A method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells

Abstract

This invention relates to the field of silkworm cocoon shell antioxidant detection technology, and provides a method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells. The method is based on the ABTS free radical scavenging method and includes a multi-frequency ultrasonic device. The multi-frequency ultrasonic device treats the silkworm cocoon shell with ultrasonic waves at three gradient frequencies, transforming the internal structure of the silkworm cocoon shell from a dense fibrous structure to a porous fibrous structure. The porous fibrous structure of the silkworm cocoon shell comes into contact with the oxidant solution and transfers mass to form a reaction solution. The reaction solution is placed in a quartz cuvette, which is equipped with an ultrasonic transducer and a temperature control module. When detecting the ABTS free radical scavenging ability of silkworm cocoon shells, the quartz cuvette is placed in a spectrophotometer, and the ABTS free radical scavenging rate of the silkworm cocoon shell is obtained by spectrophotometry.

Description

Technical Field

[0001] This invention relates to the field of silkworm cocoon shell antioxidant detection technology, and provides a method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells. Background Technology

[0002] Silkworm cocoon shells are a byproduct of silk production, possessing certain medicinal value. Furthermore, the silk from these cocoons can be used to make silk clothing, giving them significant commercial value. Silkworm cocoon shells are primarily composed of silk proteins (fibroin and sericin) along with small amounts of lipids, pigments, and minerals. Sericin contains numerous active groups such as hydroxyl and amino groups, which can scavenge free radicals (such as ROS and DPPH free radicals), exhibiting antioxidant activity. The antioxidant properties of silkworm cocoon shells make them suitable for applications in food preservatives, cosmetic antioxidants, and medical dressings, demonstrating their potential commercial value.

[0003] Currently, for the detection of antioxidant activity in biological materials, the ABTS free radical scavenging method, as an internationally standardized method, is widely used in the detection of liquid biological samples (such as serum and plant extracts). The detection of ABTS free radical scavenging ability is a commonly used method for assessing the antioxidant activity of substances. Its detection principle is that ABTS generates stable blue-green free radical cations under the action of oxidants, which have a maximum absorption peak at 734 nm. When antioxidants react with these blue-green free radical cations, the free radicals are scavenged, and the absorbance decreases. The free radical scavenging ability of the sample can be calculated by observing the change in absorbance.

[0004] However, existing ABTS free radical scavenging methods have certain technical limitations when detecting silkworm cocoon shell samples: 1. Solubility limitations: Standardized methods require the sample to be completely soluble in the reaction solution, but silkworm cocoon shells, as solid biological materials, are difficult to dissolve in solution. 2. Interface effects: Silkworm cocoon shells are solid, while the reaction solution is liquid; low solid-liquid contact efficiency leads to low free radical scavenging efficiency, making it difficult to accurately detect the free radical scavenging ability of silkworm cocoon shells. 3. After the solid-liquid reaction, the presence of solid macromolecules causes light scattering, leading to errors in subsequent absorbance detection and making it difficult to control the accuracy of the detection results. Summary of the Invention

[0005] To address the aforementioned deficiencies, the present invention aims to provide a method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells, thereby solving the problems mentioned in the background art and detecting the antioxidant properties of solid silkworm cocoon shell samples.

[0006] A method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells, the specific steps of which are as follows:

[0007] S1. Prepare the oxidant solution.

[0008] S2. Mix silkworm cocoon shell powder with oxidant solution to form a mixture.

[0009] S3. The mixture is subjected to three-level gradient frequency ultrasonic treatment by a multi-frequency ultrasonic device to transform the internal structure of the silkworm cocoon shell from a dense fibrous structure to a porous fibrous structure. The porous fibrous structure is mixed and reacted in the oxidant solution to form a reaction solution.

[0010] S4. Place the reaction solution into a spectrophotometer and obtain the ABTS free radical scavenging rate of the silkworm cocoon shell through the spectrophotometer.

