Method for evaluating quality of minced fillet based on muscle microstructure and endogenous enzyme activity

Abstract

The invention discloses a method for evaluating the quality of surimi on the basis of muscle tissue microstructure and endogenous enzyme activity, which comprises the following steps: acquiring the muscle joint length of raw material fish muscle tissue and the activity data of muscle fiber-bound cathepsin L, and constructing a correlation model between the muscle joint length and the activity data of muscle fiber-bound cathepsin L and the gel strength of surimi; and predicting to obtain the gel strength after the raw material fish is processed into the gel, so that the special raw material fish for surimi is classified and screened. According to the method disclosed by the invention, the sarcomedo length and the activity of the muscle fiber-bound cathepsin L are selected to have significant correlation with the quality of the minced fillet gel, and the parameter combination provides objective and reliable data support and decision basis for raw material screening and quality control; besides, the method can realize rapid prediction of the gel quality based on the raw material fish without preparing the minced fillet gel, greatly simplifies the process, saves time, cost, raw materials and the like, has important application value and wide popularization prospect, and can provide reliable quality prediction technical support for the minced fillet product industry.

Description

Technical Field

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

[0002] Surimi, a standardized intermediate raw material in the frozen and processed surimi product processing chain, has a gelling property that determines the quality characteristics of surimi products. Generally speaking, evaluating surimi gel quality requires processing fresh fish raw material into an evaluable surimi gel product, followed by evaluating the gel elasticity of the surimi raw material through gel penetration testing. This multi-step, high-loss processing and the attendant testing complexity further highlight the unique nature of quality evaluation. According to the national standard GB / T 36187-2014, the quality assessment of surimi and its products must be based on a complete gelling process. Sensory and physicochemical indicators must be measured only after the surimi has been standardized and processed into surimi gel. Gel strength, a key indicator, comprehensively reflects the textural properties of the surimi gel, including elasticity, hardness, and water retention, and is the core basis for determining surimi quality grade.

[0003] Specifically, processing raw fish into surimi products requires going through multiple cumbersome process steps. First, the fish meat is obtained through the meat collection process, and then it is rinsed in multiple stages (15-20 tons of water is consumed per ton of surimi) to remove lipids, impurities and fishy substances. Subsequently, it is filtered and dehydrated, and antifreeze is added to make standardized frozen surimi that can be frozen and stored. These processing steps will produce a large amount of by-products and rinsing wastewater, which is time-consuming and energy-consuming. Furthermore, the frozen surimi is processed for the second time, and through processes such as adding auxiliary materials, pounding to produce pulp, and forming and gelling, an elastin gel product that can be tested is formed. As can be seen from the above description, the entire process of processing raw fish into surimi products is time-consuming and energy-consuming, and the sewage treatment burden is heavy; some processes require professional equipment, and there are minimum requirements for the processing volume of raw fish. Furthermore, significant human error exists in key steps during the preparation of surimi and its products, such as process parameters (chopping intensity, time, and temperature control) and enema techniques. These errors can affect gel strength measurements, making it difficult to guarantee the repeatability and accuracy of traditional evaluation methods. This can lead to different results obtained by different technical operators performing the same test, a common phenomenon in the industry. Therefore, it is crucial to predict gel quality by directly evaluating the raw fish. This approach can, on the one hand, directly predict gel quality based on the characteristics of different batches of raw fish; on the other hand, it can help us screen raw materials suitable for surimi product processing.

