Extraction and purification method of type Ⅰ / Ⅱ myosin isoforms
Through a multi-step extraction and purification method, the liquid composition and operating conditions were optimized, and the type I/II myosin subtype was successfully extracted and purified from the bovine masseter muscle and trunk skin muscle, solving the problems of difficulty and inefficiency in the existing technology, achieving the acquisition of high-purity myosin, and providing technical support for meat quality research.
Patent Information
- Application Number
- CN202311847383.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2043-12-29
AI Technical Summary
It is difficult to extract and purify different myosin subtypes efficiently at the same time, especially to isolate high-purity type I and type II myosin subtypes from bovine masseter muscles and trunk skin muscles, which has problems of difficulty and inefficiency in purification.
A multi-step extraction and purification method is adopted, including homogenization, crude extraction, ammonium sulfate precipitation and dialysis steps. By optimizing liquid composition, stirring conditions and saturation control, efficient extraction and purification of type I/II myosin subtype is achieved.
The purity of type I and 93% of type II myosin was successfully obtained from the bovine masseter muscle and the trunk skin muscle, respectively, which improved the purification efficiency and quality of myosin subtypes and provided technical support for studying the relationship between myosin subtypes and meat quality characteristics.
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Figure CN118027174B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for extracting and purifying myosin, in particular to a method for extracting and purifying type I / II myosin subtypes. Background Art
[0002] Meat products, as high-quality animal proteins, are an indispensable and important part of a balanced human diet. With the improvement of living standards, people's demand for high-quality meat products continues to increase. There are many studies on meat quality at home and abroad, but most of them only focus on the effects of external conditions such as additives and processing methods on the functional properties of meat protein, ignoring the differences in the raw meat itself. The muscle fiber types and compositions of meat from different species or parts vary greatly, which fundamentally determines the processing characteristics of the raw meat and the quality characteristics of the final product.
[0003] The type of muscle fiber is determined by the heavy chain subtype of myosin. Myosin is the most abundant structural protein in myofibrillar protein and is also the main functional protein that affects important processing properties of meat protein, such as gelation, emulsification, and water retention. Myosin can be divided into two major categories, type I and type II, according to its heavy chain subtype, corresponding to the two types of slow-twitch and fast-twitch muscle fibers, respectively. In recent years, with the continuous deepening of research, the research on meat quality characteristics at home and abroad has also been increasingly in-depth to the level of myosin molecules. A large number of studies have reported the effects of exogenous small molecule additives such as salt and amino acids on the functional characteristics of myosin from different species such as chicken, pig, and aquatic products, but there is still a lack of research on the differences in the functional characteristics of different myosin subtypes. The main reason is that the muscle fiber composition of most meat pieces is a mixed type, containing both type I and type II myosin. Since the physical and chemical properties of different myosin isoforms, such as molecular weight, isoelectric point, and charge, are very similar, it is difficult to separate and purify different myosin isoforms from mixed muscle fibers using conventional methods such as gel filtration chromatography, isoelectric precipitation, and electrophoresis.
[0004] After practice and improvement, the extraction and purification methods of myosin have been developed into extraction methods that can be applied to meat from different species. At present, the existing technologies include: purifying myosin from silver carp back muscle, with a purity of more than 90%; extracting myosin from longissimus dorsi of cattle, with a purity of more than 80%; extracting bovine semimembranous muscle myosin with a purity of 95%; extracting high-purity myosin from longissimus dorsi of pig as raw material. The basic principles of the extraction methods of myosin from different species are roughly the same, but the specific operating conditions of the key steps are quite different. The first key step is homogenization, adding a dilute salt solution to the meat sample for homogenization, and discarding the supernatant after centrifugation to remove soluble myoplasmic protein. The differences between different methods in this step are mainly reflected in the differences in buffer ion strength, pH value, material-liquid ratio, and homogenization conditions; the second key step is crude extraction, using the property of myosin being easily soluble in high salt solution, dissolving myosin in a high salt solution, and then reducing the salt concentration by dilution to promote the precipitation of myosin. The main differences in this step are the ionic strength, pH value, and actomyosin dissociation agent of the high-salt solution; the third key step is purification, which involves re-dissolving the crude myosin extract in a high-salt buffer and purifying the myosin by ammonium sulfate fractionation precipitation, column chromatography separation, dialysis, etc. In this step, different methods differ greatly mainly in ammonium sulfate saturation and dialysate composition.