[0011] Preferably, in step S2, the method for preparing the silkworm cocoon shell powder is as follows:

[0012] The silkworm cocoon shell samples were placed in a vacuum drying oven and dried until the samples were completely dehydrated. The dried cocoon shells were then pulverized using a low-temperature pulverizer and passed through an 80-100 mesh sieve to obtain a uniform powder structure.

[0013] Preferably, in step S1, preparing the oxidant solution includes the following steps:

[0014] S2.1 Weigh ABTS, dilute to volume with ultrapure water, vortex for ≥30 seconds, store at 4℃ in the dark to prepare ABTS stock solution;

[0015] S2.2 Weigh potassium persulfate, dilute to volume with ultrapure water, and dissolve the potassium persulfate solid in ultrapure water with ultrasonic assistance to prepare an oxidant stock solution.

[0016] S2.3. Mix ABTS stock solution and oxidant stock solution at a volume ratio of 1:1 and let stand in the dark for 12 ± 0.5 hours; dilute with pH 7.4 phosphate buffer to an absorbance of 734 nm.

[0017] Preferably, in step S2, the liquid-to-solid ratio of the oxidant solution to the silkworm cocoon shell powder is 175:1.

[0018] Preferably, based on a multi-frequency ultrasonic device, the three-level gradient frequency ultrasonic processing includes the following steps:

[0019] S3.1 When the silkworm cocoon shell and the oxidant solution are just mixed, the multi-frequency ultrasonic device is adjusted to a frequency of 35kHz and a power density of 0.5W / cm², and continues to work for 3 minutes to expand the internal pore size of the silkworm cocoon shell to 5-8μm.

[0020] S3.2 After the silkworm cocoon shell is pretreated by ultrasound, and its internal structure changes from a dense fiber structure to a porous fiber structure, the multi-frequency ultrasonic device is adjusted to a frequency of 40kHz and a power density of 0.8W / cm², and continues to work for 5 minutes.

[0021] S3.3 After the silkworm cocoon shell has reacted with the oxidant solution, the multi-frequency ultrasonic device is adjusted to a frequency of 28kHz and a power density of 0.3W / cm², and continues to work for 2 minutes.

[0022] Preferably, in step S3, the reaction solution is placed in a low-temperature centrifuge and centrifuged at 12000×g for 10 minutes at 4°C, and the supernatant is taken in step S4.

[0023] Preferably, in step S4, the reaction solution is placed in a quartz cuvette, and the absorbance value is measured at a wavelength of 734 nm using a spectrophotometer. The measurement is performed in parallel at least 4 times, and the arithmetic mean is taken to obtain the ABTS free radical scavenging rate of the silkworm cocoon shell.

[0024] Preferably, the quartz cuvette is equipped with an ultrasonic transducer and a temperature control module. The ultrasonic transducer is connected to a multi-frequency ultrasonic device, and the temperature control module is connected to an external power supply.

[0025] Therefore, the beneficial effects of this method are as follows:

[0026] By incorporating a multi-frequency ultrasonic device during the mixing and reaction of silkworm cocoon shells with a free radical working solution, and utilizing a three-stage gradient ultrasonic treatment of the cocoon shells, the following steps are employed: First, the pretreatment stage of ultrasonic treatment expands the cavitation bubbles inside the cocoon shells, forming a porous fiber structure. Second, the main reaction stage of ultrasonic treatment accelerates the solid-liquid mass transfer between the cocoon shells and the free radical working solution, thus speeding up the reaction. Finally, the post-stabilization stage of ultrasonic treatment eliminates microbubbles generated by ultrasound in the reaction solution, thereby eliminating interference from microbubbles on absorbance detection and improving detection accuracy.

[0027] By placing the clear reaction solution obtained from centrifugation into a quartz cuvette, the solid and liquid molecules are vibrated uniformly using an ultrasonic transducer. A temperature control component is set to control the temperature, promoting the movement and activity between solid and liquid molecules and accelerating uniform mixing. The quartz cuvette with a polished inner wall is then used to enhance light transmittance and reduce the influence of light scattering on absorbance detection, thereby further improving the accuracy of the detection results. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] In the natural environment, free radicals are reactive molecules. Due to the presence of unpaired electrons, free radicals are extremely unstable and need to acquire external electrons or release their own electrons to reach a stable state. Therefore, free radicals have strong oxidizing or reducing properties. Free radicals generally play an important role in the immune system of living organisms, and some biological materials have antioxidant properties. When using these biological materials as raw materials to generate antioxidants or preservatives, free radical detection can be performed on the biological materials to assess their antioxidant properties.