[0004] At present, the prediction technology of surimi gel strength still has certain limitations. For example, Chinese patent CN119643477A discloses an online monitoring method for surimi thermal processing based on hyperspectral imaging technology. This technology realizes the prediction of gel strength by heating the pretreated surimi in a water bath in two stages and collecting hyperspectral data in the VNIR (400-1000nm) and NIR (900-1700nm) bands. However, this technology still has the following significant defects in practical application: First, this method must process the raw materials into surimi gel before prediction can be made, and it is impossible to achieve early quality assessment at the raw material stage, which is different from the demand for quality control of processed raw materials in industrial production. Summary of the Invention

[0005] To address the limitations of existing technologies, which cannot determine the suitability of raw fish for surimi processing based solely on their inherent properties before processing, this present invention provides a method for evaluating surimi quality based on muscle microstructure and endogenous enzyme activity. This method directly predicts the gel strength of processed surimi using muscle histological characteristics and endogenous enzyme activity parameters from the raw fish, thus avoiding the waste of raw materials, energy, and time associated with traditional evaluation methods.

[0006] The technical solutions of the present invention are as follows:

[0007] The present invention aims 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 headed, tailed, eviscerated, filleted, skinned, and trimmed to obtain fish fillets;

[0009] (2) taking dorsal muscle tissue from the fish fillet obtained in step (1), fixing it in glutaraldehyde fixative and osmium tetroxide fixative successively, washing it with buffer, performing gradient dehydration, then sectioning and staining it, observing the muscle tissue with a transmission electron microscope, and quantifying the sarcomere length of the muscle tissue using image processing software;

[0010] (3) taking back muscle tissue from the fish fillet obtained in step (1), extracting crude enzyme solution from the muscle tissue, fully incubating the crude enzyme solution with an enzyme reaction substrate, and measuring the activity of myofiber-bound cathepsin L in the fish fillet using a fluorescence spectrophotometer;

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

[0012] The correlation model is as follows:

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

[0014] Wherein, X1 is the sarcomere length quantified by ImageJ, in μm;

[0015] X2 is the activity of myofiber-bound cathepsin L in raw fish, in U / g;

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

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

[0018] In one embodiment of the present invention, in step (2), the muscle tissue is taken from the back white muscle 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 respectively.

[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 solution used for the buffer wash is 0.1 M 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 out and no obvious residue is left.

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

[0022] In one embodiment of the present invention, in step (2), the sectioning is performed to obtain an ultrathin section with a thickness of 50 to 100 nm; the staining is first performed using a 0.2% to 0.4% lead citrate solution for staining at room temperature for 5 to 10 minutes, followed by staining using a 50% ethanol saturated solution of uranyl acetate for staining at room temperature for 5 to 10 minutes.

[0023] In one embodiment of the present invention, in step (3), enzyme activity refers to the enzyme's catalytic ability to convert micromoles of substrate per second under specific enzyme reaction conditions. The unit (U) represents the enzyme's activity at catalytic conversion of 1 micromole of substrate per second under the 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, in step (3), the method for extracting the crude enzyme solution from the muscle tissue is as follows: mince the muscle tissue and homogenize it with three times its weight of 20 mM phosphate buffer (pH 7.5), and then incubate at 4°C, 8000

[0025] Centrifuge at × g for 15 min;

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

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

[0028] The obtained supernatant was adjusted to pH 5.5 using 2M HCl and centrifuged again to remove the precipitate. The pH value of the supernatant was adjusted to 7.5 using 1M NaOH and filtered to obtain the filtrate 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), and 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 is incubated with 500 μL of 150 mM Bis-Tris buffer at 37°C for 10 min, followed by the addition of 500 μL of enzyme reaction substrate, followed by incubation for 15 min, and the reaction is terminated with 3 mL of 50 mM Bis-Tris;

[0031] In one embodiment of the present invention, 150 mM Bis-Tris buffer contains 30 mM EDTA (ethylenediaminetetraacetic acid), 6 mM DTT (dithiothreitol), and has a pH of 6.0; 50 mM 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 is processed by meat extraction, rinsing, fine filtering and dehydration, its moisture content is uniformly adjusted to 80%;

[0034] The finely filtered surimi with uniform moisture content is mixed with ingredient additives (based on the mass of the finely filtered surimi with uniform moisture content, 7.5% sucrose, 0.125% sodium tripolyphosphate and 0.125% sodium pyrophosphate are added), and the mixture is stored at -18°C to prepare frozen surimi.

