A method for evaluating the quality of surimi based on muscle microstructure and endogenous enzyme activity

Abstract

This invention discloses a method for evaluating the quality of surimi based on the microstructure of muscle tissue and endogenous enzyme activity. The method includes: collecting data on the sarcomere length and myofibril-bound cathepsin L activity of raw fish muscle tissue; constructing a correlation model between these data and the surimi gel strength; predicting the gel strength of the raw fish after processing into a gel; and achieving graded screening of raw fish specifically for surimi production. This invention selects sarcomere length and myofibril-bound cathepsin L activity, which show a significant correlation with surimi gel quality. This parameter combination provides objective and reliable data support and decision-making basis for raw material screening and quality control. Furthermore, this invention achieves rapid prediction of gel quality based solely on the raw fish without the need for surimi gel preparation, greatly simplifying the process and saving time, cost, and raw materials. It has significant application value and broad prospects for promotion, providing reliable quality prediction technology support for the surimi product industry.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for evaluating the quality of surimi based on muscle microstructure and endogenous enzyme activity. Background Technology

[0002] As a standardized intermediate raw material in the frozen prepared surimi product processing industry chain, the gelation properties of surimi determine the quality characteristics of surimi products. Generally speaking, evaluating the quality of surimi gel requires processing fresh fish raw materials into assessable surimi gel products, and then assessing the gel elasticity of the surimi raw materials through a gel puncture test. This multi-step, high-loss processing characteristic and the accompanying complexity of testing further highlight the special nature of quality evaluation. Referring to the national standard GB / T 36187-2014, the quality assessment of surimi and its products must be based on a complete gelation process; the determination of its sensory and physicochemical indicators can only be carried out after the surimi has been standardized into surimi gel. Among these, gel strength, as a key indicator, comprehensively reflects the elasticity, hardness, water retention, and other textural properties of the surimi gel, and is the core basis for determining the quality grade of surimi.

[0003] Specifically, processing raw fish into surimi products involves multiple complex steps. First, fish meat is obtained through a harvesting process, followed by multi-stage rinsing (consuming 15-20 tons of water per ton of surimi) to remove lipids, impurities, and fishy odors. Then, through fine filtration and dehydration, and the addition of antifreeze agents, standardized frozen surimi for freezing and storage is produced. These processing steps generate a large amount of byproducts and rinsing wastewater, consuming significant time and energy. Further processing involves adding auxiliary materials, pounding and extracting the slurry, and gelling to form a testable elastin gel product. As described above, the entire process of processing raw fish into surimi products is time-consuming, energy-intensive, and places a heavy burden on wastewater treatment; some steps require specialized equipment, and there are minimum requirements for the amount of raw fish processed. Furthermore, significant human error exists in key aspects of the preparation of surimi and its products, such as process parameters (chopping intensity, time, and temperature control) and enema techniques. These factors can affect the gel strength measurement results, making it difficult to guarantee the repeatability and accuracy of traditional evaluation methods. This can lead to different operators obtaining different results when performing the same testing procedures, a very common phenomenon in the industry. Therefore, it is essential to predict the gel quality of raw fish directly through evaluation. On the one hand, this allows for the prediction of gel quality based on the characteristics of different batches of raw fish; on the other hand, it helps in screening suitable raw materials for surimi product processing.

[0004] Currently, the technology for predicting the strength of surimi gel still has certain limitations. For example, Chinese patent CN119643477A discloses an online monitoring method for the thermal processing of surimi based on hyperspectral imaging technology. This technology predicts gel strength by collecting hyperspectral data in the VNIR (400-1000nm) and NIR (900-1700nm) bands after pre-treated surimi is heated in a two-stage water bath. However, this technology still has the following significant drawbacks in practical applications: First, this method requires the raw material to be processed into surimi gel before prediction can be performed, making it impossible to achieve early quality assessment at the raw material stage, which is inconsistent with the requirements of industrial production for the quality control of processed raw materials. Summary of the Invention

[0005] To address the limitations of existing technologies that cannot determine the suitability of raw fish for surimi processing based solely on the characteristics of the raw fish before processing, this invention provides a method for evaluating surimi quality based on muscle microstructure and endogenous enzyme activity. This invention directly predicts the gel strength of the processed surimi by analyzing the muscle histological characteristics and endogenous enzyme activity parameters of the raw fish, thereby evaluating surimi quality and avoiding the waste of raw materials, energy, and time caused by traditional evaluation methods.

