Application of alpha-ketobutyric acid in promoting conversion of skeletal muscle fibers from fast muscle to slow muscle

Abstract

The invention discloses application of alpha-ketobutyric acid in promoting conversion of skeletal muscle fibers from fast muscles to slow muscles, and belongs to the technical field of animal nutrition. The porcine skeletal muscle satellite cells are treated by adopting alpha-ketobutyric acid, the mRNA expression level of slow muscle genes MyHC I and MyHC IIa of the porcine skeletal muscle satellite cells is remarkably increased, and the expression level of fast muscle genes MyHC IIb is remarkably decreased. The alpha-ketobutyric acid treatment significantly increases slow muscle protein and reduces expression of fast muscle protein. In myotubes of an alpha-ketobutyric acid treatment group, the area of MyHC-flow positive staining is obviously higher than that of a control group, and the area of MyHC-fast positive staining is obviously lower than that of the control group. The results show that the alpha-ketobutyric acid treated by the alpha-ketobutyric acid can promote conversion of muscle fibers from fast muscles to slow muscles. Therefore, the invention provides a brand new technical means and thought for regulating and controlling muscle physiology and improving meat quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal nutrition, and particularly relates to application of alpha-ketobutyric acid in promoting transformation of animal skeletal muscle fiber type from fast muscle to slow muscle. BACKGROUND

[0002] Skeletal muscle is an important motor organ and meat source of animal body, and its function and quality are determined by muscle fiber type. Muscle fibers are mainly divided into four types according to the expressed myosin heavy chain subtypes: MyHC I (slow oxidative type), MyHC IIa (fast oxidative-glycolytic type), MyHC IIx (fast glycolytic type) and MyHC IIb (fast glycolytic type). Among them, slow muscle fibers (MYHC I) contract slowly but have strong endurance, are rich in mitochondria and rely on aerobic metabolism; fast muscle fibers (IIa, IIx and IIb) contract quickly and have great strength, but are prone to fatigue and mainly rely on anaerobic glycolysis for energy supply. The proportion of muscle fiber types directly affects the animal's exercise performance, energy metabolism efficiency, and the flavor, juiciness and nutritional value of meat products. In the livestock industry, increasing the proportion of slow muscle fibers and oxidative fast muscle fibers (MyHC I and MyHC IIa) in livestock through nutritional regulation and other means is an effective strategy to improve meat quality and produce high-end meat products.

[0003] Alpha-ketobutyric acid (alpha-KB), also known as 2-oxobutyric acid, is a natural endogenous ketone acid produced in the metabolic process of amino acids (such as threonine and methionine). Currently, its known functions mainly focus on participating in the tricarboxylic acid cycle (TCA cycle) and the generation of succinyl coenzyme A as a metabolic intermediate. Currently, there is no research report on the specific regulation of alpha-ketobutyric acid on skeletal muscle fiber type transformation. As a potential feed additive that can be applied to improve meat quality, its function and value have not been fully explored.

[0004] Currently, there are very limited means to effectively and safely regulate the transformation of muscle fiber type to slow muscle. Long-term low-intensity endurance training can induce slow muscle transformation, but it is difficult to implement in large-scale breeding. Some drugs or hormones can affect muscle growth, but there are problems of drug residues, safety and ethical controversy, and their application is strictly limited. Therefore, the livestock industry urgently needs to develop a new type of nutritional regulator that is natural, safe and can effectively promote the transformation of fast muscle to slow muscle. SUMMARY

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide an application of alpha-ketobutyric acid in promoting the transformation of animal skeletal muscle fiber type from fast muscle to slow muscle, so as to provide a new type of nutritional regulator which is natural, safe and can effectively promote the transformation of fast muscle to slow muscle, and improve the proportion of slow muscle fiber and oxidative fast muscle fiber in livestock by means of nutritional regulation, thereby improving pork quality.

[0006] The technical scheme for solving the above-mentioned technical problems of the present application is as follows: providing an application of alpha-ketobutyric acid in promoting the transformation of animal skeletal muscle fiber type from fast muscle to slow muscle.

[0007] Further, the animal is a pig.

[0008] The present application provides a method for promoting the transformation of pig skeletal muscle fiber type from fast muscle to slow muscle, comprising the following steps: (1) cell culture and differentiation: culturing pig skeletal muscle satellite cells in a growth medium until fusion, and then transferring the pig skeletal muscle satellite cells to a differentiation medium for continuous culture; (2) when the pig skeletal muscle satellite cells start to differentiate, adding an alpha-ketobutyric acid solution to the culture medium and continuing to culture for 48-96h.