[0035] The frozen surimi was heated in two stages (first stage: 40°C, 1 hour, second stage: 90°C, 15 minutes) to prepare surimi gel, and the gel strength was measured.

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

[0037] The beneficial technical effects of the present invention are:

[0038] The present invention predicts the gel strength value by measuring the microstructure and endogenous enzyme activity of fish meat raw materials. The gel strength can be quickly predicted without the need to prepare fish paste and fish paste products through cumbersome processes, thereby scientifically screening freshwater fish raw materials with different levels of gel strength. This will effectively solve the blindness problem in the traditional fish species screening process, significantly improve the efficiency and scientificity of fish paste raw material selection, greatly simplify the screening process, save time, cost and raw materials, etc., have important application value and broad promotion prospects, and can provide reliable prediction technology support for the fish paste products industry.

[0039] In the evaluation method of the present invention, there is a significant correlation between the muscle microstructure and endogenous enzyme activity indicators and the gel strength, which breaks away from the limitations of relying on experience or subjective sensory judgment. The former reflects the basic structural state of the raw material, embodies the integrity, arrangement characteristics and initial network structure of the myofibrils, and has high stability and representativeness; the latter represents the potential ability to degrade proteins, especially myofiber-bound cathepsin L, which is not easily lost during the rinsing process and can continuously reflect the quality changes of the raw materials. Its role has an important influence on the degradation of proteins and the formation of the gel network. The selection of the above two indicators provides objective and reliable data support and decision-making basis for raw material screening and quality control, greatly improving the scientificity and accuracy of the evaluation.

[0040] The present invention directly uses raw fish muscle tissue as the research object, establishes a correlation model between the sarcomere length of muscle tissue, the activity of myofiber-bound cathepsin L and the gel strength of surimi, and realizes the prediction and calculation of the gel strength based on the raw material indicators without processing the surimi into a product, thereby directly evaluating the gel quality of the surimi.

[0041] Compared with the existing technology, there are the following substantial differences:

[0042] 1. Innovative testing method: Direct testing of raw fish muscle tissue (not finished surimi gel) enables early evaluation of raw materials before processing to predict gel quality, avoiding the cumbersome process of traditional preparation methods.

[0043] 2. Innovation in prediction parameter selection: For the first time, the ultrastructural parameter (sarcomere length) and the enzyme activity parameter (myofiber-bound cathepsin L activity) were combined for modeling. The prior art did not disclose the correlation between this parameter combination and gel strength. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0045] Figure 2 The muscle fiber-bound cathepsin L activity of three tilapia species was determined in Examples 1, 2, and 3 of the present invention.

[0046] Figure 3 The gel strength of three tilapia fish paste gels prepared in Examples 1, 2 and 3 of the present invention was measured. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below 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, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] Determination of gel strength of fish meat-based surimi gel

[0051] After the raw fish was minced, rinsed, and finely filtered, its moisture content was adjusted to 80%. The finely filtered surimi was mixed with additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18°C to produce frozen surimi. The frozen surimi was then heated in a two-stage process (first stage: 40°C for 1 hour, second stage: 90°C for 15 minutes) to prepare surimi gel. The surimi gel was cut into 25 mm high cylinders and equilibrated at 25°C for 2 hours. The breaking force and breaking distance of the surimi 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).

