Preparation method of tylorrhynchus antioxidative peptide

Abstract

The invention belongs to the technical field of bioactive peptide preparation, and discloses a preparation method of a tylorrhynchus antioxidant peptide, which comprises the following steps: mixing tylorrhynchus homogenate with enzyme, and carrying out enzymolysis to obtain the tylorrhynchus antioxidant peptide, the enzyme comprises flavourzyme and pancreatin. The invention provides the preparation method of the tylorrhynchus antioxidant peptide which is high in antioxidant activity and suitable for industrial mass production, the protein hydrolysis degree is improved, and the oxidation resistance of the tylorrhynchus antioxidant peptide is improved. The method can effectively solve the problems of low efficiency, high cost and inapplicability to large-scale extraction and preparation of the tylorrhynchus antioxidative peptide in the existing polypeptide extraction process.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically relating to a method for preparing an antioxidant peptide from the worm *Clerodendrum tympany*. Background Technology

[0002] Bioactive peptides are small protein fragments derived from plant and animal tissues, typically containing 2-20 amino acids. Compared to large protein molecules, their molecular weight advantage makes them more easily absorbed by the intestines. More importantly, bioactive peptides have significant regulatory functions in animals, exhibiting good affinity, low toxicity, and high stability, and displaying various physiological activities such as antioxidant, hypoglycemic, immunomodulatory, anti-fatigue, antibacterial, and anticancer effects. These characteristics have collectively made them a current research hotspot. Among them, antioxidant peptides are a class of small protein fragments with the ability to scavenge free radicals and inhibit oxidative damage. Due to their high efficiency, safety, and easy absorption, they have received widespread attention in the food, pharmaceutical, and cosmetic fields, and are considered an ideal alternative to synthetic antioxidants. The mechanisms of action of antioxidant peptides are diverse and synergistic. They can directly donate hydrogen atoms or electrons to effectively neutralize reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, thereby interrupting the chain reaction initiated by free radicals and protecting key biomolecules such as lipids, proteins, and DNA in cells from oxidative damage. Simultaneously, they can chelate transition metal ions such as iron and copper ions, preventing these metal ions from catalyzing the Fenton reaction to produce highly destructive hydroxyl radicals. More importantly, many antioxidant peptides can activate the Nrf2 / ARE signaling pathway, a core cellular defense pathway, upregulating the expression of endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and promoting the synthesis of reduced glutathione (GSH), thus comprehensively enhancing the cell's own antioxidant capacity. Furthermore, they can inhibit the activity of some pro-oxidative enzymes (such as xanthine oxidase), reducing the production of free radicals at the source.

[0003] The wartyrhynchus heterochaetus, commonly known as the sandworm or the mudworm, belongs to the class Polychaete in the phylum Annelida. It is an important benthic organism distributed in the brackish water areas of the Pearl River Delta in my country (such as rice paddies and mangrove wetlands). It plays a key role in maintaining the balance of the regional ecosystem (such as nutrient cycling). Notably, the wartyrhynchus is not only a highly nutritious aquatic food resource (rich in protein, essential amino acids, and trace elements), but it has also been used in traditional Chinese medicine for a long time. It is believed to have the effects of nourishing yin and yang, replenishing qi and blood, and strengthening the body, suggesting that it has anti-fatigue and restorative effects. Therefore, fully exploring the unique biological resource of *Nereiscus wartii*, focusing on its bioactive peptide components, and utilizing modern biotechnological methods (such as enzymatic hydrolysis, separation and purification, mass spectrometry identification, activity screening, and cell and animal model verification) to systematically discover, identify, and verify *Nereiscus wartii* peptides with high efficiency and safe bioactivity can not only fill research gaps in this field but also provide core material basis and technical support for the development of next-generation safe, natural, and efficient functional foods, health products, and even drug lead compounds, which has significant scientific and application value. However, how to refine *Nereiscus wartii* to prepare highly active *Nereiscus wartii* products, enabling its wider development and utilization, remains an urgent problem to be solved. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. The present invention proposes a method for preparing anti-oxidative peptides from *Clerodendrum chinense*. The anti-oxidative peptides prepared by this method have high antioxidant properties and can be used as raw materials for pharmaceuticals and cosmetics, which is beneficial to the development and utilization of *Clerodendrum chinense*.