[0005] Among them, the masseter muscle (100% type I muscle fibers) and the dermal muscle (93% type II muscle fibers) of cattle are very different in protein composition and quality characteristics. First, the total collagen and soluble collagen content of the masseter muscle are higher than those of the dermal muscle of the trunk, and the sarcomere length of the masseter muscle is shorter than that of the dermal muscle of the trunk, resulting in the masseter muscle being far less tender than the dermal muscle, so the masseter muscle is more difficult to crush. Secondly, the meat color of the masseter muscle is redder than that of the dermal muscle of the trunk. This is because the content of myoglobin, hemoglobin, mitochondria and other colored metabolites in type I myofibrils is higher, making it more difficult to remove the sarcoplasmic protein of the masseter muscle, which greatly increases the difficulty of purification. Finally, the pH of the masseter muscle after slaughter is significantly higher than that of the dermal muscle of the trunk, which is essentially due to the different metabolic types of the two muscle fibers. The glycolysis rate of type II muscle fibers (dermal muscle of the trunk) is fast, and more lactic acid is produced, which rapidly reduces the pH of the meat and the ATP content. Therefore, the degree of tight binding between myosin and actin in the skin muscle of the trunk after slaughter is greater, and the formed actomyosin complex is more than that of the masseter muscle, which makes the actin in the skin muscle of the trunk more difficult to dissociate and remove during the purification process. Therefore, how to optimize an extraction and purification method suitable for both type I and type II myosin is very challenging. Summary of the invention
[0006] Purpose of the invention: The present invention aims to provide a method for extracting and purifying type I / II myosin isoforms with great differences in meat characteristics and capable of obtaining high purity (greater than 93%).
[0007] Technical solution: The method for extracting and purifying type I / II myosin subtypes of the present invention comprises the following steps:
[0008] (1) Homogenization: Take the masseter muscle and the skin muscle of the trunk and mince them respectively, add liquid A1, beat and pick out the insoluble matter visible to the naked eye, beat again, centrifuge and collect the precipitate, disperse the precipitate in liquid A1 again, homogenize, centrifuge and collect the precipitate, replace liquid A1 with liquid A2 and liquid A3 respectively, and repeat the above homogenization steps once, and collect the precipitate; the liquid A1 is 20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, 2mmol / L MgCl2, 1mmol / L EGTA, pH 7.0, the liquid A2 is 20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, pH 6.25, and the liquid A3 is 20mmol / L K2HPO4 / KH2PO4, pH 6.0;
[0009] (2) Crude extraction: The obtained precipitate is dispersed again in solution B for extraction and centrifugation. The obtained supernatant is diluted with water, allowed to stand, and centrifuged. 0.1 mol / L KCl is added to the precipitate to dissolve it and then centrifuged to obtain the precipitate. The solution B comprises 0.675 mol / L KCl, 7.5 mmol / L MgCl2, 0.15 mmol / L DTT, 7.5 mmol / L ATP, and pH 6.4.
[0010] (3) Ammonium sulfate precipitation: The obtained precipitate was dissolved in solution C, and then solution D was added. Saturated ammonium sulfate solution was added dropwise while stirring, and the mixture was centrifuged after stirring. The volume of the supernatant was accurately measured, and saturated ammonium sulfate was added dropwise while stirring, and the mixture was centrifuged after stirring. The solution C contained 3 mol / L KCl, 0.6 mmol / L DTT, 0.12 mol / L Tris-maleate, pH 7.5; the solution D contained 110 mmol / L ATP, 55 mmol / L MgCl2, 5.5 mmol / L EGTA, pH 7.5.
[0011] (4) Dialysis: Dissolve the precipitate and dialyze overnight, then centrifuge and take the supernatant.
[0012] Furthermore, in step (1), the whipping speed is 6000 r / min-7000 r / min, the whipping time is 30 s-40 s, and the precipitate is redispersed in 5 to 6 times the liquid A1.
[0013] Furthermore, in step (2), the amount of solution B added is 2 to 3 times the volume of the bovine masseter muscle or trunk cutaneous muscle, the obtained supernatant is diluted with 8 to 10 times water and then allowed to stand for 2 to 4 hours; 0.1 to 0.2 mol / L KCl is added to the precipitate again to dissolve it and then centrifuged to obtain the precipitate.
[0014] Furthermore, in step (3), the amount of solution C added is 3 to 4 times the volume of the bovine masseter muscle or 2 to 3 times the volume of the trunk skin muscle, and the amount of solution D added is 2 to 3 times the volume of the bovine masseter muscle or 1 to 2 times the volume of the trunk skin muscle; in step (3), a saturated ammonium sulfate solution is added dropwise while stirring until the saturation reaches 37%, and the mixture is centrifuged after stirring, the volume of the supernatant is accurately measured, and saturated ammonium sulfate is added dropwise while stirring until the final saturation is 48%.
[0015] Furthermore, in step (4), the dialysate is 0.5 mol / L NaCl, 0.1 mmol / L DTT, 20 mmol / L Tris-maleate, pH 7.5.
