A method for in vitro preparation of bovine adipocytes enriched in highly unsaturated fatty acids

Abstract

The application discloses a method for preparing bovine adipocytes rich in high unsaturated fatty acids in vitro and belongs to the technical field of cell culture and cell culture meat. The application provides a BSA-fatty acid compound for promoting the adipogenic differentiation of bovine mesenchymal cells in vitro, the compound is prepared from bovine serum albumin and fatty acids, the molar ratio of the two is 1:4, and the compound is prepared through a constant-temperature water bath ultrasonic process, and the compound has high combination efficiency and good stability. The fatty acids are selected from one or more compounds of oleic acid, linoleic acid, linolenic acid, arachidonic acid, palmitic acid and arachidic acid. The compound is added into a basic culture medium containing horse serum and insulin in proportion, is used for the differentiation culture of bovine mesenchymal cells, can efficiently induce the adipogenic differentiation of cells in 5 days, and the obtained adipocytes are high in unsaturated fatty acid content. The application has the advantages of simple operation, short period and suitability for the preparation of bovine adipocytes rich in high unsaturated fatty acids in the field of cell culture meat.

Description

Technical Field

[0001] This invention relates to a method for preparing bovine fat cells rich in highly unsaturated fatty acids in vitro, belonging to the field of cell culture and cell-cultured meat technology. Background Technology

[0002] Cultured meat is a novel meat product obtained by culturing animal stem cells in vitro and inducing them to differentiate into muscle and adipose tissue, which are then assembled. Adipocytes are the core component determining the flavor, juiciness, and nutritional quality of the meat. Natural beef fat is rich in saturated and monounsaturated fatty acids, and their ratio directly affects the flavor, texture, and health value of the meat.

[0003] Currently, systems for inducing bovine adipocyte differentiation in vitro generally suffer from insufficient lipid accumulation, a limited fatty acid composition, and a low proportion of unsaturated fatty acids. Studies have confirmed that oleic acid is a key substrate and regulator of adipogenic differentiation; however, the solubility of free fatty acids directly added to the culture medium is low, resulting in low cellular uptake.

[0004] Bovine serum albumin (BSA) is a natural carrier of fatty acids, forming soluble complexes with fatty acids through hydrophobic binding sites. Existing BSA-fatty acid complexes used in adipogenesis induction systems are mostly prepared using conventional stirring incubation methods, which suffer from drawbacks such as long binding time, low binding efficiency, and poor product stability, leading to uncontrollable effective concentrations and poor reproducibility in differentiation experiments. Furthermore, the BSA-fatty acid complexes used in existing adipogenesis induction systems are typically composed of BSA, oleic acid, and linoleic acid, which limits adipogenesis differentiation efficiency and fails to effectively increase the fatty acid and unsaturated fatty acid content in differentiated adipocytes, nor can the proportion of unsaturated fatty acids be controlled, thus failing to fully optimize the flavor and nutrition of cultured fat. Developing a BSA-fatty acid complex with high binding efficiency, good stability, and a reasonable fatty acid composition, and establishing a corresponding efficient bovine adipocyte differentiation method, is of great significance for improving the quality and production efficiency of cultured fat. Summary of the Invention

[0005] To address the problems of insufficient lipid accumulation, monotonous fatty acid composition, low proportion of unsaturated fatty acids, and poor solubility of free fatty acids when directly added in existing in vitro adipogenic induction systems for bovine adipocytes, this invention provides a BSA-fatty acid complex, its preparation method, an adipogenic differentiation medium containing the complex, and a method for preparing bovine adipocytes with high unsaturated fatty acids using the medium.

[0006] The first objective of this invention is to provide a BSA-fatty acid complex for inducing adipogenic differentiation of bovine mesenchymal cells, the complex comprising bovine serum albumin and fatty acids. Bovine serum albumin, as a fatty acid carrier, improves the dispersibility and stability of fatty acids in aqueous culture systems, reducing precipitation and aggregation problems caused by the direct addition of free fatty acids. Fatty acids, as substrates for adipogenic differentiation and regulators of lipid composition, promote intracellular lipid droplet formation and regulate the fatty acid composition of differentiated adipocytes. The molar ratio of bovine serum albumin to fatty acids is 1:2 to 1:8.

[0007] In one embodiment of the present invention, the molar ratio of bovine serum albumin to fatty acids is 1:4 to 1:8.

[0008] In one embodiment of the present invention, the fatty acid is oleic acid; the molar ratio of bovine serum albumin to oleic acid is 1:4.

[0009] In one embodiment of the present invention, the fatty acid includes one or more of oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, palmitic acid, stearic acid, and arachidic acid.

[0010] In a preferred embodiment of the present invention, the fatty acids include oleic acid, linoleic acid and α-linolenic acid.

[0011] In a more preferred embodiment of the present invention, based on the total molar amount of fatty acids, oleic acid accounts for 50%–90%, linoleic acid accounts for 5%–40%, and α-linolenic acid accounts for 5%–40%.

[0012] In one specific embodiment of the present invention, based on the total molar amount of fatty acids, oleic acid accounts for 60%, linoleic acid accounts for 30%, and α-linolenic acid accounts for 10%.