[0006] The technical solution of the present invention is as follows:

[0007] The purpose of this invention is to provide a method for evaluating the quality of surimi based on muscle microstructure and endogenous enzyme activity, comprising the following steps:

[0008] (1) The raw fish is processed by removing the head, tail, internal organs, slicing, skinning, and trimming to obtain fish fillets;

[0009] (2) Take back muscle tissue from the fish fillets obtained in step (1), fix it in glutaraldehyde fixative and osmium tetroxide fixative, wash it with buffer and then perform gradient dehydration treatment, then slice it and stain it, observe the muscle tissue by transmission electron microscope and quantify the sarcomere length of the muscle tissue using image processing software.

[0010] (3) Take back muscle tissue from the fish fillets obtained in step (1), extract crude enzyme solution from the muscle tissue, incubate the crude enzyme solution and enzyme reaction substrate thoroughly, and use a fluorescence spectrophotometer to determine the activity of myofibril-binding cathepsin L in the fish fillets.

[0011] (4) Based on the correlation model between the sarcomere length of muscle tissue, the activity of myofibril-bound cathepsin L and the gel strength of surimi, the gel strength of the selected raw fish is calculated, and the gel quality of the surimi is evaluated.

[0012] The correlation model is as follows:

[0013] Y=3462.716-525.776×X1-15463.897×X2;

[0014] Where X1 is the sarcomere length after ImageJ quantization, in μm;

[0015] X2 represents the activity of myofibril-bound cathepsin L in the raw fish, expressed in U / g.

[0016] Y represents the gel strength, measured in g·cm.

[0017] In one embodiment of the present invention, in step (1), the raw fish is a fresh freshwater fish, including one of Nile tilapia, red tilapia, Oreochromis aureus, and Nile tilapia.

[0018] In one embodiment of the present invention, in step (2), the muscle tissue is taken from the white muscle on the back of the fish fillet and cut along the fiber direction, with a thickness of 1.0 to 1.5 cm and a length and width of 2 to 2.5 cm.

[0019] In one embodiment of the present invention, in step (2), the concentration of the glutaraldehyde fixative is 2% to 3%, and the fixation treatment conditions are: fixation at 4°C in the dark for 24 hours; the concentration of the osmium tetroxide fixative is 1% to 2%, and the fixation treatment conditions are: fixation at 4°C in the dark for 2 hours.

[0020] In one embodiment of the present invention, in step (2), the buffer used for washing is 0.1M phosphate buffer (PBS, pH 7.4), and the muscle tissue is washed 3 to 5 times, each time for 5 to 10 minutes, until the fixative in the muscle tissue is basically rinsed clean and there is no obvious residue.

[0021] In one embodiment of the present invention, in step (2), the gradient dehydration treatment uses gradient ethanol concentrations of 30%, 50%, 70%, 80%, 90%, and 100%, with each dehydration time being 15 minutes.

[0022] In one embodiment of the present invention, in step (2), the cutting surface treatment produces an ultrathin slice with a thickness of 50-100 nm; the staining treatment first uses 0.2%-0.4% lead citrate solution to stain at room temperature for 5-10 min, and then uses 50% ethanol saturated solution of uranium acetate to stain at room temperature for 5-10 min.

[0023] In one embodiment of the present invention, in step (3), enzyme activity refers to the enzyme's catalytic capacity to convert micromoles of substrate per second under specific enzyme reaction conditions. The unit (U) represents the enzyme activity that catalyzes the conversion of 1 micromole of substrate per second under enzyme reaction conditions; the unit (U / g) represents the total enzyme activity per gram of muscle tissue.

[0024] In one embodiment of the present invention, the method for extracting the crude enzyme solution from the muscle tissue in step (3) is as follows: the muscle tissue is chopped and homogenized with three times its weight of 20mM phosphate buffer (pH 7.5), and then heated at 4°C for 8000 minutes.

[0025] Centrifuge for 15 min under ×g conditions;

[0026] After repeating the above process three times, the precipitate was homogenized with 20mM phosphate buffer (pH 6.4) containing 0.5M KCl, 1mM MgCl2 and 5mM Na4P2O7 and placed in an ice bath for 30 min.

[0027] The mixture was then placed in a boiling water bath and incubated at 55°C for 10 min. The mixture was then centrifuged at 4°C and 10,000×g for 15 min to obtain the supernatant.

