Preparation method of toad-fed maggot antioxidant polypeptide

Abstract

A preparation method of a toad-fed maggot antioxidant polypeptide belongs to the technical field of polypeptide preparation, and the preparation method of the polypeptide comprises the following steps: adding neutral protease into a toad-fed maggot protein crude extract obtained by extraction, carrying out enzymolysis, centrifuging after the enzymolysis is completed, taking supernate, and freeze-drying to obtain the toad-fed maggot antioxidant polypeptide. Wherein the enzymolysis conditions are as follows: the enzymolysis temperature is 35-55 DEG C, the pH value is 5-7, the enzyme addition amount is 4000-6000 U / g, and the enzymolysis time is 2-4 hours. The research finds that the enzymolysis temperature, the enzyme addition amount, the pH and the time have obvious influence on the DPPH clearance rate of the toad-fed maggot enzymatic hydrolysate.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of polypeptides from Tenebrio molitor fed with toad, and particularly relates to a method for preparing an antioxidant polypeptide from Tenebrio molitor fed with toad. Background Art

[0002] The antioxidant polypeptide from Tenebrio molitor fed with toad is a kind of animal macromolecular polypeptide extracted from Tenebrio molitor fed with toad. So far, the widely used antioxidant products mainly include vitamins, carotenoids, polyphenols, etc., but they are likely to cause a certain degree of gastrointestinal reactions and allergic reactions. The polypeptide product obtained by enzymolysis of Tenebrio molitor fed with toad can not only inhibit and scavenge free radicals in the human body, but also is safer, non-toxic and has no side effects compared with drugs. At present, there is little research on the extraction process of the antioxidant polypeptide from Tenebrio molitor fed with toad. Therefore, it is urgent to study the extraction process of the antioxidant polypeptide from Tenebrio molitor fed with toad to provide a certain basis for the wider application of the antioxidant polypeptide from Tenebrio molitor fed with toad. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for preparing an antioxidant polypeptide from Tenebrio molitor fed with toad, so as to provide a basis for the wider application of the antioxidant polypeptide from Tenebrio molitor fed with toad.

[0004] A method for preparing an antioxidant polypeptide from Tenebrio molitor fed with toad according to the present invention is carried out as follows: Add neutral protease to the crude extract of Tenebrio molitor fed with toad protein obtained by extraction, carry out enzymolysis, after the enzymolysis is completed, centrifuge, take the supernatant, and freeze-dry to obtain the antioxidant polypeptide of Tenebrio molitor fed with toad; wherein, Tenebrio molitor fed with toad is Tenebrio molitor obtained by feeding on toad corpses; the enzymolysis conditions are enzymolysis temperature 35-55 °C, pH = 5-7, enzyme addition amount 4000-6000 U / g, and enzymolysis time 2-4 h.

[0005] Further, the enzymolysis temperature is 45-55 °C, pH = 6-7, enzyme addition amount 4000-5000 U / g, and enzymolysis time 2-4 h.

[0006] Further, the enzymolysis temperature is 35-45 °C, pH = 5-7, enzyme addition amount 5000-6000 U / g, and enzymolysis time 2-4 h.

[0007] Further, the enzymolysis temperature is 40-50 °C, pH = 6-7, enzyme addition amount 4500-5500 U / g, and enzymolysis time 2-4 h.

[0008] Further, the enzymolysis conditions are enzymolysis temperature 43 °C, pH = 6.4, enzyme addition amount 5300 U / g, and enzymolysis time 3.2 h.

[0009] Further, the method for extracting the crude extract of Tenebrio molitor fed with toad protein is: Step 1: Powder the toad-fed five-grain insects, degrease them using Soxhlet extraction with petroleum ether as the solvent, and air-dry to obtain defatted toad-fed five-grain insect powder. Among them, the mass-volume ratio of the toad-fed five-grain insect powder to petroleum ether is 20 g: 100 mL. Step 2: Take the defatted toad-fed five-grain insect powder obtained in Step 1, use water as the solvent, adjust the pH to 9 - 11, perform ultrasonic extraction 3 times, combine the extraction solutions and filter to obtain a crude protein extract.

[0010] Furthermore, the ultrasonic temperature is 30 - 35 °C, and the extraction time for each time is 0.5 - 1.5 h.

