthesis processes, and replication factors are mainly involved in cell cycle regulation and induction of myoblast proliferation. Fetal calf serum provides nutrients for cell proliferation, such as hormones, amino acids, proteins, growth factors, trace elements, carbohydrates, and the like. Compared with fetal bovine serum, a part of protein functional factors of the single-cell green algae aqueous extract replaces active ingredients such as growth factors in FBS to promote muscle cell proliferation.
The single-cell green alga combined functional factor is applied to cell culture meat instead of serum. Can completely replace serum to be used for culturing muscle cells, and achieves the same culture effect as 10% FBS.
Further, the cell culture meat is fish muscle.
The culture conditions required for proliferation and differentiation of different cells vary. Embryonic stem cells can proliferate indefinitely, but the directed differentiation into muscle cells has uncertainty, and cannot be directly applied to the production of cell culture meat. Cell types that do not have myogenic cell fates themselves need to be induced to act as seed cells. The fish muscle cells used in the invention are mature immortalized muscle cells after the differentiation of the fish muscle stem cells, can express a muscle cell characteristic factor Myf5, and are further differentiated to form myotubes, thereby forming cell culture meat. The single-cell green alga combined functional factor is added into a serum-free culture medium, and fish muscle cells are cultured in a 96-well plate for 3-7d, so that the fish muscle cells can achieve the growth effect of adding a 10% Fetal Bovine Serum (FBS) culture medium, the stability of cell gene expression is not influenced, and the expansion culture can be realized until the continuous subculture is carried out in a culture bottle for more than 4 generations.
A culture solution added with the single-cell green alga combined functional factor contains 0.5-1.5 mg/mL of the single-cell green alga combined functional factor. The addition amount of the single-cell green alga combined functional factor is too low, and the muscle cell proliferation promoting effect of the single-cell green alga combined functional factor replacing FBS is not obvious enough; the addition amount is too high, so that the cell culture cost is increased, and the osmotic pressure of cells is easily increased, so that cell wall shrinkage is caused to cause apoptosis.
Further, the method comprises the following steps: 80-90% of a basic culture medium, 10% of fish muscle extract, 0-10% of serum, 1% of penicillin and streptomycin double antibody, 1% of amphotericin B and the single-cell green alga combined functional factor freeze-dried powder are dissolved in the culture solution, and the culture solution containing the single-cell green alga combined functional factor is obtained after filtration and sterilization; wherein the concentration of the culture solution containing the unicellular chlorella combined functional factor is 0.5-1.5 mg/mL.
Further, the basic culture medium is prepared by adding triple-distilled water into an L-15 dry powder culture medium in an aseptic mode, the fish muscle extracting solution is a supernatant of muscle homogenate above a fish siding line, and the serum dosage is 10%, 5%, 2% and 0%.
The culture solution can be used for in vitro long-term expansion culture of muscle cells. The in vitro long-term expansion culture of the cells needs more fetal calf serum to maintain the cell proliferation, the cell culture cost is increased, and if the serum dosage is reduced, the cells are difficult to maintain for multiple subcultures. In this example, the proliferation activity of aqueous extract of unicellular green algae was first verified in 96-well plates, and the density of 96-well plates was 1X 10 in the case of plating muscle cells 5 /mL, CO free at 28 ℃ 2 And (3) performing medium-adherence culture for 24 hours, replacing with a culture solution, and continuing to culture for 3-7 days to realize the expanded culture of the muscle cells, namely further transferring the cell culture to 24-pore plates, 12-pore plates, 6-pore plates and T25 culture bottles for continuous passage and expanded culture to realize the expanded culture of the fish muscle cells, and realizing the continuous passage to more than 4 generations.
The invention has the beneficial effects that:
(1) the unicellular chlorella combined functional factor capable of completely replacing serum to promote muscle cell proliferation is obtained by screening in a 96-well plate according to a CCK8 method, the main components are a protein functional factor and a nucleic acid functional factor, no adverse effect is caused on the growth of muscle cells, and the requirements of long-term stable growth, proliferation and differentiation of the muscle cells can be met.
(2) The single-cell green alga combined functional factor serving as a serum substitute can stimulate the proliferation of muscle cells and maintain the differentiation potential of the muscle cells, can realize the aims of completely replacing the serum and stably expanding the cultured cells for a long time, and can be applied to the large-scale production process of cell cultured meat.
