Active peptide of phascolosoma esox and preparation method thereof, chewable tablet

Abstract

The application relates to the technical field of polypeptide extraction processes, in particular to a Phascolosoma esculenta active peptide, a preparation method thereof and a chewable tablet. The preparation method of the Phascolosoma esculenta active peptide comprises the following steps: crushing and treating washed Phascolosoma esculenta in water to obtain Phascolosoma esculenta slurry; performing first enzymolysis treatment on the Phascolosoma esculenta slurry by using neutral protease to obtain first enzymolysis liquid; performing first enzyme inactivation treatment on the first enzymolysis liquid, then adding flavor protease to perform second enzymolysis treatment to obtain second enzymolysis liquid; performing second enzyme inactivation treatment on the second enzymolysis liquid, then performing centrifugal separation to obtain supernatant; and performing filtration treatment on the supernatant to obtain Phascolosoma esculenta active peptide with a molecular weight of less than 10 kDa. The Phascolosoma esculenta active peptide obtained by the preparation method has better antioxidant activity, can relieve the oxidation stress of a body caused by D-galactose, and has a certain protective effect on liver injury caused by subacute aging.

Description

Technical Field

[0001] This application belongs to the field of polypeptide extraction technology, and particularly relates to a palatable active peptide from Sipunculus nudus, its preparation method, and chewable tablets. Background Technology

[0002] Under normal circumstances, enzymatic antioxidants in the body, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), protect the body from damage by scavenging free radicals. When reactive oxygen species (ROS) increase or the body's antioxidant capacity decreases, the body's oxidation and antioxidation become unbalanced. Excessive ROS oxidizes cellular fatty acids, producing lipid peroxides and lipid hydroperoxides. These lipid peroxides can diffuse across the cell membrane, altering its structure and function and leading to a loss of cellular homeostasis. Furthermore, proteins are also easily attacked by ROS directly or indirectly through lipid peroxidation, resulting in changes in enzyme activity and signaling pathways. High concentrations of ROS directly act on DNA, causing DNA strand breaks, point mutations, abnormal cross-linking, and mutations in proto-oncogenes and tumor suppressor genes, thereby promoting the transformation of normal cells into tumors. ROS interacts with and modifies cellular proteins, lipids, and DNA, leading to abnormal gene expression, impaired intercellular communication, and alterations in the second messenger system, ultimately resulting in oxidative stress (OS). Numerous studies have shown that OS is involved in the development of diseases such as cancer, diabetes, atherosclerosis, Alzheimer's disease, and aging.

[0003] To reduce the harm of antioxidants (OS) to the human body, various antioxidants have been added to food and pharmaceuticals. These include many synthetic antioxidants such as di-tert-butyl-p-cresol (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), but they have certain toxic side effects. With the deepening research on natural antioxidants, attention is increasingly turning to highly active and less toxic antioxidant peptides for the prevention and treatment of human diseases. Antioxidant peptides are bioactive peptides that scavenge free radicals, inhibit lipid peroxidation, maintain free radical balance, and enhance the body's anti-aging functions. Enzymatic hydrolysis is the primary method for obtaining antioxidant peptides. Different proteases have different cleavage sites, thus producing different polypeptides. Enzymatic hydrolysis is simple, convenient, easy to operate, and low in cost, therefore it has become a common method for preparing bioactive peptides.

[0004] *Phascolosoma esculenta*, commonly known as the palatable sipunculid worm, is widely distributed along the coastal mudflats of Zhejiang, Fujian, and Guangdong provinces. Besides being edible, *Phascolosoma esculenta* also has medicinal uses. For example, the *Chinese Marine Materia Medica* records that *Phascolosoma esculenta* is sweet and salty in taste, and cold in nature; it enters the spleen and kidney meridians; and is used to treat qi and blood deficiency. Modern pharmacological studies have confirmed that *Phascolosoma esculenta* possesses various biological activities. Xiang Huan et al. optimized the papain hydrolysis process using response surface methodology, obtaining enzymatically hydrolyzed *Phascolosoma esculenta* antioxidant peptides with a high scavenging rate against 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals. Peng Yixin et al. extracted collagen from *Phascolosoma esculenta*, using total antioxidant capacity and superoxide anion scavenging rate as indicators. Through single-factor experiments and response surface methodology optimization, they determined that the optimal hydrolysis scheme was a combination of papain and neutral protease, which could improve the superoxide anion scavenging rate.

[0005] Sipunculus nudus has great potential for development into antioxidant functional foods, but the discovery of Sipunculus nudus antioxidant peptides with even better effects remains to be explored. Summary of the Invention

[0006] The purpose of this application is to provide a palatable active peptide from Sipunculus nudus, its preparation method, and chewable tablets, aiming to solve the problem of how to extract palatable antioxidant peptides from Sipunculus nudus with better antioxidant activity.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] In a first aspect, this application provides a method for preparing palatable active peptides from Sipunculus nudus, comprising the following steps:

[0009] The cleaned Sipunculus nudus was crushed in water to obtain Sipunculus nudus slurry.

