CN111876458A - Urechis unicinctus antioxidant peptide and preparation and application thereof - Google Patents
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Abstract
The invention provides a preparation method and application of novel urechis unicinctus antioxidant peptide based on a spherical mesoporous material. The preparation method mainly comprises the following steps: (1) pretreating urechis unicinctus; (2) carrying out enzymolysis on the wall of the urechis unicinctus by using ultrasonic protease; (3) and (3) enriching the spherical mesoporous material of the enzymolysis liquid and separating by high performance liquid chromatography. The invention uses ultrasonic-assisted pepsin to carry out enzymolysis on the urechis unicinctus body wall, and the urechis unicinctus peptide with antioxidant activity can be obtained after separation and purification.
Description
Technical Field
The invention belongs to the field of deep processing of protein, and particularly relates to separation and purification of urechis unicinctus antioxidant peptide and antioxidation of the urechis unicinctus antioxidant peptide.
Background
Marine organisms have a different composition of matter from terrestrial organisms due to the particular growing environment. The marine bioactive peptide has wide biological activities of resisting oxidation, treating hypertension, inhibiting bacteria, resisting inflammation, resisting thrombus and the like due to different structural characteristics, amino acid compositions and sequences, wherein the antioxidant activity is closely related to human diseases and aging, and is widely applied to the food industry, cosmetics and medicines. Compared with the bioactive peptide from terrestrial organisms, the bioactive peptide from marine sources has higher biological safety, higher nutritional value and richer sources.
Urechis unicinctus (Urechis unicinctus) is commonly called as Urechis unicinctus, is an animal of Urechis unicinctus of urechidaceae (urechioidea) of urechistoidea of urechidaceae (xenocypria), is distributed in russia, korean peninsula, japanese north sea road, state and sea areas near the bohai sea and the yellow sea of China, and is the only Urechis unicinctus distributed along the sea of China. The urechis unicinctus has large individuals, rich and delicious meat, and is rich in protein and a plurality of amino acids necessary for human body in the body wall (the body wall obtained after the internal organs of the urechis unicinctus are removed), thereby having higher nutritional value and medicinal value. Analysis of the basic components of the urechis unicinctus wall dry powder shows that the fat and total sugar content is low, and the protein content is as high as 70%. Therefore, the method for preparing the antioxidant peptide by using the urechis unicinctus as the raw material can obviously improve the economic value of the antioxidant peptide.
Spherical Mesoporous Materials (SMMs) are spherical porous materials assembled by taking a supermolecular structure formed by a surfactant as a template and utilizing a sol-gel process through the directional action between organic and inorganic interfaces. The SMMs have the open characteristics of mesopores and short channels, and the density of active sites is increased, so that the SMMs play a great role in the fields of catalysis, adsorption, energy storage conversion and the like, and become one of the materials which are concerned in the last decade. The particle size/pore size adjustability of the SMMs based on reaction kinetics and thermodynamics enables the SMMs to be prepared controllably by regulating different templates and regulating reaction conditions (time, temperature, pH value and the like); in addition, the unique spherical structure, pressure resistance, heat resistance and chemical stability of SMMs also make the SMMs have great application potential on chromatographic packing (especially size-screened chromatographic packing).
The antioxidant peptide can be prepared by utilizing the urechis unicinctus body wall protein through enzymolysis, separation and purification. In order to carry out effective enzymolysis, the patent utilizes pepsin to carry out enzymolysis under the assistance of ultrasound, and the antioxidant peptide of a proper peptide segment can be obtained by controlled enzymolysis. The efficient separation of the enzymolysis mixture is the key to obtain the high-activity antioxidant peptide. According to the method, the SMMs are firstly utilized to enrich the antioxidant peptides, and then the urechis unicinctus antioxidant peptides can be obtained through high performance liquid chromatography separation. The preparation method of the urechis unicinctus antioxidant peptide provided by the specialty has the advantages of simple steps, convenience in operation and easiness in industrialization.
Disclosure of Invention
The invention provides a preparation, separation and purification method and antioxidation of urechis unicinctus antioxidant peptide. The method mainly comprises the following steps:
1. the spherical porous material with the aperture of 3-20 nm is assembled by a sol-gel process and the directional action between an organic matter and an inorganic matter interface according to the property of the urechis unicinctus antioxidant peptide.