(3) The single-cell green alga combined functional factor provided by the invention has the advantages of simple preparation process, high production yield and good substitution effect, and can be applied to large-scale production of serum-free cell culture meat.
(4) When the single-cell chlorella combined functional factor is applied to fish muscle cell culture, the single-cell chlorella combined functional factor can be directly dissolved in a basic culture medium to achieve the purpose of replacing serum, is simple in compounding mode with the culture medium, has no toxic or side effect on the growth of fish muscle cells, and can completely replace serum to be used for fish muscle cell culture.
Drawings
FIG. 1 shows GO analysis results of protein functional factors in Chlorella combined functional factors.
FIG. 2 is a comparison of the proliferation activity of chlorella combined functional factor in place of 100% Fetal Bovine Serum (FBS) in cultured fish muscle cells for 5 days, control group: 10% FBS group, 0% FBS group, experimental group: 1mg/mL chlorella combined functional factor + 0% FBS.
FIG. 3 shows the result of the immunofluorescence assay for detecting Myf5 gene characteristic of fish muscle cells (20X), A: 0% FBS group, B: 10% FBS group, C: experimental groups: 1mg/mL chlorella combined functional factor + 0% FBS.
FIG. 4 shows the growth of chlorella combined functional factor in place of 50% Fetal Bovine Serum (FBS) at passage 4 of cultured fish muscle cells expanded (10X), control group: 10% FBS group, 5% FBS group, experimental group: 1mg/mL chlorella combined functional factor + 5% FBS.
FIG. 5 shows comparison of proliferation activities of chlorella combined functional factor protease degradation products and nuclease degradation products in place of 100% Fetal Bovine Serum (FBS) in cultured fish muscle cells for 5 days, in a control group: 1mg/mL chlorella combined functional factor + 0% FBS, experimental group: 1mg/mL chlorella combined functional factor protease degradation product + 0% FBS; 1mg/mL of chlorella combined with functional factor nuclease degradation product + 0% FBS.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a single-cell green algae combined functional factor is prepared by the following steps:
a) freeze-thaw treatment: adding water into 1kg of chlorella powder according to the mass ratio of 1:15, freezing at-20 ℃ overnight, thawing at room temperature, and repeatedly freezing and thawing for 3 times.
b) Centrifuging: and (b) taking the algae liquid treated in the step (a), centrifuging for 30min, and collecting supernatant.
c) Column assembling: and (3) loading the nano-scale shrimp and crab shell powder into a chromatographic column to obtain the special adsorption chromatographic column device.
d) Refining and purifying: and c, taking the supernatant obtained by the treatment in the step b, flowing the supernatant through the adsorption chromatography column prepared in the step c, and collecting effluent.
e) And (3) drying: and d, taking the supernatant processed in the step d, and freeze-drying to obtain the chlorella combined functional factor freeze-dried powder.
The experimental results are as follows: the yield of the chlorella combined functional factor is 8.3 percent. By performing GO analysis on protein functional factors in the chlorella combined functional factors, the chlorella combined functional factors are found to contain various growth factors such as elongation factors (elongation factors), transcription factors (transcription factors), replication factors (replication factors) and DNA replication factors (DNARRreplication factors) which are involved in cell cycle regulation and cell metabolic processes, and contain kinases such as serine/threonine protein kinase, phosphoglycerate kinase and the like which regulate the proliferation process of fish muscle cells (FIG. 1).
Example 2:
a) taking fish muscle cells with the fusion rate of 80-90%, digesting a cell monolayer into a cell suspension by 300 mu L of trypsin, adjusting the cell inoculation density of an L-15 complete culture solution (79% of L-15 basic culture solution, 10% of FBS, 10% of fish muscle extract, 1% of penicillin, streptomycin double antibody and 1% of amphotericin B) to be 1 x 105/mL, paving 100 mu L of each hole to a 96-hole plate, and culturing for 24h at 28 ℃ in an incubator. Control group: 10% FBS group, 2% FBS group, experimental group: 1mg/mL chlorella combined functional factor, 87% L-15%, 2% FBS, 10% fish muscle extract, 1% penicillin, streptomycin double-resistant and 1% amphotericin B. The culture was continued for 3-7 days by replacing 100. mu.L of the above culture medium. Under the condition of keeping out of the light, the old culture solution is discarded, 100 mu L of CCK8 and L-15 basic culture solution (1:10) are added into each well after mixing, incubation is carried out for 2h, and the absorbance values of the cells under 450nm are compared.