[0013] In another specific embodiment of the present invention, based on the total molar amount of fatty acids, oleic acid accounts for 60%, linoleic acid accounts for 10%, and α-linolenic acid accounts for 30%.

[0014] A second objective of this invention is to provide a method for preparing the aforementioned BSA-fatty acid complex. The method includes preparing a BSA aqueous solution from bovine serum albumin, preparing a fatty acid stock solution from fatty acids, mixing the two in a predetermined ratio, subjecting the mixture to constant temperature water bath ultrasonic treatment, cooling, and filtering for sterilization to obtain the BSA-fatty acid complex.

[0015] In one embodiment of the present invention, the mass volume fraction of the BSA aqueous solution is 5%–30%.

[0016] In a preferred embodiment of the present invention, the mass-volume fraction of the BSA aqueous solution is 20%.

[0017] In one embodiment of the present invention, the total concentration of the fatty acid stock solution is 50–200 mM.

[0018] In a preferred embodiment of the present invention, the total concentration of the fatty acid stock solution is 100 mM.

[0019] In one embodiment of the present invention, the molar ratio of bovine serum albumin to fatty acids is 1:2–1:8.

[0020] In a preferred embodiment of the present invention, the molar ratio of bovine serum albumin to fatty acids is 1:4.

[0021] In one embodiment of the present invention, the constant temperature water bath ultrasonic treatment temperature is 40–60℃, the ultrasonic power is 100–500 W, and the ultrasonic time is 5–40 min.

[0022] In a preferred embodiment of the present invention, the constant temperature water bath ultrasonic treatment temperature is 50°C, the ultrasonic power is 300 W, and the ultrasonic time is 20–30 min.

[0023] A third objective of this invention is to provide a culture medium for preparing adipogenic differentiation of bovine fat cells with high levels of unsaturated fatty acids. The adipogenic differentiation culture medium comprises basal culture medium, serum, insulin, and the aforementioned BSA-fatty acid complex.

[0024] In one embodiment of the present invention, the basal culture medium is DMEM medium, the serum is horse serum with a volume fraction of 1%–5%, and the final insulin concentration is 10–200 nM.

[0025] In a preferred embodiment of the present invention, the adipogenic differentiation medium includes DMEM medium, 2% horse serum, 50 nM insulin, and BSA-fatty acid complex.

[0026] In one embodiment of the present invention, the final concentration of total fatty acids in the adipogenic differentiation medium is 0.03–0.6 mM.

[0027] In a preferred embodiment of the present invention, the final concentration of total fatty acids in the adipogenic differentiation medium is 0.06–0.6 mM.

[0028] A fourth objective of this invention is to provide a method for promoting adipogenic differentiation of bovine mesenchymal cells in vitro, comprising the following steps: (1) Bovine mesenchymal cells were inoculated into proliferation medium for proliferation culture; (2) When the cell fusion rate reaches 70%–95%, replace it with an adipogenic differentiation medium containing BSA-fatty acid complex; (3) Continuous induction culture for 3–7 days to obtain bovine fat cells with high unsaturated fatty acids.

[0029] In a preferred embodiment of the present invention, bovine mesenchymal cells are used at a concentration of 0.5 × 10⁻⁶. 4 –3×10 4 Inoculate cells per cm² and culture for 1–3 days. When the cell confluence reaches 80%–90%, replace with adipogenic differentiation medium and continue induction culture for 5 days, changing the medium every 1–2 days.

[0030] In one embodiment of the present invention, the bovine mesenchymal cells are mesenchymal cells derived from bovine adipose tissue, bovine muscle tissue, bovine bone marrow, or bovine umbilical cord.

[0031] The present invention also provides the application of the BSA-fatty acid complex in improving the fatty acid composition of bovine adipocytes in vitro, wherein the BSA-fatty acid complex is added to the differentiation culture medium of bovine mesenchymal cells to induce differentiation.

[0032] In one embodiment of the present invention, improving the fatty acid composition of bovine fat cells refers to increasing the total fatty acid content, increasing the unsaturated fatty acid content, and / or decreasing the saturated fatty acid content; the unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids.

[0033] The present invention also provides the application of the BSA-fatty acid complex in the preparation of cell-cultured fat, cell-cultured meat, in vitro adipose tissue models, or in regulating the fatty acid composition of cultured fat.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a BSA-fatty acid complex that promotes adipogenic differentiation of bovine mesenchymal cells in vitro. Using BSA as a fatty acid carrier, the complex is prepared via a constant-temperature water bath ultrasonic process, which improves the dispersibility and solubility of fatty acids in the culture system and reduces precipitation and aggregation caused by the direct addition of free fatty acids. This invention, through the combination of oleic acid, linoleic acid, and α-linolenic acid, can not only promote adipogenic differentiation of bovine mesenchymal cells but also regulate the fatty acid composition within differentiated adipocytes, increasing the content of monounsaturated and polyunsaturated fatty acids. It can be used for cell-cultured fat, cell-cultured meat, and the construction of in vitro adipose tissue models. Attached Figure Description