[0005] The first objective of this invention is to provide a method for preparing worm antioxidant peptides.

[0006] A second aspect of the present invention is to provide a worm antioxidant peptide.

[0007] The third aspect of this invention aims to provide a preparation method of the first aspect of this invention or the application of the anti-oxidative peptide of the second aspect of this invention in the preparation of antioxidant products.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing worm antioxidant peptides, the method comprising the following steps: mixing worm homogenate with an enzyme, enzymatically hydrolyzing, to obtain worm antioxidant peptides; The enzymes include flavor proteases and pancreatic enzymes.

[0009] In some embodiments of the present invention, the enzyme activity of the flavor protease is 150,000-160,000 U / g.

[0010] In some embodiments of the present invention, the enzyme activity of the pancreatic enzyme is 1:(4000-5000).

[0011] In some embodiments of the present invention, the mass ratio of the flavor protease to the trypsin is 1:(0.1-5).

[0012] In some preferred embodiments of the present invention, the mass ratio of the flavor protease to the trypsin is 1:(0.1-3).

[0013] In some more preferred embodiments of the present invention, the mass ratio of the flavor protease to the trypsin is 1:(0.3-3).

[0014] In some embodiments of the present invention, the worm homogenate is prepared by mixing worms with water and homogenizing them to obtain the worm homogenate.

[0015] In some embodiments of the present invention, the mass-to-volume ratio (g / mL) of the worm to water is 1:(1-10).

[0016] In some preferred embodiments of the present invention, the mass-to-volume ratio (g / mL) of the worm to water is 1:(1-5).

[0017] In some more preferred embodiments of the present invention, the mass-to-volume ratio (g / mL) of the worm to water is 1:1.

[0018] In some embodiments of the present invention, the pH value of the worm homogenate is adjusted to 6-9.

[0019] In some preferred embodiments of the present invention, the pH value of the worm homogenate is adjusted to 6.5-8.5.

[0020] In some preferred embodiments of the invention, the pH of the worm homogenate is adjusted to 7-8.

[0021] In some embodiments of the present invention, the amount of enzyme added is 1%-5% of the mass of the rice worm homogenate.

[0022] In some preferred embodiments of the present invention, the amount of enzyme added is 1%-4% of the mass of the rice worm homogenate.

[0023] In some more preferred embodiments of the present invention, the amount of enzyme added is 2%-4% of the mass of the rice worm homogenate.

[0024] In some embodiments of the present invention, the enzymatic hydrolysis conditions are 40-60℃ for 2-6 hours.

[0025] In some preferred embodiments of the present invention, the enzymatic hydrolysis conditions are 40-55℃ for 3-6 h.

[0026] In some more preferred embodiments of the present invention, the enzymatic hydrolysis conditions are 45-55°C for 4-5 hours.

[0027] In some embodiments of the present invention, before the enzymatic hydrolysis, the mixture of worm homogenate and enzyme is sonicated under the following conditions: 40-60°C, 200-400W for 8-15 minutes.

[0028] Ultrasound-assisted enzymatic hydrolysis can improve the DPPH free radical scavenging rate of the hydrolysis products.

[0029] In some embodiments of the present invention, the preparation method further includes an enzyme inactivation step, wherein the enzyme inactivation conditions are a boiling water bath for 10-20 min.

[0030] In some embodiments of the present invention, solid-liquid separation is performed after enzyme inactivation, and the supernatant is collected. If centrifugation is used, the centrifugation conditions are 8000 rpm and 4°C for 20 min.

[0031] In some embodiments of the present invention, the preparation method further includes ultrafiltration to obtain trefoil antioxidant peptides with a permeate molecular weight <3 kDa and a retentate molecular weight >3 kDa.

[0032] The ultrafiltration fraction showed a better DPPH radical scavenging rate, indicating that the ultrafiltration process effectively enriched small molecule components with stronger antioxidant activity (such as low molecular weight peptides, phenolic substances, etc.) and may have removed large molecular impurities or inhibitory components.

[0033] In some embodiments of the present invention, the preparation method further includes a freeze-drying step.

