Marine fish cell culture fish oil and preparation method thereof

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a marine fish cell culture fish oil and a preparation method thereof. It is characterized in that: first, the cells are amplified under 2D conditions, and then they are inoculated on microcarriers for culture, and adipogenic transdifferentiation induction is performed after the cells are fully grown, and adipogenic transdifferentiation induction of muscle satellite cells can be achieved after 6-8 days of induction. The present invention adjusts the adipogenic transdifferentiation culture medium, adds a lipid mixture to the culture medium, and utilizes the mutual synergistic effect between various components to achieve the adipogenic transdifferentiation of large yellow croaker muscle satellite cells, brown spot grouper and humpback seabass muscle cells, and prepares cell cultured large yellow croaker fish oil, which lays a foundation for further obtaining cell cultured fish meat.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a marine fish cell culture fish oil and a preparation method thereof. Background Art

[0002] With the increase in population and the improvement of living standards, the global demand for meat is growing. However, traditional animal husbandry has serious problems such as environmental pollution, greenhouse gas emissions, and animal welfare, and the same is true for aquaculture. Cellular agriculture is an alternative method that uses biotechnology to produce artificial meat from in vitro cultured meat cells. It is a solution to the sustainability problem of traditional meat production. Since marine food plays an indispensable role in providing high-quality protein, omega-3 fatty acids and micronutrients, people are paying more and more attention to the development and utilization of cell-cultured fish meat and its derivatives.

[0003] Most of the current research on cell-cultured meat focuses on muscle cells and tissues, but fat is also an important component of meat. Fat can give cultured meat species-specific flavor, texture and tenderness, and is the key to reproducing the characteristics of specific animals. Cell-cultured fish fat is also called cell fish oil. Fish oil is rich in polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have many health benefits.

[0004] Traditional cell culture fat is produced by direct differentiation of adipose mesenchymal stem cells cultured on a large scale. However, as the number of passages increases, their proliferation capacity decreases significantly, limiting their further application in large-scale culture fat production. At the same time, due to the variety of culture conditions for seed cells, the culture and differentiation of different cells also increase the burden of large-scale cultured meat production. Therefore, the selection of stable cultured meat seed cell lines (such as muscle stem cells) has become the key to producing high-quality cell fish oil. Summary of the invention

[0005] The technical problem to be solved by the present invention is that there is currently a lack of suitable cell fish fat seed cell lines and adipogenic differentiation methods, which seriously hinders the development of cell fish meat and its derivative products.

[0006] In view of the problems of the prior art, the present invention adjusts the adipogenic transdifferentiation culture medium, adds a lipid mixture to the culture medium, and utilizes the synergistic effect of various components to achieve the adipogenic transdifferentiation of large yellow croaker muscle satellite cells, brown-spotted grouper and humpback seabass muscle cells, and prepares cell-cultured large yellow croaker fish oil, laying a foundation for further obtaining cell-cultured fish meat.

[0007] To achieve the above-mentioned purpose, the technical scheme of the present invention is: a method for preparing marine fish cell oil, comprising the following steps: using complete culture medium to expand muscle cells, and then using adipogenic differentiation medium to induce adipogenic differentiation to obtain marine fish cell oil;

[0008] Among them, the lipid conversion differentiation medium consists of 82-95% DMEM / F12 ( v / v )、3-12%( v / v ) Horse serum, 1% ( v / v ) Penicillin-streptomycin (100X), 0.25-1.5 μM dexamethasone, 5-25 μM rosiglitazone, 2.5-15 μg / mL insulin, 25-125 μM indomethacin, 125-750 μM IBMX, 1-5% ( v / v ) lipid mixture, 1-5 mM L-glutamine, 50-250 μM vitamin C, and 10-50 μM glucose; the differentiation time is 6-8 days.

