Evaluation method for antioxidant activity of tenebrio molitor protein and application thereof

Abstract

The invention provides a method for evaluating the antioxidant activity of tenebrio molitor protein and application of the method, and belongs to the technical field of evaluation of the antioxidant activity of insect protein. The tenebrio molitor protein degrades the cumene hydroperoxide under the catalysis of glutathione peroxidase 4, the localized surface plasma effect of the silver nanoparticles enhances the ultraviolet-visible absorption spectrum of the silver nanoparticles, the degradation rate is calculated by measuring the ultraviolet spectrum absorbance change of the cumene hydroperoxide, and the antioxidant activity of the tenebrio molitor protein is evaluated. According to the method, the biological oxidation resistance of tenebrio molitor protein is evaluated by utilizing the degradation degree of cumyl hydroperoxide, so that a complex metabolic process can be simulated more truly, and the actual oxidation resistance is closer to that of the tenebrio molitor protein; the silver nanoparticles enhance molecular vibration, the detection limit of the reaction is reduced, and the sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of evaluating the antioxidant activity of insect proteins, and particularly relates to a method for evaluating the antioxidant activity of Tenebrio molitor proteins and its application. Background Art

[0002] Tenebrio molitor has a high protein content and antioxidant activity, and has broad application prospects in industries such as food, health products, and cosmetics. Regarding the antioxidant activity of Tenebrio molitor, the existing evaluation methods mainly evaluate it through its ability to scavenge and inhibit various free radicals in vitro, including scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, and inhibiting superoxide anion radical (O2 - ) ability, etc. However, the existing in vitro evaluation system has significant limitations: First, the in vitro free radical scavenging evaluation can only reflect the direct antioxidant activity of Tenebrio molitor proteins and cannot simulate the complex metabolic processes in vivo; Second, the traditional detection index is the free radical scavenging rate, which is difficult to comprehensively characterize the comprehensive action mechanism of the multi-component antioxidant system of Tenebrio molitor proteins, especially for more biotoxic reactive oxygen species (ROS), resulting in a deviation between the evaluation results and the actual effects of Tenebrio molitor proteins. Therefore, developing an accurate, efficient and comprehensive evaluation method is of great significance for in-depth study of the antioxidant activity of Tenebrio molitor proteins.

[0003] Cumene hydroperoxide (CHP) is an organic peroxide. Compared with inorganic H2O2, it can generate more reactive hydroxyl groups, can specifically cause continuous oxidative stress damage to biomacromolecules such as membrane phospholipids and proteins, and has a more significant impact on the mitochondrial respiratory chain. While H2O2 does not have damage specificity and does not cause damage to biomacromolecules such as phospholipids. Therefore, it is more ideal to evaluate the biological antioxidant activity of Tenebrio molitor using the degradation degree of CHP. Glutathione peroxidase 4 (GPx4) is the only enzyme in the body that scavenges peroxides, and substances with reducing properties all have the potential to participate in the process of catalyzing peroxide degradation as GPx4 reductants. However, existing research mostly focuses on the evaluation of enzyme activity, while ignoring that the process of the enzyme catalyzing the degradation of CHP can be used to evaluate the biological reducing properties of substances. Therefore, using reducing substances as substrates to participate in the degradation process of organic peroxides catalyzed by GPx4 can more realistically simulate complex metabolic processes and be closer to the actual antioxidant activity of substances. This patent uses Tenebrio molitor protein powder as a reductant for GPx4 to scavenge CHP, and measures the scavenging rate of CHP by ultraviolet-visible absorption spectroscopy (UV-Vis) to evaluate the antioxidant activity of Tenebrio molitor protein powder. This will provide an important scientific basis for the development of antioxidant products of Tenebrio molitor proteins and is of great significance for promoting the extension and value addition of the insect industry. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a method for evaluating the antioxidant activity of mealworm protein and its application. The present invention can be used to evaluate the activity of antioxidants with higher accuracy and closer to the actual physiological level.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] A method for evaluating the antioxidant activity of mealworm protein and its application, characterized in that the method comprises the following steps:

[0007] Step 1: Preparation of mealworm protein freeze-dried powder and working solution

[0008] Dry the mealworm at 105°C until the weight is constant, remove the fat by Soxhlet extraction, and prepare the crude protein by enzymatic hydrolysis. After centrifugation, obtain the supernatant protein solution, freeze it in liquid nitrogen, and then freeze-dry it to obtain the protein powder for use. Take an appropriate amount of mealworm protein freeze-dried powder and dissolve it in ultrapure water to obtain a protein working solution with a concentration of 1-50 mg / L.