[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: taking bovine masseter muscle and bovine trunk skin muscle as the objects, an extraction and purification method that can be simultaneously applied to two myosin subtypes is established, and the protein composition and myosin subtype purity of the extract are analyzed by liquid chromatography-mass spectrometry technology and sodium dodecyl sulfate polyacrylamide electrophoresis. Type I and type II myosin with a purity of up to 95% and 93% can be obtained from bovine masseter muscle and trunk skin muscle. This method can provide technical feasibility for promoting the in-depth research on meat quality to the myosin subtype level, and lay the foundation for promoting the research on the relationship between myosin subtypes and meat quality characteristics. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The SDS-PAGE electrophoresis patterns of type II myosin extracts obtained in Comparative Example 1 (lane 4) and Comparative Example 2 (lanes 2-3), where M indicates the standard of protein molecular weight;
[0018] Figure 2 The SDS-PAGE electrophoresis patterns of the type II myosin extracts obtained in Comparative Example 3 (lanes 2 to 4) and Comparative Example 4 (lanes 5 to 8), where M indicates the standard of protein molecular weight;
[0019] Figure 3 is the SDS-PAGE electrophoresis pattern of the myosin extract obtained in Comparative Example 5, where M indicates the standard of protein molecular weight;
[0020] Figure 4 This is the SDS-PAGE electrophoresis pattern of the myosin subtype extract obtained in the example of the present invention, and M indicates the standard of protein molecular mass. DETAILED DESCRIPTION
[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.
[0022] Example
[0023] Step 1: Homogenization: Take 100g of bovine masseter and trunk skin muscle and mince them, add 6 times the volume of liquid A1 (20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, 2mmol / L MgCl2, 1mmol / L EGTA, pH 7.0), beat with a tissue crusher (masseter 7000r / min 35s; trunk skin muscle 6000r / min 30s) and pick out the insoluble substances visible to the naked eye such as fascia. Beat with a tissue crusher again at 10000r / min for 20s and stop for 20s, repeat 3 times, centrifuge (4℃, 11500×g, 10min) and collect the precipitate. The precipitate was redispersed in 6 volumes of liquid A1 (20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, 2mmol / L MgCl2, 1mmol / L EGTA, pH7.0), and homogenized again with an IKA homogenizer at 10000r / min for 20s and stopped for 20s, repeated 3 times, and centrifuged (4°C, 11500×g, 10min) to collect the precipitate. The above homogenization steps were repeated once each with liquid A2 (20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, pH 6.25) and liquid A3 (20mmol / L K2HPO4 / KH2PO4, pH 6.0) replacing liquid A1.
[0024] The second step is crude extraction: the obtained precipitate is dispersed again in a solution B containing 2 times the volume (0.675mol / L KCl, 7.5mmol / L MgCl2, 0.15mmol / L DTT, 7.5mmol / L ATP, pH 6.4), extracted for 12 minutes, and then centrifuged (4°C, 11500×g, 10min). The obtained supernatant is diluted with 9 times the volume of cold distilled water, allowed to stand for 2 hours, and centrifuged (4°C, 11500×g, 10min). 3 times the volume of 0.1mol / L KCl aqueous solution is added to the precipitate to dissolve it, and then centrifuged (4°C, 11500×g, 10min) to obtain the precipitate.
[0025] Step 3: Ammonium sulfate precipitation: The obtained precipitate is dissolved with solution C (3mol / LKCl, 0.6mmol / L DTT, 0.12mol / L Tris-maleate, pH 7.5) with a volume of 4 times that of the masseter muscle (2 times that of the trunk skin muscle), and then solution D (110mmol / L ATP, 55mmol / L MgCl2, 5.5mmol / L EGTA, pH 7.5) with a volume of 2 times that of the masseter muscle (1 times that of the trunk skin muscle) is added. Saturated ammonium sulfate solution is added dropwise while stirring until the saturation reaches 37%, and then stirred for 10 minutes and centrifuged (4°C, 16000×g, 15min). Accurately measure the volume of the supernatant, and add saturated ammonium sulfate dropwise while stirring until the final saturation is 48%, and then stirred for 10 minutes and centrifuged (4°C, 16000×g, 15min).
[0026] The fourth step was dialysis: the precipitate was dissolved with dialysate E (0.5 mol / L NaCl, 0.1 mmol / L DTT, 20 mmol / L Tris-maleate, pH 7.5) and dialyzed overnight, and then centrifuged (4°C, 12000×g, 20 min) to obtain the supernatant.
[0027] The solvent of solutions A1, A2, B, C, D, and E is all water.
[0028] Comparative Example 1
[0029] Referring to the prior art method for extracting myosin from the back muscle of bighead carp, the myosin was extracted from the skin muscle of the bovine trunk, and the myosin purification effect was tested by SDS-PAGE electrophoresis. (Refer to Shi Tong. Study on the mechanism of the effect of exogenous arginine on the thermal aggregation behavior of silver carp myosin and its application [D]. Zhenjiang: Jiangsu University, 2022. DOI: 10.27170 / d.cnki.gjsuu.2021.000075.)