2. Removing viscera of fresh urechis unicinctus, taking the body wall of the urechis unicinctus, homogenizing, performing Soxhlet extraction degreasing by using acetone, and performing freeze drying and crushing to obtain dry powder of the body wall of the urechis unicinctus;
3. carrying out enzymolysis on the wall of the urechis unicinctus by pepsin under the assistance of ultrasonic waves, inactivating enzymes, and centrifuging to obtain supernatant;
4. and (4) enriching and eluting the centrifuged urechis unicinctus enzymatic hydrolysate by using a spherical mesoporous material.
5. Separating and purifying the eluted enzymatic hydrolysate by using high performance liquid chromatography to obtain a component with the highest antioxidant activity, and freeze-drying to obtain the urechis unicinctus antioxidant peptide.
The Soxhlet extraction degreasing in the step 1 is carried out at the temperature of 45-55 ℃ for 4-8h.
And 2, the power of ultrasonic pretreatment is 300-540W, the temperature is 25-50 ℃, and the time is 5-30 min. The protease is pepsin, the addition amount of the protease is 0.2-4% of the body wall, the reaction time is 2-24 hours, the reaction temperature is 35-40 ℃, and the material-liquid ratio is 1: 10-1: 50.
According to the spherical mesoporous material obtained in the step 3, according to the urechis unicinctus antioxidant peptide, the aperture is 3-30nm, acetonitrile water solution is used as eluent, and the volume of acetonitrile is 20% -80%.
And 4, carrying out gradient elution by using 0-40% acetonitrile on the high performance liquid chromatography column BDS-C18 in the step 4 at the flow rate of 1-10 mL/min, collecting an elution peak, and detecting an absorption value at 280nm by using an ultraviolet detector.
The antioxidant activity is DPPH free radical scavenging rate, hydroxyl free radical scavenging rate and superoxide anion scavenging rate.
The invention has the following advantages:
1. the preparation method of the urechis unicinctus antioxidant peptide can prepare the antioxidant active peptide with high purity and strong activity, and compared with the original enzymolysis liquid, the antioxidant activity of the antioxidant active peptide is obviously improved, so that the antioxidant active peptide has higher commercial value;
2. the preparation method of the urechis unicinctus antioxidant peptide is simple in process, strong in operation controllability and easy for industrial production;
3. the invention utilizes urechis unicinctus to prepare the antioxidant active peptide, changes the traditional edible mode, deeply processes protein and obviously improves the economic value of the protein.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto. The body wall in the text refers to the body wall of fresh urechis unicinctus, which is obtained by cutting off the liquid and internal organs of the body cavity of fresh urechis unicinctus from one end, and then cleaning the urechis unicinctus with clear water.
Washing with water, shearing, and homogenizing.
Examples
The spherical porous material with the aperture of 3-30nm is assembled by a sol-gel process through the directional action between an organic matter-inorganic matter interface according to the molecular weight property (300-3000Da) of the urechis unicinctus antioxidant peptide.
Removing viscera of fresh urechis unicinctus, taking the body wall of the urechis unicinctus, homogenizing, carrying out Soxhlet extraction and degreasing by using acetone, and then carrying out freeze drying and crushing to obtain dry powder of the body wall of the urechis unicinctus; under the assistance of ultrasonic waves, carrying out enzymolysis on the wall of the urechis unicinctus by pepsin, centrifuging after enzyme deactivation, taking supernatant, and sequentially carrying out enrichment by Spherical Mesoporous Materials (SMMs) and separation and purification by high performance liquid chromatography to obtain the component, namely the urechis unicinctus antioxidant peptide component. After each step of treatment, the antioxidant activity of each component is measured, and the component with the highest antioxidant activity is subjected to next step of treatment.
In the examples, DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) radical scavenging, hydroxyl radical scavenging and superoxide anion scavenging were performed as follows.
Determination of DPPH radical scavenging Rate: active peptide solutions were prepared with deionized water at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively. VC solutions were prepared at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively, as controls. Accurately weighing 4.0mg of DPPH, dissolving with a small amount of absolute ethyl alcohol, transferring into a 100mL volumetric flask, fixing the volume to a scale mark with the absolute ethyl alcohol, and shaking up for later use.