[0011] Furthermore, before centrifugation, the enzymatic hydrolysate is placed in a water bath at 100 °C to inactivate the enzyme for 10 min.

[0012] Furthermore, the centrifugation conditions are centrifugation at 5000 r / min for 5 min.

[0013] Application of the antioxidant polypeptide of toad-fed five-grain insects prepared by the present invention, wherein the antioxidant polypeptide of toad-fed five-grain insects is used for preparing anti-tumor drugs.

[0014] The antioxidant polypeptide of toad-fed five-grain insects of the present invention is used for preparing anti-tumor drugs, and the tumors are lung cancer cell line A549 and breast cancer cell line MDA-MB-231.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The research of the present invention's scheme finds that the enzymatic hydrolysis temperature, enzyme addition amount, pH, and time have significant effects on the DPPH scavenging rate of the enzymatic hydrolysate of toad-fed five-grain insects. Through single-factor experiments and response surface methodology, the optimal enzymatic hydrolysis conditions are determined as 43 °C, enzyme addition amount of 5300 U / g, pH = 6.4, and enzymatic hydrolysis time of 3.2 h. Under these conditions, the DPPH scavenging rate obtained is 70.218%. The method of the present invention can effectively improve the DPPH scavenging rate. The present invention can provide a certain reference basis for the preparation of the antioxidant polypeptide of toad-fed five-grain insects. Description of the Drawings

[0016] Figure 1 Graph showing the effects of four variables (a) enzymatic hydrolysis temperature, (b) enzyme addition amount, (c) pH, and (d) time on the DPPH scavenging rate.

[0017] Figure 2 Effects of the interaction of (a) temperature and enzyme addition amount, (b) temperature and time, (c) temperature and pH, (d) enzyme addition amount and time, (e) enzyme addition amount and pH, and (f) pH and time on the DPPH scavenging rate.

[0018] Figure 3 Molecular weight distribution map of the original protein of toad-fed five-grain insects.

[0019] Figure 4 Molecular weight distribution diagram of enzymatically hydrolyzed polypeptides from Bufo-fed Tenebrio molitor

[0020] Figure 5 Microscopic observation diagram of the original protein of Bufo-fed Tenebrio molitor; the left figure is 200X, and the right figure is 1000X.

[0021] Figure 6 Microscopic observation diagram of enzymatically hydrolyzed polypeptides from Bufo-fed Tenebrio molitor; the left figure is 200X, and the right figure is 1000X.

[0022] Figure 7 Tumor tissue and tumor weight diagrams of mice in each group; A is the tumor tissue diagram of mice in each group; B is the tumor tissue weight diagram of mice in each group.

[0023] Figure 8 Diagram of the effect of polypeptides on the pathological morphology of tumor tissues in H22-bearing mice (×200).

[0024] Figure 9 Diagram of the effect of polypeptides on the contents of SOD, MDA, and GSH-Px in the serum of H22-bearing mice. Detailed implementation manners

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art in the technical field understands the embodiments of the content of the present invention, they can make changes and modifications based on the techniques taught by the content of the present invention, and it does not deviate from the spirit and scope of the content of the present invention.

[0026] The schematic embodiments of the present invention and their descriptions are used to explain the present invention, but are not used to limit the present invention. Embodiment 1

[0027] A processing technology for optimizing the enzymatic hydrolysis of Bufo-fed Tenebrio molitor by response surface in this embodiment. The enzymatic hydrolysis conditions for optimizing the enzymatic hydrolysis processing technology of Bufo-fed Tenebrio molitor by response surface method are temperature 45°C, enzyme addition amount 5000 U / g, pH 6, and enzymatic hydrolysis time 3 h.