[0028] The supernatant was adjusted to pH 5.5 with 2M HCl and centrifuged again to remove the precipitate. The pH of the supernatant was then adjusted to 7.5 with 1M NaOH and filtered. The filtrate was collected to obtain the crude enzyme solution.

[0029] In one embodiment of the present invention, in step (3), the enzyme reaction substrate is Z-Phe-Arg-AMC (benzyloxycarbonyl-phenylalanyl-arginyl-7-amino-4-methylcoumarin), the substrate is prepared into a 9 mM stock solution using DMSO (dimethyl sulfoxide), and diluted to 90 μM before use.

[0030] In one embodiment of the present invention, in step (3), the incubation process is as follows: 500 μL of crude enzyme solution and 500 μL of 150 mM Bis-Tris buffer are incubated at 37°C for 10 min, then 500 μL of enzyme reaction substrate is added and incubated for 15 min, and the reaction is terminated with 3 mL of 50 mM Bis-Tris.

[0031] In one embodiment of the present invention, the 150mM Bis-Tris buffer contains 30mM EDTA (ethylenediaminetetraacetic acid), 6mM DTT (dithiothreitol), and has a pH of 6.0; the 50mM Bis-Tris buffer contains 1% SDS (sodium dodecyl sulfate) and has a pH of 7.0.

[0032] In one embodiment of the present invention

[0033] After the raw fish undergoes meat extraction, rinsing, fine filtration, and dehydration, its moisture content is uniformly adjusted to 80%.

[0034] Frozen fish paste is prepared by mixing finely filtered fish paste with uniform moisture content and additives (7.5% sucrose, 0.125% sodium tripolyphosphate and 0.125% sodium pyrophosphate are added based on the quality of finely filtered fish paste with uniform moisture content) and storing it at -18°C.

[0035] Frozen fish paste was prepared into fish paste gel using a two-stage heating method (first stage: 40℃, 1h, second stage: 90℃, 15min), and the gel strength was then measured.

[0036] The accuracy of the evaluation method of this application can be assessed by comparing the gel strength measured above with the gel strength calculated by substituting the sarcomere length of muscle tissue and the activity of myofibril-bound cathepsin L into the constructed correlation model.

[0037] The beneficial technical effects of this invention are as follows:

[0038] This invention enables the prediction of gel strength by measuring the microstructure and endogenous enzyme activity of fish raw materials. It allows for rapid prediction of gel strength without the need for cumbersome processes in preparing surimi and surimi products. This facilitates the scientific screening of freshwater fish raw materials with different gel strength levels, effectively solving the problem of blind selection in traditional fish species screening processes. It significantly improves the efficiency and scientific rigor of surimi raw material selection, greatly simplifies the screening process, and saves time, costs, and raw materials. It has significant application value and broad prospects for promotion, providing reliable predictive technology support for the surimi product industry.

[0039] In this invention's evaluation method, muscle microstructure and endogenous enzyme activity indicators show a significant correlation with gel strength, overcoming the limitations of relying on experience or subjective sensory judgment. The former reflects the basic structural state of the raw material, embodying the integrity, arrangement characteristics, and initial network structure of myofibrils, exhibiting high stability and representativeness. The latter represents the potential for protein degradation, particularly myofibril-binding cathepsin L, which is not easily lost during the rinsing process, continuously reflecting changes in raw material quality, and its role significantly influences protein degradation and gel network formation. The selection of these two indicators provides objective and reliable data support and decision-making basis for raw material screening and quality control, greatly improving the scientific rigor and accuracy of the evaluation.

[0040] This invention directly uses raw fish muscle tissue as the research object, and establishes a correlation model between the sarcomere length, myofibril-binding cathepsin L activity and surimi gel strength. This allows for the prediction and calculation of surimi gel strength based on raw material indicators without the need for processing into surimi products, and can directly evaluate the gel quality of surimi.

[0041] The following are substantial differences compared to existing technologies:

[0042] 1. Breakthrough in innovative testing methods: Direct testing of raw fish muscle tissue (not finished fish paste gel) enables prediction of gel quality through early assessment of raw materials before processing, avoiding the cumbersome process of traditional preparation methods;

[0043] 2. Innovative selection of prediction parameters: For the first time, ultrastructural parameters (sarcomere length) and enzyme activity parameters (myofibrillar-binding cathepsin L activity) are combined for modeling. Existing technologies have not disclosed the correlation between this parameter combination and gel strength. Attached Figure Description

[0044] Figure 1 The ultrastructure of muscle tissue and the quantified sarcomere length of three types of tilapia were determined in Examples 1, 2 and 3 of this invention.