[0035] Figure 1Oil Red O staining images of bovine mesenchymal cells induced by different oleic acid addition methods in Example 1 and Comparative Examples 1-3, where (a) Comparative Example 1: BSA blank group, (b) Comparative Example 2: free oleic acid group, (c) Example 1: 0.3 mM BSA-oleic acid group, (d) Comparative Example 3: 0.03 mM BSA-oleic acid group, (e) Comparative Example 3: 0.06 mM BSA-oleic acid group, (f) Comparative Example 3: 0.6 mM BSA-oleic acid group; Figure 2 The graph shows the statistical analysis of lipid droplet area in bovine mesenchymal cells induced by different oleic acid addition methods in Example 1 and Comparative Examples 1-3. Among them, (a) Comparative Example 1: BSA blank group, (b) Comparative Example 2: free oleic acid group, (c) Example 1: 0.3 mM BSA-oleic acid group, (d) Comparative Example 3: 0.03 mM BSA-oleic acid group, (e) Comparative Example 3: 0.06 mM BSA-oleic acid group, (f) Comparative Example 3: 0.6 mM BSA-oleic acid group; Figure 3 Oil Red O staining images of bovine mesenchymal cells induced to adipogenic differentiation at different treatment times in the water bath treatment group and the water bath ultrasound treatment group in Example 2, where (a) is water bath BSA blank, (b) is water bath for 10 min, (c) is water bath for 20 min, (d) is water bath for 30 min, (e) is water bath ultrasound BSA blank, (f) is water bath ultrasound for 10 min, (g) is water bath ultrasound for 20 min, and (h) is water bath ultrasound for 30 min. Figure 4 The graph shows the statistical analysis of lipid droplet area induced by BSA-oleic acid complex in adipogenic differentiation of bovine mesenchymal cells prepared by different treatment methods and treatment times in Example 2. (a) is water bath BSA blank, (b) is water bath for 10 min, (c) is water bath for 20 min, (d) is water bath for 30 min, (e) is water bath ultrasound BSA blank, (f) is water bath ultrasound for 10 min, (g) is water bath ultrasound for 20 min, and (h) is water bath ultrasound for 30 min. Figure 5 Oil Red O staining images of bovine mesenchymal cells induced by different fatty acid systems in Comparative Example 1, Example 1, and Example 3, where (a) Comparative Example 1: BSA blank group, (b) Example 1: BSA-oleic acid group, (c) Example 3: BSA-oleic acid / linoleic acid group, OA:LA=1:1, (d) Example 3: BSA-α-linolenic acid group, (e) Example 3: BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:3:1, (f) Example 3: BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:1:3; Figure 6The graph shows the statistical analysis of lipid droplet area inducing adipogenic differentiation of bovine mesenchymal cells in different fatty acid systems in Comparative Example 1, Example 1, and Example 3. Among them, (a) Comparative Example 1: BSA blank group, (b) Example 1: BSA-oleic acid group, (c) Example 3: BSA-oleic acid / linoleic acid group, OA:LA=1:1, (d) Example 3: BSA-α-linolenic acid group, (e) Example 3: BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:3:1, (f) Example 3: BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:1:3; Figure 7 This is a statistical chart showing the content of major fatty acid categories in bovine fat cells after induction with different fatty acid systems in Example 4. Figure 8 This is a statistical chart showing the composition of major fatty acids in bovine fat cells after induction with different fatty acid systems in Example 4. Detailed Implementation

[0036] The implementation of the present invention will be described in detail below with reference to the embodiments and accompanying drawings. The following embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0037] In this embodiment of the invention, the proliferation medium is DMEM medium containing 10% fetal bovine serum; the adipogenic differentiation basal medium is DMEM medium containing 2% horse serum and 50 nM insulin. Oil Red O staining was used to observe intracellular lipid droplet formation, and lipid droplet quantification was performed using ImageJ software to statistically analyze Oil Red O positive areas. Unless otherwise specified, cells in all groups were cultured at 37°C and 5% CO2, and the adipogenic induction time was 5 days.

[0038] The BSA described in this embodiment of the invention is obtained by fermentation and expression of Pichia pastoris, purification by Ni-NTA, concentration by dialysis or ultrafiltration, and sterilization by filtration. For specific preparation methods, please refer to the literature: Li Yan, "High-efficiency recombinant expression of bovine serum albumin in Pichia pastoris", Master's thesis of Jiangnan University, 2026.

[0039] The bovine mesenchymal cells described in this embodiment of the invention were obtained by separating bovine adipose tissue, digesting with type I collagenase, filtering, lysing red blood cells, and adhering to the culture vessel. For specific separation methods, please refer to the literature: Tang Haohao, "Preparation of Cultured Bovine Fat Cells Based on Serum-Depleted Culture Medium and Food-Grade Inducer", Master's Thesis, Jiangnan University, 2025.