[0028] The steps of the specific processing technology for optimizing the enzymatic hydrolysis of Bufo-fed Tenebrio molitor by response surface in this embodiment are as follows: Step 1: Powder the Bufo-fed Tenebrio molitor, degrease it with petroleum ether (30-60), and dry it to obtain defatted Bufo-fed Tenebrio molitor powder; Step 2: Take the Bufo-fed Tenebrio molitor powder obtained in Step 1, use water as a solvent, adjust the pH to 10, perform ultrasonic extraction, the ultrasonic temperature is 35°C, extract for 1 h each time, extract 3 times, combine the extraction solutions and filter to obtain a crude protein extract; Step 3: Take the crude protein extract in Step 2, adjust the pH of the crude extract, add neutral protease according to the enzyme addition ratio, mix evenly, carry out enzymatic hydrolysis in a constant temperature water bath, immediately take it out after the enzymatic hydrolysis is completed, place the enzymatic hydrolysate in a water bath at 100 °C to inactivate the enzyme for 10 min; centrifuge at 5000 r / min for 5 min, take the supernatant, and freeze-dry. Example 2

[0029] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 35 °C, the enzyme addition amount is 5000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 63.15%. Example 3

[0030] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 35 °C, the enzyme addition amount is 5000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 67.16%. Example 4

[0031] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 55 °C, the enzyme addition amount is 5000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 60.78%. Example 5

[0032] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 55 °C, the enzyme addition amount is 5000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 63.07%. Example 6

[0033] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 45 °C, the enzyme addition amount is 4000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 67.09%. Example 7

[0034] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 45 °C, the enzyme addition amount is 6000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions is measured to be 67.47%. Example 8

[0035] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 45 °C, the enzyme addition amount is 4000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 68.39%. Example 9

[0036] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 45 °C, the enzyme addition amount is 6000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 68.55%. Example 10

[0037] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 45 °C, the enzyme addition amount is 5000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 63.62%. Example 11

[0038] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 45 °C, the enzyme addition amount is 5000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 66.94%. Example 12

[0039] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 45 °C, the enzyme addition amount is 5000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 65.95%. Example 13

[0040] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 45 °C, the enzyme addition amount is 5000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 68.23%. Example 14

[0041] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 35 °C, the enzyme addition amount is 4000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 65.35%. Example 15

[0042] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 55 °C, the enzyme addition amount is 4000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under this enzymatic hydrolysis condition is measured to be 63.87%. Example 16

[0043] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 35 °C, the enzyme addition amount is 6000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 67.51%. Example 17

[0044] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 55 °C, the enzyme addition amount is 6000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 64.60%. Example 18

[0045] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 45 °C, the enzyme addition amount is 4000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 63.79%. Example 19

[0046] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 45 °C, the enzyme addition amount is 4000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 63.09%. Example 20

[0047] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 2 h, the temperature is 45 °C, the enzyme addition amount is 6000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 65.09%. Example 21

[0048] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 4 h, the temperature is 45 °C, the enzyme addition amount is 6000 U / g, and the pH is 6. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 68.28%. Example 22

[0049] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 35 °C, the enzyme addition amount is 5000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed five-grain insects under these enzymatic hydrolysis conditions is measured to be 66.10%. Example 23

[0050] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 55 °C, the enzyme dosage is 5000 U / g, and the pH is 5. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions was measured to be 63.15%. Example 24

[0051] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 35 °C, the enzyme dosage is 5000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions was measured to be 68.30%. Example 25

[0052] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the enzymatic hydrolysis time is 3 h, the temperature is 55 °C, the enzyme dosage is 5000 U / g, and the pH is 7. Finally, the DPPH scavenging rate of the toad-fed G. mellonella under these enzymatic hydrolysis conditions was measured to be 65.55%.

[0053] I. Optimization of the processing technology for enzymatic hydrolysis of toad-fed G. mellonella

[0054] Next, the above specific example processes were optimized and analyzed by response surface: Based on the results of the single-factor experiments, the effects of four variables, namely enzymatic hydrolysis time (A), temperature (B), enzyme dosage (C), and pH (D), on the DPPH scavenging rate were investigated next, and the coefficients of the second-order polynomial equation were calculated. The response surface experimental design and results are shown in Table 1, and the regression equation corresponding to the DPPH scavenging rate can be expressed by the following quadratic equation: DPPH scavenging rate (%) = 70.82 + 1.20A - 1.38B + 0.8264C + 0.8840D - 0.4288AB + 0.9730AC - 0.2586AD - 0.3545BC + 0.0496BD - 0.0531CD - 3.64A 2 - 3.71B 2 - 1.90C 2 - 1.12D 2 .

