Green preparation method of hilsa herring roe source peptide, hilsa herring roe source peptide and application of hilsa herring roe source peptide

Abstract

The invention discloses a green preparation method of hilsa herring roe-derived peptide, the hilsa herring roe-derived peptide and application of the hilsa herring roe-derived peptide, the hilsa herring roe-derived peptide is prepared by the following steps: cleaning and dehydrating hilsa herring roes with 10% saline water to reduce the water content of the roes, then freeze-drying and crushing to prepare freeze-dried hilsa herring roe powder, and degreasing the protein of the hilsa herring roes by a supercritical CO2 extractor to obtain the hilsa herring roe-derived peptide. Rancidity of the hilsa herring roes caused by lipid oxidation is effectively prevented; a hilsa herring egg solution is subjected to ultrasonic treatment, the protein structure of the hilsa herring eggs is uniformly damaged, and specific protease is adopted for enzymolysis, so that the enzymolysis time can be effectively shortened, the enzymolysis efficiency can be improved, and the hilsa herring egg source peptide with antioxidant activity and ACE inhibitory activity is obtained. The anti-aging test of the hilsa herring roe-derived peptide in a nematode animal model shows that the hilsa herring roe-derived bioactive peptide can obviously prolong the life of nematodes, and the average life of the nematodes can be prolonged by 33.33%. The method disclosed by the invention is of great significance in fully utilizing shad roe resources, developing functional foods and anti-aging products and realizing high-valued utilization of characteristic aquatic resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of the development of functional bioactive peptides, and particularly relates to a green preparation method of macrura reevesii egg-derived peptides, macrura reevesii egg-derived peptides and applications thereof. Background Art

[0002] Since the Ming Dynasty, as a precious aquatic product with high nutritional value, macrura reevesii has always been a "tribute to the court", and its precious medicinal value has been included in medicinal classics such as "Supplements to Compendium of Materia Medica". Macrura reevesii is a fish with remarkable specificity. It belongs to deep-sea migratory fish, lays eggs in fresh water and grows in seawater, and its migration path is as long as 2,000 kilometers; macrura reevesii is the only fish that can receive ultra-low frequency waves and is very sensitive to changes in the external environment. Its fast swimming day and night can avoid the attacks of large animals such as sharks and dolphins. Its powerful vitality, osmotic pressure and adaptation mechanism of stress resistance are still a mystery so far. Macrura reevesii eggs contain all the nutrients for the early development of macrura reevesii, which are used to support its specific biological habits and accept the challenges of changes and uncertainties in the external environment. We found in the study of the whole genome of macrura reevesii that macrura reevesii has 193 specific genomes, which are related to its antioxidant stress and repair functions. By extracting relevant active substances from macrura reevesii eggs and studying the powerful biological adaptation mechanism and biological mechanism of stress resistance of macrura reevesii, it has important commercial value and theoretical significance for the development of medicine and food homologous biological medicines.

[0003] Fertilized eggs of macrura reevesii are semi-buoyant eggs with a high water content of more than 90%. The crude protein and crude fat contents are 5.33% and 1.14% respectively. The total amino acid content and total essential amino acid content in the dry egg sample are 61.41% and 27.05% respectively. The types and contents of essential amino acids are rich. Coupled with its medicinal value in traditional medicine, it has become a high-quality raw material for preparing bioactive peptides. At present, there is no research on the development and utilization of macrura reevesii eggs.

[0004] During the normal metabolic process of the human body, reactive oxygen species (ROS) are produced. In a healthy state, the production and removal of ROS are in a long-term balance. When too much ROS is produced, it will cause the human body to develop complications such as cancer, cognitive decline, inflammation, hypertension and diabetes. At present, industrial synthetic antioxidants have advantages such as high efficiency and low price, but these synthetic antioxidants have potential toxicity and carcinogenic and other side effects and cannot be added to food for consumption. As a natural antioxidant, antioxidant active peptides are widely used in the food field because they meet the characteristics of "natural, safe, non-toxic, and high nutritional value", and have become one of the natural products with the highest usage rate in functional foods. Developing high-value antioxidant active peptides with nutritional and safety properties using low-cost biological resources has always been a research hotspot in the polypeptide field.