[0010] The first enzymatic hydrolysate was obtained by first enzymatic hydrolysis of Sipunculus nudus slurry with neutral protease.

[0011] The first enzymatic hydrolysate is subjected to a first enzyme inactivation treatment, and then flavor protease is added for a second enzymatic hydrolysis treatment to obtain a second enzymatic hydrolysate.

[0012] The second enzyme hydrolysate was subjected to a second enzyme inactivation treatment, and then centrifuged to obtain the supernatant.

[0013] The supernatant was filtered to obtain palatable active peptides of Sipunculus nudus with a molecular weight of <10kDa.

[0014] Secondly, this application provides an oral active peptide of Sipunculus nudus, which is prepared by the preparation method described above in this application.

[0015] Thirdly, this application provides a chewable tablet comprising the palatable active peptide of Sipunculus nudus of this application and pharmaceutically acceptable excipients.

[0016] The method for preparing palatable *Sipunculus nudus* bioactive peptides provided in the first aspect of this application involves enzymatically hydrolyzing *Sipunculus nudus* using a combination of neutral and flavor proteases. The supernatant obtained after centrifugation is then filtered to extract bioactive peptides with a molecular weight <10 kDa. The bioactive peptides obtained by this method exhibit better antioxidant activity, can alleviate oxidative stress induced by D-galactose, and have a certain protective effect against subacute aging-related liver damage. Therefore, the bioactive peptides prepared by this method have promising applications in antioxidant functional foods.

[0017] The palatable Sipunculus cuspidatus active peptides provided in the second aspect of this application are prepared by a unique preparation method of this application. Specifically, the palatable Sipunculus cuspidatus active peptides of this application are obtained by extracting and separating palatable Sipunculus cuspidatus active peptides with a molecular weight of <10kDa through complex enzymatic hydrolysis of Sipunculus cuspidatus using neutral protease and flavor protease. Such palatable Sipunculus cuspidatus active peptides have better antioxidant activity, can alleviate oxidative stress caused by D-galactose, and have a certain protective effect against subacute aging-related liver damage. They can be used to make antioxidant functional foods.

[0018] The chewable tablets provided in the third aspect of this application include the palatable *Sipunculus nudus* active peptide obtained by the preparation method unique to this application and pharmaceutically acceptable excipients. Based on the fact that the palatable *Sipunculus nudus* active peptide has better antioxidant activity, such chewable tablets can alleviate oxidative stress caused by D-galactose and have a certain protective effect against subacute aging-related liver damage. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 These are comparative images of HE staining results of liver tissue containing the bioactive peptides of Sipunculus nudus from the present application.

[0021] Figure 2 This is a comparison image of the HE staining results of brain tissue containing the bioactive peptides of Sipunculus nudus from the present application. Detailed Implementation

[0022] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0023] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0024] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0025] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0026] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0027] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0028] The first aspect of this application provides a method for preparing palatable active peptides from Sipunculus nudus, comprising the following steps:

[0029] S01: The cleaned Sipunculus nudus is crushed in water to obtain Sipunculus nudus slurry.

[0030] S02: The first enzymatic hydrolysate was obtained by first enzymatic hydrolysis of Sipunculus nudus slurry with neutral protease.

[0031] S03: The first enzymatic hydrolysate is subjected to a first enzyme inactivation treatment, and then flavor protease is added for a second enzymatic hydrolysis treatment to obtain a second enzymatic hydrolysate;

[0032] S04: The second enzyme hydrolysate is subjected to a second enzyme inactivation treatment, and then centrifuged to obtain the supernatant;

[0033] S05: Filter the supernatant to obtain palatable active peptides of Sipunculus nudus with a molecular weight of <10kDa.

[0034] The method for preparing palatable *Sipunculus nudus* active peptides provided in this application involves enzymatically hydrolyzing *Sipunculus nudus* using a combination of neutral and flavor proteases. The supernatant obtained after centrifugation is then filtered to extract palatable *Sipunculus nudus* active peptides with a molecular weight <10 kDa. The palatable *Sipunculus nudus* active peptides prepared by this method exhibit better antioxidant activity, can alleviate oxidative stress induced by D-galactose, and have a certain protective effect against subacute aging-related liver damage. Therefore, the palatable *Sipunculus nudus* active peptides prepared by this method have excellent application prospects in antioxidant functional foods.

[0035] Step S01 is the preparation step of the palatable Sipunculus nudus pulp. Specifically, fresh Sipunculus nudus can be selected first, and then the selected fresh Sipunculus nudus can be washed and crushed. For example, select fresh Sipunculus nudus with a body length of 8-12cm, wash off the surface dirt with water; cut open the body of the Sipunculus nudus, remove the body cavity fluid and internal organs, wash again with pure water and drain for later use; cut the washed Sipunculus nudus ...

[0036] Step S02 is the neutral protease hydrolysis step.