[0030] In existing detection techniques, the ABTS free radical scavenging method is generally used to detect the antioxidant properties of sample materials. The principle is that ABTS generates stable blue-green free radical cations under the action of an oxidant, which have a maximum absorption peak at 734 nm. When an antioxidant is added and reacts with the green free radicals, the free radicals are scavenged, and the absorbance decreases. The free radical scavenging ability of the sample can be calculated by observing the change in absorbance, thus detecting the antioxidant properties of the sample material.

[0031] Example 1

[0032] This invention is based on the ABTS free radical scavenging method. In order to detect the antioxidant properties of silkworm cocoon shells, a multi-frequency ultrasonic device is set up to treat the silkworm cocoon shells with a three-level gradient ultrasonic treatment. This modulates the pore size and fiber structure inside the silkworm cocoon shells, which are solid biomaterials. The porous structure increases the mass transfer efficiency between the silkworm cocoon shells and the oxidant solution, thereby improving the reaction efficiency.

[0033] Furthermore, the multi-frequency ultrasonic device mainly consists of an ultrasonic generator and an ultrasonic transducer, which are existing equipment. The ultrasonic generator produces electrical signals of different frequencies, and the ultrasonic transducer converts electrical energy into high-frequency mechanical vibrations through the piezoelectric effect, which are then transmitted to the load (liquid or solid).

[0034] In this embodiment, since the silkworm cocoon shell is a solid structure, it needs to be mixed and reacted with the oxidant solution. After mixing, the internal pores are expanded by a multi-frequency ultrasonic device. Therefore, the working principle of the multi-frequency ultrasonic device is to generate cavitation bubbles in the reaction solution by vibration (ultrasonic cavitation effect). The bursting of several cavitation bubbles is used to expand the dense fibrous structure of the silkworm cocoon shell and form a porous fibrous structure inside it.

[0035] In the process of mixing and reacting the silkworm cocoon shell with the oxidant solution, the multi-frequency ultrasonic device uses a three-level gradient frequency ultrasonic treatment to treat the silkworm cocoon shell, thereby matching the entire reaction process.

[0036] When the silkworm cocoon shell is first mixed with the oxidant solution, it is the pretreatment stage: the multi-frequency ultrasonic device is adjusted to a frequency of 35kHz and a power density of 0.5W / cm², and works continuously for 3 minutes to expand the internal pore size of the silkworm cocoon shell to 5-8μm.

[0037] Preferably, 35kHz falls within the low-to-medium frequency range. Low-frequency ultrasound generates larger cavitation bubbles with stronger impact, which can effectively expand the aperture. By utilizing a long-term (3min) low-power effect, the aperture is gradually opened through the long-term cumulative effect of cavitation, reducing the occurrence of cocoon silk breakage caused by instantaneous high-power cavitation.

[0038] After the silkworm cocoon shell is pretreated with ultrasound to produce a porous fiber structure, the main reaction stage begins: the multi-frequency ultrasonic device is adjusted to a frequency of 40kHz and a power density of 0.8W / cm², and continues to work for 5 minutes to accelerate the solid-liquid mass transfer process between the silkworm cocoon shell and the oxidant solution.

[0039] Preferably, the ultrasonic frequency is increased to 40kHz to enhance cavitation intensity and promote the penetration of liquid microfluidics into the cocoon layer, thereby improving the mass transfer efficiency between solid and liquid and making the two mix more evenly. At this time, because a porous fiber structure has already been formed inside the cocoon shell, increasing the cavitation intensity will not cause the cocoon silk to break. Then, under the action of a longer time (5min) and higher power (0.8W), the diffusion effect of the antioxidant solution in the porous fiber structure inside the cocoon shell is accelerated, and the reaction efficiency is improved.