[0040] Example 1: Preparation of high-concentration BSA-oleic acid complex by water bath sonication and induction of adipogenic differentiation of bovine mesenchymal cells Weigh the BSA prepared above and add sterile deionized water to prepare a 20% (w / v) BSA aqueous solution. Dissolve oleic acid in anhydrous ethanol to prepare a 100 mM oleic acid stock solution. Mix the BSA aqueous solution and the oleic acid stock solution at a bovine serum albumin to oleic acid molar ratio of 1:4, place the mixture in a water bath ultrasonic bath, and sonicate at 50℃ and 300 W for 30 min. After ultrasonication, cool to room temperature and filter through a 0.22 μm sterile filter membrane to obtain the BSA-oleic acid complex for later use.

[0041] Bovine mesenchymal cells were used at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 1 / cm² in 6-well plates and cultured on proliferation medium at 37°C and 5% CO₂ for 2 days. When cell confluence reached 80%-90%, adipogenesis was induced. The proliferation medium was discarded, and the cells were washed once with PBS and replaced with adipogenic differentiation medium containing a BSA-oleic acid complex. Induction culture was continued for 5 days, with medium changes every 2 days. The final concentration of oleic acid in the adipogenic differentiation medium was 0.3 mM, the final concentration of BSA was 0.075 mM, and the remainder was the basal adipogenic differentiation medium, named the 0.3 mM BSA-oleic acid group. Oil Red O staining and quantitative analysis of lipid droplet area were performed 5 days after induction.

[0042] Comparative Example 1: BSA blank induction group without added fatty acids Bovine mesenchymal cells were induced to become adipogenic through the method described in Example 1, except that only BSA was added to the adipogenic differentiation medium, without oleic acid. The final BSA concentration was 0.075 mM, and the other culture conditions were the same as in Example 1. This group was named the BSA blank group. Figure 1 (a) Figure 5 (a), and statistically analyzed the corresponding lipid droplet areas. Figure 2 , Figure 6 The control group was used as a blank induction group for comparison. Oil Red O staining and lipid droplet area quantification were performed 5 days after induction differentiation.

[0043] Comparative Example 2: Direct addition of free oleic acid induces bovine mesenchymal cell differentiation Bovine mesenchymal cells were induced to become adipogenic through the method described in Example 1, except that only 0.3 mM oleic acid was added to the adipogenic differentiation medium, and BSA pre-complexation was not performed. All other culture conditions were the same as in Example 1. This group was named the free oleic acid group. This group corresponds to... Figure 1 (b) Oil Red O staining and lipid droplet area quantification were performed 5 days after induction of differentiation.

[0044] Comparative Example 3: Effects of different final oleic acid concentrations on adipogenic differentiation of bovine mesenchymal cells To further investigate the effect of final oleic acid concentration on the adipogenic differentiation of bovine mesenchymal cells, BSA-oleic acid complexes were prepared according to the method in Example 1 and added to the basal adipogenic differentiation medium to achieve final oleic acid concentrations of 0.03 mM (0.03 mM BSA-oleic acid group), 0.06 mM (0.06 mM BSA-oleic acid group), and 0.6 mM (0.6 mM BSA-oleic acid group). Figure 1 (d)- Figure 1 (f). Except for the different final concentrations of oleic acid, the molar ratio of BSA to fatty acids, cell seeding density, culture conditions, and induction time were the same as in Example 1 for each group.

[0045] Bovine mesenchymal cells were used at a rate of 1×10⁻⁶. 4 Cells were seeded per cm² in 6-well plates and cultured on proliferation medium at 37°C and 5% CO₂ for 2 days. When the cell confluence reached 80%-90%, the medium was replaced with adipogenic differentiation medium containing different final concentrations of BSA-oleic acid complex. Induction culture was continued for 5 days, with medium changes every 2 days. Oil Red O staining was performed 5 days after induction, and the area of ​​Oil Red O-positive lipid droplets was analyzed using ImageJ software.

[0046] The results are as follows Figure 1 As shown, after treatment with the BSA-oleic acid complex in Example 1, a large number of Oil Red O-positive lipid droplets appeared around the cells, and the lipid droplets were relatively densely distributed. Figure 1 c). and Figure 1 (a) Comparative Example 1 without added fatty acids, Figure 1 Compared to the group with direct addition of free oleic acid shown in Comparative Example 2 (b), the group with the 0.3 mM BSA-oleic acid complex in this example showed the most significant lipid droplet formation. The statistical results of the lipid droplet area are as follows: Figure 2 As shown, the Oil Red O positive area in Example 1 group (c) reached 21%, which was significantly higher than that in Comparative Example 1 and Comparative Example 2, indicating that BSA pre-complexation treatment and appropriate oleic acid concentration are beneficial to improving the adipogenic induction effect of bovine mesenchymal cells.