[0052] Example 1

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

[0054] (1) Fresh GIFT tilapia is chopped, tailed, eviscerated, filleted, skinned, and trimmed to obtain fillets;

[0055] (2) The dorsal muscle tissue was taken from the obtained fish fillets, fixed in glutaraldehyde fixative and osmium tetroxide fixative successively, washed with buffer solution, and then dehydrated by gradient treatment. The muscle tissue was then sliced ​​and stained. The myometrium length X1 of GIFT tilapia was 1.81±0.02 μm ( Figure 1 A and Figure 1 D);

[0056] (3) The back muscle tissue was taken from the fish fillet, and the crude enzyme solution in the muscle tissue was extracted by heating method. The crude enzyme solution and the enzyme reaction substrate were fully incubated, and the activity of myofiber-bound cathepsin L in the fish fillet was determined by fluorescence spectrophotometry. The content of myofiber-bound cathepsin L in GIFT tilapia was 0.021±3.4992E-5U / g( Figure 2 ).

[0057] (4) The values ​​of X1 and X2 obtained in steps (2) and (3) were substituted into the multiple regression model, i.e., Y = 3462.716-525.776×X1-15463.897×X2, and the gel strength of GIFT tilapia surimi was calculated to be 2186.32 g·cm.

[0058] (5) Fresh GIFT tilapia was minced, rinsed, and finely filtered, and its moisture content was uniformly adjusted to 80%. The finely filtered surimi was mixed with ingredient additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18°C to produce frozen surimi. The frozen surimi was then heated in two stages (first stage: 40°C for 1 hour, second stage: 90°C for 15 minutes) to prepare surimi gel. The surimi gel was cut into 25 mm high cylinders and equilibrated at 25°C for 2 hours. The breaking force and breaking distance of the surimi 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±98.11 g·cm, and the relative error between the predicted and tested results was 3.77%.

[0059] Example 2

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

[0061] (1) Fresh Aurelia tilapia is chopped, tailed, eviscerated, filleted, skinned, and trimmed to obtain fillets;

[0062] (2) The dorsal muscle tissue was taken from the obtained fish fillets, fixed in glutaraldehyde fixative and osmium tetroxide fixative successively, washed with buffer solution, and then dehydrated by gradient treatment. The tissue was then sliced ​​and stained. The myometrium length X1 of the Aureus tilapia was 2.32±0.07 μm ( Figure 1 B and Figure 1 D).

[0063] (3) The back muscle tissue was taken from the fish fillet, and the crude enzyme solution in the muscle tissue was extracted by heating method. The crude enzyme solution and the enzyme reaction substrate were fully incubated, and the activity of muscle fiber-bound cathepsin L in the fish fillet was determined by fluorescence spectrophotometer. The content X2 of muscle fiber-bound cathepsin L in Aurelia tilapia was 0.054±3.30289E-4U / g( Figure 2 ).

[0064] (4) The values ​​of X1 and X2 obtained in steps (2) and (3) were substituted into the multiple regression model, i.e., Y = 3462.716-525.776×X1-15463.897×X2, and the gel strength of the tilapia surimi was calculated to be 1407.86 g·cm.

[0065] (5) Fresh tilapia (Aurelia tilapia) was minced, rinsed, and finely filtered, and its moisture content was uniformly adjusted to 80%. The finely filtered surimi was mixed with ingredient additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18°C to produce frozen surimi. The frozen surimi was then heated in two stages (first stage: 40°C for 1 hour, second stage: 90°C for 15 minutes) to prepare surimi gel. The surimi gel was cut into 25 mm high cylinders and equilibrated at 25°C for 2 hours. The breaking force and breaking distance of the surimi 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±55.79 g·cm, and the relative error between the predicted and tested results was 2.47%.

[0066] Example 3

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

[0068] (1) Fresh red tilapia is chopped, tailed, eviscerated, filleted, skinned, and trimmed to obtain fillets;

[0069] (2) The back muscle tissue was taken from the obtained fish fillet, fixed in glutaraldehyde fixative and osmium tetroxide fixative successively, washed with buffer solution, and then dehydrated by gradient, then sliced ​​and stained. The myometrium length X1 of red tilapia was 2.04±0.09 μm ( Figure 1 C and Figure 1 D).