[0055] Table 1 Box-Behnken experimental design and results

[0056] Using Design Expert software, the experimental results in Table 1 were fitted by quadratic multiple regression and analyzed by variance, and the results are shown in Table 2. The significance of the influence of each variable in the regression equation on the response value was determined by the F test. The smaller the probability p value, the higher the degree of the corresponding variable. p < 0.05 indicates that the variable term is significant, and p > 0.1 represents that the variable term is not significant.

[0057] Table 2 Analysis of Variance Results

[0058] As can be seen from Table 2, the F value of the quadratic term model selected in the present invention is 19.76, which is highly significant (p < 0.0001). Among all variables, A, B, C, D, AC, A 2 , B 2 , C 2 and D 2 , and the corresponding p values are all less than 0.05, indicating that they have a significant impact on the experimental results. The F value of 1.2 indicates that the influence of the lack-of-fit term is not significant. The multiple correlation coefficient R 2 is 0.9518, which indicates that 95.18% of the change in DPPH scavenging rate comes from the selected variables, and the predictive correlation coefficient of 0.7709 is consistent with the corrected correlation coefficient value of 0.9036. Therefore, the regression equation can well describe the true relationship between each variable and the response value, and this regression equation can be used to determine the optimal enzymatic hydrolysis conditions. In addition, a signal-to-noise ratio greater than 4 means that the established model is credible. The signal-to-noise ratio of the present invention, 15.0116, is much greater than 4, further indicating that the experimental model is reliable and can well reflect the relationship between the DPPH scavenging rate and temperature, enzyme dosage, pH, and time. Within the selected range of factor levels, the influence of each variable on the experimental results is in the order of: temperature > time > pH > enzyme dosage.

[0059] By fixing the values of two variables and examining the influence of the other two variables on the DPPH scavenging rate to draw a response surface plot, the influence results of temperature, enzyme dosage, pH, and time on the DPPH scavenging rate are shown in Figure 2。As the variable changes, the DPPH scavenging rate first shows an upward trend and then starts to decline after reaching the highest point. The decrease in the DPPH scavenging rate caused by the simultaneous change of temperature and enzyme dosage can be attributed to the inhibitory effect on enzyme activity with the increase in temperature and the gradual saturation of the enzyme and substrate concentrations after the increase in enzyme dosage. The decrease in the DPPH scavenging rate caused by the simultaneous change of temperature and time can be attributed to the decrease in enzyme activity or the hydrolysis of the enzyme with the increase in temperature and the prolongation of time. The decrease in the DPPH scavenging rate caused by the simultaneous change of temperature and pH can be attributed to the decrease in enzyme activity due to the increase in temperature and pH. The decrease in the DPPH scavenging rate caused by the change of enzyme dosage and time may be due to the decrease in enzyme activity caused by enzyme hydrolysis and the saturation of the enzyme and substrate concentrations with the prolongation of time. The decrease in the DPPH scavenging rate caused by the simultaneous change of enzyme dosage and pH can be attributed to the saturation of the enzyme and substrate concentrations and the decrease in enzyme activity due to the decrease in pH. The decrease in the DPPH scavenging rate caused by the simultaneous change of pH and time can be attributed to the decrease in enzyme activity and the hydrolysis of the enzyme caused by the increase in pH and the prolongation of time. According to the literature report, the response surface substrate presents elliptical contour lines, indicating a significant interaction between the variables. The results show that the interaction between time and enzyme dosage is the strongest. The response surface plot shows extremely high accuracy in predicting the significant interaction between the DPPH scavenging rate and multiple combined variables, and the results are consistent with the regression model.

[0060] According to the analysis results, the optimal enzymatic hydrolysis conditions are as follows: when the temperature is 42.918 °C, the enzyme dosage is 5283.455 U / g, the reaction time is 3.202 h, and the pH is 6.360, the predicted highest DPPH scavenging rate is 71.360%. Considering the convenience of actual operation, the optimal enzymatic hydrolysis conditions are modified to a temperature of 43 °C, an enzyme dosage of 5300 U / g, a time of 3.2 h, and a pH of 6.4. Under these conditions, the obtained DPPH scavenging rate is 70.218%. Compared with the predicted value, the relative error is 1.60%. This indicates that the established regression equation can highly reflect the effects of temperature, enzyme dosage, pH, and time on the DPPH scavenging rate of Bufo-fed Gryworms, and the DPPH scavenging rate can be effectively improved through this optimization model. II. Application of Antioxidant Polypeptides from Bufo-fed Gryworms