[0047] Comparative Example 3 investigated the effect of different final oleic acid concentrations on the adipogenic differentiation of bovine mesenchymal cells. Figure 1 (d) shows that a small number of Oil Red O-positive lipid droplets were observed in the cells of the 0.03 mM group, indicating that low concentrations of oleic acid can induce a certain degree of lipid accumulation, but the induction effect is weak; as the final concentration of oleic acid increases, Figure 1 (c) Figure 1 (e) Figure 1 (f) shows that the number and staining intensity of intracellular lipid droplets gradually increased in the 0.06 mM, 0.3 mM, and 0.6 mM groups, with more pronounced lipid droplet formation in the 0.3 mM and 0.6 mM groups. The statistical results of lipid droplet area are shown below. Figure 2 As shown, the lipid droplet areas in the 0.06 mM, 0.3 mM, and 0.6 mM groups were significantly higher than those in the 0.03 mM group, and the lipid droplet area increased with increasing final oleic acid concentration. These results indicate that the final oleic acid concentration affects the adipogenic differentiation of bovine mesenchymal cells, promoting lipid droplet formation within the range of 0.06-0.6 mM. Since there was no statistically significant difference between 0.3 mM and 0.6 mM, 0.3 mM can be considered a more preferred adipogenic induction concentration.

[0048] Example 2: Effect of water bath sonication time on the lipoproliferative effect of BSA-fatty acid complex To compare the effects of different treatment methods and times on the lipogenic induction of the BSA-oleic acid complex, the BSA-oleic acid complex was prepared according to the method in Example 1, with only the treatment method and time during the complex preparation process being changed. A water bath treatment group and a water bath ultrasonic treatment group were set up for comparison.

[0049] Only the water bath treatment group and the water bath ultrasonic treatment group had a BSA blank control, with no oleic acid added, only BSA added; the other treatments were 10 min, 20 min, and 30 min, respectively. The specific grouping criteria are as follows: (a) Water bath BSA blank: Based on Example 1, the addition of oleic acid was omitted and the treatment method was changed. The BSA aqueous solution was treated in a water bath at 50°C for 30 min. (b) Water bath for 10 min: Mix the BSA aqueous solution and oleic acid stock solution at a molar ratio of bovine serum albumin to oleic acid of 1:4, and place them in a water bath at 50°C for 10 min (based on Example 1, replace the water bath ultrasound with a water bath and shorten the treatment time).

[0050] (c) Water bath for 20 min: Mix BSA aqueous solution and oleic acid stock solution at a molar ratio of bovine serum albumin to oleic acid of 1:4, and place in a water bath at 50°C for 20 min (based on Example 1, replace the water bath ultrasound with a water bath and shorten the treatment time).

[0051] (d) Water bath for 30 min: Mix BSA aqueous solution and oleic acid stock solution at a molar ratio of bovine serum albumin to oleic acid of 1:4, and place in a water bath at 50°C for 30 min (based on Example 1, replace water bath ultrasound with water bath).

[0052] (e) Water bath ultrasonic BSA blank: Based on Example 1, omit the addition of oleic acid and ultrasonically treat the BSA aqueous solution at 50°C and 300 W for 30 min; (f) Water bath sonication for 10 min: Mix BSA aqueous solution and oleic acid stock solution according to the molar ratio of bovine serum albumin to oleic acid of 1:4, and sonicate at 50°C and 300 W for 10 min (the sonication time was changed based on Example 1).

[0053] (g) Water bath sonication for 20 min: Mix BSA aqueous solution and oleic acid stock solution at a molar ratio of bovine serum albumin to oleic acid of 1:4, and sonicate at 50°C and 300 W for 20 min (the sonication time was changed based on Example 1).

[0054] (h) Water bath sonication for 30 min: Mix BSA aqueous solution and oleic acid stock solution according to the molar ratio of bovine serum albumin to oleic acid of 1:4, and sonicate at 50°C and 300 W for 30 min (same as the treatment method in Example 1).

[0055] After treatment, the BSA obtained from groups (a) and (e) was added to the adipogenic differentiation basal medium at a final concentration of 0.075 mM; the complexes obtained from the other treatment groups were added to the adipogenic differentiation basal medium at a final concentration of 0.3 mM oleic acid and 0.075 mM BSA, and bovine mesenchymal cells were induced to differentiate for 5 days according to the method described in Example 1.

[0056] After induction, Oil Red O staining was performed, and microscopic imaging and lipid droplet area quantification were conducted according to the method described in Example 1.

[0057] The results are as follows Figure 3 As shown, Figure 3 (a) and Figure 3 (e) As shown in the BSA blank control, no obvious lipid droplet accumulation was observed, indicating that BSA itself cannot induce significant adipogenic differentiation. Figure 3 (b)- Figure 3 (d) shows the comparison between the water bath treatment group and the group treated for the same amount of time. Figure 3 (f)- Figure 3 As shown in (h), Oil Red O-positive lipid droplets were more prominent in the water bath ultrasound treatment group, with greater lipid droplet accumulation in the 20 min and 30 min water bath ultrasound groups. The statistical results of lipid droplet area are as follows: Figure 4 As shown, the lipid droplet areas in the water bath sonication groups for 10 min, 20 min, and 30 min were significantly higher than those in the water bath-only group under the same treatment time. The lipid droplet areas in the water bath sonication groups for 20 min and 30 min were also higher. These results indicate that, under the same treatment time, water bath sonication is more conducive to the formation of BSA-oleic acid complexes that can be utilized by cells than water bath treatment alone; and that sonication at 50℃ and 300 W for 20-30 min can achieve a better lipid-promoting effect.