[0037] In one embodiment, the conditions for the first enzymatic hydrolysis treatment include: a temperature of 43–47°C, a pH of 6.3–6.7, and a time of 3.5–4.5 h. Specifically, a neutral protease is subjected to isothermal enzymatic hydrolysis under the above conditions. A neutral protease is added to the obtained *Sipunculus nudus* slurry, and the first enzymatic hydrolysis treatment is performed according to the conditions for the first enzymatic hydrolysis treatment, thereby better utilizing the activity of the neutral protease to obtain the first enzymatic hydrolysate. Further, the conditions for the first enzymatic hydrolysis treatment include: a temperature of 45°C, a pH of 6.5, and a time of 4 h.

[0038] In one embodiment, in the first enzymatic hydrolysis step of Sipunculus nudus slurry with neutral protease, the amount of neutral protease added is 0.4 to 0.6% of the weight of Sipunculus nudus. Specifically, neutral protease is added at a weight ratio of 0.5% (w / w) for isothermal enzymatic hydrolysis, so that Sipunculus nudus is fully hydrolyzed by neutral protease.

[0039] Step S03 is the enzymatic hydrolysis step of flavor protease.

[0040] In one embodiment, before adding the flavor protease for enzymatic hydrolysis, the first hydrolysate obtained after neutral protease hydrolysis can be subjected to a first enzyme inactivation treatment. This inactivates the neutral protease, thereby allowing the flavor protease to exert its activity more stably. Specifically, the first enzyme inactivation treatment includes: inactivating the enzyme in the first hydrolysate at 98–102°C for 8–10 minutes, for example, at 100°C for 10 minutes; this completely inactivates the neutral protease.

[0041] In one embodiment, the conditions for the second enzymatic hydrolysis treatment include: a temperature of 38–42°C, a pH of 5.3–5.7, and a time of 2.5–3.5 h. Specifically, the flavor protease is subjected to isothermal enzymatic hydrolysis under the above conditions. In the first enzymatic hydrolysate after enzyme inactivation, a second enzymatic hydrolysis treatment is performed according to the conditions for the second enzymatic hydrolysis treatment, thereby better utilizing the activity of the flavor protease to obtain the second enzymatic hydrolysate. Further, the conditions for the second enzymatic hydrolysis treatment include: a temperature of 40°C, a pH of 5.5, and a time of 3 h.

[0042] In one embodiment, in the step of performing a second enzymatic hydrolysis on the first enzymatic hydrolysate after enzyme inactivation with flavor protease, the amount of flavor protease added is 0.4 to 0.6% of the weight of Sipunculus nudus. Specifically, flavor protease is added at a weight ratio of 0.5% (w / w) for isothermal enzymatic hydrolysis, so that the Sipunculus nudus polypeptides that have been hydrolyzed by neutral protease are fully hydrolyzed again by flavor protease.

[0043] Furthermore, in the preparation method of the palatable Sipunculus nudus active peptide of this application, the amounts of neutral protease and flavor protease can be 1:1.

[0044] In the first and second enzymatic hydrolysis steps, the pH of the homogenate is adjusted to the optimal pH for protease hydrolysis using NaOH (1M) and HCl (1M).

[0045] Step S04 is the supernatant extraction step.

[0046] In one embodiment, before centrifugation, the second enzyme hydrolysate can be subjected to a second enzyme inactivation treatment to deactivate the flavor protease, thereby obtaining stable and palatable Sipunculus nudus active peptides. Specifically, the second enzyme inactivation treatment includes: inactivating the enzyme in the first enzyme hydrolysate at 98–102°C for 8–10 min, for example, at 100°C for 10 min; this completely inactivates the flavor protease.

[0047] In one embodiment, the centrifugation step includes: centrifuging the second enzymatic hydrolysate after the second enzyme inactivation treatment at 2–4°C at a speed of 8000–12000 r / min for 15–25 min. Centrifugation under these conditions separates the peptides from the combined enzymatic hydrolysis of neutral protease and flavor protease to the supernatant, generally yielding palatable active peptides of *Sipunculus nudus* with a molecular weight <10 kDa.

[0048] Step S05 is the separation step of active peptides from Sipunculus nudus with a molecular weight of <10kDa.

[0049] In one embodiment, the step of filtering the supernatant includes: pretreating the supernatant by passing it through an aqueous membrane with a pore size of 0.45 μm, and then separating it by passing it through a filter membrane with a molecular weight cutoff of 10 kDa. Specifically, the supernatant obtained by enzymatic hydrolysis and centrifugation is first pretreated using an aqueous membrane with a pore size of 0.45 μm, and then the pretreated supernatant is separated by passing it through a filter membrane with a molecular weight cutoff of 10 kDa using a membrane separation system. The outlet pressure of the controlled filter membrane is kept below 5 Bar, and the bioactive peptides of *Sipunculus nudus* with a molecular weight <10 kDa are collected. The obtained bioactive peptides of *Sipunculus nudus* are then freeze-dried and stored at -20°C for later use.