b) Collecting fish muscle cells with fusion rate of 80-90%, digesting cell monolayer into cell suspension with 300 μ L of trypsin, adjusting cell inoculation density to 1 × 10 with L-15 complete culture solution 5 one/mL, 100. mu.L per well were plated into 96-well plates and incubated in an incubator at 28 ℃ for 24 h. Control group: 10% FBS group, 0% FBS group, experimental group: 1mg/mL chlorella combined functional factor, 89% L-15+ 10% fish muscle extract, 1% penicillin, streptomycin double-resistant and 1 ‰ amphotericin B. The culture was continued for 3-7 days by replacing 100. mu.L of the above culture medium. Under the condition of keeping out of the light, the old culture solution is discarded, 100 mu L of CCK8 and L-15 basic culture solution (1:10) are added into each well after mixing, incubation is carried out for 2h, and the absorbance values of the cells under 450nm are compared.
The experimental results are as follows: the condition that the substance is added into a low-serum culture medium is 0.5-1.5 mg/m, and in an experiment for replacing 100% FBS (figure 2), an experimental group can reach the cell absorbance equivalent to 10% FBS, so that the chlorella combined functional factor can reach the same growth effect as 10% FBS, and can be completely used for preparing cell culture meat by replacing serum.
Example 3:
collecting fish muscle cells with fusion rate of 80-90%, digesting cell monolayer into cell suspension with 300 μ L of trypsin, and adjusting cell suspension density to 2 × 10 with L-15 complete culture solution 5 Each well was inoculated at 500. mu.L/mL in a 24-well plate and incubated at 28 ℃ for 24 hours.
Control group: 10% FBS group, 0% FBS group, experimental group: 1mg/mL chlorella combined functional factor, 89% L-15+ 10% fish muscle extract, 1% penicillin, streptomycin double-resistant and 1 ‰ amphotericin B. Changing 500 mu L of the culture solution, culturing at 28 ℃ for 3d, and detecting the expression condition of the characteristic gene Myf5 of the fish muscle cells in the cell nucleus by an immunofluorescence method.
Immunofluorescence detection the following procedure was followed:
(1) cell fixation: mu.L of cell fixative was added to each well, incubated at room temperature for 15min and discarded, and washed 2 times with 200. mu.L of PBS.
(2) Cell permeation: 150 μ L of cell permeabilizing agent was added to each well, incubated at room temperature for 15min and discarded, and washed 2 times with 200 μ L of PBS.
(3) Cell sealing: mu.L of cell blocking solution (10% goat serum) was added to each well, incubated at room temperature for 60min and discarded, and 200. mu.L of PBS was washed 2 times.
(4) Primary antibody incubation: mu.L of primary antibody was added to each well and incubated overnight at 4 ℃.
(5) And (3) secondary antibody incubation: equilibrate 24 well plate for 30min at room temperature, discard primary antibody, wash 2 times with PBS, add 150. mu.L secondary antibody in dark place, incubate for 1h in dark place.
(6) DAPI staining: the secondary antibody was discarded, washed 2 times with PBS, 150. mu.L of DAPI was added in the dark, and incubated for 5min at room temperature in the dark. The DAPI was discarded, washed 2 times with PBS and photographed under a fluorescent microscope.
The experimental results are as follows: in the proliferation process of fish muscle cells, immunofluorescence results show that the muscle stem cell characteristic factor Pax 7 is not expressed in the fish muscle cells, but the muscle cell characteristic gene Myf5 can be normally expressed in cell nuclei, and the stability of the expression of the fish muscle cell gene is not influenced by the addition of the chlorella combined functional factor (figure 3).
Example 4:
control group: 10% FBS group, 5% FBS group, experimental group: 1mg/mL chlorella combined functional factor, 84% L-15%, 5% FBS, 10% fish muscle extract, 1% penicillin, streptomycin double-resistant and 1 ‰ amphotericin B.
Generation 1: taking 1 bottle of fish muscle cells with fusion rate of 90%, cleaning with PE for 2 times to obtain cell monolayer, digesting cells with pancreatin, blowing L-15 complete culture solution to obtain cell suspension, counting, adjusting the cell suspension to 1 × 10 with L-15 complete culture solution 5 And (4) subpackaging cell suspension, centrifugally collecting cells, resuspending the cells by using the culture solution, inoculating the cells into a 24-well plate for culture, performing half-liquid change for 1-2 days, and continuously photographing to observe the adherent condition and the growth condition of the cells.