[0009] Among them, DMEM / F12 is the basic culture medium, which provides basic nutrition for cells; horse serum contains serum proteins, growth factors, amino acids, sugars, inorganic salts, etc., which can provide basic nutrition and energy for cells. Horse serum is not as nutritious as fetal bovine serum, but it contains some hormones and factors that can guide cells to differentiate into specific cell types; penicillin-streptomycin (100X) can prevent microbial contamination; L-glutamine can provide energy and nitrogen source for cells and improve cell health; dexamethasone is a synthetic glucocorticoid that promotes C / EBPs expression; rosiglitazone is an insulin sensitizer that can Activate PPARγ; insulin can activate CREB and a series of downstream transcriptional activators, and promote the synthesis of triglycerides from free fatty acids; indomethacin can block the differentiation of cells into osteoclasts and osteoblasts, and is a PPARγ activator; IBMX can activate the cAMP-dependent protein kinase pathway to enhance the expression of C / EBPs and PPARγ; the lipid mixture contains a variety of fatty acids such as oleic acid, linoleic acid, linolenic acid, and stearic acid, which can provide synthetic precursors for lipid synthesis. Many fatty acids such as oleic acid, as natural ligands for PPARγ, can promote the translocation of PPARγ into the cell nucleus and regulate the expression of downstream genes. Fatty acids can also increase the expression of SREBP1 and C / EBPα, and increase the activity of fatty acid desaturase and elongase, thereby promoting lipid production; vitamin C can upregulate the transcription levels of adipogenesis-related genes such as PPARγ, retinoic acid X receptor α, and lipoprotein esterase, and can also promote collagen synthesis, upregulate the expression of LPL and GPDH, downregulate the expression of adiponectin, promote triglyceride accumulation, and accelerate the process of cell differentiation; glucose provides energy and carbon source for cells through glycolysis.

[0010] Furthermore, the muscle cells are muscle satellite cells of large yellow croaker, muscle cells of brown-spotted grouper or muscle cells of humpback seabass. Large yellow croaker and grouper have high economic benefits and market demand, and their muscle cells usually show good uniformity when cultured in vitro. Compared with other cells used for adipogenesis, such as dedifferentiated adipocytes, adipose-derived stem cells, preadipocytes, and pluripotent stem cells, muscle satellite cells are easier to obtain, and their adipogenic efficiency and proliferation ability will not decrease with the increase in the number of passages.

[0011] Furthermore, the complete culture medium is a DMEM / F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X). DMEM / F12 is a mixture of DMEM and F-12 in a 1:1 ratio. It combines the advantages of the two culture media, making the culture medium more comprehensive and providing basic energy and nutrition for cell growth and proliferation. Fetal bovine serum provides various growth factors, hormones, proteins, amino acids, vitamins, minerals and trace elements required for cell growth, which can promote cell proliferation, differentiation and maintain normal physiological functions. Cells can maintain normal cell morphology and proliferation efficiency in 10% fetal bovine serum. If the concentration of fetal bovine serum is too low, the cell proliferation rate will decrease, the morphology will deteriorate, and cavitation will occur easily. If the concentration is too high, the cost will increase and it will easily promote cell differentiation.

[0012] Further, the lipid mixture consists of the following ingredients:

[0013] .

[0014] Furthermore, 2D adipogenic differentiation includes the following steps: inoculating muscle cells into a 6-well plate, adding complete culture medium, and replacing it with adipogenic differentiation medium after the cells are fully grown, and performing differentiation.

[0015] Furthermore, 3D adipogenic transdifferentiation includes the following steps:

[0016] (1) Add microcarriers to a 6-well plate and add basal culture medium to swell;

[0017] (2) After standing, discard the upper basal medium, add basal medium for washing, and after standing, discard the upper basal medium;

[0018] (3) Inoculate muscle cells and add complete culture medium to cover the microcarriers. Let stand for 2 h. Add culture medium to 5 mL and change the medium every other day.

[0019] (4) After the cells have grown all over the microcarriers, discard the complete medium and add the transdifferentiation medium. Change the medium every other day.

