Method for detecting capability of removing ABTS free radicals from silkworm cocoon shells

Abstract

The invention is suitable for the technical field of silkworm cocoon shell oxidation resistance detection, and provides a silkworm cocoon shell ABTS free radical scavenging capability detection method, which is based on an ABTS free radical scavenging method, and comprises a multi-frequency ultrasonic device, the multi-frequency ultrasonic device performs three-stage gradient frequency ultrasonic treatment on silkworm cocoon shells, and the silkworm cocoon shells are subjected to three-stage gradient frequency ultrasonic treatment on the silkworm cocoon shells. The internal structure of the silkworm cocoon shell is converted into an aperture fiber structure from a compact fiber structure; the aperture fiber structure of the silkworm cocoon shell is in contact with the oxidant solution for mass transfer to form a reaction solution; the reaction liquid is placed in a quartz cuvette, and an ultrasonic transduction head and a temperature control module are arranged in the quartz cuvette; when the silkworm cocoon shell ABTS free radical scavenging capacity is detected, the quartz cuvette is placed in a spectrophotometer, and the silkworm cocoon shell ABTS free radical scavenging rate is obtained through the spectrophotometer.

Description

Technical Field

[0001] The present invention is applicable to the technical field of antioxidant property detection of silkworm cocoon shells, and provides a method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells. Background Art

[0002] Silkworm cocoon shells are products in silk production, have certain medicinal values, and their silk can be used to make silk clothes, having high commercial values. Silkworm cocoon shells mainly consist of silk proteins (fibroin and sericin), as well as a small amount of lipids, pigments and minerals. Sericin contains a large number of active groups such as hydroxyl groups and amino groups, can scavenge free radicals (such as ROS, DPPH free radicals), and has antioxidant activity. And the antioxidant property of silkworm cocoon shells enables them to be applied in fields such as food preservatives, antioxidant components of cosmetics, and medical dressings, having potential commercial values.

[0003] Nowadays, for the antioxidant detection of biological materials, the ABTS free radical scavenging method, as an international standard method, has been widely applied to the detection of liquid biological samples (such as serum, plant extracts, etc.). The detection of the ABTS free radical scavenging ability is a common method for evaluating the antioxidant activity of substances. Its detection principle is that ABTS generates a stable blue-green free radical cation under the action of an oxidant, which has a maximum absorption peak at 734 nm. When an antioxidant substance reacts with the blue-green free radical cation, the free radical is scavenged, and the absorbance decreases. The free radical scavenging ability of the sample can be calculated through the change in absorbance.

[0004] However, the existing ABTS free radical scavenging method has certain technical limitations when detecting silkworm cocoon shell samples: 1. Solubility limitation; the standard method requires that the test sample can be completely dissolved in the reaction solution, while silkworm cocoon shells, as solid biological materials, are difficult to be dissolved by the solution. 2. Interface effect; silkworm cocoon shells are solid, and the reaction solution is liquid. The 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, due to the existence of solid macromolecules, light scattering phenomenon will occur, resulting in errors in the subsequent detection of absorbance and making it difficult to control the accuracy of the detection results. Summary of the Invention

[0005] Aiming at the above defects, the purpose of the present invention is to provide a method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells, aiming to solve the problems raised in the background art and detect the antioxidant property of solid silkworm cocoon shell samples.

[0006] A method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells is as follows: S1. Prepare an oxidant solution.

[0007] S2. Mix the silk cocoon shell powder with the oxidant solution to form a mixed solution.

[0008] S3. Subject the mixed solution to three - level gradient frequency ultrasonic treatment through a multi - frequency ultrasonic device, transforming the internal structure of the silk cocoon shell from a dense fiber structure into a pore - fiber structure. The pore - fiber structure reacts in the oxidant solution to form a reaction solution.

[0009] S4. Place the reaction solution into a spectrophotometer, and obtain the ABTS free - radical scavenging rate of the silk cocoon shell through the spectrophotometer.

[0010] Preferably, in step S2, the preparation method of the silk cocoon shell powder is as follows: Place the silk cocoon shell sample in a vacuum drying oven and dry it until the sample is completely dehydrated; use a low - temperature grinder to crush the dried cocoon shell, and pass it through an 80 - 100 - mesh sieve to obtain a uniform powder structure.