[0040] After the silkworm cocoon shell reacts with the oxidant solution, the post-stabilization stage begins: the multi-frequency ultrasonic device is adjusted to a frequency of 28kHz and a power density of 0.3W / cm², and operates continuously for 2 minutes to eliminate microbubbles generated by cavitation during the main reaction stage.

[0041] Preferably, a lower ultrasonic frequency of 28kHz is used, which makes it easier to generate large-sized bubbles. The microbubbles generated during the reaction stage will merge and float together under the induction of the large-sized bubbles, thus leaving the pore fiber structure of the silkworm cocoon shell. In addition, the low power of 0.3W can reduce the generation of bubbles and avoid generating too many bubbles. At the same time, the acoustic flow effect can dissipate residual bubbles, eliminate microbubble interference, reduce the impact of microbubbles on the subsequent reaction detection process, and improve the accuracy of the detection results.

[0042] Furthermore, after the silkworm cocoon shell and oxidant solution are reacted using a multi-frequency ultrasonic device to generate a reaction solution, the reaction solution is placed in a centrifuge for solid-liquid separation. The clear liquid in the centrifuged reaction solution is then placed in a quartz cuvette. Because solid-liquid mass transfer occurs in the reaction solution, the clear liquid contains mixed solid macromolecules. These solid macromolecules cause uneven distribution of the medium in the clear liquid, resulting in light scattering and reducing the accuracy of absorbance detection.

[0043] Therefore, in order to improve the accuracy of detecting the antioxidant properties of silkworm cocoon shells, in this embodiment, a cuvette container made of high-transmittance quartz material is used to hold the clear liquid. Based on the existing quartz cuvette, its inner wall is polished to further increase the light transmittance of the quartz cuvette. An ultrasonic transducer and temperature control module are quickly attached to the quartz cuvette to ensure uniform distribution of the medium in the clear liquid and reduce light scattering. Then, the quartz cuvette is placed separately in a spectrophotometer, and its absorbance value is measured at a wavelength of 734nm to detect the antioxidant properties of the silkworm cocoon shell.

[0044] Polishing the inner wall of the quartz cuvette increases its light transmittance and prevents it from interfering with light transmission. A quick-release ultrasonic transducer is attached to the cuvette to uniformly mix the various media in the solution through vibration. A quick-release temperature control module (Parl patch) controls the temperature in the solution, promoting the movement of molecules and accelerating mixing. These three elements work together to effectively reduce light scattering.

[0045] Furthermore, in this embodiment, the antioxidant capacity of silkworm cocoon shells is detected based on the ABTS free radical scavenging method. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells is as follows:

[0046] S1. Place the silkworm cocoon shell sample in a vacuum drying oven, set the temperature to 60±2℃ and the vacuum degree to -0.1MPa, and dry until the sample is completely dehydrated to avoid moisture affecting subsequent experiments; use a low-temperature pulverizer (to avoid thermal degradation) to pulverize the dried cocoon shell and pass it through an 80-100 mesh sieve to obtain a uniform powder structure. The powder structure increases the contact area between the cocoon shell and the solution, thus accelerating the reaction.

[0047] S2. Prepare the free radical working solution (oxidant solution).

[0048] ABTS generates stable blue-green ABTS+ free radical cations (free radical working solution) under the action of potassium persulfate, which have a characteristic absorption peak at 734 nm. When the antioxidant (silkworm cocoon shell) comes into contact with the ABTS+ free radical working solution, free radicals are removed through electron transfer mechanism, resulting in a decrease in the absorbance of the system. The greater the decrease in absorbance, the stronger the antioxidant activity of the silkworm cocoon shell.

[0049] The specific steps for preparing a free radical working solution are as follows:

[0050] S2.1 Accurately weigh ABTS, dilute to volume with ultrapure water (to reduce the content of free ions in the water), vortex for ≥30 seconds, store at 4℃ in the dark, and prepare ABTS stock solution.