[0058] Example 3: Effects of changing fatty acid types and concentrations on adipogenic differentiation of bovine mesenchymal cells Based on Example 1, the types and ratios of fatty acids were changed to investigate the effects of fatty acid types and ratios on adipogenic differentiation of bovine mesenchymal cells. The specific groups are as follows: group c and group d are the control groups, and group e and group f are the ternary compound experimental groups.

[0059] Group A: Same as Comparative Example 1; Group b: Same as Example 1; Group C: BSA-fatty acid complexes were prepared and used to induce adipogenic differentiation of bovine mesenchymal cells according to the method in Example 1, except that oleic acid was replaced with a complex fatty acid composed of oleic acid and linoleic acid. Oleic acid and linoleic acid were separately prepared into 100 mM fatty acid ethanol stock solutions, and then mixed at a molar ratio of 1:1 to obtain a complex fatty acid stock solution. A 20% BSA aqueous solution was mixed with the complex fatty acid stock solution at a molar ratio of bovine serum albumin to complex fatty acids of 1:4. The mixture was then sonicated at 50°C and 300 W for 30 min, cooled, and filtered through a 0.22 μm sterile filter membrane to obtain the BSA-oleic acid / linoleic acid complex (BSA-oleic acid / linoleic acid group).

[0060] Bovine mesenchymal cells were cultured according to the method in Example 1. When cell confluence reached 80%-90%, the medium was replaced with adipogenic differentiation medium containing a BSA-oleic acid / linoleic acid complex. The final concentration of total fatty acids in the adipogenic differentiation medium was 0.3 mM, with oleic acid at 0.15 mM, linoleic acid at 0.15 mM, and BSA at 0.075 mM; the remainder was the basal adipogenic differentiation medium. The cells were continuously induced and cultured for 5 days, with the medium changed every 2 days.

[0061] Group d: The specific implementation method is the same as in Example 1, except that α-linolenic acid is used instead of oleic acid. α-Linolenic acid was prepared into a 100 mM fatty acid ethanol stock solution. A 20% BSA aqueous solution was mixed with the α-linolenic acid stock solution at a molar ratio of bovine serum albumin to α-linolenic acid of 1:4. The mixture was then sonicated at 50°C and 300 W for 30 min. After cooling, it was filtered through a 0.22 μm sterile filter membrane to obtain the BSA-α-linolenic acid complex (BSA-α-linolenic acid group).

[0062] Bovine mesenchymal cells were cultured according to the method in Example 1. When the cell confluence reached 80%-90%, the medium was replaced with adipogenic differentiation medium containing a BSA-α-linolenic acid complex. The final concentration of α-linolenic acid in the adipogenic differentiation medium was 0.3 mM, the final concentration of BSA was 0.075 mM, and the remainder was the basal adipogenic differentiation medium. The cells were continuously induced and cultured for 5 days, with the medium changed every 2 days.

[0063] Group E: The specific implementation method is the same as in Example 1, except that oleic acid is replaced with a complex fatty acid composed of oleic acid, linoleic acid, and α-linolenic acid. Oleic acid, linoleic acid, and α-linolenic acid are each prepared into 100 mM fatty acid ethanol stock solutions. These are mixed at a molar ratio of 6:3:1 to obtain the complex fatty acid stock solution. A 20% BSA aqueous solution is mixed with the complex fatty acid stock solution at a molar ratio of bovine serum albumin to complex fatty acid of 1:4. The mixture is then sonicated at 50°C and 300 W for 30 min, cooled, and filtered through a 0.22 μm sterile filter membrane to obtain the BSA-complex fatty acid complex (BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:3:1, high linoleic acid complex group).

[0064] Bovine mesenchymal cells were cultured according to the method in Example 1. When the cell confluence reached 80%-90%, the medium was replaced with adipogenic differentiation medium containing BSA-complex fatty acid complex. The final concentration of total fatty acids in the adipogenic differentiation medium was 0.3 mM, including 0.18 mM oleic acid, 0.09 mM linoleic acid, 0.03 mM α-linolenic acid, and 0.075 mM BSA; the remainder was the basal adipogenic differentiation medium. The cells were continuously induced and cultured for 5 days, with the medium changed every 2 days.

[0065] Group F: The specific implementation method is the same as in Example 1, except that oleic acid is replaced with a complex fatty acid composed of oleic acid, linoleic acid, and α-linolenic acid. Oleic acid, linoleic acid, and α-linolenic acid are each prepared into 100 mM fatty acid ethanol stock solutions. These are mixed at a molar ratio of 6:1:3 to obtain the complex fatty acid stock solution. A 20% BSA aqueous solution is mixed with the complex fatty acid stock solution at a molar ratio of bovine serum albumin to complex fatty acid of 1:4. The mixture is then ultrasonically treated at 50°C and 300 W for 30 min. After cooling, it is filtered through a 0.22 μm sterile filter membrane to obtain the BSA-complex fatty acid complex (BSA-oleic acid / linoleic acid / α-linolenic acid group, OA:LA:ALA=6:1:3, high α-linolenic acid complex group).