[0005] As of 2019, there were 1.3 billion hypertension patients globally. Some studies predict that by 2025, the global number of hypertension patients will exceed 1.5 billion. As a chronic disease, hypertension is an inducement for many other diseases, seriously affecting health and longevity, such as stroke, vascular lesions, heart failure, arteriosclerosis, and kidney diseases. Angiotensin-converting enzyme (ACE) mainly raises blood pressure through two channels. One is to catalyze the conversion of inactive angiotensin I into angiotensin II, which causes blood vessels to constrict and blood pressure to rise. The other is to inactivate bradykinin, which has the ability to lower blood pressure. Currently, clinically used antihypertensive drugs such as chemically synthesized ACE inhibitors like captopril and enalapril have obvious therapeutic effects, but long-term use is likely to cause adverse reactions such as kidney damage, angioedema, and hyperkalemia. Therefore, it is particularly urgent to find natural and safe ACE inhibitors to prevent chronic diseases, improve health levels, and prolong life.

[0006] When ultrasonic waves propagate in a medium, they will produce thermal effects, mechanical effects, and cavitation effects, causing some characteristic changes in the medium. Research shows that ultrasound has significant effects in promoting the extraction of active ingredients, assisting enzymatic hydrolysis, and promoting protein emulsification. Among them, the most significant is in ultrasonic-assisted enzymatic hydrolysis, which can shorten the enzymatic hydrolysis time, improve the enzymatic hydrolysis efficiency, increase the product yield and uniformity.

[0007] Currently, there are no relevant reports on the preparation of antioxidant and ACE-inhibitory active peptides from tenpounder eggs. Tenpounder eggs are different from other fish eggs, with a water content of more than 90%. Using ordinary water extraction methods to extract egg peptides will cause a large amount of nutrient loss. Based on this, if a dry powder extraction method can be proposed without using any chemical additives, it is of great significance to efficiently prepare a safe and reliable tenpounder egg bioactive peptide with both antioxidant activity and ACE inhibitory ability and the effect of prolonging life. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide a green preparation method for tenpounder egg-derived peptides.

[0009] Another technical problem to be solved by the present invention is to provide tenpounder egg-derived peptides with antioxidant activity and ACE inhibitory activity prepared by the above method.

[0010] Finally, the technical problem to be solved by the present invention is to provide the application of the described tenpounder egg-derived peptides in the preparation of antioxidant, antihypertensive, life-prolonging, and / or anti-aging products.

[0011] Technical solution: To solve the above technical problems, the present invention provides a green preparation method for hilsa egg-derived peptides, comprising the following steps: First, wash and dehydrate the hilsa eggs to reduce the water content of the eggs, then perform freeze-drying, pulverize them to prepare hilsa egg freeze-dried powder; after extracting the oil by supercritical CO2, obtain defatted hilsa egg powder; add distilled water and perform ultrasonic treatment to obtain a hilsa egg solution, then add protease for enzymatic hydrolysis, perform enzyme inactivation and centrifugation on the enzymatic hydrolysate, take the supernatant and perform freeze-drying to obtain hilsa egg-derived peptides.

[0012] Specifically, the present invention first washes with a brine with a mass percentage of 10-15%, and at the same time, the high osmotic pressure of the brine can dehydrate the fish eggs, and the water content can be reduced to 68-76%. The measure of brine dehydration not only disinfects and prevents biological contamination, but also creates a hypertonic environment, causing the water inside the egg membrane to overflow - "dehydration", reducing the water content of the fish eggs, thereby greatly reducing the time of freeze-drying and reducing energy consumption. However, brine exceeding 20% will cause damage to the egg membrane and loss of nutrients.

[0013] Among them, the conditions for supercritical CO2 extraction are: the extraction pressure is 12 MPa, the extraction temperature is 40 °C, and the extraction time is 30 min.

[0014] Among them, the ratio of the hilsa egg protein powder to distilled water is 1:70 (mg / mL).

[0015] Among them, the ultrasonic conditions are: the ultrasonic power is 135-205 W, and the ultrasonic treatment time is 7

[0016] ~19 min.

[0017] Among them, after ultrasonic treatment, it also includes adjusting the pH of the hilsa egg solution to 7.0-8.0 and then performing enzymatic hydrolysis.

[0018] Among them, the protease includes one or more of alkaline protease, trypsin, papain, flavor protease or compound protease.

[0019] Among them, the addition amount of the protease is 1%-5%.

[0020] Among them, the enzyme inactivation temperature is a water bath at 90-100 °C for 15 min, the centrifugation speed is 9000 r / min, and the centrifugation time is 15 min.

[0021] The present invention also includes the hilsa egg-derived peptides prepared by the above method.