[0020] (5) Let the cells stand and discard the transdifferentiation medium. Collect the samples, wash twice with PBS, add cell lysis buffer, centrifuge, and collect the supernatant.

[0021] Furthermore, in step (1), the addition ratio of the microcarrier is 2-5 mg / mL based on the volume of the total culture system; the swelling time is 12 h. Swelling the microcarrier in advance is beneficial to cell attachment and proliferation.

[0022] Furthermore, the basic culture medium in step (1) and step (2) is DMEM / F12 culture medium, and the amount added is 50-100 mL. Substances that may be residual in the microcarrier and have an effect on the cells are washed away as much as possible.

[0023] Furthermore, in step (3), the density of inoculating muscle cells is 50,000-200,000 / mL; and the cells are left to stand at 28° C. If the cell inoculation density is too low, the cells will proliferate slowly, the state will be poor, and the culture time will be long; if the inoculation density is too high, the cell growth will be restricted, the competition between cells will intensify, the cell growth and proliferation will be affected, and the utilization efficiency of the microcarriers will be reduced.

[0024] Furthermore, in step (5), the cell lysis time is 2-3 min at room temperature; the centrifugation time is 5 min at 5000 rpm. The microcarriers are treated in the lysis solution for 2-3 min to separate the cells from the microcarriers. The microcarriers are then precipitated to the bottom by centrifugation, while the cells remain in the supernatant for easy collection.

[0025] A marine fish cell fish oil prepared by the above method. The obtained cell culture fish oil has a large difference in lipid composition from the fish oil extracted from the actual fish belly fat, and contains a higher phosphatidylcholine. The lipid mixture in the culture medium contains a variety of fatty acids and cholesterol, and fatty acids are an important component in the synthesis of phosphatidylcholine. In the synthesis process of phosphatidylcholine, fatty acids combine with glycerol to form phosphatidic acid, and phosphatidic acid is then combined with choline or other alcohol compounds to form different types of phospholipids, including phosphatidylcholine. In addition, cholesterol may indirectly affect the synthesis rate of phosphatidylcholine by regulating the activity of enzymes related to phosphatidylcholine synthesis. Under certain conditions, cholesterol can be converted into an intermediate product in the synthesis process of phosphatidylcholine. The cell culture fish oil obtained by the present invention contains a higher phosphatidylcholine than the lipid composition of the fish oil extracted from the actual fish belly fat.

[0026] The beneficial effects of the present invention are:

[0027] By optimizing the transdifferentiation culture medium, adipogenic transdifferentiation of large yellow croaker muscle satellite cells, brown-spotted grouper and humpback seabass muscle cells can be achieved, and the preparation of cell-cultured fish oil can be realized, laying the foundation for the subsequent preparation of cell-cultured fish oil and cell-cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The effect diagram of inducing differentiation for 12 days without adding lipid mixture in Example 1.

[0029] Figure 2 : The effect diagram of adding 2% lipid mixture to induce differentiation for 10 days in Example 1.

[0030] Figure 3 : The effect diagram of adding 2% lipid mixture and 25 μM glucose to induce differentiation for 10 days in Example 1.

[0031] Figure 4 : The effect diagram of adding 2% lipid mixture, 25 μM glucose and 100 μM vitamin C to induce differentiation for 8 days in Example 1.

[0032] Figure 5 : Schematic diagram of the 2D adipogenic transdifferentiation effect of large yellow croaker muscle satellite cells in Example 2.

[0033] Figure 6 : Comparative Example 1 shows the effect of adding oleic acid, linoleic acid and linolenic acid at the same concentration as the 2% lipid mixture to induce differentiation for 10 days.

[0034] Figure 7 : Diagram of the proliferation effect of 3D culture of large yellow croaker muscle satellite cells in Example 3.

[0035] Figure 8 : Graph showing the effect of 3D adipogenic transdifferentiation of large yellow croaker muscle satellite cells in Example 3.