[0050] This application uses fresh *Sipunculus nudus* as raw material, and applies a combination of neutral protease and flavor protease to enzymatically hydrolyze the *Sipunculus nudus*. The supernatant from the hydrolysis is then prepared by centrifugation, and a membrane separation system is used to separate the supernatant into fractions with molecular weights <10 kDa and >10 kDa. Simultaneously, experiments demonstrate that the *Sipunculus nudus* bioactive peptides with molecular weights <10 kDa extracted in this application exhibit better antioxidant activity than the fractions with molecular weights >10 kDa.

[0051] The second aspect of this application provides an oral active peptide from Sipunculus nudus, which is prepared by the preparation method described above in the embodiments of this application.

[0052] The palatable Sipunculus cuspidatus active peptides provided in this application are prepared by a unique preparation method of this application. Specifically, the palatable Sipunculus cuspidatus active peptides of this application are obtained by enzymatically hydrolyzing Sipunculus cuspidatus with neutral protease and flavor protease, and then extracting and separating palatable Sipunculus cuspidatus active peptides with a molecular weight of <10kDa. Such palatable Sipunculus cuspidatus active peptides have better antioxidant activity, can alleviate oxidative stress caused by D-galactose, and have a certain protective effect against liver damage caused by subacute aging. They can be used to make antioxidant functional foods.

[0053] A third aspect of this application provides a chewable tablet comprising a palatable active peptide from Sipunculus nudus, unique to this application, and pharmaceutically acceptable excipients.

[0054] The chewable tablets provided in this application include the palatable *Sipunculus nudus* active peptide obtained by the preparation method unique to this application and pharmaceutically acceptable excipients. Based on the fact that the palatable *Sipunculus nudus* active peptide has better antioxidant activity, such chewable tablets can alleviate oxidative stress caused by D-galactose and have a certain protective effect against subacute aging-related liver damage.

[0055] This application screened for palatable Sipunculus nudus active peptide components with molecular weight <10kDa that exhibited the highest antioxidant activity by measuring DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, superoxide anion free radical scavenging capacity, and total antioxidant capacity. After freeze-drying the component with the highest antioxidant activity and combining it with excipients possessing certain physicochemical functions, palatable Sipunculus nudus chewable tablets were prepared. These chewable tablets are characterized by stable quality, convenient consumption, rapid absorption, and high bioavailability, making them suitable for a wide range of people, with a unique flavor suitable for all ages.

[0056] Furthermore, the excipients include thickeners such as maltodextrin, and flavoring agents such as erythritol and citric acid. In each tablet, the amount of active peptides from Sipunculus nudus is approximately 1-3%, the amount of citric acid is approximately 4-6%, and the remaining amount of maltodextrin:erythritol is (4-5):(4-5).

[0057] Specifically, the preparation of chewable tablets includes: pulverizing maltodextrin, erythritol, active peptides of Sipunculus nudus with a molecular weight of <10kDa, and citric acid, and passing them through an 80-mesh sieve, then sealing and storing them for later use; using the direct compression method of powder, all materials are first mixed evenly according to the addition amount, and then compressed into tablets using a single punch tablet press.

[0058] The following description is based on specific embodiments.

[0059] Example 1

[0060] A method for preparing palatable active peptides from Sipunculus nudus, comprising the following steps:

[0061] 1. Raw material preparation: Select fresh and palatable Sipunculus nudus from Suixi, Zhanjiang, Guangdong. The worms are 8-12cm long. First, wash off the mud and sand on the surface with tap water, then wash twice with pure water. Use stainless steel scissors to cut open the worms, remove the body fluid and internal organs, wash twice more with pure water, and drain for later use.

[0062] 2. Preparation of delicious Sipunculus nudus pulp: Cut the cleaned and drained insects into sections, add an appropriate amount of pure water to a tissue homogenizer, and pulverize to obtain a delicious Sipunculus nudus pulp with a material-to-liquid ratio of 1:3.

[0063] 3. Neutral protease isothermal hydrolysis: Adjust the temperature of the Sipunculus nudus slurry to 45℃ (the optimal hydrolysis temperature for neutral protease). Under the conditions of pH 6.5 and temperature 45℃, add 0.5% (w / w) neutral protease and perform isothermal hydrolysis for 4 hours. Adjust the pH every 1 hour to stabilize it at 6.5.

[0064] 4. First enzyme inactivation: After constant temperature enzymatic hydrolysis of neutral protease, inactivate the enzyme in the hydrolysate at 100℃ for 10 min, and then allow it to cool naturally to room temperature (25~27℃);

[0065] 5. Isothermal enzymatic hydrolysis of flavor protease: The enzymatic hydrolysate that has been inactivated and cooled to room temperature is heated to 40°C (the optimal enzymatic hydrolysis temperature of flavor protease). Under the conditions of pH 5.5 and temperature 40°C, 0.5% (w / w) flavor protease is added and isothermal enzymatic hydrolysis is carried out for 3 hours. The pH is adjusted every 1 hour to stabilize it at 5.5.