[0034] In a second aspect, the present invention provides a worm antioxidant peptide, which is prepared by the preparation method of the first aspect of the present invention.

[0035] A third aspect of the present invention provides the preparation method of the first aspect of the present invention or the application of the anti-oxidative peptide of the second aspect of the present invention in the preparation of antioxidant products.

[0036] In some embodiments of the present invention, the antioxidant product includes reagents, pharmaceuticals, or cosmetics.

[0037] The beneficial effects of this invention are: This invention provides a method for preparing worm-derived antioxidant peptides with high hydrolysis degree and suitable for large-scale industrial production. By optimizing the enzymatic hydrolysis process (such as enzyme selection and ratio), the degree of protein hydrolysis is improved, thereby enhancing the antioxidant activity of the worm-derived antioxidant peptides. This effectively solves the problems of low efficiency, high cost, and unsuitability for large-scale extraction and preparation of worm-derived antioxidant peptides in current peptide extraction processes.

[0038] The anti-oxidative peptides from *Clerodendrum chinense* provided by this invention possess excellent antioxidant properties and exhibit significant scavenging effects against DPPH free radicals. When the concentration of the anti-oxidative peptides from *Clerodendrum chinense* increases from 1 mg / mL to 10 mg / mL, its DPPH free radical scavenging rate approaches that of vitamin C. This makes it widely applicable in cosmetics, nutritional foods, functional foods, health products, feed, and the preparation of antioxidant pharmaceuticals, thus benefiting the development and utilization of *Clerodendrum chinense*. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of the degree of hydrolysis test are for the antioxidant peptides of *Hemiberlesia lataniae* prepared by the extraction methods of Examples 1-9 of this invention.

[0040] Figure 2 The results of the degree of hydrolysis test are for the antioxidant peptides of *Hemiberlesia lataniae* prepared by the extraction methods of Examples 9-13 of this invention.

[0041] Figure 3 The results of the degree of hydrolysis test of the antioxidant peptides prepared by the extraction methods of Examples 14-29 of this invention are shown.

[0042] Figure 4 The results show the DPPH free radical scavenging rate of the anti-oxidative peptides prepared by the extraction methods of Examples 1-9 of this invention.

[0043] Figure 5 The results of the DPPH free radical scavenging rate test of the anti-oxidative peptides of *Clerodendrum typhimurium* prepared by the extraction method in Examples 9-13 of this invention are shown.

[0044] Figure 6 The results show the DPPH free radical scavenging rate of the anti-oxidative peptides prepared by the extraction methods of Examples 14-29 of this invention.

[0045] Figure 7 The results show the DPPH free radical scavenging rate of the anti-oxidative peptides prepared by the extraction method in Example 31 of this invention. Detailed Implementation

[0046] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0047] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0048] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] Example 1 An extraction method for antioxidant peptides from *Clerodendrum tigrinum* with antioxidant activity suitable for large-scale industrial production includes the following steps: (1) Take 10 g of fresh sandworms and 10 mL of distilled water and homogenize them into a paste using a tissue homogenizer, then adjust the pH to 8.0 with NaOH; (2) Add 3wt% alkaline protease (Shanghai Maclean Biochemical Technology Co., Ltd., product number: P824138, enzyme activity: 200 U / mg) and enzymatically hydrolyze at 50℃ for 4 h; (3) After hydrolysis, inactivate the enzyme at 100℃ for 15 min, centrifuge at 8000 rpm at 4℃ for 20 min, and then take the supernatant; (4) Collect the supernatant after centrifugation and freeze-dry it (vacuum freeze-drying below 50 Pa for 48 h) to obtain the crude peptide solution of worm, which is the worm antioxidant peptide.

[0050] Example 2 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 1 only in that: the pH value is adjusted to 7.0 in step (2), and the enzyme in step (3) is a neutral protease (Shanghai Maclean Biochemical Technology Co., Ltd., catalog number: D915910, enzyme activity: 100 U / mg, the same below).

[0051] Example 3 An extraction method for antioxidant peptides from *Clerodendrum trichotomum* with antioxidant activity suitable for large-scale industrial production, differing from Example 1 only in that: the pH value is adjusted to 7.5 in step (2), and the enzyme used in step (3) is bromelain (Shanghai Yuanye Biotechnology Co., Ltd., product number: [missing information]). S10009 Enzyme activity: 300 U / g (the same applies below).