[0061] 2.1 Preparation of Gryworm Protein and Bufo-fed Gryworm Protein The Gryworms and Bufo-fed Gryworms are separately powdered, passed through a No. 5 sieve, defatted with petroleum ether (30 - 60), dried, and the defatted powders of both are obtained. They are mixed with water as a solvent according to a liquid-to-material ratio of (40:1), the pH is adjusted to 10, and ultrasonic extraction is carried out at an ultrasonic temperature of 35 °C for 1 h each time, for 3 times. The extraction solutions are combined and filtered to obtain a protein extraction solution, which is freeze-dried to obtain the protein powders of the two samples of Gryworms and Bufo-fed Gryworms.

[0062] 2.2 Comparison of Antioxidant Activities 2.2.1 Determination of DPPH Scavenging Rate Take anhydrous ethanol, and prepare solutions of protein from larvae of Lucilia sericata and protein from larvae of Lucilia sericata fed with toads at concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg / mL respectively, and a DPPH solution at a concentration of 0.25 mg / mL. Take 800 μL of the DPPH solution, add 200 μL of the sample solution, place it in the dark at room temperature for 10 min, then centrifuge at 8000 r / min for 5 min. Take the supernatant and measure the absorbance at a wavelength of 519 nm.

[0063] Formula for calculating DPPH scavenging rate

[0064] In the formula: A1 is the sample + DPPH; A2 is the sample + ethanol; A3 is water + DPPH; A4 is water + ethanol.

[0065] 2.2.2 Results The results of the in vitro antioxidant activity experiment showed that the IC50 values of the scavenging activity of polypeptides from larvae of Lucilia sericata against DPPH free radicals were 1.24 mg / mL respectively, while the IC50 values of the scavenging activity of polypeptides from larvae of Lucilia sericata fed with toads against DPPH free radicals were 1.12 mg / mL respectively. Their in vitro antioxidant activity was significantly better than that of polypeptides from larvae of Lucilia sericata.

[0066] Table 3 Comparison of IC50 values of the scavenging activity of polypeptides from larvae of Lucilia sericata and polypeptides from larvae of Lucilia sericata fed with toads against DPPH free radicals

[0067] 2.3 Comparison of in vitro antitumor activities Take cells with good growth and adjust the concentration to 6 × 10 4 cells / mL. In a 96-well plate, add 200 μL of PBS to the peripheral wells, and add 100 μL of the above cell suspension to the remaining wells, and culture in an incubator for 24 h. Set up a blank group, groups of polypeptides from larvae of Lucilia sericata at different concentrations, and groups of polypeptides from larvae of Lucilia sericata fed with toads at different concentrations. Add 100 μL of complete medium to the blank group, and add 100 μL of complete medium containing drugs at different concentrations to the groups of polypeptides from larvae of Lucilia sericata at different concentrations and the groups of polypeptides from larvae of Lucilia sericata fed with toads at different concentrations respectively. Each group has 6 replicates. Culture in an incubator for 48 h. After reaching the time, discard the medium, wash twice with PBS, add CCK-8 detection solution. Another 6 peripheral wells are discarded of the contained PBS and added with CCK-8 detection solution as the zero-well group, and culture in a cell incubator for 1 h, and measure the absorbance at a wavelength of 450 nm. The formula for cell viability is as follows:

[0068] In the formula: A is the average absorbance of the drug administration group; B is the average absorbance of the blank group; C is the average absorbance of the zero-well group.

[0069] The results of in vitro anti-tumor activity experiments showed that the IC50 values of the polypeptides from Lucilia sericata against lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were 682.1 mg / mL and 102.2 mg / mL respectively, while the IC50 values of the polypeptides from toad-fed Lucilia sericata against the two cell lines were 45.2 mg / mL and 50.1 mg / mL respectively, and its in vitro anti-tumor activity was significantly better than that of the polypeptides from Lucilia sericata.