[0066] 6. Second enzyme inactivation: After constant temperature enzymatic hydrolysis of flavor protease, the hydrolysate is inactivated at 100℃ for 10 minutes, and then naturally cooled to room temperature;

[0067] 7. Centrifugation: After cooling the enzyme hydrolysate after the second enzyme inactivation, centrifuge at 10000 r / min and 4℃ for 20 min. Store the supernatant in a -20℃ refrigerator.

[0068] 8. Ultrafiltration: First, the supernatant above is pretreated using an aqueous membrane with a pore size of 0.45 μm. Then, the pretreated supernatant is separated by a membrane separation system through a filter membrane with a molecular weight cutoff of 10 kDa. The outlet pressure of the control system filter membrane is controlled to be less than 5 Bar. The active peptides of *Sipunculus nudus* with a molecular weight of <10 kDa are collected, freeze-dried, and stored in a -20°C refrigerator for later use.

[0069] Example 2

[0070] A method for preparing a chewable tablet containing palatable active peptides from Sipunculus nudus, comprising the following steps:

[0071] The active peptides of Sipunculus nudus with a molecular weight of <10kDa prepared in Example 1 were pulverized with maltodextrin, erythritol and citric acid and passed through an 80-mesh sieve. All raw and auxiliary materials were mixed evenly according to the addition amount and then tableted using a single punch tablet press.

[0072] Each chewable tablet weighs 0.7g ± 0.1g. The ratio of maltodextrin to erythritol in each tablet is 5:4, the amount of polypeptide is 2%, and the amount of citric acid is 5%. Under this formula, the chewable *Sipunculus nudus* polypeptide chewable tablets have neat edges, are not broken, have a uniform milky white color, a delicate texture, a pleasantly sweet and sour taste, and a slight umami flavor from seafood.

[0073] Performance testing

[0074] Example 1: Study on the in vitro antioxidant activity of palatable Sipunculus nudus bioactive peptides

[0075] Reagents and Instruments

[0076] reagents

[0077] Neutral protease (50000U / g) Guangxi Pangbo Biotechnology Co., Ltd.

[0078] Flavor protease (50000U / g) Guangxi Pangbo Biotechnology Co., Ltd.

[0079] Hydrochloric acid (analytical grade) Guangdong Guanghua Technology Co., Ltd.

[0080] Sodium hydroxide (analytical grade) Xilong Chemical Co., Ltd.

[0081] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) Shanghai Maclean Biochemical Technology Co., Ltd.

[0082] Anhydrous ethanol (analytical grade) Guangdong Guanghua Technology Co., Ltd.

[0083] Carnosine Hefei Bomei Biotechnology Co., Ltd.

[0084] Hydroxyl radical reagent kit (A018-1-1) Nanjing Jiancheng Bioengineering Institute

[0085] Total Antioxidant Capacity Reagent Kit (A015-2-1) Nanjing Jiancheng Bioengineering Institute

[0086] Superoxide Anion Assay Kit (A052-1-1) Nanjing Jiancheng Bioengineering Institute

[0087] instrument

[0088] GCM-FT-01 Membrane Separation System, Guochu Technology (Xiamen) Co., Ltd.

[0089] JJ-2B Tissue Homogenizer, manufactured by Xicheng Xinrui Instrument Factory, Jintan District

[0090] DF-101S Thermostatic Heating Magnetic Stirrer, Gongyi Yuhua Instrument Co., Ltd.

[0091] 3H16RI Intelligent High-Speed ​​Refrigerated Centrifuge, Hunan Hexi Instrument Equipment Co., Ltd.

[0092] S1010E Handheld Centrifuge from Scilogex (USA)

[0093] XW-80A Vortex Mixer, Shanghai Jingke Industrial Co., Ltd.

[0094] 1900614S Freeze Dryer, Beijing Boyikang Experimental Instrument Co., Ltd.

[0095] Milli Ultrapure Water Preparation Systems, by Millipore (USA)

[0096] Ice maker Guangzhou Feidi Biotechnology Co., Ltd.

[0097] Pipettes Qingdao Jiading Analytical Instruments Co., Ltd.

[0098] Kaifeng Electronic Weighing Scales Group Co., Ltd.

[0099] Shanghai Xiaohan Industrial Development Co., Ltd.

[0100] Multi-station magnetic stirring water bath, Changzhou Jintan Liangyou Instrument Co., Ltd.

[0101] Epoch microplate spectrophotometer, BioTec, USA

[0102] 1.1 Determination of DPPH free radical scavenging ability

[0103] Take 500 μl of 0.2 mM DPPH solution and mix it with 500 μl of the enzymatically hydrolyzed Sipunculus nudus active peptide. After shaking and mixing, let it stand at room temperature in the dark for 30 min. Take the supernatant and measure the absorbance at a wavelength of 517 nm, and record it as A0. Take 500 μl of DPPH solution and mix it with 500 μl of distilled water, and measure the absorbance at a wavelength of 517 nm, and record it as A1. Take 500 μl of sample solution and mix it with 500 μl of anhydrous ethanol, and measure the absorbance at a wavelength of 517 nm, and record it as A2. The scavenging ability is expressed as: DPPH scavenging rate (%) = [1 - (A0 - A2) / A1] × 100.