[0036] Fig. 9 : Analysis of lipid composition of 3D adipogenic transdifferentiation samples of large yellow croaker muscle satellite cells in Example 3.

[0037] Fig.10 : Schematic diagram of the 2D adipogenic transdifferentiation effect of brown-spotted grouper muscle cells in Example 4.

[0038] Fig.11 : Schematic diagram of the 2D adipogenic transdifferentiation effect of humpback seabass muscle cells in Example 5. DETAILED DESCRIPTION

[0039] The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0040] The following examples can be understood as a part of the local structure or method of the present invention alone, and can also be understood as the combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are obtained from commercial sales.

[0041] Embodiment 1:

[0042] Large yellow croaker muscle satellite cells were seeded into 6-well plates and added with complete culture medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)). After the cells were confluent, the medium was replaced with adipogenic differentiation medium (consisting of 87% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX and 2 mM L-glutamine). The effects of additional addition of lipid mixture, vitamin C and glucose on adipogenic differentiation were compared.

[0043] The lipid mixture was purchased from Gibco with a product number of 11905031. It consists of the following ingredients:

[0044] Table 1: Lipid mixture composition:

[0045] .

[0046] The present invention uses large yellow croaker muscle satellite cells to undergo adipogenic transdifferentiation to produce cell culture large yellow croaker fish oil. Figure 1 As shown, in the group without lipid mixture, no lipid droplets were produced in the cells after 12 days of adipogenic differentiation, indicating a poor adipogenic differentiation effect. Figure 2 As shown in the figure, 2% lipid mixture was added to the culture medium. After 10 days of transdifferentiation induction, changes in cell morphology were observed, and small lipid droplets were produced in some cells, indicating a significant adipogenic transdifferentiation effect. Figure 3 As shown, compared with the group without glucose addition ( Figure 2 ), a significant adipogenic transdifferentiation effect was observed after 10 days of induction of differentiation by adding 25 μM glucose to the 2% lipid mixture, with higher lipid accumulation in the cells, indicating that the addition of glucose further improved the differentiation efficiency and shortened the differentiation time. On this basis, an additional 100 μΜ vitamin C was added to the culture medium, which was significantly different from the group without addition ( Figure 3 ) can further shorten the differentiation time. Cells can accumulate higher lipids after 8 days of induction of transdifferentiation, achieving the ideal differentiation effect. Figure 4 shown.

[0047] Comparative Example 1:

[0048] Only oleic acid (0.70804 μM), linolenic acid (0.71828 μM), and linoleic acid (0.71314 μM) at the same concentration as the 2% lipid mixture were added to the culture medium, and transdifferentiation was induced for 10 days. The results were observed by Nile red fluorescence staining. Figure 6 As shown, only a small number of cells can be stained with weak fluorescence, and the transdifferentiation effect is not as good as the 2% lipid mixture induction group ( Figure 2The better adipogenic transdifferentiation effect of mixed lipids may be the result of the combined action of multiple fatty acids.