[0045] Figure 2 The activity of myofiber-binding cathepsin L in three types of tilapia was determined in Examples 1, 2 and 3 of this invention.

[0046] Figure 3 The gel strength of the three types of tilapia prepared into fish paste gel was measured in Examples 1, 2 and 3 of this invention. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Determine the gel strength of fish meat after it has been prepared into surimi gel.

[0051] After the raw fish is processed through meat extraction, rinsing, and fine filtration, its moisture content is uniformly adjusted to 80%. The finely filtered fish paste is mixed with additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18℃ to obtain frozen fish paste. The frozen fish paste is then prepared into fish paste gel using a two-stage heating method (first stage: 40℃, 1 hour; second stage: 90℃, 15 minutes). The fish paste gel is cut into 25 mm high cylinders and equilibrated at 25℃ for 2 hours. The breaking force and breaking distance of the fish paste gel are measured using a texture analyzer equipped with a P / 5S spherical probe. The gel strength (g·cm) is calculated by multiplying the breaking force (g) by the breaking distance (cm).

[0052] Example 1

[0053] A method for evaluating tilapia based on muscle microstructure and endogenous enzyme content includes the following steps:

[0054] (1) Fresh tilapia is cut into fillets by removing the tail, internal organs, fillets, skin, and trimming.

[0055] (2) Dorsal muscle tissue was extracted from the obtained fish fillets and fixed successively in glutaraldehyde fixative and osmium tetroxide fixative. After washing with buffer, it underwent gradient dehydration, followed by sectioning and staining. Observation was performed using transmission electron microscopy and quantitative analysis based on ImageJ, and the myosal length X1 of Nile tilapia was found to be 1.81±0.02μm. Figure 1 A and Figure 1 D);

[0056] (3) Take dorsal muscle tissue from fish fillets, extract crude enzyme solution from the muscle tissue using a heating method, incubate the crude enzyme solution and enzyme reaction substrate thoroughly, and determine the activity of myofibril-bound cathepsin L in the fish fillets using a fluorescence spectrophotometer. The content of myofibril-bound cathepsin L in Nile tilapia was determined to be 0.021±3.4992E-5U / g (X2). Figure 2 ).

[0057] (4) Substitute the values ​​of X1 and X2 obtained in steps (2) and (3) into the multiple regression model, i.e. Y = 3462.716 - 525.776 × X1 - 15463.897 × X2, and calculate the gel strength of tilapia surimi as 2186.32 g·cm.

[0058] (5) Fresh tilapia, after being processed by meat extraction, rinsing, and fine filtration, had its moisture content uniformly adjusted to 80%. The finely filtered fish paste was mixed with additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18℃ to obtain frozen fish paste. The frozen fish paste was then prepared into fish paste gel using a two-stage heating method (first stage: 40℃, 1h; second stage: 90℃, 15min). The fish paste gel was cut into 25mm high cylinders and equilibrated at 25℃ for 2h. The breaking force and breaking distance of the fish paste gel were measured using a texture analyzer equipped with a P / 5S spherical probe. The gel strength (g·cm) was calculated by multiplying the breaking force (g) by the breaking distance (cm). The gel strength tested using the texture analyzer was 2268.83±98.11g·cm, and the relative error between the predicted result and the test result was 3.77%.

[0059] Example 2

[0060] A method for evaluating Oreochromis aureus based on muscle microstructure and endogenous enzyme activity includes the following steps:

[0061] (1) Fresh Oreo tilapia are cut into fillets by removing the tail, internal organs, fillets, skin, and trimming.

[0062] (2) Dorsal muscle tissue was extracted from the obtained fish fillets and fixed successively in glutaraldehyde fixative and osmium tetroxide fixative. After washing with buffer, it underwent gradient dehydration, followed by sectioning and staining. Observation was performed using transmission electron microscopy and quantitative analysis based on ImageJ, and the myosal length X1 of the Oreochromis aureus was found to be 2.32±0.07μm. Figure 1 B and Figure 1 D).

[0063] (3) Back muscle tissue was taken from the fish fillets, and crude enzyme solution was extracted from the muscle tissue using a heating method. The crude enzyme solution and enzyme reaction substrate were fully incubated, and the activity of myofibril-bound cathepsin L in the fish fillets was determined using a fluorescence spectrophotometer. The content of myofibril-bound cathepsin L in Oreochromis aureus was determined to be 0.054±3.30289E-4U / g (X2). Figure 2 ).