[0011] Preferably, in step S1, the preparation of the oxidant solution includes the following steps: S2.1. Weigh ABTS, make up the volume with ultrapure water, vortex for ≥30 seconds, and store it in the dark at 4°C to prepare an ABTS stock solution. S2.2. Weigh potassium persulfate, make up the volume with ultrapure water, and dissolve the solid potassium persulfate in ultrapure water by ultrasonic assistance to prepare an oxidant stock solution. S2.3. Mix the ABTS stock solution and the oxidant stock solution in a volume ratio of 1:1, let it stand in the dark for 12 ± 0.5 hours; dilute it with a pH 7.4 phosphate buffer solution to an absorbance of 734 nm.

[0012] Preferably, in step S2, the liquid - to - solid ratio of the oxidant solution to the silk cocoon shell powder is 175:1.

[0013] Preferably, based on the multi - frequency ultrasonic device, the three - level gradient frequency ultrasonic treatment includes the following steps: S3.1. When the silk cocoon shell and the oxidant solution are just mixed, adjust the multi - frequency ultrasonic device to a frequency of 35 kHz and a power density of 0.5 W / cm², and keep working for 3 minutes to expand the internal pore diameter of the silk cocoon shell to 5 - 8 μm.

[0014] S3.2. After the silk cocoon shell undergoes ultrasonic pretreatment and its internal structure changes from a dense fiber structure to a pore - fiber structure, adjust the multi - frequency ultrasonic device to a frequency of 40 kHz and a power density of 0.8 W / cm², and keep working for 5 minutes.

[0015] S3.3. After the reaction between the silk cocoon shell and the oxidant solution is completed, adjust the multi - frequency ultrasonic device to a frequency of 28 kHz and a power density of 0.3 W / cm², and keep working for 2 minutes.

[0016] Preferably, in step S3, the reaction solution formed is placed in a low-temperature centrifuge and centrifuged at 12,000×g for 10 minutes at 4°C, and the supernatant is taken for step S4.

[0017] Preferably, in step S4, the reaction solution is put into a quartz cuvette, and the absorbance value is measured at a wavelength of 734 nm by a spectrophotometer. It is measured in parallel at least 4 times, and the arithmetic mean is taken to obtain the ABTS free radical scavenging rate of the cocoon shell.

[0018] Preferably, an ultrasonic transducer and a temperature control module are arranged inside the quartz cuvette. The ultrasonic transducer is connected to a multi-frequency ultrasonic device, and the temperature control module is connected to an external power supply.

[0019] Thus, the beneficial effects of this method are as follows: During the mixing reaction process of the cocoon shell and the free radical working solution, a multi-frequency ultrasonic device is set up, and the cocoon shell is treated with three-level gradient ultrasound. First, through the ultrasonic treatment in the pretreatment stage, the inside of the cocoon shell is expanded by cavitation bubbles to form a porous fiber structure; then, through the ultrasonic treatment in the main reaction stage, the solid-liquid mass transfer effect between the cocoon shell and the free radical working solution is accelerated, and the reaction is accelerated; finally, through the ultrasonic treatment in the post-stabilization stage, the microbubbles generated by the ultrasonic waves in the reaction solution are eliminated, the interference of the microbubbles on the absorbance detection is eliminated, and the detection accuracy is improved.

[0020] By putting the supernatant of the reaction solution obtained by centrifugation into a quartz cuvette, the ultrasonic transducer is used to vibrate the solid-liquid molecules evenly, and the temperature control component is set to control the temperature, promoting the movement activity between the solid-liquid molecules, accelerating the uniform mixing, and then using the quartz cuvette with a polished inner wall to enhance the light transmittance and reduce the influence of the light scattering phenomenon on the absorbance detection, further improving the accuracy of the detection result. Detailed implementation mode

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0022] In the natural environment, free radicals are active molecules. Due to the presence of unpaired electrons in themselves, free radicals are extremely unstable and need to capture 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 organisms, and some biological materials have antioxidant properties. When using these biological materials as raw materials to produce antioxidants or preservatives, etc., the biological materials can be subjected to free radical detection to detect their antioxidant properties.

[0023] In the existing detection techniques, the ABTS free radical scavenging method is generally used to detect the antioxidant property of sample materials. The principle is that ABTS generates stable blue-green radical cations under the action of an oxidant, which has a maximum absorption peak at 734 nm. When an antioxidant substance is added to react with the green radicals, the radicals are scavenged and the absorbance decreases. The free radical scavenging ability of the sample can be calculated through the change in absorbance, thereby detecting the antioxidant property of the sample material.