[0022] The present invention also includes the application of the hilsa egg-derived peptides in the preparation of antioxidant, blood pressure-lowering and / or anti-aging products.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages: Different from other fish egg extraction methods, in order to obtain better bioactive peptides, the present invention uses the fertilized eggs of Tenualosa reevesii with good biological activity. The fertilized eggs of Tenualosa reevesii are semi-buoyant eggs with a relatively high water content, more than 90%. Moreover, during the extraction process, no chemical additives are used, and mild extraction measures are adopted to maximize the protection of the biological activity of the extract and avoid nutrient loss. First, the Tenualosa reevesii eggs are washed and dehydrated with 10% brine to reduce the water content of the eggs, and then freeze-dried to make freeze-dried powder to prevent nutrient loss; degreasing treatment is carried out by a supercritical CO2 extraction instrument to effectively avoid the rancidity of Tenualosa reevesii eggs caused by lipid oxidation; the Tenualosa reevesii egg solution is ultrasonically treated to destroy the protein structure of Tenualosa reevesii eggs, and specific protease is used for enzymatic hydrolysis, effectively shortening the enzymatic hydrolysis time and improving the enzymatic hydrolysis efficiency. The peptide content of the Tenualosa reevesii egg peptide prepared under the optimal process conditions is (552.84 ± 2.77) mg / g, the oxygen radical absorbance capacity is (603.99 ± 7.32) μmol TE / g, and the ACE inhibitory ability is (96.93 ± 0.35)%, and the IC 50 is 0.48 mg / mL. The present invention can obtain Tenualosa reevesii egg-derived peptides with antioxidant activity and ACE inhibitory activity. The anti-aging test in the nematode animal model shows that the Tenualosa reevesii egg-derived bioactive peptides can significantly increase the lifespan of nematodes, and the average lifespan increase rate is 33.33%, higher than the average lifespan increase rate of the American anti-aging D+Q combination (the latter is 30.34%). The present invention lays a foundation for the development of Tenualosa reevesii egg-derived functional foods and anti-aging biomedicines, and has important significance for the development of high-value utilization of characteristic aquatic resources and meeting the health product needs of the special elderly population for prolonging life. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the preparation flow chart of the Tenualosa reevesii egg-derived peptide of the present invention;

[0025] Figure 2 is the influence of different types of protease treatments on the peptide content, ORAC and ACE inhibition rate of the enzymatic hydrolysate;

[0026] Figure 3 is the influence of different ultrasonic powers on the peptide content, ORAC and ACE inhibition rate of the enzymatic hydrolysate;

[0027] Figure 4 is the influence of different ultrasonic times on the peptide content, ORAC and ACE inhibition rate of the enzymatic hydrolysate;

[0028] Figure 5 is the influence of different enzyme addition amounts on the peptide content, ORAC and ACE inhibition rate of the enzymatic hydrolysate;

[0029] Figure 6Effect of different enzymatic hydrolysis times on peptide content, ORAC and ACE inhibition rate in the enzymatic hydrolysate;

[0030] Figure 7 Graph showing the experimental results of the effect of macrura reevesii egg peptides on the lifespan of Caenorhabditis elegans. Detailed implementation mode

[0031] The detection methods used in the embodiments of the present invention are as follows:

[0032] Determination of peptide content: Take the enzymatic hydrolysis supernatant I and mix it evenly with 10 g / mL trichloroacetic acid solution in equal volume. After standing for 10 min, centrifuge at 9000 r / min for 10 min to obtain supernatant II. Dilute the obtained supernatant II 5 times with 5 g / mL trichloroacetic acid solution and mix it with biuret reagent in a ratio of 3:2 for reaction. After standing for 10 min, centrifuge at 4000 r / min for 10 min. Take 200 μL of the supernatant and measure the OD value at 540 nm with an enzyme-labeling instrument. Each sample is measured three times repeatedly, and the obtained results are averaged.

[0033] P=(2C×5×30) / m.............................................(1)

[0034] In formula (1), P is the content of polypeptides in the macrura reevesii egg enzymatic hydrolysate, mg / g; 2 is the multiple of the volume expansion after mixing the supernatant I in equal volume with 10 g / mL trichloroacetic acid solution; C is the mass concentration of polypeptides in the enzymatic hydrolysate, mg / mL; 5 is the dilution multiple of supernatant II; 30 is the volume of the enzymatic hydrolysate, mL; m is the mass of the freeze-dried powder of macrura reevesii eggs, g.

[0035] The determination method of oxygen radical absorbance capacity (ORAC) refers to the article Evaluation of antioxidant activity of polyphenolic compounds based on ORAC [J]. Subtropical Plant Science, Chang Qiang, Su Minghua, Chen Qingxi,. 2016, 45(3): 225-229.