[0052] Example 4 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 1 only in that: the pH value is adjusted to 7.0 in step (2), and the enzyme in step (3) is flavor protease (Shanghai Maclean Biochemical Technology Co., Ltd., catalog number: F768979, enzyme activity: 150,000 U / g, the same below).

[0053] Example 5 An extraction method for antioxidant peptides from *Clerodendrum trichotomum* with antioxidant activity suitable for large-scale industrial production is described. The only difference from Example 1 is that the pH value is adjusted to 7.0 in step (2), and the enzyme used in step (3) is papain (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: [missing information]). S10011 Enzyme activity: 800 U / mg (the same applies below).

[0054] Example 6 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 1 only in that: the pH value is adjusted to 7.0 in step (2), and the enzyme in step (3) is trypsin (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10031, enzyme activity: 1:4000, the same below).

[0055] Example 7 An extraction method for antioxidant peptides from *Clerodendrum tigrinum* with antioxidant activity suitable for large-scale industrial production includes the following steps: (1) Take 10 g of fresh sandworms and 10 mL of distilled water and homogenize them into a paste using a tissue homogenizer, then adjust the pH to 7.0 with NaOH; (2) Add 3wt% of compound enzyme (flavor protease and bromelain mixed at a mass ratio of 1:1) and hydrolyze at 50℃ for 4 h; (3) After hydrolysis, inactivate the enzyme at 100℃ for 15 min, centrifuge at 8000 rpm at 4℃ for 20 min, and then take the supernatant; (4) Collect the supernatant after centrifugation, freeze dry it to obtain the crude peptide solution of worm, which is the antioxidant peptide of worm.

[0056] Example 8 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 7 only in that the compound enzyme is a mixture of trypsin and bromelain in a 1:1 mass ratio.

[0057] Example 9 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 7 only in that the compound enzyme is a mixture of flavor protease and trypsin in a 1:1 mass ratio.

[0058] Example 10 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the compound enzyme is a mixture of flavor protease and trypsin at a mass ratio of 1:2.

[0059] Example 11 An extraction method for antioxidant peptides from worms with antioxidant activity suitable for large-scale industrial production, differing from Example 9 only in that the compound enzyme is a mixture of flavor protease and trypsin at a mass ratio of 1:3.

[0060] Example 12 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the compound enzyme is a mixture of flavor protease and trypsin at a mass ratio of 2:1.

[0061] Example 13 An extraction method for antioxidant peptides from worms with antioxidant activity suitable for large-scale industrial production, differing from Example 9 only in that the compound enzyme is a mixture of flavor protease and trypsin in a mass ratio of 3:1.

[0062] Example 14 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the ratio of worm pulp to distilled water is 1:2 (10g / 20mL).

[0063] Example 15 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the ratio of worm pulp to distilled water is 1:3 (10g / 30mL).

[0064] Example 16 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the ratio of worm pulp to distilled water is 1:4 (10g / 40mL).

[0065] Example 17 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, differs from Example 9 only in that the ratio of worm slurry to distilled water is 1:5 (10g / 50mL).

[0066] Example 18 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the pH value is adjusted to 6.5 in step (1).

[0067] Example 19 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the pH value is adjusted to 7.5 in step (1).

[0068] Example 20 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for industrial mass production, which differs from Example 9 only in that the pH value is adjusted to 8 in step (1).

[0069] Example 21 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the pH value is adjusted to 8.5 in step (1).

[0070] Example 22 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis time in step (2) is 2 h.

[0071] Example 23 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis time in step (2) is 3 h.

[0072] Example 24 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis time in step (2) is 5 h.

[0073] Example 25 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis time in step (3) is 6 h.

[0074] Example 26 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis temperature in step (2) is 40°C.

[0075] Example 27 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis temperature in step (2) is 45°C.

[0076] Example 28 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis temperature in step (2) is 55°C.

[0077] Example 29 An extraction method for antioxidant peptides from worms with antioxidant activity and suitable for large-scale industrial production, which differs from Example 9 only in that the hydrolysis temperature in step (2) is 60°C.