[0070] Table 4 IC50 values of polypeptides from Lucilia sericata and polypeptides from toad-fed Lucilia sericata in inhibiting the proliferation of lung cancer cell line A549 and breast cancer cell line MDA-MB-231

[0071] 2.4 Structural characterization 2.4.1 Determination of particle size and Zeta potential The enzymatically hydrolyzed polypeptide and the original protein solution were accurately diluted to a concentration of 1.0 mg / mL with distilled water. Then each protein sample was dispensed into a dedicated cuvette. The particle size distribution and zeta potential were comprehensively analyzed using a laser light scattering in-situ particle size analyzer (NanoBrook90Plus, Brookhaven Co., Ltd., New York, NY, USA), and maintained at a constant temperature of 25 °C. To ensure accuracy and reliability, each sample was tested in triplicate.

[0072] 2.4.2 Determination of molecular weight distribution The enzymatically hydrolyzed polypeptide and the original protein solution were prepared at a concentration of 2.0 mg / mL, filtered through a 0.22 μm membrane (Shenghan Co., Ltd., Qingdao, China) to ensure the removal of impurities. These filtered solutions were separately added to a high performance gel chromatography system (LC-20AD, Shimadzu, Kyoto, Japan) for molecular weight distribution analysis. The high performance gel chromatography system used a G-3000 PWXL superdex linear gel filtration column (Tosoh Co., Ltd., Tokyo, Japan) combined with an ultraviolet detector for accurate detection and quantification. Deionized water was used as the eluent for sample elution. To accurately determine the average molecular weight (Mw) of each sample, a calibration curve was plotted using a series of standards with known molecular weights (13.050, 36.800, 64.650, 135.350, 300, 600, and 2,000,000 Da). This precise calibration process enabled the accurate and detailed evaluation of the average molecular weight of each sample.

[0073] 2.5 Pharmacodynamic experiments 2.5.1 Establishment of H22 tumor-bearing mouse model Take the H22 cells after ascites passage, centrifuge at 1000 r·min -1 for 5 min, discard the supernatant, and adjust the cell concentration to 1×107 cells / mL, and placed in an ice bath for later use. The hair on the left abdomen of the mice was shaved, wiped and disinfected with alcohol cotton. Then, the above cell suspension was taken and inoculated subcutaneously into the left abdomen of the mice at a dose of 0.2 mL / mouse, and 30 mice were inoculated.

[0074] 2.5.2 Experimental grouping and drug administration The mice inoculated with H22 cells were randomly divided into 5 groups, with 6 mice in each group, namely the model group (M), the cyclophosphamide group (Y, 26 mg / kg), the high-dose toad-fed larvae polypeptide group (PH, 2.60 g / kg), the medium-dose group (PM, 1.30 g / kg), and the low-dose group (PL, 0.65 g / kg). Another 6 mice had their hair on the left abdomen shaved, wiped and disinfected with alcohol cotton, and were subcutaneously injected with PBS at a dose of 0.2 mL / mouse as the blank group (K).

[0075] 24 hours after the modeling and grouping, the blank group and the model group were intragastrically administered normal saline at a dose of 10 mL / kg, and the administration groups were intragastrically administered according to the above doses, once a day for 10 consecutive days. During the experiment, the mice were allowed to eat and drink freely. On the 11th day, after weighing, blood was collected from the eyeballs, the mice were sacrificed, and samples were taken.

[0076] 2.5.3 Determination of tumor inhibition rate 24 hours after the last administration, the mice were sacrificed by cervical dislocation, and the tumors were removed and weighed. The formula for the tumor inhibition rate is as follows:

[0077] 2.5.4 Histopathological observation of tumor tissues in H22 tumor-bearing mice The tumor tissues of the mice were taken, fixed with 4% paraformaldehyde, trimmed, dehydrated, embedded, made into 5-μm paraffin sections, then dewaxed with xylene, rehydrated, stained with H&E, dehydrated, and sealed. The processed sections were placed under a microscope to observe the histopathological changes of the tissues.

[0078] 2.5.5 Contents of SOD, MDA and GSH-Px in the serum of H22 tumor-bearing mice Blood was collected from the eyeballs of the mice, left to stand at 4°C for 1 h, and centrifuged at 3000 r·min -1 centrifuged at 4°C for 5 min to obtain the serum, and then the determination was carried out according to the instructions of the kit.