[0104] 1.2 Determination of hydroxyl radical scavenging ability

[0105] The hydroxyl radical assay kit, obtained from Nanjing Jiancheng Biotechnology Institute, was used for the assay. The specific operating procedures were performed according to the kit instructions.

[0106] 1.3 Determination of superoxide anion radical scavenging ability

[0107] The assay was performed using a kit for the inhibition and generation of superoxide anion free radicals, sourced from Nanjing Jiancheng Bioengineering Institute. Specific operating procedures were performed according to the kit's instruction manual.

[0108] 1.4 Determination of Total Antioxidant Capacity (T-AOC)

[0109] Weigh an appropriate amount of Sipunculus nudus active peptides to prepare a 20 mg / mL solution for later use. Then, determine the total antioxidant capacity (T-AOC) according to the specific steps of the kit, which was obtained from Nanjing Jiancheng Biotechnology Institute.

[0110] The antioxidant activity studies described above tested the oral active peptides of Sipunculus nudus obtained in Example 1, with a molecular weight <10 kDa. Three control groups were also set up:

[0111] Control group 1: The active peptide of Sipunculus nudus with a molecular weight >10kDa obtained by combined enzymatic hydrolysis of neutral protease and flavor protease (i.e. the remaining polypeptide component after obtaining the active peptide of Sipunculus nudus with a molecular weight <10kDa from Example 1).

[0112] Control group 2: Active peptides of Sipunculus nudus with a molecular weight of <10kDa obtained by enzymatic hydrolysis with neutral protease alone (i.e., active peptides of Sipunculus nudus with a molecular weight greater than <10kDa obtained by enzymatic hydrolysis with neutral protease alone in Example 1).

[0113] Control group 3: Flavorful Sipunculus bioactive peptides with a molecular weight of <10kDa obtained by enzymatic hydrolysis with flavor protease alone (i.e., flavorful Sipunculus bioactive peptides with a molecular weight of <10kDa obtained by enzymatic hydrolysis with flavor protease alone in Example 1).

[0114] The results show that the DPPH free radical scavenging capacity is shown in Table 1, the hydroxyl free radical scavenging capacity is shown in Table 2, the superoxide anion free radical scavenging capacity is shown in Table 3, and the total antioxidant capacity is shown in Table 4.

[0115] Table 1. DPPH free radical scavenging rate (%)

[0116] Grouping 1mg / ml 2mg / ml 4mg / ml 6mg / ml 8mg / ml 10mg / ml Example 1 43.38±3.11 55.21±2.69 71.46±3.19 83.26±3.87 91.67±3.63 93.58±3.12 Control group 1 41.11±2.03 46.83±2.71 57.28±1.96 62.36±4.17 79.17±4.65 86.67±2.72 Control group 2 23.38±3.11 35.21±2.69 41.46±3.19 53.26±3.87 61.67±3.63 73.58±3.12 Control group 3 27.11±2.03 36.83±2.71 47.28±1.96 52.36±4.17 69.17±4.65 76.67±2.72

[0117] Table 2. Hydroxyl radical scavenging rate (%)

[0118]

[0119]

[0120] Table 3. Superoxide anion radical scavenging rate (%)

[0121] Grouping 10mg / ml 20mg / ml 30mg / ml 40mg / ml 50mg / ml Example 1 2.36±1.01 8.69±1.35 27.57±2.11 53.96±3.73 67.83±3.61 Control group 1 1.89±0.94 6.14±2.06 11.43±3.38 34.77±1.85 49.13±3.07 Control group 2 1.36±1.01 4.69±1.35 12.57±2.11 23.96±3.73 37.83±3.61 Control group 3 1.89±0.94 6.14±2.06 11.43±3.38 34.77±1.85 39.13±3.07

[0122] Table 4 Total antioxidant capacity T-AOC (mmol / g)

[0123] Components T-AOC (mmol / g) Example 1 0.0165 Control group 1 0.0116 Control group 2 0.0101 Control group 3 0.0117

[0124] Example 1: Study on the in vivo antioxidant activity of the oral Sipunculus nudus bioactive peptide

[0125] Experimental materials

[0126] Experimental animals: 50 ICR mice, SPF grade, weighing 18 - 20 g, half male and half female, provided by the Guangdong Provincial Center for Medical Experimental Animals, animal license number: SCXK(Guangdong)2018 - 0002. The mice were housed in an environment with a temperature maintained at 22 ± 1 °C and a relative humidity of 55 ± 10%, and were subjected to 12 h light-dark alternation. During the feeding period, the mice could eat and drink freely. The feed and drinking water were strictly disinfected, and the cages and bedding were cleaned and replaced in a timely manner.

[0127] Polypeptides in the complex enzyme hydrolysis experimental group: The active peptides of Phascolosoma esculenta with a molecular weight < 10 kDa prepared in Example 1 were stored in a -20 °C refrigerator for later use.