[0078] Example 30 An extraction method for antioxidant peptides from *Clerodendrum tigrinum* with antioxidant activity suitable for large-scale industrial production includes the following steps: (1) Take 10g of fresh sandworms and 10mL of distilled water and homogenize them into a paste using a tissue homogenizer, then adjust the pH to 7.0 with NaOH; (2) Add 3% of compound enzyme (flavor protease and trypsin mixed at a mass ratio of 1:1) and sonicate for 10 min at an ultrasonic temperature of 50℃ and a power of 300W to promote better binding of enzyme and substrate. (3) After the ultrasound is completed, continue hydrolysis at 50℃ for 4 hours; (4) After hydrolysis, inactivate the enzyme at 100℃ for 15 min, centrifuge at 8000 rpm at 4℃ for 20 min, and then take the supernatant; (5) Collect the supernatant after centrifugation, freeze dry it to obtain the crude peptide solution of worm, that is, worm antioxidant peptide; The DPPH free radical scavenging rate was measured (using the same method as in the effect example), and the final measured value was 86.78%.

[0079] Example 31 A method for extracting antioxidant peptides from *Clerodendrum trichotomum* with antioxidant activity suitable for large-scale industrial production is disclosed. Compared with Example 30, the only difference is that after extracting the crude peptide solution from *Clerodendrum trichotomum*, the antioxidant peptides with a cutoff of <3 kDa are dialyzed. The specific process is as follows: the crude peptide solution from *Clerodendrum trichotomum* is dialyzed, and ultrafiltration is performed using ultrafiltration membranes with different cutoff values. The permeate with a cutoff of <3 kDa and the retentate with a cutoff of >3 kDa are collected. The permeate and the retentate are freeze-dried to obtain antioxidant peptides from *Clerodendrum trichotomum* with cutoffs of <3 kDa and >3 kDa.

[0080] Effect Example (1) Determination of the degree of hydrolysis (DH) of anti-oxidative peptides from trevally The degree of hydrolysis is a key indicator for measuring the extent to which proteins are broken during hydrolysis, usually expressed as the percentage of broken peptide bonds in a protein molecule relative to the total number of peptide bonds. Essentially, it reflects the degree of protein hydrolysis and is commonly used in the preparation of bioactive peptides through enzymatic and chemical hydrolysis. The formaldehyde titration method was used to determine the content of free amino acids: 8.0 mL of the enzyme-inactivated hydrolysate was placed in a 200 mL beaker, 60 mL of distilled water was added, a magnetic stirrer was turned on, a small amount of alkali solution was added to adjust the pH to 8.2, then 10 mL of neutral formaldehyde solution was added. The pH of the system was adjusted to 9.2 with 0.5 mol / L NaOH standard solution, and the volume of NaOH standard solution consumed was recorded as V (mL). Simultaneously, 80 mL of water was taken, first adjusted to pH 8.2 with sodium hydroxide standard solution, then 10.0 mL of formaldehyde solution was added, and titrated to pH 9.2 with sodium hydroxide standard titration solution. A reagent blank test was performed, and the volume of NaOH standard solution consumed was recorded as V0 (mL).

[0081] The total nitrogen content was determined using the method specified in GB 5009.5—2016.

[0082] The degree of hydrolysis (DH) is calculated using the following formula: .

[0083] In the formula: C is the concentration of NaOH standard solution (mol / L); V is the volume of NaOH standard solution consumed by the enzymatic hydrolysate (mL); V0 is the volume of NaOH standard solution consumed by the blank solution (mL); 0.014 is the nitrogen milliequivalent; N is the total nitrogen content of the substrate sample (g).

[0084] The results are as follows Figures 1-3As shown, different proteases were used to hydrolyze *Clerodendrum chinense*, and flavor protease and trypsin showed the best degree of hydrolysis. Different compound enzymes were used to hydrolyze *Clerodendrum chinense*, and the results showed that the combination of flavor protease and trypsin resulted in the best degree of hydrolysis; subsequently, a compound enzyme of flavor protease and trypsin was used. Experiments were conducted on the material-to-liquid ratio, the ratio of flavor protease to trypsin, pH value, enzymatic hydrolysis temperature, and time (i.e., Examples 9-29) in the extraction method of *Clerodendrum chinense* antioxidant peptides. The results showed that with increasing enzymatic hydrolysis time, the degree of hydrolysis of the *Clerodendrum chinense* hydrolysate first increased and then decreased, with the best degree of hydrolysis at 4 hours. Temperature and pH showed similar effects, with the best hydrolysis effect at 50℃ and pH 7.5. However, with increasing material-to-liquid ratio, the degree of hydrolysis decreased, with the best hydrolysis effect at a ratio of 1:1, which may be because the enzyme can bind to more substrate.