Taking 1mL of sample solution and VC control solution with different concentrations, adding 1mL of DPPH, shaking uniformly, standing for 30min, and measuring the absorbance value A1 at 517 nm. 1mL of sample solution and VC control solution with different concentrations are taken, 1mL of absolute ethyl alcohol is added, the mixture is shaken evenly and kept stand for 30 minutes, and the absorbance value A0 at 517nm is measured. 1mL of deionized water was added to 1mL of DPPH solution, shaken well and left to stand for 30 minutes, and then the absorbance at 517nm, A2, was measured. Each group is parallelly processed into three groups
DPPH clearance ═ a2- (a1-a0))/a2 × 100%
Determination of hydroxyl radical clearance: active peptide solutions were prepared with deionized water at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively. VC solutions were prepared at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively, as controls. Respectively preparing 9mmol/L FeSO4Solution, 9mmol/L salicylic acid-ethanol solution and 8.8mmol/L H2O2And (3) solution.
Taking 0.5mL of sample solution and 0.5mL of VC control solution with different concentrations, and respectively adding 0.5mL of FeSO4Solution, 0.5mL salicylic acid-ethanol solution and 0.5mL H2O2The solution was subjected to 37 ℃ water bath for 30min, and the absorbance value A1 at 510nm was measured. Taking 0.5mL of sample solution and 0.5mL of VC control solution with different concentrations, and respectively adding 0.5mL of FeSO4The solution, 0.5mL of salicylic acid-ethanol solution and 0.5mL of distilled water were subjected to water bath at 37 ℃ for 30min, and the absorbance value A2 at 510nm was measured. In 0.5mL FeSO4Solution, 0.5mL salicylic acid-ethanol solution and 0.5mL H2O2To the solution was added 0.5ml of distilled water. The mixture is subjected to water bath at 37 ℃ for 30min, and the absorbance A0 is measured at the wavelength of 510 nm. Samples of each concentration were divided into three parallel groups.
OH clearance ═ [ (a0- (a1-a2))/a0] × 100%
Determination of superoxide anion scavenging: active peptide solutions were prepared with deionized water at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively. VC solutions were prepared at concentrations of 2, 4, 6, 8 and 10mg/mL, respectively, as controls. Preparing 10mmol/L HCl solution and Tris-HCl (pH 8.2) buffer solution respectively; accurately weighing 0.142g of pyrogallol, dissolving the pyrogallol in a small amount of 10mmol/LHCl solution, transferring the solution into a 25ml volumetric flask, fixing the solution on a scale mark by using the 10mmol/LHCl solution, and shaking up to obtain the pyrogallol hydrochloride solution for later use.
Taking sample solutions with different concentrations and VC control solution 0.5mL, adding 1mL of distilled water, 1.5mL of Tris-HCl (pH 8.2) buffer solution, carrying out water bath at 25 ℃ for 10min, adding 50 mu L of pyrogallol-HCl solution, rapidly shaking, measuring the absorbance value at 325nm, and recording the value once every 10 seconds. The slope K0 is calculated with wavelength as the ordinate and time as the ordinate. Samples of each concentration were divided into three parallel groups.
Taking 1.5mL of distilled water and 1.5mL of Tris-HCl (pH 8.2) buffer solution, carrying out water bath at 25 ℃ for 10min, adding 50 mu L of pyrogallol-HCl solution, rapidly shaking, measuring the absorbance value at 325nm, recording a value every 10 seconds, and calculating the slope K1 by taking the wavelength as the ordinate and the time as the abscissa.
O2-Clearance ═ [ (K0-K1)/K0]×100
Example 1: preparation of urechis unicinctus body wall dry powder
Removing viscera from fresh urechis unicinctus, cleaning, freeze-drying, pulverizing, and defatting by Soxhlet extraction. The used solvent is acetone, the temperature is 50 ℃, the degreasing time is 6h, freeze drying is carried out again to obtain degreased urechis unicinctus body wall dry powder, and the next step of enzymolysis reaction is carried out.