[0030] The specific steps are as follows: 100g muscle was minced, 10 times the volume of solution A (0.1mol / L KCl, 20mmol / L Tris-HCl, pH 7.5) was added and homogenized at 10000r / min for 3min, and then centrifuged (4℃, 3000×g, 6min) after standing for 15min. 5 times the volume of solution B (0.45mol / L KCl, 5mmol / Lβ-ME, 0.2mol / L Mg(CH3COO)2, 1mmol / L EGTA, 20mmol / L Tris-maleate, pH 6.8) was added to the precipitate, and then ATP was added to a final concentration of 10mmol / l, and the reaction was performed for 90min before centrifugation (4℃, 12000×g, 13min). The supernatant was slowly diluted with 7 times the volume of 1mmol / L KHCO3, and then centrifuged again (4℃, 12000×g, 13min) after standing for 25min. Take the precipitate and add 2.5 times the volume of solution C (0.5mol / L KCl, 5mmol / Lβ-ME, 20mmol / L Tris-HCl, pH 7.5) to react for 10 minutes, then add 5 times the volume of 1mmol / L KHCO3 to slowly dilute, and then add MgCl2 to a final concentration of 10mmol / L. The above mixture was centrifuged at 4℃ overnight (4℃, 17700×g, 25min), and the obtained precipitate was dissolved in solution D (0.5mol / L NaCl, 20mmol / L Tris-HCl, pH 7.0) and centrifuged (4℃, 5000×g, 13min). The supernatant obtained is the myosin solution. The solvents of solutions A, B, C, and D are all water.
[0031] Comparative Example 2
[0032] The method for extracting myosin from the longissimus dorsi muscle of cattle in the prior art was slightly modified and the trunk skin muscle was used as the raw material for extraction. (Refer to Zhang Liping. Na + / K + Study on the influence of beef myosin system[D]. Zhengzhou: Henan Agricultural University, 2021.DOI:10.27117 / d.cnki.ghenu.2021.000080.)
[0033] The specific steps are as follows: 100g of muscle was minced and then added with 5 times the volume of solution A (0.1mol / L Tris-HCl, 20mmol / L EDTA, pH 7.0) and stirred for 1min, centrifuged at 4℃, 3000×g, 10min and repeated once, then added with solution B (0.1mmol / L Tris-HCl, 20mmol / L EDTA, pH 7.5) and stirred for 1min, centrifuged at 4℃, 3000×g, 10min and repeated once. The precipitate was collected and added with 3 times the volume of solution C (0.5mol / L KCl, 0.1mol / L KH2PO4 / K2HPO4, 10mmol / L Na4P2O7, 1mmol / L MgCl2, 2mmol / L EGTA, pH 6.4), and centrifuged at 4℃, 5000×g, 10min. Take the supernatant and add 9 times the volume of cold ultrapure water at 4℃ overnight, then centrifuge (4℃, 12000×g, 12min). Collect the precipitate and add 3 times the volume of solution D (0.1mol / L KCl, pH 7.0), stir evenly and centrifuge (4℃, 1500×g, 10min). Collect the lower precipitate and add 1mL of solution E (0.6mol / L KCl, 0.15mol / L KH2PO4 / K2HPO4, 5mmol / L Na4P2O7, 5mmol / L MgCl2, 1mmol / L EGTA, 2mmol / L ATP, pH 6.5) and stir for 30min. Slowly add ammonium sulfate solid particles to make the ammonium sulfate saturation concentration reach 35% and then centrifuge (4℃, 10000×g, 25min). Weigh the sodium sulfate solid particles again according to the volume of the supernatant to make the ammonium sulfate saturation concentration reach 48%, stir evenly with a glass rod for 10 minutes, and then centrifuge (4°C, 10000×g, 25 minutes). The precipitate is the myosin precipitate. The solvent of solutions A, B, C, D, and E is water.