[0009] Further, the concentration of the alpha-ketobutyric acid solution in step (2) is 1-30μmol / L.

[0010] The present application provides a nutritional regulator for promoting the transformation of pig fast muscle to slow muscle, and the nutritional regulator comprises alpha-ketobutyric acid.

[0011] Further, the dosage form of the nutritional regulator comprises any one of tablets, capsules, oral liquids, granules and sustained-release preparations.

[0012] The present application also provides a method for improving the proportion of slow muscle in pig skeletal muscle fiber, which adds the above-mentioned nutritional regulator to the basal daily ration of pigs to improve the proportion of slow muscle in pig skeletal muscle fiber.

[0013] The application has the following beneficial effects: the application adopts alpha-ketobutyric acid to treat porcine skeletal muscle satellite cells. RT-PCR results show that, compared with a control group, the mRNA expression levels of slow muscle genes MyHC I and MyHC IIa in a 20 μmol / L alpha-ketobutyric acid treatment group are significantly up-regulated, and the expression level of fast muscle gene MyHC IIb is significantly down-regulated to 50% of the control group. Western Blot results show that alpha-ketobutyric acid treatment significantly increases the expression of slow muscle protein and reduces the expression of fast muscle protein, which is consistent with the change in gene expression. Immunofluorescence results show that the area of MyHC-slow positive staining in the myotube of the alpha-ketobutyric acid treatment group is significantly higher than that of the control group, and the area of MyHC-fast positive staining is significantly lower than that of the control group, indicating that alpha-ketobutyric acid treatment can promote the transformation of muscle fibers from fast muscle to slow muscle, thereby increasing the proportion of slow muscle in skeletal muscle fibers. Therefore, the application first discloses that the natural metabolite alpha-ketobutyric acid has the biological function of promoting the transformation of fast muscle to slow muscle, provides a new technical means and idea for regulating muscle physiology and improving meat quality, and fills the gap in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure for identification of porcine skeletal muscle satellite cells isolated and cultured; Figure 2 Figure for the effect of different concentrations of alpha-ketobutyric acid on the activity of porcine skeletal muscle satellite cells; Figure 3 Figure for the effect of alpha-ketobutyric acid on the expression of muscle fiber type related genes in porcine skeletal muscle satellite cells detected by real-time fluorescence quantitative PCR; Figure 4 Figure for the effect of alpha-ketobutyric acid on the expression of muscle fiber type related proteins in porcine skeletal muscle satellite cells detected by Western Blot; Figure 5 Figure for the change of MyHC-slow positive staining area of myotube observed by immunofluorescence staining after alpha-ketobutyric acid treatment; Figure 6 Figure for the change of MyHC-fast positive staining area of myotube observed by immunofluorescence staining after alpha-ketobutyric acid treatment. DETAILED DESCRIPTION

[0015] The following examples are only used to explain the application, and are not used to limit the scope of the application. If specific conditions are not indicated in the examples, conventional conditions or manufacturer's recommended conditions are used. If the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be purchased on the market.

[0016] Example 1: In vitro culture of porcine skeletal muscle satellite cells The longissimus dorsi muscle samples of healthy piglets (Duroc × Landrace × Yorkshire) were collected and placed in DMEM / F12 serum-free medium. In a clean bench, the tissue was cut into about 1 mm 3 pieces and added with collagenase type II for digestion. The cell suspension after digestion was filtered through 200 mesh and 400 mesh cell screens, and the filtrate was collected and obtained by centrifugation. The cells were resuspended and inoculated in cell culture vessels and cultured in a 37℃, 5% CO2 incubator. Satellite cells can be further purified by differential adhesion method. The cells were inoculated in a 96-well plate, fixed with 4% paraformaldehyde at room temperature for 30 min, permeated for 10 min, added with BSA liquid blocking for 2 h, washed with PBS; incubated with pig skeletal muscle satellite cell marker protein Pax7 antibody, washed with PBS; incubated with the same species secondary antibody for 2 h, washed with PBS; incubated with DAPI for 10 min, washed with PBS, and then mounted, photographed under a microscope, and the cell culture was proved to be successful by detecting pig skeletal muscle satellite cell marker protein Pax7 Figure 1 ).

[0017] Example 2: Verification of the effect of α-ketobutyric acid on the fiber type transformation of pig skeletal muscle satellite cells The MTT method was used to screen the effect of different concentrations (0, 1, 5, 10, 20 and 30 μmol / L) of α-ketobutyric acid on the viability of pig skeletal muscle satellite cells. The results showed that the use of no more than 30 μmol / L α-ketobutyric acid to treat cells for 48 h or 96 h had no significant inhibitory effect on cell viability Figure 2 A and 2B).