Example 2: enzymolysis of urechis unicinctus body wall dry powder
Weighing 1g of degreased urechis unicinctus wall dry powder, adding deionized water according to different material-liquid ratios (mL of deionized water/g of the wall dry powder), adjusting the pH to 1.9, preheating to the reaction temperature, adding different amounts of pepsin, carrying out enzymolysis, inactivating enzyme in boiling water at 100 ℃ for 15min, centrifuging in a refrigerated centrifuge at 4 ℃ for 20min at the rotating speed of 8000rap/min, taking supernatant, and freeze-drying to obtain the urechis unicinctus peptide powder. The oxidation resistance was measured as shown in table 1.
TABLE 1
The preferable conditions of the enzymolysis reaction are 3# conditions, the enzyme dosage is 1%, the reaction temperature is 37 ℃, the material-liquid ratio is 1:20, and the reaction time is 4h, so that the urechis unicinctus antioxidant peptide is obtained.
Example 3: urechis unicinctus body wall dry powder enzymolysis assisted by ultrasonic pretreatment
Before enzymolysis, ultrasonic pretreatment under different conditions is carried out, and enzymolysis is carried out according to the 3# enzymolysis condition in the example 1. The antioxidant activity of urechis unicinctus peptide is shown in table 2.
TABLE 2
The preferable conditions of the ultrasonic-assisted enzymolysis are 6#, the ultrasonic power is 360W, the ultrasonic temperature is 37 ℃, and the ultrasonic time is 15min, so that the urechis unicinctus antioxidant peptide is obtained.
Example 4: enrichment of Spherical Mesoporous Materials (SMMs) in enzymatic hydrolysate
By means of improvements
Method for preparing SMMs (small molecule metal-organic semiconductors) matrix with controllable pore diameter: tetraethoxysilane (TEOS) is used as an inorganic precursor; taking a nonionic surfactant and fatty alcohol-polyoxyethylene ether (AEO-9) as a template agent; by NH3·H2O and NaOH created alkaline conditions. Firstly, under different ratios of TEOS/alkaline catalyst/AEO-9, TEOS is hydrolyzed to form silicic acid and alcohol, further the silicic acid and TEOS are induced to generate polycondensation reaction, and micro-nuclei are formed on the surface of AEO-9 micelle. Secondly, controlling the reaction temperature and time to enable the micro-cores to collide with each other, and further depositing residual TEOS in the system on the micro-cores to form the silica microspheres. Finally, regulating and controlling the post-treatment calcining condition, removing AEO-9 and finally forming SMMs. By N2Physical adsorption desorption instrument, Transmission Electron Microscope (TEM) and other means characterize the particle size distribution and the pore structure of the SMMs.
In 100ml of an ethanol-water mixed solution (volume ratio of ethanol to water 1: 1), AEO was dissolved9Ammonia water and TEOS at final concentrations of 0.02mol/L, 0.5mol/L and 0.1mol/L, respectively, in the formation reaction solution, and then reacted for 30 minutes. Then, the spherical mesoporous material with the aperture of 3 +/-0.2 nm (the average aperture of 3nm) is baked at 600 ℃ after being cleaned for 3 times by ethanol and deionized water respectively and dried.
In 100ml of an ethanol-water mixed solution (volume ratio of ethanol to water 1: 1), AEO was dissolved9Ammonia water and TEOS at final concentrations of 0.02mol/L, 0.45mol/L and 0.2mol/L, respectively, in the formation reaction solution, and then reacted for 50 minutes. Then, the spherical mesoporous material with the aperture of 5 +/-0.2 nm (the average aperture is 5nm) is obtained by respectively cleaning 3 times with ethanol and deionized water, drying and baking at 650 ℃.
In 100ml of an ethanol-water mixed solution (volume ratio of ethanol to water 1: 1), AEO was dissolved9Ammonia water and TEOS, the final concentrations of which in the formed reaction solution are respectively 0.02 mol/L; 0.40mol/L and 0.3mol/L, followed by reaction for 80 minutes. Then, the spherical mesoporous material with the aperture of 10 +/-0.3 nm (the average aperture is 10nm) is baked at 700 ℃ after being respectively cleaned for 3 times by ethanol and deionized water and dried.
In 100ml of an ethanol-water mixed solution (volume ratio of ethanol to water 1: 1), AEO was dissolved9Ammonia and TEOS, their final concentrations in the reaction solutionRespectively is 0.02 mol/L; 0.35mol/L and 0.4mol/L, and then reacted for 120 minutes. Then, the spherical mesoporous material with the aperture of 20 +/-0.3 nm (the average aperture of 20nm) is baked at 700 ℃ after being respectively cleaned for 3 times by ethanol and deionized water and dried.