[0009] Step 2: Preparation of GPx4 working solution

[0010] The GPx4 working solution is composed of GPx4 recombinant protein, glutathione reductase (GR), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The volume ratio of GPx4, GR, and NADPH is 1:1:1-1:1:4, wherein the concentrations of GPx4 and GR are 100-200 μg / mL, and the concentration of NADPH is 62.5 nM.

[0011] Step 3: Preparation of Ag NPs

[0012] AgNO3 is dispersed in ultrapure water, heated to boiling, and then an appropriate amount of sodium citrate is added. After the temperature is appropriately lowered, a gray-green colloid is obtained, which is cooled to room temperature for use. The ultraviolet absorption peak of Ag NPs is between 400nm and 450nm, and the particle size is greater than or less than or equal to 60nm.

[0013] Step 4: Test of Antioxidant Activity of Mealworm Protein

[0014] Take 1 μL of the protein working solution in step 1, add 2 μL of GPx4 working solution, incubate at 25°C for 15 minutes, add 1 μL of CHP, add pure water to 10 μL, add 10 μL of Ag NPs. Use a UV-visible spectrophotometer to detect the absorbance between 200-300 nm, where the CHP concentration is 30 mM.

[0015] Step 5: Calculation of antioxidant activity of mealworm protein

[0016] A standard curve was established using a blank sample to calculate the clearance rate of Tenebrio molitor protein against CHP, which was calculated according to the following formula (1):

[0017]

[0018] The detection principle of the present invention is as follows:

[0019] First, a freeze-dried sample of Tenebrio molitor protein was prepared in the present invention. GPx4 working solution was used to degrade CHP, and at the same time, AgNPs were added to enhance the absorption of ultraviolet-visible spectroscopy and Raman spectroscopy, and the antioxidant activity was measured. Under the reaction conditions at room temperature, Tenebrio molitor protein degrades CHP catalyzed by GPx4, while Ag NPs enhance the peak intensity. The occurrence of the reaction was corroborated by the determination of ultraviolet-visible spectroscopy and surface-enhanced Raman spectroscopy. A standard curve was established using SERS to calculate the clearance rate of Tenebrio molitor protein against organic peroxides. In the present invention, AgNPs are enhancement reagents for ultraviolet-visible absorption spectroscopy and Raman spectroscopy. Through its local surface plasmon effect, Ag NPs enhance the absorption peak of the peroxide bond in the CHP molecule, achieving the purpose of improving sensitivity and reducing the detection limit.

[0020] The detection process of the present invention is as follows: preparation of freeze-dried powder of Tenebrio molitor protein and working solution, preparation of GPx4 working solution, preparation of Ag NPs, antioxidant activity test and calculation of Tenebrio molitor protein.

[0021] The beneficial effects of the present invention: The evaluation method for the antioxidant activity of Tenebrio molitor protein provided by the present invention mixes the freeze-dried powder of Tenebrio molitor protein with GPx4 working solution, degrades CHP at room temperature, and adds Ag NPs to increase the absorbance of the ultraviolet-visible absorption spectrum and the intensity of the Raman spectrum of the substance, playing the role of improving sensitivity and reducing the detection limit.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) Using biological protease as a catalyst can better reflect the antioxidant activity under physiological conditions (the protective effect on biological proteins). Antioxidants with antioxidant activity detected by chemical methods may not show antioxidant activity towards biological macromolecules or may not show the expected antioxidant activity. The present invention can truly detect the antioxidant activity of Tenebrio molitor protein and its protective effect on biological proteins;

[0024] (2) CHP has a characteristic peak between 200 - 300 nm in the ultraviolet spectrum, and the concentration can be determined by ultraviolet spectroscopy and the clearance rate can be calculated;

[0025] (3) The AgNPs used in the present invention as a spectral enhancement reagent have the effects of improving sensitivity and reducing the detection limit. Description of the Drawings

[0026] Figure 1 Schematic diagram of the ultraviolet spectral absorption peak of the degradation of cumene hydroperoxide enhanced by AgNPs according to the present invention;

[0027] Figure 2 Inhibitory rate of Tenebrio molitor protein on DPPH radicals;

[0028] Figure 3 Inhibitory rate of Tenebrio molitor protein on ABTS radicals; Detailed Description of the Invention

[0029] In order to make the objectives, features, and advantages of the present invention more obvious and understandable, the embodiments of the present invention will be clearly and completely described below in conjunction with the specific embodiments involved in the drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts, such as embodiments that only change the use without changing the basic principles involved in the claims, fall within the scope of protection of the present invention.