[0064] (4) Substitute the values ​​of X1 and X2 obtained in steps (2) and (3) into the multiple regression model, i.e. Y = 3462.716 - 525.776 × X1 - 15463.897 × X2, and calculate the gel strength of the Oreochromis surimi as 1407.86 g·cm.

[0065] (5) Fresh Oreochromis aquatic tilapia, after being processed by meat extraction, rinsing, and fine filtration, had its moisture content uniformly adjusted to 80%. The finely filtered fish paste was mixed with additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18℃ to obtain frozen fish paste. The frozen fish paste was then prepared into fish paste gel using a two-stage heating method (first stage: 40℃, 1h; second stage: 90℃, 15min). The fish paste gel was cut into 25mm high cylinders and equilibrated at 25℃ for 2h. The breaking force and breaking distance of the fish paste gel were measured using a texture analyzer equipped with a P / 5S spherical probe. The gel strength (g·cm) was calculated by multiplying the breaking force (g) by the breaking distance (cm). The gel strength tested using the texture analyzer was 1442.61±55.79g·cm, and the relative error between the predicted result and the test result was 2.47%.

[0066] Example 3

[0067] A method for evaluating red tilapia based on muscle microstructure and endogenous enzyme activity includes the following steps:

[0068] (1) Fresh red tilapia are cut into fillets after the tail is removed, the internal organs are removed, the fillets are sliced, the skin is removed and the fillets are trimmed.

[0069] (2) Dorsal muscle tissue was extracted from the obtained fish fillets and fixed successively in glutaraldehyde fixative and osmium tetroxide fixative. After washing with buffer, it underwent gradient dehydration, followed by sectioning and staining. Observation was performed using transmission electron microscopy and quantitative analysis based on ImageJ, and the myosalp length X1 of the red tilapia was found to be 2.04±0.09μm. Figure 1 C and Figure 1 D).

[0070] (3) Take dorsal muscle tissue from fish fillets, extract crude enzyme solution from the muscle tissue using a heating method, incubate the crude enzyme solution and enzyme reaction substrate thoroughly, and determine the activity of myofibril-bound cathepsin L in the fish fillets using a fluorescence spectrophotometer. The content of myofibril-bound cathepsin L in red tilapia was determined to be 0.021±3.4992E-5U / g (X2). Figure 2 ).

[0071] (4) Substitute the values ​​of X1 and X2 obtained in steps (2) and (3) into the multiple regression model, i.e. Y = 3462.716 - 525.776 × X1 - 15463.897 × X2, and calculate the gel strength of red tilapia surimi as 2065.39 g·cm.

[0072] (5) Fresh red tilapia, after being processed by meat extraction, rinsing, and fine filtration, had its moisture content uniformly adjusted to 80%. The finely filtered fish paste was mixed with additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18℃ to obtain frozen fish paste. The frozen fish paste was then prepared into fish paste gel using a two-stage heating method (first stage: 40℃, 1h; second stage: 90℃, 15min). The fish paste gel was cut into 25mm high cylinders and equilibrated at 25℃ for 2h. The breaking force and breaking distance of the fish paste gel were measured using a texture analyzer equipped with a P / 5S spherical probe. The gel strength (g·cm) was calculated by multiplying the breaking force (g) by the breaking distance (cm). The gel strength tested using the texture analyzer was 2016.29±146.46g·cm, and the relative error between the predicted result and the test result was 2.38%.

[0073] The results of the fish surimi quality evaluation method of the present invention were compared with the gel strength measured by a texture analyzer. The gel strength of fish surimi gel samples I, II, and III prepared in each step (5) of Examples 1, 2, and 3 was measured by a texture analyzer and compared with the results of the fish surimi quality evaluation method of the present invention. The results are shown in Table 1.

[0074] Table 1 Comparison of the predicted results of the method of the present invention with the results of gel strength determination by the standard method.

[0075] sample <![CDATA[X1(μm)]]> <![CDATA[X2(U / g)]]> Predicting gel strength (g·cm) Measured gel strength (g·cm) Relative error (%) Sample I 1.81 0.021 2186.32 2268.83 3.77 Sample II 2.32 0.054 1407.86 1442.61 2.47 Sample III 2.04 0.021 2065.39 2016.29 2.38

[0076] As can be seen from the data in Table 1, the gel strength predicted by the evaluation method constructed in this invention has good consistency with the results measured by the texture analyzer.