[0024] Example 1 Based on the ABTS free radical scavenging method, in order to detect the antioxidant property of mulberry silkworm cocoonshells, the present invention sets up a multi-frequency ultrasonic device, uses three-stage gradient ultrasonic treatment on the cocoonshells, regulates the pore fiber structure inside the cocoonshells as a solid biological material, and increases the mass transfer efficiency between the cocoonshells and the oxidant solution by using the porous structure to improve the reaction efficiency.

[0025] Furthermore, the multi-frequency ultrasonic device mainly includes an ultrasonic generator and an ultrasonic transducer head, which are existing devices. The ultrasonic generator generates electrical signals of different frequencies, and the ultrasonic transducer head converts electrical energy into high-frequency mechanical vibrations through the piezoelectric effect, and the vibrations are then transmitted to the load (liquid or solid).

[0026] In this embodiment, since the cocoonshell has a solid structure, it needs to be mixed and reacted with the oxidant solution, and the pores inside it are enlarged by the multi-frequency ultrasonic device after mixing. Therefore, the working principle of the multi-frequency ultrasonic device is to generate cavitation bubbles (ultrasonic cavitation effect) in the reaction solution through vibration, and use the rupture of several cavitation bubbles to expand the tight fiber structure of the cocoonshell and form a pore fiber structure inside it.

[0027] Among them, during the mixing and reaction process of the cocoonshell and the oxidant solution, the multi-frequency ultrasonic device uses three-stage gradient frequency ultrasonic treatment on the cocoonshell to match the entire reaction process.

[0028] When the cocoonshell and the oxidant solution are just mixed, it is the pretreatment stage: the multi-frequency ultrasonic device is adjusted to a frequency of 35 kHz and a power density of 0.5 W / cm², and continuously works for 3 min to expand the internal pore diameter of the cocoonshell to 5 - 8 μm.

[0029] Preferably, 35 kHz belongs to the medium and low frequency range. The cavitation bubbles generated by low-frequency ultrasonic waves are larger in size and have strong impact force, which can effectively expand the pore diameter. Then, by using the low-power effect for a long time (3 min), the pore diameter can be gradually opened through the long-term cumulative effect of cavitation, reducing the occurrence of cocoon silk breakage caused by the instantaneous high-power cavitation effect.

[0030] After the pore fiber structure is generated by ultrasonic pretreatment of the silkworm cocoon shell, it enters the main reaction stage: the multi-frequency ultrasonic device is adjusted to a frequency of 40 kHz and a power density of 0.8 W / cm², and it works continuously for 5 min to accelerate the solid-liquid mass transfer process between the silkworm cocoon shell and the oxidant solution.

[0031] Preferably, the ultrasonic frequency is increased to 40 kHz to enhance the cavitation intensity, promote the penetration of the liquid microflow into the cocoon layer, thereby improving the mass transfer efficiency between the solid and the liquid, making the mixture more uniform. At this time, since the pore fiber structure has been formed inside the silkworm cocoon shell, increasing the cavitation intensity will not cause the cocoon filaments to break; then, under the action of a longer time (5 min) and a higher power (0.8 W), the diffusion effect of the antioxidant solution in the pore fiber structure inside the silkworm cocoon shell is accelerated, and the reaction efficiency is improved.

[0032] After the reaction between the silkworm cocoon shell and the oxidant solution is completed, it enters the post-stabilization stage: the multi-frequency ultrasonic device is adjusted to a frequency of 28 kHz and a power density of 0.3 W / cm², and it works continuously for 2 min to eliminate the microbubbles generated by the cavitation effect in the main reaction stage.

[0033] Preferably, a lower ultrasonic frequency of 28 kHz is used, which is more likely to generate large-sized bubbles. The tiny bubbles generated in the reaction stage will merge and float upward 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 0.3 W can reduce the generation of bubbles, avoid generating too many bubbles, and at the same time disperse the residual bubbles together through the acoustic streaming effect, eliminate the interference of microbubbles, reduce the influence of microbubbles on the subsequent reaction detection process, and improve the accuracy of the detection results.

[0034] Furthermore, after the reaction solution is generated by reacting the silkworm cocoon shell with the oxidant solution using the multi-frequency ultrasonic device, the reaction solution is placed in a centrifuge for solid-liquid separation. The supernatant in the reaction solution after centrifugation is taken out and placed in a quartz cuvette container. Since solid-liquid mass transfer occurs in the reaction solution, the supernatant is mixed with solid macromolecules, which will cause uneven distribution of the medium in the supernatant, resulting in light scattering phenomena and reducing the accuracy of absorbance value detection.