[0128] Polypeptides in the neutral protease hydrolysis group: The active peptides of Phascolosoma esculenta with a molecular weight < 10 kDa obtained by single enzyme hydrolysis with neutral protease (that is, the active peptides of Phascolosoma esculenta with a molecular weight > < 10 kDa obtained by only using neutral protease for enzyme hydrolysis in Example 1).

[0129] Polypeptides in the flavor protease hydrolysis group: The active peptides of Phascolosoma esculenta with a molecular weight < 10 kDa obtained by single enzyme hydrolysis with flavor protease (that is, the active peptides of Phascolosoma esculenta with a molecular weight < 10 kDa obtained by only using flavor protease for enzyme hydrolysis in Example 1).

[0130] Experimental methods

[0131] After one week of adaptive feeding, 50 ICR mice were randomly divided into 4 groups: blank group, model group, complex enzyme hydrolysis experimental group (400 mg / Kg), neutral protease hydrolysis group (400 mg / Kg), and flavor protease hydrolysis group (400 mg / Kg). Except for the blank group, the mice in the other groups were subcutaneously injected with 100 mg / Kg of D-galactose in the neck and back once a day for 8 consecutive weeks. The blank group was injected with an equal amount of normal saline. At the same time of D-galactose modeling, intragastric administration was carried out according to the above dosage once a day for 8 consecutive weeks. The mice in the blank group and the model group were intragastrically administered distilled water (10 mL / Kg). Then, some liver tissues and brain tissues were taken, fixed with 4% paraformaldehyde for 24 h, dehydrated, paraffin-embedded, sectioned (4 μm), stained with hematoxylin-eosin (HE), and sealed with neutral gum. The pathological changes of the liver and brain tissues of the mice were observed under an optical microscope.

[0132] 2.1 Determination of antioxidant-related indicators

[0133] The protein content in mouse serum, brain tissue, and liver tissue was determined using a BCA reagent kit. MDA, SOD, CAT, and GSH-Px levels in serum, brain tissue, and liver tissue were measured according to the corresponding methods described in the kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0134] 2.2 Pathological observation of liver and brain tissue

[0135] Partial liver and brain tissues were collected, fixed with 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin, sectioned (4μm), stained with hematoxylin and eosin (HE), and mounted with neutral resin. The pathological changes of mouse liver tissue were observed under an optical microscope.

[0136] 2.3 Statistical Processing Methods

[0137] Data analysis was performed using SPSS 26.0 statistical software. One-way ANOVA was used for comparisons between groups, and t-tests were used for pairwise comparisons. P < 0.05 was considered statistically significant. Results are expressed as mean ± standard deviation. express.

[0138] Finally, the MDA measurement data are shown in Table 5, the SOD measurement data are shown in Table 6, the GSH-Px measurement data are shown in Table 7, and the CAT measurement data are shown in Table 8.

[0139] Table 5. MDA activity in mouse serum

[0140] Grouping MDA (nmol / ml) Blank control group 7.68±0.45 Model group <![CDATA[13.31±0.52 ## ]]> Compound enzyme hydrolysis experimental group <![CDATA[8.30±0.30 #** ]]> Neutral protease digestion group <![CDATA[10.36±1.01 ##** ]]> Flavor protease hydrolysis group <![CDATA[9.89±0.94 ##** ]]>

[0141] Table 6. Serum SOD activity in mice

[0142] Grouping SOD (U / ml) Blank control group 86.88±6.12 Model group <![CDATA[73.12±4.82 ## ]]> Compound enzyme hydrolysis experimental group <![CDATA[88.30±0.30 * ]]> Neutral protease digestion group <![CDATA[76.36±1.01 ## <!-- 9 -->]]> Flavor protease hydrolysis group <![CDATA[73.89±0.94 ## ]]>

[0143] Table 7. Serum GSH-Px activity in mice

[0144]

[0145] Table 8. Serum CAT activity in mice

[0146] Grouping CAT (U / ml) Blank control group 11.40±2.20 Model group <![CDATA[6.95±0.72 ## ]]> Compound enzyme hydrolysis experimental group <![CDATA[9.01±0.76 #* ]]> Neutral protease digestion group <![CDATA[8.73±0.82 ##* ]]> Flavor protease hydrolysis group <![CDATA[8.44±0.79 ##* ]]>

[0147] Note: Compared with the blank group, # P < 0.05 ## P < 0.01; compared with the model group, * P < 0.05, **P < 0.01.

[0148] Tables 5-8 show that the active peptides of Sipunculus nudus with a molecular weight of <10kDa prepared by enzymatic hydrolysis in Example 1 have the best in vivo antioxidant activity.