[0051] S2.2 Accurately weigh potassium disulfate, dilute to volume with ultrapure water, and dissolve the solid potassium disulfate in water with ultrasonic assistance (40kHz, 5 minutes) to prepare an oxidant stock solution.

[0052] S2.3 Mix the ABTS stock solution and the oxidant stock solution at a volume ratio of 1:1 and let stand in the dark for 12 ± 0.5 hours; dilute with pH 7.4 phosphate buffer (0.1 mol / L) to an absorbance of 734 nm. The working solution should be used within 4 hours to reduce the possibility of changes in ions and absorbance within the solution over a long period of time.

[0053] S3. Mix the free radical working solution with the silkworm cocoon shell powder at a liquid-to-solid ratio of 175:1.

[0054] S4. The mixed solid and liquid are subjected to three-level gradient ultrasonic treatment to obtain the reaction solution generated after the free radical working solution reacts with the silkworm cocoon shell.

[0055] S5. Place the reaction solution in a low-temperature centrifuge and centrifuge at 12000xg for 10 minutes at 4°C. Take the supernatant to avoid interference from suspended particles.

[0056] S6. Using a quartz cuvette (optical path 4.00±0.01mm), measure the absorbance value at a wavelength of 734nm using a spectrophotometer. Perform at least four parallel measurements and take the arithmetic mean (to avoid random errors) to obtain the ABTS free radical scavenging rate of the silkworm cocoon shell.

[0057] Experimental Example 1

[0058] Compared with the traditional ABTS free radical detection method, the above-mentioned method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells uses a multi-frequency ultrasonic device to treat the silkworm cocoon shells and utilizes a three-level gradient ultrasonic treatment to improve the solid-liquid mass transfer efficiency of the silkworm cocoon shell solids in the free radical working solution, accelerate the reaction time, and thus reduce the time of the entire detection process.

[0059] In the traditional ABTS free radical detection method, when detecting solid substances, the solid substances and free radical working solution need to be mixed and shaken at a constant temperature for at least 4 hours to ensure that the solid substances and free radical working solution are fully mixed and reacted.

[0060] In this embodiment, please refer to Tables 1 and 2. In order to determine the optimal time range for the solid material and the free radical working fluid to fully mix and react during the entire three-level gradient ultrasonic treatment process, multiple control groups with different ultrasonic device working times were set up to determine the optimal three-level gradient ultrasonic treatment time.

[0061] The configuration conditions for each experimental group are as follows: 0.02 g of silkworm cocoon shell powder per test, 3.5 mL of free radical working solution per test, and the experiment at different times for each group is repeated 4 times. The detection time is based on the 4 parallel measurements.

[0062] Table 1: ABTS radical detection time under three-level gradient ultrasonic treatment

[0063]

[0064] As shown in Table 1, when the total ultrasonic time of the ultrasonic device reaches 30 minutes, the free radical scavenging rate of the entire silkworm cocoon sample reaches about 59.4%. Compared with the data at subsequent times, the scavenging rate tends to be stable, indicating that the reaction time is sufficient for accurate detection. Therefore, the total ultrasonic time of the ultrasonic device should be controlled at 30 minutes to achieve the detection effect.

[0065] Table 2: ABTS radical detection time under three-level gradient ultrasonic treatment

[0066]

[0067] Within a time range of 5-30 minutes, the detection interval was further reduced, resulting in the data in Table 2. As shown in Table 2, when the overall ultrasonic time of the ultrasonic device reached 10 minutes, the free radical scavenging rate of the entire silkworm cocoon shell sample reached approximately 59.3%. Compared with the data from subsequent times, the scavenging rate tended to stabilize, indicating that this reaction time was sufficient for accurate detection. Therefore, the overall ultrasonic time of the ultrasonic device should be controlled at 10 minutes to achieve the desired detection effect.

[0068] Comparative experimental results show that, under three-level gradient ultrasonic treatment, the total reaction time of mixing solid substances with free radical working fluid is reduced by about 96% compared with the traditional reaction time, thus shortening the overall detection time.