[0035] Therefore, in order to improve the accuracy of the antioxidant detection of the mulberry silkworm cocoon shell, in this embodiment, a cuvette container made of high-transparency quartz is set to hold the supernatant. On the basis of the existing quartz cuvette, its inner wall is polished to further increase the light transmittance of the quartz cuvette, and a quick-release ultrasonic transducer and a temperature control module are adapted to the quartz cuvette, so that the medium distribution in the supernatant is uniform and the occurrence of light scattering is reduced. At this time, the quartz cuvette is then placed alone in a spectrophotometer, and its absorbance value is measured at a wavelength of 734 nm in the spectrophotometer to detect the antioxidant property of the silkworm cocoon shell.

[0036] Among them, the inner wall of the quartz cuvette is polished to increase the light transmittance of its inner wall and avoid the interference of the inner wall with the propagation of light. The ultrasonic transducer is quickly disassembled and adapted to the quartz cuvette to utilize the vibration of the ultrasonic transducer to uniformly mix various media in the clear liquid through vibration. The temperature control module (Peltier patch) is quickly disassembled and adapted to the quartz cuvette to control the temperature in the clear liquid by using the temperature control component, promote the movement between the molecules of each medium in the clear liquid, and accelerate the mixing. The combined action of the three can effectively reduce the occurrence of light scattering.

[0037] Furthermore, in this embodiment, the antioxidant property of the silkworm cocoon shell is detected based on the ABTS free radical scavenging method. The detection method for the ABTS free radical scavenging ability of the silkworm cocoon shell is as follows: S1. Place the silkworm cocoon shell sample in a vacuum drying oven, set the temperature at 60 ± 2 °C and the vacuum degree at -0.1 MPa, and dry until the sample is completely dehydrated to avoid the influence of moisture on subsequent experiments. Use a low-temperature grinder (to avoid thermal degradation) to crush the dried cocoon shell, and pass it through an 80-100 mesh sieve to obtain a uniform powder structure, and increase its contact area with the solution through the powder structure to accelerate the reaction.

[0038] S2. Prepare the free radical working solution (oxidant solution).

[0039] ABTS generates stable blue-green ABTS+ free radical cations (free radical working solution) under the action of potassium persulfate, which has a characteristic absorption peak at 734 nm. When the antioxidant substance (silkworm cocoon shell) contacts the ABTS+ free radical working solution, the free radicals are scavenged through the 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.

[0040] The specific steps for preparing the free radical working solution are as follows: S2.1. Weigh ABTS precisely, make up the volume with ultrapure water (to reduce the content of free ions in water), vortex for ≥ 30 seconds, and store in the dark at 4 °C to prepare the ABTS stock solution.

[0041] S2.2. Weigh potassium persulfate precisely, make up the volume with ultrapure water, and ultrasonically assist in dissolving the potassium persulfate solid in water (40 kHz, 5 minutes) to prepare the oxidant stock solution.

[0042] S2.3. Mix the ABTS stock solution and the oxidant stock solution in a volume ratio of 1:1, and let it stand in the dark for 12 ± 0.5 hours; dilute it to an absorbance of 734 nm with a pH 7.4 phosphate buffer solution (0.1 mol / L). The working solution needs to be used within 4 hours to reduce the possibility of changes in the internal ions and absorbance of the solution over a long time.

[0043] S3. Mix the radical working solution with the silkworm cocoon shell powder at a liquid-solid ratio of 175:1.

[0044] S4. Perform three-stage gradient ultrasonic treatment on the mixed solid-liquid to obtain the reaction solution generated after the reaction of the radical working solution with the silkworm cocoon shell.

[0045] S5. Place the reaction solution in a low-temperature centrifuge and centrifuge at 4°C at 12000 xg for 10 minutes. Take the supernatant to avoid interference from suspended particles.

[0046] S6. Use a quartz cuvette (optical path 4.00 ± 0.01 mm) to measure the absorbance value at a wavelength of 734 nm on a spectrophotometer. Measure at least 4 times in parallel and take the arithmetic mean (to avoid accidental errors) to obtain the ABTS radical scavenging rate of the silkworm cocoon shell.