[0149] In addition, mouse livers were stained with hematoxylin and eosin (HE) and observed under a microscope, such as... Figure 1 HE staining (×40) of mouse liver tissue in each group is shown. A is the blank group, B is the model group, C is the neutral protease digestion group, D is the flavor protease digestion group, and E is the compound enzyme digestion experimental group. Results showed that in the blank group, the liver cells were neatly arranged, with normal nuclei, uniform cytoplasm, intact lobular structure, and hepatic cords arranged radially around the central vein with clear boundaries. No obvious inflammatory infiltration was observed in the portal areas. In the model group, hepatocytes were significantly swollen, with small fat granules visible in the cytoplasm of some hepatocytes. The connections between hepatocytes were loose, and binucleated hepatocytes appeared. Hepatic sinusoids were dilated, the cord-like structure of hepatocytes around the central vein was not obvious, and a large number of Kupffer cells with large bodies and black nuclei proliferated at the edge of hepatocytes and within the hepatic sinusoids. Compared with the model group, in the compound enzyme digestion experimental group with a molecular weight <10kDa, the hepatocytes were neatly arranged, the number of hepatocytes with fat granules was reduced, the hepatic sinusoids were shrunken, and the number of binucleated hepatocytes was reduced. The hepatic sinusoidal dilation phenomenon was slightly improved in the neutral protease hydrolysis group and the flavor protease hydrolysis group compared with the model group, but some binuclear hepatocytes were still present, hepatocyte swelling was obvious, hepatocyte connections were loose, and the cord-like structure of hepatocytes around the central vein was not obvious. Therefore, the experimental group with complex enzymes with a molecular weight <10kDa had better results.

[0150] Mouse brain tissue stained with hematoxylin and eosin (HE) and observed under a microscope, such as... Figure 2 HE staining (×40) of the CA3 region of the hippocampus in mice of each group is shown. A is the blank group, B is the model group, C is the neutral protease digestion group, D is the flavor protease digestion group, and E is the compound enzyme digestion experimental group. The results showed that in the blank group, the nuclei of the hippocampal pyramidal neurons were uniform in size and clearly distinguishable, the cells were neatly arranged, the morphology was good, the staining was uniform, and no obvious inflammatory cell infiltration or necrosis was observed. In the model group, the hippocampal cells were loosely arranged, the morphology was not fixed, some neurons were lost, and the neurons were loosely and randomly arranged. In the compound enzyme digestion experimental group with a molecular weight of <10kDa, the hippocampal pyramidal cells were relatively neatly arranged, the nuclei were clearly visible, the cell morphology was uniform, the boundaries were clear, a small number of neurons were lost, and the cell layers were rich. In the neutral protease digestion group and the flavor protease digestion group, the hippocampal pyramidal cells were loosely arranged, most of the neurons did not show obvious morphological changes, the morphology of the mouse hippocampal cells was basically intact, the cell outlines were clear, but some neurons were still lost, and the cell layers were reduced. Therefore, the enzymatic hydrolysis experimental group with a molecular weight <10kDa showed better results.

[0151] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing palatable active peptides from Sipunculus nudus, characterized in that, Includes the following steps: The cleaned Sipunculus nudus was crushed in water to obtain Sipunculus nudus slurry. The first enzymatic hydrolysate was obtained by first enzymatic hydrolysis of the *Sipunculus nudus* slurry with a neutral protease. The conditions for the first enzymatic hydrolysis included: a temperature of 43–47°C, a pH of 6.3–6.7, and a time of 3.5–4.5 h. The amount of neutral protease added was 0.4–0.6% of the weight of the *Sipunculus nudus*. The first enzymatic hydrolysate is subjected to a first enzyme inactivation treatment, and then a flavor protease is added for a second enzymatic hydrolysis treatment to obtain a second enzymatic hydrolysate. The first enzyme inactivation treatment includes: inactivating the enzyme at 98-102℃ for 8-10 min. The conditions for the second enzymatic hydrolysis treatment include: a temperature of 38-42℃, a pH of 5.3-5.7, and a time of 2.5-3.5 h. The amount of flavor protease added is 0.4-0.6% of the weight of the palatable Sipunculus nudus. The second enzyme hydrolysate is subjected to a second enzyme inactivation treatment, and then centrifuged to obtain a supernatant. The second enzyme inactivation treatment includes: inactivating the enzyme at 98-102℃ for 8-10 min. The centrifugation step includes: centrifuging the second enzyme hydrolysate after the second enzyme inactivation treatment at 2-4℃ at a speed of 8000-12000 r / min for 15-25 min. The supernatant is filtered to obtain palatable active peptides of Sipunculus nudus with a molecular weight of <10kDa; the step of filtering the supernatant includes: pre-treating the supernatant by passing it through an aqueous membrane with a pore size of 0.45μm, and then separating it by passing it through a filter membrane with a molecular weight cutoff of 10kDa.

2. A palatable active peptide from Sipunculus nudus, characterized in that, The palatable Sipunculus nudus active peptide is prepared by the preparation method described in claim 1.

3. A chewable tablet, characterized in that, It includes the palatable Sipunculus nudus active peptide as described in claim 2, and pharmaceutically acceptable excipients.

4. The chewable tablet as described in claim 3, characterized in that, The excipients include thickeners and flavoring agents.

Images