[0018] After pig skeletal muscle satellite cells were cultured in the medium to fusion, the medium was replaced with differentiation medium to induce myotube formation. At the beginning of differentiation, 20 μmol / L of α-ketobutyric acid was added to the medium of the experimental group, and an equal amount of PBS was added to the control group. After 96 hours of differentiation, total RNA was extracted and reverse transcribed into cDNA. Real-time fluorescent quantitative PCR was performed using the SYBR Green method to detect the expression changes of muscle fiber type marker genes. Total protein was extracted, and Western Blot technology was used to detect the expression changes of fast and slow muscle proteins. The results showed that compared with the control group, the mRNA expression levels of slow muscle genes MyHC I and MyHC IIa in the 20 μmol / L α-ketobutyric acid treatment group were significantly up-regulated (P<0.05), while the expression level of fast muscle gene MyHC IIb was significantly down-regulated to 50% of the control group (P<0.05) Figure 3 ). Western Blot found that α-ketobutyric acid treatment significantly increased the expression of slow muscle protein and decreased the expression of fast muscle protein (P<0.05), which was consistent with the changes in gene expression Figure 4 A and 4B).

[0019] Immunofluorescence results: The area of MyHC-slow positive staining in the muscle tube of the α-ketobutyric acid treatment group was significantly higher than that of the control group Figure 5 , and the area of MyHC-fast positive staining was significantly lower than that of the control group Figure 6 , indicating that α-ketobutyric acid treatment can promote the transformation of muscle fibers from fast muscle to slow muscle.

[0020] Prior art research shows that slow muscle fibers have higher myoglobin content, stronger oxidative metabolism capacity and richer mitochondrial density, which can significantly improve meat color redness, increase muscle water retention and reduce shear force, thereby improving tenderness (see: ①Joo ST, Kim GD, Hwang YH, Ryu YC. Control of fresh meat quality through manipulation of muscle fiber characteristics. Meat Sci. 2013;95(4):828-836. doi:10.1016 / j.meatsci.2013.04.044.; ②Li Y, Feng Y, Chen X, et al. Dietary short-term supplementation of grape seed proanthocyanidin extract improves pork quality and promotes skeletal muscle fiber type conversion in finishing pigs. Meat Sci. 2024;210:109436. doi:10.1016 / j.meatsci.2024.109436). In addition, slow muscle fibers are usually positively correlated with intramuscular fat deposition, which together improves the flavor and juiciness of meat (see: Zhang L, Li F, Guo Q, et al. Balanced branched-chain amino acids modulate meat quality by adjusting muscle fiber type conversion and intramuscular fat deposition in finishing pigs. J Sci Food Agric. 2022;102(9):3796-3807. doi:10.1002 / jsfa.11728.). Therefore, the method of the present application for treating muscle fibers with α-ketobutyric acid to promote the transformation of fast muscle to slow muscle provides a new strategy for improving pork quality.

[0021] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of α-ketobutyric acid in promoting the conversion of animal skeletal muscle fiber type from fast-twitch to slow-twitch.

2. The application according to claim 1, characterized in that, The animal in question is a pig.

3. A method for promoting the transformation of porcine skeletal muscle fiber type from fast-twitch to slow-twitch muscle, characterized in that, Includes the following steps: (1) Cell culture and differentiation: porcine skeletal muscle satellite cells were cultured in growth medium until they fused, and then the porcine skeletal muscle satellite cells were transferred to differentiation medium for further culture; (2) When porcine skeletal muscle satellite cells begin to differentiate, add α-ketobutyric acid solution to the culture medium and continue culturing for 48-96 hours.

4. The method according to claim 3, characterized in that, The concentration of the α-ketobutyric acid solution in step (2) is 1-30 μmol / L.

5. A nutritional regulator that promotes the conversion of fast-twitch muscle fibers in pigs to slow-twitch muscle fibers, characterized in that, The nutrient regulator includes α-ketobutyric acid.

6. The nutrient regulator according to claim 5, characterized in that, The dosage form of the nutritional regulator includes any one of tablets, capsules, oral liquids, granules, and sustained-release formulations.

7. A method for increasing the proportion of slow-twitch muscle fibers in porcine skeletal muscle fibers, characterized in that, The proportion of slow-twitch muscle fibers in pig skeletal muscle fibers can be increased by adding the nutritional regulator described in claim 5 or 6 to the basal diet of pigs.

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