[0070] (3) The back muscle tissue was taken from the fish fillet, and the crude enzyme solution in the muscle tissue was extracted by heating method. The crude enzyme solution and the enzyme reaction substrate were fully incubated, and the activity of myofiber-bound cathepsin L in the fish fillet was determined by fluorescence spectrophotometry. The content of myofiber-bound cathepsin L in red tilapia was 0.021±3.4992E-5U / g ( Figure 2 ).

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

[0072] (5) Fresh red tilapia was minced, rinsed, and finely filtered, and its moisture content was uniformly adjusted to 80%. The finely filtered surimi was mixed with ingredient additives (7.5% sucrose, 0.125% sodium tripolyphosphate, and 0.125% sodium pyrophosphate) and stored at -18°C to produce frozen surimi. The frozen surimi was then heated in a two-stage process (first stage: 40°C for 1 hour, second stage: 90°C for 15 minutes) to prepare surimi gel. The surimi gel was cut into 25 mm high cylinders and equilibrated at 25°C for 2 hours. The breaking force and breaking distance of the surimi 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±146.46 g·cm, and the relative error between the predicted and tested results was 2.38%.

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

[0074] Table 1 Comparison of the prediction results of the method of the present invention and the gel strength determination results of the standard method

[0075] sample <![CDATA[X1(μm)]]> <![CDATA[X2(U / g)]]> Predicted 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] It can be seen from the data in Table 1 that the gel strength predicted by the evaluation method constructed in the present invention is in good agreement with the results measured by the texture analyzer.

[0077] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for evaluating surimi quality based on muscle microstructure and endogenous enzyme activity, characterized in that: The steps include: (1) The raw fish is headed, tailed, eviscerated, filleted, skinned, and trimmed to obtain fish fillets; (2) taking dorsal muscle tissue from the fish fillet obtained in step (1), fixing it in glutaraldehyde fixative and osmium tetroxide fixative successively, washing it with buffer, performing gradient dehydration, then sectioning and staining it, observing the muscle tissue with a transmission electron microscope, and quantifying the sarcomere length of the muscle tissue using image processing software; (3) taking back muscle tissue from the fish fillet obtained in step (1), extracting crude enzyme solution from the muscle tissue, fully incubating the crude enzyme solution with an enzyme reaction substrate, and measuring the activity of myofiber-bound cathepsin L in the muscle tissue using a fluorescence spectrophotometer; (4) Based on the correlation model between the sarcomere length of muscle tissue, the activity of myofiber-bound cathepsin L, and the gel strength of surimi, the gel strength of surimi made from the selected raw fish was calculated, and the gel quality of surimi was evaluated; The correlation model is as follows: Y=3462.716-525.776×X1-15463.897×X2; Wherein, X1 is the sarcomere length quantified by ImageJ, in μm; X2 is the activity of myofiber-bound cathepsin L in raw fish, in U / g; Y is the gel strength, in g·cm.

2. The method according to claim 1, characterized in that In step (1), the raw fish is fresh freshwater fish, including one of Nile tilapia, red tilapia, Aurelian tilapia and Gift tilapia.

3. The method according to claim 1, characterized in that In step (2), the muscle tissue is taken from the back white muscle 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 respectively.

4. The method according to claim 1, wherein 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.

5. The method according to claim 1, wherein In step (2), the gradient dehydration treatment uses gradient ethanol concentrations of 30%, 50%, 70%, 80%, 90%, and 100%, and the dehydration time for each level is 15 minutes.

6. The method according to claim 1, characterized in that In step (2), the section is cut to obtain an ultrathin section with a thickness of 50 to 100 nm; and the staining is performed by using a lead citrate solution and a 50% ethanol saturated solution of uranyl acetate in sequence.

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

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

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

10. The method according to claim 9, characterized in that 150 mM Bis-Tris buffer contains 30 mM EDTA, 6 mM DTT, pH 6.0; 50 mM Bis-Tris buffer contains 1% SDS, pH 7.0.

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