[0077] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for evaluating the quality of surimi based on muscle microstructure and endogenous enzyme activity, characterized by, Includes the following steps: (1) The raw fish is processed by removing the head, tail, internal organs, slicing, skinning, and trimming to obtain fish fillets; (2) Take back muscle tissue from the fish fillets obtained in step (1), fix it in glutaraldehyde fixative and osmium tetroxide fixative, wash it with buffer solution and then perform gradient dehydration treatment, then slice it and stain it, observe the muscle tissue by transmission electron microscope and use image processing software to quantify the sarcomere length of the muscle tissue. (3) Take back muscle tissue from the fish fillets obtained in step (1), extract crude enzyme solution from the muscle tissue, incubate the crude enzyme solution and enzyme reaction substrate thoroughly, and use a fluorescence spectrophotometer to determine the activity of myofibril-binding cathepsin L in the muscle tissue. (4) Based on the correlation model between the sarcomere length of muscle tissue, the activity of myofibril-bound cathepsin L and the gel strength of surimi, the gel strength of the selected raw fish is calculated, and the gel quality of the surimi is evaluated. The correlation model is as follows: Y= 3462.716 - 525.776 × X1- 15463.897× X2; Where X1 is the sarcomere length after ImageJ quantization, in μm; X2 represents the activity of myofibril-bound cathepsin L in the raw fish, expressed in U / g. Y represents gel strength, measured in g·cm. The raw fish is one of the following: Nile tilapia, red tilapia, Oreochromis aureus, or Nile tilapia.

2. The method of claim 1, wherein, In step (2), the muscle tissue needs to be taken from the white muscle on the back of the fish fillet and cut along the fiber direction, with a thickness of 1.0 ~ 1.5cm and a length and width of 2 ~ 2.5cm respectively.

3. The method of claim 1, wherein, In step (2), the concentration of the glutaraldehyde fixative is 2% to 3%, and the fixation conditions are: fixation at 4°C in the dark for 24 hours; the concentration of the osmium tetroxide fixative is 1% to 2%, and the fixation conditions are: fixation at 4°C in the dark for 2 hours.

4. The method of claim 1, wherein, In step (2), the gradient dehydration process uses gradient ethanol concentrations of 30%, 50%, 70%, 80%, 90%, and 100%, with each dehydration stage lasting 15 minutes.

5. The method of claim 1, wherein, In step (2), the cutting surface treatment produces ultrathin slices with a thickness of 50 ~ 100 nm; staining is performed by sequentially staining with lead citrate solution and 50% ethanol saturated solution of uranium acetate.

6. The method of claim 1, wherein, In step (3), the method for extracting crude enzyme solution from muscle tissue is as follows: after cutting the muscle tissue into small pieces, homogenize it with three times the weight of 20 mM pH 7.5 phosphate buffer, and then centrifuge it at 4℃ and 8000 × g for 15 min. After repeating the above process three times, the precipitate was homogenized with 20 mM pH 6.4 phosphate buffer containing 0.5 M KCl, 1 mM MgCl2 and 5 mM Na4P2O7 and placed in an ice bath for 30 min. The mixture was then incubated in a water bath at 55°C for 10 min, and centrifuged at 4°C and 10,000 × g for 15 min to obtain the supernatant. The supernatant was adjusted to pH 5.5 with 2 M HCl and centrifuged again to remove the precipitate. The pH of the supernatant was then adjusted to 7.5 with 1 M NaOH and filtered. The filtrate was collected to obtain the crude enzyme solution.

7. The method of claim 1, wherein, In step (3), the enzyme reaction substrate is Z-Phe-Arg-AMC. The substrate is prepared into a 9 mM stock solution using DMSO and diluted to 90 μM before use.

8. The method of claim 1, wherein, In step (3), the incubation process is as follows: 500 μL of crude enzyme solution and 500 μL of 150 mM Bis-Tris buffer are incubated at 37°C for 10 min, then 500 μL of enzyme reaction substrate is added and incubated for 15 min, and the reaction is terminated with 3 mL of 50 mM Bis-Tris.

9. The method of claim 8, wherein, 150 mM Bis-Tris buffer contains 30 mM EDTA, 6 mM DTT, and pH 6.0; 50 mM Bis-Tris buffer contains 1% SDS and pH 7.0.

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