[0049] PPARγ, SREBP1 and C / EBPα are important transcription factors related to fat differentiation and fatty acid synthesis. PPARγ is a member of the ligand-activated nuclear transcription factor superfamily. It consists of four different functional domains. After being activated by exogenous or endogenous ligands, it participates in regulating the activation of adipocytes and regulating glucose metabolism. Muscle satellite cells can activate adipocytes through PPARγ agonists. SREBP is a family of transcription factors that regulate lipid balance and can control the expression of enzymes related to fat synthesis. The SREBP1 gene induces adipogenesis and triglyceride accumulation by changing cell metabolism, and SREBP1 can change its own expression by regulating the transcriptional activation process of various enzymes related to fat synthesis, thereby regulating adipogenesis. C / EBPα can strongly promote the expression of PPARγ and initiate the differentiation of preadipocytes, and overexpression of C / EBPα can induce the synthesis of lipid droplets in the cytoplasm. The lipid mixture contains a variety of fatty acids such as oleic acid, linoleic acid, linolenic acid, and stearic acid (the specific components are shown in Table 1), which can provide synthetic precursors for lipid synthesis. Many fatty acids, such as oleic acid, are natural ligands of PPARγ, which can promote the translocation of PPARγ into the nucleus and regulate the expression of downstream genes. Fatty acids can also increase the expression of SREBP1 and C / EBPα, increase the activity of fatty acid desaturase and elongase, and thus promote lipid production. Therefore, the addition of lipid mixtures is essential for the adipogenic transdifferentiation of muscle satellite cells. The utilization of glucose in cells is divided into two parts, one is glycolysis, and the other is the energy production through the end products of glycolysis entering the tricarboxylic acid cycle. The intermediate product of glycolysis, acetyl-CoA, is a precursor for the synthesis of fatty acids and provides a carbon source for lipid production. Glucose can also activate the expression and activity of lipogenic transcription factors such as ChREBP, leading to increased lipid production. Vitamin C has the dual effects of promoting triglyceride accumulation and lipolysis. Vitamin C can upregulate the transcriptional levels of adipogenesis-related genes such as PPARγ, retinoic acid X receptor α, and lipoprotein esterase. Vitamin C can also promote collagen synthesis, upregulate the expression of LPL and GPDH, downregulate the expression of adiponectin, promote triglyceride accumulation, and accelerate cell differentiation.

[0050] Embodiment 2:

[0051] Large yellow croaker muscle satellite cells were seeded into 6-well plates and complete culture medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)) was added. After the cells were confluent, the medium was replaced with adipogenic differentiation medium (consisting of 87% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2% lipid mixture, 2 mM L-glutamine, 100 μM vitamin C, 25 μM glucose). Oil red staining was performed after 8 days of differentiation.

[0052] like Figure 5 As shown, after the optimization of differentiation medium, the adipogenic transdifferentiation effect of large yellow croaker muscle satellite cells was better and the lipid content was higher.

[0053] Embodiment 3:

[0054] Add 20 mg of microcarriers to a 6-well plate, add a small amount of DMEM / F12 basal medium, and swell for 12 h; discard the upper basal medium after standing, add a small amount of basal medium to wash the microcarriers, and discard the upper basal medium after standing; add a small amount of complete medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)) to cover the microcarriers, inoculate 50 million cells, and stand for 2 h; add medium to 5 mL, culture in a 28°C incubator, and change the medium half every other day; after the cells are fully grown, stand and remove the complete medium, add transdifferentiation medium (consisting of 87% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2% lipid mixture, 2 mM L-glutamine, 100 μM Vitamin C and 25 μM glucose), differentiation was induced for 6 days, during which samples were taken for Nile red staining. After the differentiation was completed, the differentiation medium was discarded and the samples were collected, washed twice with PBS, added with cell lysis buffer, lysed at room temperature for 3 min, centrifuged at 5000 rpm for 5 min, and the supernatant was collected for lipid composition determination.

[0055] like Figure 7 As shown, large yellow croaker muscle satellite cells attached and proliferated on the microcarriers. After about 6 days of culture, the cells were fully grown and the cell density could reach 600,000 / mL.

[0056] like Figure 8 As shown, large yellow croaker muscle satellite cells can achieve a good differentiation effect after 6 days of transdifferentiation induction on microcarriers, which is two days shorter than the differentiation time under 2D culture conditions, and the differentiation efficiency is further improved.

[0057] like Fig. 9 As shown, the lipid content of fish oil obtained by transdifferentiation of large yellow croaker cell culture is lower than that of fish oil extracted from the abdomen of large yellow croaker, and the lipid composition is quite different, but the phosphatidylcholine (PC) content is higher than that of actual extracted fish oil.