[0047] Experimental Example 1 Compared with the traditional ABTS radical detection method, the above detection method for the ABTS radical scavenging ability of the silkworm cocoon shell improves the solid-liquid mass transfer efficiency of the silkworm cocoon shell solid in the radical working solution and accelerates the reaction time by setting up a multi-frequency ultrasonic device to treat the silkworm cocoon shell and using three-stage gradient ultrasonic treatment, thereby reducing the time in the whole detection process.

[0048] When the traditional ABTS radical detection method detects solid substances, after the solid substances are mixed with the radical working solution, it is necessary to shake at a constant temperature for at least 4 hours to ensure sufficient mixing and reaction between the solid substances and the radical working solution.

[0049] In this embodiment, please refer to Table 1 and Table 2. In order to determine the optimal time range for the whole three-stage gradient ultrasonic treatment process to ensure sufficient mixing and reaction between the solid substances and the radical working solution, multiple different control groups of the working time of the ultrasonic device are set to determine the optimal three-stage gradient ultrasonic treatment time.

[0050] Among them, the configuration conditions of each experimental group are as follows: the amount of silkworm cocoon shell powder is 0.02 g / time, the radical working solution is 3.5 mL / time, the experiments at different times in each group are repeated 4 times, and the detection time-consuming is based on the 4 parallel measurement values.

[0051] Table 1: Detection time of ABTS radicals under three-stage gradient ultrasonic treatment

[0052] As can be seen from Table 1, when the overall ultrasonic time of the ultrasonic device reaches 30 minutes, the radical scavenging rate of the whole silkworm cocoon shell sample reaches about 59.4%. Compared with the data at later times, the scavenging rate value tends to be stable, indicating that at this reaction time, it is sufficient for accurate detection. Therefore, the overall ultrasonic time of the ultrasonic device should be controlled at 30 minutes to achieve the detection effect.

[0053] Table 2: Detection time of ABTS free radicals under three - level gradient ultrasonic treatment

[0054] In the time range of 5 - 30 min, further narrowing the detection interval time, the data in Table 2 were obtained. As can be seen from Table 2, when the overall ultrasonic time of the ultrasonic device reaches 10 minutes, the free radical scavenging rate of the entire silkworm cocoon shell sample reaches about 59.3%. Compared with the data at later times, the scavenging rate value tends to be stable, indicating that at this reaction time, it is sufficient for accurate detection. Therefore, the overall ultrasonic time of the ultrasonic device should be controlled at 10 minutes to achieve the detection effect.

[0055] It can be seen from the comparison of experimental results that under three - level gradient ultrasonic treatment, the total time consumption of the mixing reaction time between solid substances and the free radical working solution is reduced by about 96% compared with the traditional reaction time, shortening the overall detection time.

[0056] Experimental Example 2 In this embodiment, the three - level 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 pore - sized fiber structures 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 solution; the post - stabilization stage (frequency 28 kHz) is used to eliminate the micro - bubbles generated by ultrasound in the reaction solution, eliminate micro - bubble interference, and improve the detection accuracy.

[0057] Among them, in order to improve the detection accuracy of the antioxidant properties of the silkworm cocoon shell 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, therefore, please refer to Table 3 and Table 4, and a comparative experiment is carried out with the ultrasonic treatment containing the post - stabilization stage and the ultrasonic treatment without the post - stabilization stage as a control.

[0058] Among them, the configuration conditions of each experimental group are as follows: the amount of silkworm cocoon shell powder is 0.02 g / time, the free radical working solution is 3.5 mL / time. Before detecting the ABTS free radical scavenging ability, the average absorbance of the silkworm cocoon shell sample is set as A0, and after detecting the ABTS free radical scavenging ability, the average absorbance of the silkworm cocoon shell sample is set as A1. Each group of experiments is repeated 6 times, and a blank control group is added to eliminate the occurrence of accidental outliers.

[0059] Table 3: Three - level ultrasonic treatment (pretreatment + main reaction + post - stabilization)

[0060] As can be seen from Table 3, in the three-stage ultrasonic treatment with a post-stabilization stage, 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).

[0061] Table 4: Three-stage ultrasonic treatment (pretreatment + main reaction, without post-stabilization stage)

[0062] As can be seen from 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).

[0063] From the comparison of the experimental results, it can be seen that in the experimental group without a post-stabilization stage, due to the interference of suspended microbubbles, the average value of the absorbance A1 of the experimental group was on the high side, resulting in a slight decrease in the scavenging rate. This shows that the residual microbubbles in the ultrasonic treatment will affect the accuracy of the detection of the ABTS free radical scavenging ability of the whole sample. Therefore, it is necessary to set up an ultrasonic treatment process with a post-stabilization stage to improve the accuracy of the detection data.