[0034] Comparative Example 3
[0035] The method for extracting bovine semimembranous muscle myosin in the prior art was slightly modified and the trunk skin muscle was used as the raw material for extraction. (Refer to FRETHEIM K, EGELANDSDAL B, HARBITZ O, et al. Slow lowering of pH induces gel formation of myosin [J]. Food Chemistry, 1985, 18 (3): 169-178. DOI: 10.1016 / 0308-8146 (85) 90044-5)
[0036] The specific steps are as follows: mince 100g of muscle, add 0.3L modified GS solution (0.3mol / L KCl, 0.1mol / LKH2PO4, 0.05mol / L K2HPO4, 4mmol / L Na4P2O7, 1mmol / L EDTA, pH 6.5) and stir for 10min, then add 0.3L water while stirring and centrifuge (4℃, 3000×g, 20min). Filter the supernatant and add 6.5 times the volume of water containing 1mmol / EDTA, precipitate for 2h and centrifuge (4℃, 10000×g, 20min). Add 0.1L KCl solution (3mol / LKCl, 25mmol / L TES, pH 7.0) to each 100g precipitate and gently stir overnight. Then add 1.5 times the volume of water, and add MgCl2 and Na4P2O7 to make the final concentration 5mmol / L, stir vigorously for 10 minutes and centrifuge (4℃, 11000×g, 60min). Add 0.15L phosphate buffer (1.0mol / L KCl, 0.02mol / L NaH2PO4 / Na2HPO4, pH 7.5) to dissolve every 100g of precipitate, and retain some samples that have not been saturated with ammonium sulfate precipitation (0%). Add ammonium sulfate to the remaining samples while stirring until the saturation reaches 35%, stir for 10 minutes and centrifuge (4℃, 16000×g, 15min). Take the supernatant after centrifugation and add saturated ammonium sulfate to the saturation of 48%, stir for another 10 minutes and centrifuge (4℃, 16000×g, 15min). After overnight dialysis, the clarified myosin solution was obtained by centrifugation (4°C, 20,000×g, 30 min), and the purification effects of the three saturation precipitated myosin were compared by SDS-PAGE electrophoresis. The solvents of the GS solution and phosphate buffer were both water.
[0037] Comparative Example 4
[0038] The method for extracting myosin from the longissimus dorsi muscle of pigs in the prior art was slightly modified, and the trunk skin muscle was used as the raw material for extraction. (Refer to HAN Z, LI X, LIU Y, et al. The evolution of pork myosin aggregates and the relationship between aggregation modes and microstructures of O / Wemulsions [J]. Food Hydrocolloid, 2021, 119: 106825. DOI: 10.1016 / j.foodhyd.2021.106825. DOI: 10.1016 / j.foodchem.2006.02.054.)
[0039] The specific steps are as follows: mince 100g muscle, add 6 times phosphate buffer A (6mmol / L K2HPO4 / KH2PO4, pH 6.9), homogenize at 10000r / min on ice for 18s, centrifuge every 6s (4℃, 11500×g, 10min) and collect the precipitate. Redisperse the precipitate in 6 times volume of phosphate buffer A (6mmol / / L K2HPO4 / KH2PO4, pH 6.9), homogenize again at 10000r / min and centrifuge (4℃, 11500×g, 10min), repeat three times. The obtained precipitate was dispersed again in a twice-volume extracting solution B (0.675mol / L KCl, 7.5mmol / L MgCl2, 0.15mmol / L DTT, 7.5mmol / L ATP, pH 6.4), extracted for 12min, and then centrifuged (4°C, 11500×g, 10min). The obtained supernatant was diluted with 9 times the volume of cold distilled water and centrifuged (4°C, 11500×g, 10min). The obtained precipitate was dissolved with 4 times the volume of masseter muscle (2 times the volume of trunk skin muscle) of solution C (3mol / L KCl, 0.6mol / L DTT, 0.12mol / L Tris-maleate, pH 7.5), and then 2 times the volume of masseter muscle (1 times the volume of trunk skin muscle) of solution D (110mmol / L ATP, 55mmol / L MgCl2, 5.5mmol / L EGTA, pH 7.5) was added. Keep some samples that have not been precipitated with ammonium sulfate (0%), and add ammonium sulfate to the rest while stirring until the saturation is 20%, and then stir for 10 minutes and centrifuge (4°C, 16000×g, 15 minutes); take the supernatant and add ammonium sulfate to 35% saturation while stirring, and then stir for 10 minutes and centrifuge (4°C, 16000×g, 15 minutes); take the supernatant and add saturated ammonium sulfate to make the saturation reach 48%, and then stir for 10 minutes and centrifuge (4°C, 16000×g, 15 minutes). Dissolve the precipitate with dialysate E (0.5mol / L NaCl, 0.1mmol / L DTT, 20mmol / LTris-maleate, pH 7.5) and dialyze overnight, then centrifuge (4°C, 12000×g, 20 minutes) and take the supernatant. The purification effects of myosin obtained under four operating conditions of ammonium sulfate saturation 0% to 20%, 20% to 35%, 35% to 48% and 35% to 48% + dialysis were compared by SDS-PAGE electrophoresis. The solvents of solutions A, B, C, D and E were all water.