[0030] Example 1: Determination of the scavenging rates of Tenebrio molitor protein on ABTS and DPPH radicals

[0031] When the mass concentration of Tenebrio molitor protein is 2 - 10 mg / mL, the scavenging rates of ABTS and DPPH radicals are measured respectively.

[0032] Table 1 Scavenging rates of Tenebrio molitor protein at different concentrations on ABTS and DPPH radicals

[0033]

[0034] Example 2: Determination of the scavenging rate of Tenebrio molitor protein on CHP under the catalysis of GPx4 by ultraviolet-visible spectroscopy

[0035] When the mass concentration of Tenebrio molitor protein is 0.20 mg / mL, the degradation of CHP was continuously measured within 10 min, and the scavenging rate was calculated as shown in Table 2. At 10 min, the scavenging rate of 0.20 mg / mL Tenebrio molitor protein on CHP was 27.31%.

[0036] Table 2 Monitoring the scavenging rate of Tenebrio molitor protein on CHP under the catalysis of GPx4 within 10 min

[0037]

[0038] Example 3: Determination of the scavenging rate of Tenebrio molitor protein on H2O2 under the catalysis of GPx4 by ultraviolet-visible spectroscopy

[0039] When the protein concentration of Tenebrio molitor was 200 μg / mL, the degradation of H2O2 was continuously measured within 10 min, and the scavenging rate was calculated as shown in Table 3. At 10 min, the scavenging rate of 200 μg / mL Tenebrio molitor protein for CHP was 1.79%.

[0040] Table 3 Monitoring the scavenging rate of Tenebrio molitor protein for H2O2 catalyzed by GPx4 within 10 min

[0041]

[0042]

[0043] The combination features of components not described in detail in the specification belong to the content that can be easily thought of by well-known techniques or can be easily determined and have no objection when implementing the present invention. The above solutions are only descriptions of several preferred embodiments of this application. However, the protection scope of this application is not limited to this. Anyone familiar with this technology can easily implement it within the scope described in this application. Without changing the basic principles involved in the claims, any changes or substitutions should be covered within the protection scope of this application. That is, the protection scope of this application should be subject to the protection scope of the claims.

Claims

1. An evaluation method for the antioxidant activity of Tenebrio molitor protein, characterized in that, It includes the following steps: Step 1: Preparation of Tenebrio molitor protein freeze-dried powder and working solution Tenebrio molitor larvae are dried to a constant weight at 105 °C, defatted by Soxhlet extraction, and then crude protein is prepared by enzymatic hydrolysis. The supernatant protein solution is obtained by centrifugation, frozen in liquid nitrogen, and then freeze-dried to obtain the protein powder for standby. An appropriate amount of the freeze-dried protein powder is dissolved in ultrapure water to obtain the protein working solution; Step 2: Preparation of glutathione peroxidase 4 (GPx4) working solution The GPx4 working solution is composed of GPx4 recombinant protein, glutathione reductase (GR), and reduced nicotinamide adenine dinucleotide phosphate (NADPH); Step 3: Preparation of Ag NPs: AgNO3 is dispersed in ultrapure water. After heating to boiling, an appropriate amount of sodium citrate is added. After the temperature is appropriately reduced, a gray-green colloid is obtained and stored at room temperature for standby; Step 4: Testing the antioxidant capacity of Tenebrio molitor protein 1 μL of the protein working solution in Step 1 is pipetted, 2 μL of the GPx4 working solution is added, incubated at 25 °C for 15 minutes, 1 μL of cumene hydroperoxide (CHP) is added, made up to 10 μL with pure water, and 10 μL of Ag NPs is added. The absorbance between 200 - 300 nm is detected using a UV-visible spectrophotometer; Step 5: Calculation of the antioxidant capacity of Tenebrio molitor protein A standard curve is established using a blank sample, and the clearance rate of Tenebrio molitor protein against CHP is calculated according to the following formula (1):

2. The evaluation method according to claim 1, wherein The concentration of the protein working solution in Step 1 is 0.10 - 100 mg / mL.

3. The evaluation method according to claim 1, characterized in that, The volume ratio of GPx4, GR, and NADPH in Step 2 is 1:1:1 - 1:1:4, where the concentrations of GPx4 and GR are 100 - 200 μg / mL, and the concentration of NADPH is 62.5 nM.

4. The evaluation method according to claim 1, characterized in that, The UV absorption peak of Ag NPs in Step 3 is between 400 nm - 450 nm, and the particle size is greater than or less than or equal to 60 nm.

5. The evaluation method according to claim 1, wherein The concentration of CHP in Step 4 is 30 mM.

6. The evaluation method according to claim 1, characterized in that, In Step 5, the clearance rate of 0.20 mg / mL Tenebrio molitor protein against CHP within 10 min is 27.31%.

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