In 100ml of an ethanol-water mixed solution (volume ratio of ethanol to water 1: 1), AEO was dissolved9Ammonia water and TEOS at final concentrations of 0.02mol/L, 0.35mol/L and 0.4mol/L, respectively, in the formation reaction solution, followed by reaction for 150 minutes. Then, the spherical mesoporous material with the aperture of 30 +/-0.4 nm (the average aperture is 30nm) is baked at 750 ℃ after being respectively cleaned for 3 times by ethanol and deionized water, and dried.
The prepared spherical mesoporous materials with the aperture of 3 +/-0.2 nm, 5 +/-0.2 nm, 10 +/-0.3 nm, 20 +/-0.3 nm and 30 +/-0.4 nm are spherical with the diameter of 3-40 microns and the specific surface area of 1196m2/g，1292m2/g，730m2/g、528m2G and 492m2/g。
Weighing 30mg of urechis unicinctus peptide freeze-dried powder, placing the urechis unicinctus peptide freeze-dried powder into a 50ml centrifuge tube, adding 30ml of deionized water into the centrifuge tube, and placing the centrifuge tube into an ultrasonic cleaning machine to clean for 5min for later use. The spherical mesoporous materials with the pore diameters of 3nm, 5nm, 10nm, 20nm and 30nm are respectively used for enrichment for 2h, 50% acetonitrile water solution is used for elution, and the antioxidant activity of the eluent is measured, and is shown in table 3.
TABLE 3
The aperture of the preferred SMMs is 10nm, and the urechis unicinctus antioxidant peptide is obtained.
Example 5: elution effect of acetonitrile aqueous solutions of different concentrations
Weighing 30mg of urechis unicinctus peptide freeze-dried powder, placing the urechis unicinctus peptide freeze-dried powder into a 50ml centrifuge tube, adding 30ml of deionized water into the centrifuge tube, and placing the centrifuge tube into an ultrasonic cleaning machine to clean for 5min for later use. Enrichment was performed for 2h using 40mg of spherical mesoporous material with a pore size of 10nm, elution was performed using 20%, 50% and 80% acetonitrile in water (v/v), respectively, the eluates were collected after centrifugation, and the antioxidant activity of the eluates was measured, as shown in Table 3.
TABLE 4
The volume of the preferred eluent acetonitrile is 50 percent, and the urechis unicinctus antioxidant peptide is obtained
Example 6: contrast the enrichment effect of non-spherical mesoporous material
The 6# enzymolysis solution in the preparation example 3 is respectively enriched by non-spherical (irregular macroscopic shape) mesoporous materials with the aperture of 3nm, 5nm, 10nm and 20nm, wherein the mesoporous material with the aperture of 3nm and 5nm is MCM-41, and the non-spherical (irregular macroscopic shape) mesoporous material with the aperture of 10nm and 20nm is SBA-15. As a control of the spherical mesoporous material, 50% acetonitrile aqueous solution was used for elution, and the antioxidant activity of the eluate was measured, as shown in Table 5.
TABLE 5
The spherical mesoporous material with the aperture of 3nm, 5nm, 10nm and 20nm has more excellent enrichment effect than the amorphous mesoporous material with the aperture of 3nm, 5nm, 10nm and 20 nm.
Example 7: high performance liquid chromatography column separation of enzymatic hydrolysate
Further separating and purifying the 2# in the embodiment 5 by a high performance liquid chromatography column BDS-C18, wherein the mobile phase is eluted by 0-40% acetonitrile, and the gradient elution condition is 0-10min and 0-5% acetonitrile; 10-20min, 5% -5% acetonitrile; 20-30min, 5% -10% acetonitrile; 30-50min, 10% -40% acetonitrile; 50-60min, 40% -0% acetonitrile. Eluting at 1mL/min for 60min, detecting sample peak at 280nm with ultraviolet detector, collecting sample peak, and freeze drying to obtain six components. The antioxidant activity of each component was measured and is shown in table 6.
TABLE 6
And preferably, eluting the peak IV to obtain the urechis unicinctus antioxidant peptide.