[0066] Bovine mesenchymal cells were cultured according to the method in Example 1. When cell confluence reached 80%-90%, the medium was replaced with adipogenic differentiation medium containing a BSA-complex fatty acid complex. The final concentration of total fatty acids in the adipogenic differentiation medium was 0.3 mM, including 0.18 mM oleic acid, 0.03 mM linoleic acid, 0.09 mM α-linolenic acid, and 0.075 mM BSA. The cells were continuously induced and cultured for 5 days, with the medium changed every 2 days.

[0067] Oil Red O staining was performed on day 5 of induction, and the lipid droplet area was analyzed using ImageJ software. Comparative Example 1 (… Figure 5 a) and Example 1 ( Figure 5 b) As a control group, the BSA-oleic acid / linoleic acid group corresponds to Figure 5 (c), BSA-α-linolenic acid group corresponding to Figure 5 (d), the two are respectively in Figure 6 In the lipid droplet area statistics, the baseline group (oleic acid / linoleic acid / BSA without α-linolenic acid) and the group treated with α-linolenic acid alone were used. The results showed that both the BSA-oleic acid / linoleic acid group and the BSA-α-linolenic acid group could induce the formation of a certain number of Oil Red O positive lipid droplets in bovine mesenchymal cells, but the degree of lipid droplet formation and the lipid droplet area were lower than those of the baseline group. Figure 5 (e) The 6:3:1 ternary compound system of Example 3 shown in Example 3 and Figure 5 (f) shows the 6:1:3 ternary compound system of Example 4.

[0068] To further evaluate the synergistic promoting effect of α-linolenic acid in the oleic acid / linoleic acid / BSA system, the Bliss independent model and King's Q-value method were used, with the area of ​​Oil Red O positive lipid droplets as the lipogenic effect index. The mean lipid droplet areas of each group were as follows: blank group 1.524, BSA-oleic acid / linoleic acid group 14.110, BSA-α-linolenic acid group 6.259, 6:3:1 ternary compound group 21.918, and 6:1:3 ternary compound group 25.179. Taking the blank group as the lowest effect and the highest treatment group as the highest effect, the effect was normalized according to E = (lipid droplet area of ​​treatment group - lipid droplet area of ​​blank group) / (lipid droplet area of ​​highest treatment group - lipid droplet area of ​​blank group), resulting in the effect value E_OA+LA = 0.532 for the oleic acid / linoleic acid / BSA group and E_ALA = 0.200 for the α-linolenic acid / BSA group. According to the Bliss independent model, the theoretical additive effect E_pred = E_OA + LA + E_ALA - E_OA + LA × E_ALA = 0.626.

[0069] for Figure 5(e) shows a 6:3:1 ternary complex system with an actual effect value E_actual = 0.862 and a King's Q value Q = E_actual / E_pred = 1.378; for Figure 5 (f) shows the 6:1:3 ternary compound system, with an actual effect value E_actual = 1.000 and a King's Q value Q = 1.598. According to the criterion of Q > 1.15 for synergistic effect, both of the above ternary compound systems showed a synergistic promoting effect. This result indicates that the adipogenic effect of the simple BSA-oleic acid / linoleic acid compound system is less than that of the ternary compound system with the addition of α-linolenic acid; in the BSA delivery system where oleic acid and linoleic acid coexist, the addition of α-linolenic acid can produce a synergistic promoting effect with oleic acid and linoleic acid, thereby further improving the adipogenic differentiation effect of bovine mesenchymal cells.

[0070] Example 4: Detection of fatty acid composition in bovine adipocytes after induction with different fatty acid systems To evaluate the effects of different fatty acid systems on the fatty acid composition of differentiated bovine adipocytes, GC-MS was used to detect the fatty acid composition of the cells after induced differentiation. Bovine adipocytes from the control group (Comparative Example 1), the 10:0:0 oleic acid group (Example 1), the 6:1:3 high α-linolenic acid compound group (Example 3, group f), and the 6:3:1 high linoleic acid compound group (Example 3, group e) on day 5 of induced differentiation were collected. The culture medium was discarded, the cells were washed twice with PBS, the cells were scraped and collected in centrifuge tubes, the supernatant was removed by centrifugation, and the cell pellet was obtained.

[0071] A methanol-chloroform mixture was added to the cell pellet, vortexed thoroughly, and allowed to stand for extraction. Chloroform and water were then added for phase separation, and the lower organic phase was collected after centrifugation. The organic phase was dried under nitrogen to obtain total cellular lipids. NaOH methanol solution was added to the dried lipid sample, and the mixture was reacted in a 60 °C water bath for 30 min to convert fatty acids into fatty acid methyl esters. After the reaction, the mixture was cooled to room temperature, and n-hexane was added to extract the fatty acid methyl esters. The upper organic phase was collected after centrifugation for GC-MS analysis.

[0072] Separation was performed using a fatty acid methyl ester analytical column with helium as the carrier gas. Qualitative and quantitative analysis of different fatty acids was conducted using fatty acid methyl ester standards, with C13:0 as the internal standard. The content and relative composition of each fatty acid were calculated. The fatty acids detected included, but were not limited to, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (C18:3), and arachidonic acid (C20:4).