[0064] Experimental Example 3 In the prior art, ultrasonic treatment is also often applied to the treatment of chemical substances (such as the extraction process), and through continuous ultrasonic treatment, the solid-liquid mass transfer effect between the reactants and the reaction solution is improved.

[0065] In this embodiment, by setting up a multi-frequency ultrasonic device, the silkworm cocoon shell samples are subjected to ultrasonic treatment at different frequencies in turn using three-stage gradient ultrasound, so as to accelerate the mixing reaction between the silkworm cocoon shell samples and the free radical working solution. In order to compare the effects of the three-stage gradient ultrasonic treatment in this embodiment and the commonly used single-frequency ultrasonic treatment on the solid-liquid mixing reaction efficiency, please refer to Table 5 and Table 6. An experimental group with single-frequency ultrasonic treatment and an experimental group with three-stage gradient ultrasonic treatment are set up for a comparative experiment.

[0066] Table 5: Detection time of ABTS free radicals under single-frequency ultrasonic treatment

[0067] Table 6: Detection time of ABTS free radicals under three-stage gradient ultrasonic treatment

[0068] From the comparison of the experimental results, it can be seen that for the experimental group under single-frequency ultrasonic treatment to achieve the detection effect, at least one hour of ultrasonic treatment time is required, while for the experimental group under three-stage gradient ultrasonic treatment, the detection effect can be achieved in 10 minutes. Under the same ultrasonic treatment time, the ABTS free radical scavenging ability of the three-stage gradient ultrasonic treatment is better.

[0069] Of course, the present invention may have many other 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. However, these corresponding changes and modifications should all fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells, characterized in that ； S1, prepare oxidant solution; S2, mixing the cocoon shell powder and the oxidant solution to form a mixed solution; S3, subjecting the mixed solution to three-level gradient frequency ultrasonic treatment through a multi-frequency ultrasonic device, so as to transform the internal structure of the cocoon shell from a compact fiber structure to a porous fiber structure, and the porous fiber structure is mixed and reacted in an oxidant solution to form a reaction solution; S4. The reaction solution is placed in a spectrophotometer, and the ABTS free radical scavenging rate of the cocoon shell is obtained by the spectrophotometer.

2. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: In step S2, the method for preparing the silkworm cocoon shell powder is as follows: 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 crushed using a low-temperature pulverizer and passed through an 80-100 mesh sieve to obtain a uniform powder structure.

3. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: In step S1, preparing the oxidant solution comprises the following steps: S2.

1. Weigh ABTS, make up to volume with ultrapure water, vortex for ≥30 seconds, and store at 4°C in dark to prepare ABTS stock solution. S2.2, weigh potassium persulfate, make up to volume with ultrapure water, dissolve potassium persulfate solid in ultrapure water with the aid of ultrasound to prepare an oxidant stock solution; S2.

3. Mix the ABTS stock solution and the oxidant stock solution in a 1:1 volume ratio and keep in the dark 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 free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: In step S2, the liquid-to-solid ratio of the oxidant solution and the silkworm cocoon shell powder is 175:

1.

5. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: Based on a multi-frequency ultrasonic device, three-level gradient frequency ultrasonic treatment includes the following steps: S3.

1. When the cocoon shells 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 continuously works for 3 minutes to expand the internal pore size of the cocoon shells to 5-8μm; S3.

2. After the cocoon shell is pre-treated with ultrasound and the internal structure is transformed from a compact fiber structure to an aperture 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; S3.

3. After the reaction between the cocoon shell and the oxidant solution is completed, 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.

6. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: In step S3, the reaction solution is placed in a low-temperature centrifuge and centrifuged at 12,000×g for 10 minutes at 4° C., and the supernatant is used in step S4.

7. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 1, characterized in that: 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 for at least 4 times, and the arithmetic mean is taken to obtain the ABTS free radical scavenging rate of the silkworm cocoon shell.

8. The method for detecting the ABTS free radical scavenging ability of silkworm cocoon shells according to claim 7, characterized in that: An ultrasonic transducer head and a temperature control module are arranged in the quartz cuvette. The ultrasonic transducer head is connected to a multi-frequency ultrasonic device, and the temperature control module is connected to an external power supply.

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