[0040] Comparative Example 5
[0041] The first step was homogenization: 100 g of masseter and trunk skin muscles were minced and added to 6 volumes of phosphate buffer A (6 mmol / L K2HPO4 / KH2PO4, pH 6.9). The mixture was homogenized at 10,000 r / min for 18 s on ice, and centrifuged every 6 s (4°C, 11,500 × g, 10 min) and the precipitate was collected. The precipitate was redispersed in 6 volumes of phosphate buffer A (6 mmol / L K2HPO4 / KH2PO4, pH 6.9), homogenized again at 10,000 r / min and centrifuged (4°C, 11,500 × g, 10 min), and the process was repeated three times.
[0042] The second step of crude extraction: The obtained precipitate was dispersed again in 2 volumes of extraction solution B (0.675 mol / L KCl, 7.5 mmol / L MgCl2, 0.15 mmol / L DTT, 7.5 mmol / L ATP, pH 6.4), extracted for 12 min, and then centrifuged (4°C, 11500×g, 10 min). The obtained supernatant was diluted with 9 volumes of cold distilled water and centrifuged (4°C, 11500×g, 10 min).
[0043] Step 3: Ammonium sulfate precipitation: The obtained precipitate is dissolved with high salt solution C (3 mol / L KCl, 0.6 mmol / L DTT, 0.12 mol / L Tris-maleate, pH 7.5) with a volume of 4 times that of the masseter muscle (2 times that of the trunk skin muscle), and then solution D (110 mmol / L ATP, 55 mmol / L MgCl2, 5.5 mmol / L EGTA, pH 7.5) with a volume of 2 times that of the masseter muscle (1 times that of the trunk skin muscle) is added. Saturated ammonium sulfate solution is added while stirring to 35% saturation, and then stirred for 10 minutes and centrifuged (4°C, 16000×g, 15 minutes). The supernatant is taken again and added with saturated ammonium sulfate solution to reach 48% saturation, and then stirred for 10 minutes and centrifuged (4°C, 16000×g, 15 minutes).
[0044] The fourth step was dialysis: the precipitate was dissolved with dialysate E (0.5 mol / L NaCl, 0.1 mmol / L DTT, 20 mmol / L Tris-maleate, pH 7.5) and dialyzed overnight, followed by centrifugation (4°C, 12000×g, 20 min) to collect the supernatant.
[0045] The solvent of solutions A, B, C, D, and E is all water.
[0046] Myosin purity analysis:
[0047] The protein concentration was determined by using a BCA kit with BSA as the standard protein. Protein purity was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE conditions: gradient separation gel 4% to 12%, voltage set to 200V, current 20-50V, and Coomassie brilliant blue staining. Myosin purity was determined by analyzing the total gray value of the myosin heavy chain and two light chains divided by the total gray value of all protein bands in the lane using Image J (National Institute of Mental Health, USA) software.
[0048] Myosin Isoform Identification:
[0049] This experiment is based on the combination of nano-liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometry (Orbitrap Exploris480) to qualitatively detect the proteins in the gel spots. The required gel strips were cut into pieces and then decolorized and dehydrated. After reduction with DTT and alkylation with iodoacetamide, they were washed with 25mmol / L NH4HCO3, 25mmol / L NH4HCO3+50% ACN solution and ACN in sequence. The ACN was dehydrated until the gel particles turned completely white, and 0.5μg of trypsin was added to the solution for protein digestion. The resulting peptide products were desalted and injected into the Nano LC-Orbitrap Exploris 480 mass spectrometer for identification. The sample was placed on the analytical column ( RSLC, C18, 75μm×15cm, 3μm, The peptides were separated by gradient separation with 3% to 35% elution buffer (80% ACN, 0.1%) on an Orbitrap Exploris 480 (Thermo Scientific). The flow rate was 300 nL / min in HPLC grade water for 90 min. The eluted peptides were analyzed by Orbitrap Exploris 480. From the high-resolution MS1 scan, the parent ions with the highest intensity were selected for high-energy collision fragmentation (HCD) within a 1 s cycle time to generate MS / MS mass spectra with a normalized collision energy (NCE) of 30.0 and a resolution of 15000 (m / z 200) for the MS / MS mass spectra. According to the actual sequence of the recombinant protein, a protein database was built and protein identification was processed using Proteome Discoverer software (version 2.4, Thermo Fisher Scientific, Waltham, MA, USA). Peptide identification based on MS / MS was accepted if the confidence level was greater than 99.0% according to the Sequest HT algorithm.