[0058] Embodiment 4:

[0059] Brown-spotted grouper muscle cells were seeded into 6-well plates and complete culture medium (L-15 medium containing 15% fetal bovine serum, 1% penicillin-streptomycin (100X) and 20 ng / mL FGF-2) was added. After the cells were confluent, the medium was replaced with adipogenic differentiation medium (consisting of 88% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2% lipid mixture, 2 mM L-glutamine, 100 μM vitamin C and 25 μM glucose). Nile red staining was performed after 6 days of differentiation.

[0060] like Fig.10 As shown, the developed adipogenic transdifferentiation medium can achieve adipogenesis of brown-spotted grouper muscle cells, but the medium causes great damage to the cells, and most of the cells fall off during the differentiation process. The composition of the transdifferentiation medium needs to be further adjusted.

[0061] Embodiment 5:

[0062] Humpback seabass muscle cells were seeded into 6-well plates and complete culture medium (L-15 medium containing 15% fetal bovine serum, 1% penicillin-streptomycin (100X) and 20 ng / mL FGF-2) was added. After the cells were confluent, the medium was replaced with adipogenic differentiation medium (consisting of 88% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2% lipid mixture, 2 mM L-glutamine, 100 μM vitamin C and 25 μM glucose). Nile red staining was performed after 6 days of differentiation.

[0063] like Fig.11 As shown, the developed adipogenic differentiation medium can achieve adipogenesis of humpback seabass muscle cells with good differentiation effect.

[0064] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0065] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing marine fish cell oil, characterized in that The following steps are involved: The muscle cells are expanded using complete medium, and then adipogenic differentiation medium is used to induce adipogenic differentiation to obtain marine fish cell oil; Wherein, the lipid transdifferentiation medium is composed of 82-95% DMEM / F12, 3-12% horse serum, 1% penicillin-streptomycin (100X), 0.25-1.5 μM dexamethasone, 5-25 μM rosiglitazone, 2.5-15 μg / mL insulin, 25-125 μM indomethacin, 125-750 μM IBMX, 1-5% lipid mixture, 1-5 mM L-glutamine, 50-250 μM vitamin C, and 10-50 μM glucose; the differentiation time is 6-8 days; the muscle cells are large yellow croaker muscle satellite cells or humpback seabass muscle cells; the lipid mixture is composed of the following components:

2. The preparation method according to claim 1, characterized in that: The complete culture medium was DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin 100X.

3. The preparation method according to claim 1, characterized in that 2D adipogenic differentiation includes the following steps: inoculate muscle cells into a 6-well plate, add complete culture medium, and replace with adipogenic differentiation medium after the cells are fully grown for differentiation.

4. The preparation method according to claim 1, characterized in that 3D adipogenic transdifferentiation includes the following steps: (1) Add microcarriers to a 6-well plate and add basal culture medium to swell; (2) After standing, discard the upper basal medium, add basal medium for washing, and after standing, discard the upper basal medium; (3) Inoculate muscle cells and add complete culture medium to cover the microcarriers. Let stand for 2 h. Add culture medium to 5 mL and change the medium every other day. (4) After the cells have grown all over the microcarriers, the complete medium is discarded and the transdifferentiation medium is added, and the medium is changed every other day. (5) Let the culture medium stand and discard, collect the sample, wash twice with PBS, add cell lysis buffer, centrifuge, and collect the supernatant.

5. The preparation method according to claim 4, characterized in that: The addition ratio of the microcarrier in step (1) is 2-5 mg / mL based on the volume of the total culture system; the swelling time is 12 hours; and the basic culture medium in steps (1) and (2) is DMEM / F12 culture medium.

6. The preparation method according to claim 4, characterized in that: Step (3) The density of inoculating muscle cells is 50,000-200,000 / mL; and the cells are allowed to stand at 28°C.

7. The preparation method according to claim 4, characterized in that: In step (5), the cell lysis time is 2-3 minutes, and the operation is carried out at room temperature; the centrifugation time is 5 minutes, and the rotation speed is 5000 rpm.

8. A marine fish cell oil prepared by the method of any one of claims 1 to 7.

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