[0069] Experimental Example 2

[0070] In this embodiment, the three-stage gradient ultrasound includes a pretreatment stage, a main reaction stage, and a post-stabilization stage. The pretreatment stage (frequency 35 kHz) is used to form a porous fiber structure inside the silkworm cocoon shell; the main reaction stage (frequency 40 kHz) is used to increase the solid-liquid transfer efficiency between the silkworm cocoon shell and the free radical working fluid; and the post-stabilization stage (frequency 28 kHz) is used to eliminate microbubbles generated by ultrasound in the reaction solution, thereby eliminating microbubble interference and improving detection accuracy.

[0071] In order to improve the accuracy of detecting the antioxidant properties of silkworm cocoon shells in this embodiment, a post-stabilization stage is set in the three-level gradient ultrasound. In order to determine whether the post-stabilization stage improves the accuracy of the detection process, please refer to Tables 3 and 4 and conduct a comparative experiment with ultrasound treatment containing the post-stabilization stage and ultrasound treatment without the post-stabilization stage as controls.

[0072] The configuration conditions for each experimental group were as follows: 0.02 g of silkworm cocoon shell powder per test, 3.5 mL of free radical working solution per test. Before the ABTS free radical scavenging ability test, the average absorbance of the silkworm cocoon shell sample was set to A0. After the ABTS free radical scavenging ability test, the average absorbance of the silkworm cocoon shell sample was set to A1. Each experiment was repeated 6 times, with a blank control group added to eliminate the occurrence of random outliers.

[0073] Table 3: Three-stage ultrasonic treatment (pretreatment + main reaction + post-stabilization)

[0074]

[0075] As shown in Table 3, in the three-stage ultrasonic treatment with a post-stabilization phase, the average free radical scavenging rate of the silkworm cocoon shell samples was 59.37±0.04%, and the average turbidity was 1.2±0.1 NTU (the solution was clear and there were no visible bubbles).

[0076] Table 4: Three-stage ultrasonic treatment (pretreatment + main reaction, no post-stabilization phase)

[0077]

[0078] As shown in Table 4, in the three-stage ultrasonic treatment without a post-stabilization stage, the average free radical scavenging rate of the silkworm cocoon shell samples was 58.52±0.18%, and the average turbidity was 9.2±0.3 NTU (the solution was turbid and contained suspended bubbles).

[0079] The comparative experimental results show that the experimental group without a post-stabilization phase had a higher mean absorbance A1 due to interference from suspended microbubbles, resulting in a slight decrease in the scavenging rate. This indicates that residual microbubbles during ultrasonic treatment affect the accuracy of the ABTS free radical scavenging ability detection of the entire sample. Therefore, a post-stabilization phase ultrasonic treatment process is needed to improve the accuracy of the detection data.

[0080] Experimental Example 3

[0081] In the prior art, ultrasonic treatment is also often used in the treatment of chemical substances (such as extraction processes) to improve the solid-liquid mass transfer effect between reactants and reaction solutions through continuous ultrasonic treatment.

[0082] In this embodiment, a multi-frequency ultrasonic device is also set up to sequentially treat the silkworm cocoon shell sample with different frequencies using a three-level gradient ultrasonic device, thereby accelerating the mixing reaction between the silkworm cocoon shell sample and the free radical working solution. In order to compare the effect of the three-level gradient ultrasonic treatment in this embodiment and the commonly used single-frequency ultrasonic treatment on the efficiency of solid-liquid mixing reaction, please refer to Tables 5 and 6. A single-frequency ultrasonic treatment experimental group and a three-level gradient ultrasonic treatment experimental group were set up for comparative experiments.

[0083] Table 5: ABTS radical detection time under single-frequency ultrasonic treatment

[0084]

[0085] Table 6: ABTS radical detection time under three-level gradient ultrasonic treatment

[0086]

[0087] The comparative experimental results show that the experimental group under single-frequency ultrasound treatment needs at least one hour of ultrasound treatment time to achieve the detection effect, while the experimental group under three-gradient ultrasound treatment can achieve the detection effect in 10 minutes. Under the same ultrasound treatment time, the three-gradient ultrasound treatment has a better ABTS free radical scavenging ability.