[0050] After denaturing electrophoresis, a myosin heavy chain appears near 220KDa, and two bands appear near 20KDa, which are myosin light chain 1 and myosin light chain 2. Figure 1 It can be seen that the target band of Comparative Example 1 (lane 5) is clearly visible, but there is a thicker miscellaneous band between 135 and 180 KDa. Combining the identification results of previous studies and the proteomics of this study, it can be seen that the miscellaneous band is mainly myoplasmic proteins such as enzymes, polymers, and chaperone proteins. The reason for the appearance of this miscellaneous band may be that Comparative Example 1 is mainly suitable for extracting myosin from the back muscle of bighead carp, while the hardness of the cattle trunk skin muscle used in the present invention is relatively large, and the connective tissue content is relatively high, resulting in its tissue fragmentation difficulty being much higher than that of fish meat. It is difficult to fragment the myofiber cell membrane in beef using the homogenization step in Comparative Example 1, resulting in the inability to dissolve and remove myoplasmic proteins well. In addition, myofibrillar protein miscellaneous bands such as actin, troponin, and myosin appear in sequence between 35 and 48 KDa, which may be due to the fact that Comparative Example 1 did not perform protein purification operations such as ammonium sulfate precipitation, column chromatography separation, and dialysis.
[0051] In contrast, Comparative Example 2 ( Figure 1 Lanes 2-4 & 6-8) have significantly improved the removal of 135-180KDa bands, but the bands of actin, troponin, myosin, etc. between 35-48KDa are still clearly visible. These proteins are the main components of myofibrillar proteins. In particular, actin can form an actomyosin complex with myosin after slaughter, which is difficult to dissociate, making it difficult to remove actin. This is also a major obstacle to the purification of myosin. Figure 2 By comparing lanes 2 to 4 with lanes 6 to 8, it was found that there was no obvious difference in the ammonium sulfate graded precipitation effect at different saturations for both type I and type II muscle fibers. This may be because solid ammonium sulfate was used in the original comparative example 2, which easily caused local oversaturation of the solution and made the graded precipitation effect unclear.
[0052] Figure 2The extraction effects of Comparative Example 3 and Comparative Example 4 were compared. It can be seen from lanes 2 to 4 that when the myosin of the bovine trunk cutaneous muscle was extracted using Comparative Example 3, no ammonium sulfate precipitation (0%) was performed and the myosin extract obtained with a saturation of 0%-35% had clearly visible bands between 135 and 180 KDa, around 100 KDa, 75 KDa, 63 KDa, between 35 and 48 KDa, and below 17 KDa, while the myosin extraction obtained with a saturation of 35% to 48% effectively removed the bands of actin, troponin, myosin, etc. between 35 and 48 KDa, and weakened the bands around 100 KDa (α-actinin) and 63 KDa (enzymes), but there were still obvious bands between 135 and 180 KDa, around 75 KDa, and below 17 KDa. The purity of myosin in lane 4 was measured to be about 50% by grayscale analysis. Compared with Comparative Examples 1 and 2, Comparative Example 3 has a better effect on removing impurities such as actin, which may be due to the use of saturated ammonium sulfate solution for graded precipitation in Comparative Example 3, which is better than the use of no ammonium sulfate precipitation in Comparative Example 1 and the use of solid ammonium sulfate for precipitation in Comparative Example 2. However, the removal effect of sarcoplasmic protein in Comparative Example 3 is not ideal, which may be caused by the lack of a homogenization step at the beginning of Comparative Example 3 to remove sarcoplasmic protein. In addition, the entire extraction process of Comparative Example 3 takes a long time.
[0053] Depend on Figure 2 As can be seen from lanes 5 to 8, when comparative example 4 is used to extract the skin muscle of the bovine trunk, a more obvious myosin target band can be obtained when the ammonium sulfate saturation is in the range of 0% to 20%, but at the same time, there are more protein bands between 135 and 180 KDa, near 100 KDa, 75 KDa, 63 KDa, between 35 and 48 KDa, and below 17 KDa. In comparison, the bands near 100 KDa, 75 KDa, and 63 KDa of the extract obtained with a saturation of 20% to 35% are significantly weakened, but other bands are still clearly visible. The bands near 100 KDa, 75 KDa, and 63 KDa of the extraction with a saturation of 35% to 48% are all removed, but the bands between 135 and 180 KDa, between 35 and 48 KDa, and below 17 KDa still exist. The precipitate obtained with a saturation of 35% to 48% was further dialyzed, and it was found that the actin bands basically disappeared, the troponin and myosin bands weakened, but the bands between 135 and 180 KDa and below 17 KDa still existed. The purity of myosin was about 70% as measured by grayscale analysis. The above results preliminarily determined that the appropriate range of ammonium sulfate saturation is between 35% and 48%, and the dialysis step is essential, which can not only remove some impurities but also remove residual salt ions (ammonium sulfate, etc.) in the extract.
[0054] Figure 3The results of comparative example 5 are as follows: the masseter muscle of cattle and the skin muscle of cattle trunk were extracted. Figure 3 It can be seen that the target bands of the two myosin isoforms are clearly visible, but there are still obvious miscellaneous bands between 135 and 180 KDa and below 17 KDa. The reasons may be: 1) the tissue is not completely broken, resulting in the inability to fully dissolve the protein components in the sarcolemma; 2) the salt ion strength, pH and other conditions of the homogenate are not suitable for better separation and removal of myosin; 3) the saturation range of ammonium sulfate fractionation precipitation needs further improvement.