[0079] 2.6 Experimental results 2.6.1 Structural characterization part 2.6.1.1 Particle size and Zeta potential The particle sizes of the enzymatically hydrolyzed polypeptide and the native protein were measured to be 219.3 ± 11.1 nm and 246.4 ± 5.6 nm, respectively. This indicates that the enzymatic hydrolysis process resulted in a 11.12% reduction in the particle size of the polypeptide. This reduction indicates a significant change in the physical properties of the protein caused by enzymatic hydrolysis. The decrease in the protein particle size can significantly affect its functional and conformational properties.

[0080] Zeta potential is an important parameter for evaluating the dispersion characteristics of polymer solutions or colloids and plays an important role in evaluating the stability of polymer solutions or colloids. The higher the absolute value of the zeta potential, the smaller the protein molecules dispersed in the solution and the more stable the system. When comparing the enzymatically hydrolyzed polypeptide with the native protein, the absolute surface charge decreased significantly by 79.48%, indicating that the enzymatically hydrolyzed polypeptide should be stored in a freeze-dried form to avoid storage in a solvent. See the table below.

[0081] Table 5 Particle size distribution and zeta potential before and after enzymatic hydrolysis

[0082] 2.6.1.2 Molecular weight distribution Molecular weight, as an important physicochemical property of proteins, has an important impact on the pharmacological activity of proteins. The molecular weight distribution of each sample was determined using high-performance gel chromatography. The protein molecular weight was calculated based on the standard curve equation: LogMw = −3.1305 T + 31.548 (R2 = 0.999), where Mw represents the average molecular weight and T represents the retention time. The results showed that the protein molecular weight distributions were 9.12 × 103, 3.96 × 103, 1.70 × 103, 1.23 × 103, 8.68, 4.26, and 1.02 kDa, corresponding to retention times of 8.79, 10.10, 11.36, 11.92, 19.36, 20.60, and 22.78 min, respectively. This method showed an obvious peak pattern for the protein, and the percentage of each peak in the total peak was different for the two groups of samples. Compared with the native protein, the percentages of peaks 5, 6, and 7 of the enzymatically hydrolyzed polypeptide increased significantly, while the percentages of peaks 1, 2, and 3 decreased significantly. See Figure 3 、 4 and Table 6.

[0083] Table 6 Proportion of molecular weight distribution before and after enzymatic hydrolysis

[0084] 2.6.1.3 Electron microscopy observation Scanning electron microscopy (SEM) is a key tool for the detailed analysis of the surface structure and morphology of proteins, providing a direct way to determine the polymer structure. The microscopic morphology of proteins before and after enzymatic hydrolysis was precisely examined by SEM. At a magnification of 1000 times, the protein particles of both enzymatically hydrolyzed polypeptides and native proteins were in the shape of glass fragments, while the enzymatically hydrolyzed polypeptide fragments were smaller. When magnified to 10,000 times, the native protein particles showed cavitation, while the enzymatically hydrolyzed polypeptides were smoother, indicating that their particle size changed significantly after enzymatic hydrolysis. See below Figure 5 and 6 。

[0085] 2.6.2 Pharmacodynamics section 2.6.2.1 Inhibitory effect on tumor growth in H22 tumor-bearing mice Compared with the model group, the average tumor weight of mice decreased, and the decrease was significant in the cyclophosphamide group and the high-dose polypeptide group (p < 0.05). The tumor inhibition rates of the high-, medium-, and low-dose polypeptide groups were 33.67%, 23.18%, and 13.71% respectively, indicating that the polypeptides from Bufo-fed G. mellonella have anti-tumor effects, as shown in Table 7 and Figure 7 ;

[0086] 2.6.2.2 Effect on the pathological morphology of tumor tissues in H22 tumor-bearing mice In the model group, the cells of the tumor tissues in mice had irregular shapes, increased volumes, decreased nucleus-cytoplasm ratios, and increased nuclear fissions. Compared with the model group, the nucleus-cytoplasm ratio increased, nuclear fissions decreased, and necrotic cells increased in the cyclophosphamide group; in the polypeptide groups, as the dose increased, the nucleus-cytoplasm ratio gradually increased, nuclear fissions gradually decreased, and necrotic cells gradually increased. This indicates that the polypeptides from Bufo-fed G. mellonella have anti-tumor effects, and the effect is enhanced with the increase in dose. See Figure 8 。