Generation 2: and cleaning the cell monolayer for 2 times by using PE, digesting the cells by using pancreatin, preparing a culture solution, suspending the cells, blowing and beating the cells into a cell suspension, observing whether the cell monolayer is completely digested under an inverted microscope, inoculating all the cell monolayers into a 12-well plate for culture, supplementing the rest fresh culture solution, performing half-liquid change for 1-2 days, and continuously photographing to observe the cell adherence condition and the growth density.
Generation 3: and cleaning the cell monolayer for 2 times by using PE, digesting the cells by using pancreatin, preparing a culture solution, suspending the cells, blowing and beating the cells into a cell suspension, observing whether the cell monolayer is completely digested under an inverted microscope, inoculating all the cell monolayers into a 6-well plate for culture, supplementing the rest fresh culture solution, performing half-liquid change for 1-2 days, and continuously photographing to observe the cell adherence condition and the growth density.
Generation 4: and cleaning the cell monolayer for 2 times by using PE, digesting the cells by using pancreatin, preparing a culture solution, suspending the cells, blowing the cells into a cell suspension, observing whether the cell monolayer is completely digested under an inverted microscope, inoculating all the cell monolayers into a T25 culture bottle for culture, supplementing the residual fresh culture solution, and continuously photographing to observe the cell adherence condition and the growth density.
Generation 5: and cleaning the PE for 2 times for a cell monolayer, digesting the cells by pancreatin, stopping digestion, blowing the cells into a cell suspension, counting the cells, and comparing the number of the cells in the control group and the number of the cells in the experimental group.
The experimental results are as follows: during the continuous passage and expansion culture process of the cells, when 50% FBS is replaced and the cell number of the chlorella combined functional factor + 5% FBS experimental group is found to be higher than that of the 5% FBS group when the cell number reaches the 4 th generation, the proliferation effect of the chlorella combined functional factor is further verified. Meanwhile, the cell number of the chlorella combined functional factor and 5% FBS experimental group is higher than that of the 10% FBS group, so that the effect of replacing 50% FBS in the process of enlarging culture can be achieved (figure 4).
Example 5:
(1) and (3) proteolysis: dissolving chlorella combined functional factor powder in 1 XPBS, preparing solution with concentration of 1mg/mL, adding trypsin, reacting in water bath at 37 deg.C for 2h, and performing protein enzymolysis. After the reaction is finished, carrying out enzyme inactivation in boiling water bath for 10min, centrifuging, taking supernate to obtain a chlorella combined functional factor protease degradation product, and freeze-drying.
(2) Nucleic acid enzymolysis: dissolving chlorella combined functional factor powder in 1 xPBS to prepare a solution with the concentration of 1mg/mL, adding nucleolytic enzyme, carrying out water bath reaction at 37 ℃ for 2h, and carrying out nucleic acid enzymolysis. After the reaction is finished, carrying out enzyme inactivation in boiling water bath for 10min, centrifuging, taking supernate to obtain chlorella combined functional factor nuclease degradation product powder, and freeze-drying.
(3) Collecting fish muscle cells with fusion rate of 80-90%, digesting cell monolayer into cell suspension with 300 μ L of trypsin, adjusting cell inoculation density to 1 × 10 with L-15 complete culture solution 5 one/mL, 100. mu.L per well were plated into 96-well plates and incubated in an incubator at 28 ℃ for 24 h. Control group: 1mg/mL chlorella combined functional factor + 0% FBS culture solution, experimental group: 1mg/mL chlorella combined functional factor protease degradation product and 0% FBS culture solution; 1mg/mL chlorella combined with functional factor nuclease degradation product and 0% FBS culture solution. The culture was continued for 3-7 days by replacing 100. mu.L of the above culture medium. Under the condition of keeping out of the light, the old culture solution is discarded, 100 mu L of CCK8 and L-15 basic culture solution (1:10) are added into each well after mixing, incubation is carried out for 2h, and the absorbance values of the cells under 450nm are compared.