[0055] The results of extracting two myosin isoforms by the method described in the embodiment of the present invention are shown in Figure 4 . As can be seen from the figure, the three target bands of the two myosin subtype extracts are relatively clear, and only a thin mixed band can be seen between 135 and 180 KDa. The protein composition of lanes I and II was identified by liquid chromatography-mass spectrometry technology, confirming that the myosin subtypes extracted from bovine masseter and trunk cutaneous muscles are type I and type II, respectively. Grayscale analysis showed that the purity of type I myosin was about 95%, and the purity of type II myosin was about 93%.
[0056] The present invention uses cattle masseter muscle and trunk skin muscle as the object, optimizes the degree of tissue fragmentation, sarcoplasmic protein removal effect and ammonium sulfate graded precipitation effect in the myosin extraction and purification method, establishes an optimal myosin extraction and purification method, and obtains type I / II myosin subtypes with a purity greater than 93% through quality identification through SDS-PAGE electrophoresis and proteomics. The extraction method described in the present invention has the advantages of simple operation, low equipment requirements, and short time consumption, and can be widely used in the study of myosin subtypes. The high-purity myosin subtype extraction method established by the present invention is of great significance for promoting the research on meat quality characteristics to the myosin subtype level.
Claims
1. A method for extracting and purifying type I / II myosin isoforms, characterized in that: The steps include: (1) Homogenization: Take the masseter muscle and the skin muscle of the bovine trunk and mince them respectively, add liquid A1, use a tissue crusher at 6000-7000 r / min, beat for 30-40 s, pick out the insoluble matter visible to the naked eye, use the tissue crusher at 6000-7000 r / min again, beat for 30-40 s, centrifuge and collect the precipitate, redisperse the precipitate in liquid A1, homogenize, centrifuge, collect the precipitate, replace liquid A1 with liquid A2 and liquid A3 respectively, and repeat the above homogenization steps once, and collect the precipitate; the liquid A1 is 20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, 2mmol / L MgCl2, 1mmol / L EGTA, pH 7.0, the liquid A2 is 20mmol / L K2HPO4 / KH2PO4, 0.1mol / L NaCl, pH 6.25, and the liquid A3 is 20mmol / L K2HPO4 / KH2PO4, pH 6.0; (2) Crude extraction: The obtained precipitate is dispersed again in solution B for extraction and then centrifuged. The obtained supernatant is diluted with water and then allowed to stand and centrifuged. 0.1 mol / L KCl solution is added to the precipitate to dissolve it and then centrifuged to obtain the precipitate; the solution B comprises 0.675 mol / L KCl, 7.5 mmol / L MgCl2, 0.15 mmol / L DTT, 7.5 mmol / L ATP, and pH 6.4; (3) Ammonium sulfate precipitation: The obtained precipitate was dissolved in solution C, and then solution D was added. Saturated ammonium sulfate solution was added dropwise while stirring until the saturation reached 37%. After stirring, the mixture was centrifuged. The volume of the supernatant was accurately measured. Saturated ammonium sulfate was added dropwise while stirring until the saturation reached 48%. After stirring, the mixture was centrifuged. The solution C contained 3 mol / L KCl, 0.6 mmol / L DTT, 0.12 mol / L Tris-maleate, pH 7.5, and the solution D contained 110 mmol / L ATP, 55 mmol / L MgCl2, 5.5 mmol / L EGTA, pH 7.
5. (4) Dialysis: Dissolve the precipitate and dialyze overnight, then centrifuge and take the supernatant.
2. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: The precipitate in step (1) is redispersed in 5 to 6 times the amount of liquid A1.
3. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: The amount of solution B added in step (2) is 2 to 3 times the volume of the bovine masseter muscle or the bovine trunk cutaneous muscle.
4. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: The supernatant obtained in step (2) is diluted with 8 to 10 times of water and allowed to stand for 2 to 4 hours.
5. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: In step (2), 0.1-0.2 mol / L KCl is added to the precipitate to dissolve it and then centrifuged to obtain the precipitate.
6. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: The amount of solution C added in step (3) is 3 to 4 times the volume of the bovine masseter muscle or 2 to 3 times the volume of the bovine trunk cutaneous muscle.
7. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: The amount of solution D added in step (3) is 2 to 3 times the volume of the bovine masseter muscle or 1 to 2 times the volume of the bovine trunk skin muscle.
8. The method for extracting and purifying type I / II myosin isoforms according to claim 1, characterized in that: In step (4), the dialysate is 0.5 mol / L NaCl, 0.1 mmol / L DTT, 20 mmol / L Tris-maleate, pH 7.5.