[0087] 2.6.2.3 Effect on SOD, MDA, and GSH-Px in the serum of H22 tumor-bearing mice Compared with the blank group, SOD and GSH-Px in the model group were significantly decreased (p < 0.05), while MDA was significantly increased (p < 0.05). Compared with the model group, there were no significant differences in SOD, MDA, and GSH-Px in the low-dose polypeptide group; the indicators of the remaining groups were all significant (p < 0.05), among which the cyclophosphamide group was the most obvious, followed by the high-dose polypeptide group. The results show that as the dose of polypeptide administered increases, the levels of SOD and GSH-Px increase, and the level of MDA decreases, suggesting that the polypeptides from Bufo-fed G. mellonella can inhibit tumor growth by enhancing antioxidant capacity;

[0088] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0089] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A preparation method of antioxidant polypeptide from Bufo-fed Gonyostomum semen, characterized in that It is carried out as follows: To the crude extract of Bufo-fed Tenebrio molitor protein obtained by extraction, neutral protease is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, centrifugation is carried out, and the supernatant is taken and freeze-dried to obtain the Bufo-fed Tenebrio molitor antioxidant polypeptide. Among them, the Bufo-fed Tenebrio molitor is obtained by feeding Tenebrio molitor with the corpses of toads. The enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 35-55 °C, pH = 5-7, enzyme addition amount 4000-6000 U / g, and enzymatic hydrolysis time 2-4 h.

2. The preparation method of a toad-fed five-grain insect antioxidant polypeptide according to claim 1, characterized in that The enzymatic hydrolysis temperature is 45-55 °C, pH = 6-7, enzyme addition amount 4000-5000 U / g, and enzymatic hydrolysis time 2-4 h.

3. The preparation method of an antioxidant polypeptide from Bombyx batryticatus fed with grains according to claim 1, wherein The enzymatic hydrolysis temperature is 35-45 °C, pH = 5-7, enzyme addition amount 5000-6000 U / g, and enzymatic hydrolysis time 2-4 h.

4. The preparation method of a toad-fed five-grain insect antioxidant polypeptide according to claim 1, characterized in that The enzymatic hydrolysis temperature is 40-50 °C, pH = 6-7, enzyme addition amount 4500-5500 U / g, and enzymatic hydrolysis time 2-4 h.

5. The preparation method of an antioxidant polypeptide of Bufo-fed Gryworms according to claim 1, 2, 3 or 4, characterized in that The enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 43 °C, pH = 6.4, enzyme addition amount 5300 U / g, and enzymatic hydrolysis time 3.2 h.

6. The preparation method of an antioxidant polypeptide from Bombyx batryticatus fed with grains according to claim 1, characterized in that The method for extracting the crude extract of Bufo-fed Tenebrio molitor protein is as follows: Step 1: Powder the Bufo-fed Tenebrio molitor, use Soxhlet extraction method to defat it with petroleum ether, and air-dry it to obtain defatted Bufo-fed Tenebrio molitor powder. Among them, the mass-volume ratio of the powdered Bufo-fed Tenebrio molitor to petroleum ether is 20 g:100 mL. Step 2: Take the Bufo-fed Tenebrio molitor powder obtained in Step 1, use water as the solvent, adjust the pH to 9-11, carry out ultrasonic extraction 3 times, combine the extraction solutions and filter to obtain the crude protein extract.

7. The preparation method of an antioxidant polypeptide from Bufo-fed Tenebrio molitor according to claim 6, characterized in that The ultrasonic temperature is 30-35 °C, and the extraction time for each time is 0.5-1.5 h.

8. The preparation method of an antioxidant polypeptide from Bufo-fed Tenebrio molitor according to claim 1, characterized in that Before centrifugation, the enzymatic hydrolysis solution is placed in a water bath at 100 °C to inactivate the enzyme for 10 min.

9. The preparation method of an antioxidant polypeptide from Bufo-fed Tenebrio molitor according to claim 1 or 8, characterized in that The centrifugation conditions are centrifugation at 5000 r / min for 5 min.

10. Use of the antioxidant polypeptide of toad-fed G. mellonella prepared as claimed in claim 1, 2, 3 or 4, characterized in that The Bufo-fed Tenebrio molitor antioxidant polypeptide is used for preparing anti-tumor drugs, and the tumors are lung cancer cell line A549 and breast cancer cell line MDA-MB-231.

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