[0088] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.

Claims

1. A method for detecting the ABTS radical scavenging capacity of cocoon shell, characterized in that; S1, configure an oxidizing agent solution; S2, mix the silkworm cocoon shell powder with the oxidizing agent solution to form a mixed solution; S3, perform three-stage gradient frequency ultrasonic treatment on the mixed solution through a multi-frequency ultrasonic device, so that the internal structure of the silkworm cocoon shell is changed from a tight fiber structure to a pore fiber structure, and the pore fiber structure is mixed and reacted in the oxidizing agent solution to form a reaction solution; S4, place the reaction solution in a spectrophotometer, and obtain the ABTS free radical scavenging rate of the silkworm cocoon shell through the spectrophotometer; Based on the multi-frequency ultrasonic device, the three-stage gradient frequency ultrasonic treatment includes the following steps: S3.1, when the cocoon shell is mixed with the oxidizing agent solution, the multi-frequency ultrasonic device is adjusted to a frequency of 35 kHz and a power density of 0.5 W / cm 2 , and works for 3 min, expanding the internal pore diameter of the cocoon shell to 5-8 μm; S3.2, after the cocoon shell is pretreated by ultrasonic waves and the internal structure is changed from a tight fiber structure to a pore diameter fiber structure, the multi-frequency ultrasonic device is adjusted to a frequency of 40 kHz and a power density of 0.8 W / cm 2 , and works for 5 min; S3.3, after the cocoon shell is reacted with the oxidizing agent solution, the multi-frequency ultrasonic device is adjusted to a frequency of 28 kHz and a power density of 0.3 W / cm 2 , and works for 2 min; In step S4, the reaction solution is placed in a quartz cuvette, and the absorbance value is measured at a wavelength of 734 nm under the spectrophotometer, at least 4 times are determined in parallel, and the arithmetic mean value is taken to obtain the ABTS free radical scavenging rate of the silkworm cocoon shell; The quartz cuvette is provided with an ultrasonic transducer head and a temperature control module, the ultrasonic transducer head is connected to the multi-frequency ultrasonic device, and the temperature control module is connected to an external power supply.

2. The method for detecting the ABTS radical scavenging ability of cocoon shell according to claim 1, characterized in that, In step S2, the preparation method of the silkworm cocoon shell powder is as follows: place the silkworm cocoon shell sample in a vacuum drying box and dry until the sample is completely dehydrated; use a low-temperature pulverizer to crush the dried cocoon shell, pass through an 80-100 mesh screen, and obtain a uniform powder structure.

3. The method for detecting the ABTS radical scavenging capacity of cocoon shell according to claim 1, characterized in that, In step S1, the configuration of the oxidizing agent solution includes the following steps: S2.1, weigh the ABTS, dilute with ultrapure water, vortex for ≥30 seconds, store in the dark at 4℃, and prepare the ABTS stock solution; S2.2, weigh the potassium peroxydisulfate, dilute with ultrapure water, and dissolve the potassium peroxydisulfate solid in ultrapure water with ultrasonic assistance to prepare the oxidizing agent stock solution; S2.3, mix the ABTS stock solution and the oxidizing agent stock solution in a volume ratio of 1:1, avoid light and stand for 12±0.5 hours; dilute with pH 7.4 phosphate buffer to an absorbance of 734 nm.

4. The method for detecting the ABTS radical scavenging capacity of cocoon shell according to claim 1, characterized in that, In step S2, the liquid-solid ratio of the oxidizing agent solution and the silkworm cocoon shell powder is 175:

1.

5. The method for detecting the ABTS radical scavenging capacity of cocoon shell according to claim 1, characterized in that, In step S3, the reaction solution is placed in a low-temperature centrifuge, centrifuged at 12000×g at 4℃ for 10 minutes, and the supernatant is taken to step S4.

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