[0073] The results are as follows Figure 7As shown, in terms of total fatty acid (FA) content, compared with the control group (2.95 g / 100g), the total fatty acid content of the 10:0:0 single oleic acid group (3.78 g / 100g), the 6:1:3 high α-linolenic acid compound group (4.19 g / 100g), and the 6:3:1 high linoleic acid compound group (5 g / 100g) were significantly increased, with increases of approximately 28%, 42%, and 69%, respectively. This indicates that exogenous fatty acids, after being compounded with BSA, can promote intracellular lipid accumulation. The total fatty acid content of both compound groups was higher than that of the single oleic acid group, with the 6:3:1 group showing an increase of approximately 32% compared to the 10:0:0 group. Regarding saturated fatty acid (SFA) content, compared with the control group (1.73 g / 100g), the saturated fatty acid content of the 10:0:0 monooleic oleic acid group (0.84 g / 100g), the 6:1:3 high α-linolenic acid compound group (0.92 g / 100g), and the 6:3:1 high linoleic acid compound group (0.97 g / 100g) were all reduced, with a reduction of more than 40%. The total monounsaturated fatty acid (MUFA) content of each treatment group was significantly increased by about 200% compared with the control group in all three groups. The content of polyunsaturated fatty acids (PUFAs) also increased significantly. The PUFA content in the control group was only 0.53 g / 100g, while the PUFA content in the 10:0:0 single oleic acid group, the 6:1:3 high α-linolenic acid compound group, and the 6:3:1 high linoleic acid compound group reached 0.95 g / 100g, 1.34 g / 100g, and 1.93 g / 100g, respectively, which were approximately 79%, 153%, and 264% higher than the control group. The total PUFA content in the 6:3:1 group was approximately 73% higher than that in the 10:0:0 group.

[0074] In terms of specific components, such as Figure 8 As shown, the 6:3:1 group showed the highest upregulation of linoleic acid (C18:2) (approximately 16 times that of the control group), and the 6:1:3 group showed the highest upregulation of α-linolenic acid (C18:3n3) (approximately 9 times that of the control group), both significantly higher than the other two groups. These results indicate that this invention, by adjusting the fatty acid composition in the BSA-fatty acid complex, can regulate the fatty acid composition within differentiated bovine fat cells and increase the content of unsaturated fatty acids, especially polyunsaturated fatty acids.

[0075] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A BSA-fatty acid complex for inducing adipogenic differentiation of bovine mesenchymal cells, characterized in that, The BSA-fatty acid complex comprises bovine serum albumin and fatty acids, wherein the fatty acids include one or more of oleic acid, linoleic acid, α-linolenic acid, and arachidonic acid, and the molar ratio of bovine serum albumin to fatty acids is 1:2 to 1:

8.

2. The BSA-fatty acid complex according to claim 1, characterized in that, The molar ratio of bovine serum albumin to fatty acids is 1:4 to 1:

8.

3. The BSA-fatty acid complex according to claim 2, characterized in that, The fatty acid is oleic acid; the molar ratio of bovine serum albumin to oleic acid is 1:

4.

4. The BSA-fatty acid complex according to claim 2, characterized in that, The fatty acids are composed of oleic acid, linoleic acid and α-linolenic acid. Based on the total molar amount of fatty acids, oleic acid accounts for 50%–90%, linoleic acid accounts for 5%–40%, and α-linolenic acid accounts for 5%–40%. The molar ratio of oleic acid, linoleic acid and α-linolenic acid in the fatty acids is 6:3:1 or 6:1:

3.

5. A method for preparing the BSA-fatty acid complex according to any one of claims 1 to 4, characterized in that, The BSA solution and fatty acid solution are mixed and then subjected to ultrasonic treatment; the ultrasonic conditions are a temperature of 40~60℃, an ultrasonic power of 100~500W, and an ultrasonic time of 5~40 min.

6. A method for promoting adipogenic differentiation of bovine mesenchymal cells in vitro, characterized in that, The BSA-fatty acid complex according to any one of claims 1 to 4 is added to the adipogenic differentiation medium and the differentiation is induced for 3 to 7 days; the bovine mesenchymal cells are derived from bovine adipose tissue, bovine muscle tissue, bovine bone marrow or bovine umbilical cord.

7. The method according to claim 6, characterized in that, The adipogenic differentiation medium is DMEM medium containing horse serum and insulin; the total fatty acid concentration in the adipogenic differentiation medium is 0.06–0.6 mM.

8. The use of the BSA-fatty acid complex according to any one of claims 1 to 4 in improving the fatty acid composition of bovine fat cells in vitro, characterized in that, The BSA-fatty acid complex was added to the differentiation medium of bovine mesenchymal cells to induce adipogenic differentiation.

9. The application according to claim 8, characterized in that, The improvement of fatty acid composition in bovine fat cells refers to increasing the total fatty acid content, increasing the unsaturated fatty acid content, and / or decreasing the saturated fatty acid content; the unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids.

10. The use of the BSA-fatty acid complex according to any one of claims 1 to 4 in the preparation of cell-cultured fat, cell-cultured meat, in vitro adipose tissue models, or in regulating the fatty acid composition of cultured fat.

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