The experimental results are as follows: as shown in fig. 5, after the chlorella combined functional factor is treated by protease and nucleolytic enzyme, the cell proliferation activity is significantly reduced, which indicates that the main active ingredients in the substitute are protein functional factor and nucleic acid functional factor. The chlorella combined functional factor contains various growth factors such as elongation factors, transcription factors, replication factors and DNA replication factors which are involved in the regulation of cell cycle and the cell metabolic process, and contains kinases such as serine/threonine protein kinase, phosphoglycerate kinase and the like which regulate the proliferation process of fish muscle cells. Serine/threonine protein kinases are mainly involved in regulating muscle cell metabolic processes, transcription initiation/elongation/termination factors and the like are mainly involved in protein synthesis processes, and replication factors are mainly involved in cell cycle regulation and induction of myoblast proliferation. Therefore, part of protein functional factors in the aqueous extract of the unicellular chlorella can replace active ingredients in FBS such as growth factors to promote muscle cell proliferation.
In conclusion, the single-cell green alga combined functional factor capable of completely replacing serum to promote fish muscle cell proliferation is obtained by screening in a 96-well plate according to a CCK8 method, and the condition that the substance is added into a low-serum culture medium is 0.5-1.5 mg/mL, so that the effect equivalent to 10% Fetal Bovine Serum (FBS) can be achieved after the cells are cultured for a period of time; the single-cell green alga combined functional factor culture medium is applied to the in-vitro expanded culture of fish muscle cells, so that continuous subculture can be realized to more than 4 generations, and finally a Fetal Bovine Serum (FBS) group with the cell number higher than 10% is obtained; meanwhile, the single-cell chlorella combined functional factor does not influence the stable expression of characteristic genes of muscle cells in the process of promoting the proliferation of the muscle cells of the fish, has no adverse effect on the growth of the muscle cells of the fish, is a serum substitute and a culture medium supplement with better effect and very promising prospect, and can be applied to the preparation process of cell culture meat.
It should be noted that although the present invention has been described with respect to specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made to the present invention based on the basic idea of the invention. Without departing from the basic idea of the invention, should be considered within the scope of protection of the invention.
Claims (10)
1. A single-cell green alga combined functional factor is characterized in that the preparation method comprises the following steps:
(1) freeze-thaw treatment: weighing a single-cell green alga raw material, adding water, freezing overnight at the temperature of-20 to-80 ℃, unfreezing at room temperature, repeatedly freezing and thawing for 2-5 times, and centrifuging to obtain a supernatant;
(2) column assembling: loading the nano-scale shrimp and crab shell powder into a chromatographic column to obtain a special adsorption chromatographic column device;
(3) refining and purifying: and (3) taking the supernatant obtained by the treatment in the step (2), flowing through the adsorption chromatography column prepared in the step (3), collecting effluent liquid, and drying the effluent liquid.
2. The single-cell chlorella combinatorial functional factor of claim 1, wherein: the single-cell green algae is Chlorella.
3. The single-cell chlorella combinatorial functional factor of claim 1, wherein: adding water according to the dry basis mass ratio of 1: 5-1: 20, and repeatedly freezing and thawing for 2-5 times.
4. The single-cell chlorella composite functional factor of claim 1, wherein: and (3) adopting freeze drying.
5. The use of a serum replacement for a single-cell chlorella combined function factor according to claim 1 in cell culture meat.
6. The use of claim 5, wherein: the cell culture meat is fish muscle.
7. A culture solution to which the single-cell green alga combined functional factor of claim 1 is added, comprising 0.5-1.5 mg/mL of the single-cell green alga combined functional factor.
8. The culture solution according to claim 7, characterized by comprising the following components: 80-90% of basal culture medium, 10% of fish muscle extract, 0-10% of serum, 1% of penicillin and streptomycin double antibody, 1% of amphotericin B and the single-cell green alga combined functional factor freeze-dried powder are dissolved in the culture solution, and the culture solution containing the single-cell green alga combined functional factor is obtained after filtration and sterilization.
9. Use of a culture medium of the single-cell chlorella combined with functional factor according to claim 7 or 8 for continuous in vitro passage and expansion of muscle cells.
10. The use of claim 9, wherein: muscle cell plating 96-well plate density 1X 10 5 /mL, CO free at 28 ℃ 2 Medium adherence culture 2And 4h, replacing the culture solution for continuous culture for 3-7d to realize the enlarged culture of the muscle cells.
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