The present invention has been described in detail above, but the present invention is not limited to the specific embodiments described herein. It will be understood by those skilled in the art that other modifications and variations may be made without departing from the scope of the invention. The scope of the invention is defined by the appended claims.
Claims (10)
1. A preparation method of urechis unicinctus antioxidant peptide is characterized by comprising the following steps: the wall of the urechis unicinctus is subjected to enzymolysis through pepsin under the assistance of ultrasound, and the enzymolysis liquid is sequentially enriched by Spherical Mesoporous Materials (SMMs) and separated and purified by high performance liquid chromatography, so that the obtained component is the urechis unicinctus antioxidant peptide component.
2. The method of claim 1, wherein:
the SMMs are spherical mesoporous materials with the aperture of 3-30nm, which are assembled by a sol-gel process;
the specific process is as follows:
dissolving AEO in ethanol-water mixed solution with volume ratio of 1:1-1:39Ammonia water and TEOS, so that the final concentrations of the ammonia water and the TEOS in the formed reaction solution are 0.02mol/L, 0.35-0.5mol/L and 0.10-0.4mol/L respectively, and then the reaction is carried out for 30-150 minutes; then, the spherical mesoporous material with the average pore diameter of 3-30nm (preferably 10nm) is respectively cleaned and dried by ethanol and deionized water in turn and baked at the temperature of 600-750 ℃.
3. The method of claim 2, wherein:
the spherical mesoporous material is spherical with the diameter of 3-40 microns, and the specific surface areas are 492-1292m2/g。
4. The method of claim 1, wherein:
(1) the power of ultrasonic pretreatment is 300-540W (preferably 320-480W, more preferably 360W), the temperature is 25-50 ℃ (preferably 25-45 ℃, more preferably 37 ℃), and the time is 5-30 min (preferably 5-25 min, more preferably 15 min);
(2) the protease is pepsin, the addition amount of the protease is 0.2-4% (preferably 0.3-3%, more preferably 1%) of the mass of the body wall, the reaction time is 2-24 h (preferably 3-18 h, more preferably 4h), the reaction temperature is 35-40 ℃ (preferably 36-39 ℃, more preferably 37 ℃), and the feed-liquid ratio is 1: 10-1: 50 (preferably 1: 15-1: 40, more preferably 1: 20).
5. The method of claim 1, wherein:
(1) centrifuging the urechis unicinctus enzymatic hydrolysate, taking supernate, enriching and eluting by using spherical mesoporous materials;
(2) separating and purifying the eluted enzymatic hydrolysate by using high performance liquid chromatography to obtain a component with the highest antioxidant activity, and freeze-drying to obtain the urechis unicinctus antioxidant peptide.
6. A process according to claim 5, wherein the eluent comprises an aqueous solution of acetonitrile, the concentration of acetonitrile being from 20 to 80% by volume, preferably from 45 to 55% by volume, more preferably 50% by volume.
7. The process of claim 5, wherein the high performance liquid chromatography column is BDS-C18, eluting with 0% -40% acetonitrile at a flow rate of 1mL/min-10mL/min, collecting the elution peak, and detecting the absorbance at 280nm with an ultraviolet detector, preferably the target product of elution peak IV.
8. An urechis unicinctus antioxidant peptide prepared by the method of any one of claims 1-7.
9. An application of the urechis unicinctus antioxidant peptide of claim 8 as an antioxidant or an antioxidant active ingredient.
10. The use according to claim 9, wherein the urechis unicinctus antioxidant peptide can be used in functional food, cosmetics or biological medicament or any one of them in the production process.
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| CN113528605A (en) * | 2021-09-16 | 2021-10-22 | 中国农业大学 | Method for preparing urechis unicinctus viscera antioxidant peptide by ultrasonic-assisted enzymolysis |
| CN113583084A (en) * | 2021-07-28 | 2021-11-02 | 中日友好医院(中日友好临床医学研究所) | Urechis unicinctus leach liquor protein extract and extraction method and application thereof |
| CN114681491A (en) * | 2020-12-31 | 2022-07-01 | 烟台大学 | A product containing sea intestine body wall and its preparation method |
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| CN113528605A (en) * | 2021-09-16 | 2021-10-22 | 中国农业大学 | Method for preparing urechis unicinctus viscera antioxidant peptide by ultrasonic-assisted enzymolysis |
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