[0085] (2) Determination of DPPH free radical scavenging rate DPPH free radicals exhibit a characteristic purple absorbance at 517 nm and are widely used as a standard reagent for evaluating the antioxidant capacity of bioactive compounds. This absorbance decreases significantly when free radicals are scavenged. This invention uses anhydrous ethanol to prepare a 1 mg / mL DPPH ethanol solution. 2 mL of the sample is mixed with 2 mL of the DPPH ethanol solution and shaken well. After incubation at room temperature in the dark for 30 min, the absorbance at 517 nm is measured. 2 mL of deionized water is mixed with 2 mL of the anhydrous ethanol solution and shaken well, and the absorbance at 517 nm is measured. 2 mL of anhydrous ethanol solution is mixed with 2 mL of deionized water and shaken well, and the absorbance at 517 nm is measured. VC serves as a positive control. The DPPH free radical scavenging rate is calculated using the following formula: In the formula: A1 is the absorbance of the sample and DPPH ethanol solution; A2 is the absorbance of deionized water and DPPH ethanol solution; A3 is the absorbance of the sample and anhydrous ethanol solution.

[0086] The results are as follows Figures 5-7 As shown, the anti-oxidative peptides from *Clerodendrum tigrinum* obtained by hydrolysis using flavor protease and trypsin exhibited good DPPH radical scavenging rate. When the liquid-to-solid ratio was 1:1, the ratio of flavor protease to trypsin was 1:1, and the hydrolysis conditions were 50°C for 4 hours (i.e., the extraction method in Example 9), the extracted anti-oxidative peptides showed good DPPH radical scavenging rate. The dialysis-ultrafiltration fraction (<3 kDa) showed even better DPPH radical scavenging rate, indicating that the ultrafiltration process effectively enriched small molecule components with stronger antioxidant activity (such as low molecular weight peptides, phenolic substances, etc.) and may have removed large molecular impurities or inhibitory components.

[0087] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing an antioxidant peptide from a geophilic worm, characterized in that, The preparation method comprises the following steps: mixing the homogenate of the chironomus with enzymes, and enzymolysis to obtain chironomus antioxidant peptides. The enzymes comprise flavour protease and trypsin.

2. The production method according to claim 1, characterized by, The mass ratio of the flavour protease and the trypsin is 1: (0.1-5).

3. The production method according to claim 1, characterized by, The homogenate of the chironomus is prepared by the following method: mixing the chironomus with water, and homogenizing to obtain the homogenate of the chironomus.

4. The production method according to claim 3, characterized by, The mass-volume ratio of the chironomus and water is 1: (1-10).

5. The production method according to claim 3, wherein The pH value of the homogenate of the chironomus is adjusted to 6-9.

6. The production method according to any one of claims 1 to 5, characterized by, The added amount of the enzymes is 1%-5% of the mass of the homogenate of the chironomus; and / or, the enzymolysis conditions are 40-60℃ for 2-6 h.

7. The production method according to claim 6, wherein Before the enzymolysis, the mixture of the homogenate of the chironomus and the enzymes is subjected to ultrasonic treatment under the conditions of 40-60℃ and 200-400W for 8-15 min.

8. The production method according to any one of claims 1 to 5, characterized by, The preparation method comprises ultrafiltration, and the chironomus antioxidant peptides with a molecular weight <3 kDa are allowed to pass through, and the chironomus antioxidant peptides with a molecular weight >3 kDa are intercepted.

9. A chironomus antioxidant peptide prepared by the preparation method of any one of claims 1-8.

10. Use of the preparation method of any one of claims 1-8 or the chironomus antioxidant peptide of claim 9 in the preparation of an antioxidant product.

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