[0036] The determination method of ACE inhibition rate refers to the article Separation and identification of ACEinhibitory peptides from defatted walnut meal. [J]. European Food Research andTechnolog. Chen Y, Li J, Dong N, et al. 2020, 246(10): 2029-2038.

[0037] Example 1

[0038] A method for preparing hilsa fish egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0039] A. Freeze-drying and defatting of hilsa fish eggs:

[0040] First, wash and dehydrate the hilsa fish eggs with 10% brine to reduce the water content of the fish eggs to 76%. Then, perform freeze-drying, pulverize them to prepare freeze-dried hilsa fish egg powder, and obtain defatted hilsa fish eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0041] B. Ultrasonic treatment:

[0042] Add the defatted hilsa fish egg powder to distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 135 W, and the ultrasonic time is 13 min (set with a pulse duration of 1 s and a pulse off-time of 1 s).

[0043] C. Enzymatic hydrolysis:

[0044] Adjust the pH of the hilsa fish egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain a hydrolysate;

[0045] D. Enzyme inactivation and freeze-drying:

[0046] Boil the hydrolysate for 15 min to inactivate the enzyme. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain hilsa fish egg-derived peptides.

[0047] Analyze the experimental results as Figure 2 It can be seen that when alkaline protease is selected, the peptide content is (452.19 ± 4.56) mg / g, the oxygen radical absorbance capacity is (480.20 ± 8.14) μmol TE / g, and the ACE inhibition capacity is (93.82 ± 0.39)%.

[0048] Example 2

[0049] A method for preparing hilsa fish egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0050] A. Freeze-drying and defatting of hilsa fish eggs: First, wash and dehydrate the hilsa fish eggs with 10% brine to reduce the water content of the fish eggs to 76%. Then, perform freeze-drying, pulverize them to prepare freeze-dried hilsa fish egg powder, and obtain defatted hilsa fish eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0051] B. Ultrasonic treatment: Add the defatted tenpounder egg powder into distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 135 W and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse off time of 1 s).

[0052] C. Enzymolysis: Adjust the pH of the tenpounder egg solution after ultrasonic treatment to 8.0, add 1% trypsin, and carry out enzymatic hydrolysis in a magnetic stirring water bath at 37 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0053] D. Enzyme inactivation and freeze-drying: Boil the enzymatic hydrolysate for 15 min to inactivate the enzyme. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0054] Analysis of the experimental results shows that Figure 2 when trypsin is selected, the peptide content is (447.63 ± 5.48) mg / g, the oxygen radical absorbance capacity is (450.00 ± 7.14) μmol TE / g, and the ACE inhibition capacity is (90.88 ± 0.76)%.

[0055] Example 3

[0056] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibition activity, comprising the following steps:

[0057] A. Freeze-drying and defatting of tenpounder eggs:

[0058] First, wash and dehydrate the tenpounder eggs with 15% brine until the water content of the eggs drops to 68%. Then carry out freeze-drying, pulverize to prepare freeze-dried tenpounder egg powder, and obtain defatted tenpounder eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0059] B. Ultrasonic treatment:

[0060] Add the defatted tenpounder egg powder into distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 135 W and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse off time of 1 s).

[0061] C. Enzymolysis: Adjust the pH of the tenpounder egg solution after ultrasonic treatment to 7.0, add 1% papain, and carry out enzymatic hydrolysis in a magnetic stirring water bath at 50 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0062] D, Enzyme inactivation and freeze-drying: Boil the enzymolysis solution for 15 min to inactivate the enzyme. After the temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain the hilsa egg-derived peptide.

[0063] Analysis of the experimental results shows that Figure 2 when papain is selected, the peptide content is (414.90 ± 6.84) mg / g, the oxygen radical absorbance capacity is (400.74 ± 9.12) μmol TE / g, and the ACE inhibition capacity is (88.89 ± 0.45)%.

[0064] Example 4

[0065] A method for preparing hilsa egg-derived peptide with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0066] A, Freeze-drying and defatting of hilsa eggs:

[0067] First, wash and dehydrate the hilsa eggs with 12% brine until the water content of the eggs drops to 70%. Then carry out freeze-drying, pulverize to prepare freeze-dried hilsa egg powder, and obtain defatted hilsa eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0068] B, Ultrasonic treatment:

[0069] Add the defatted hilsa egg powder to distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasound is lower than 40 °C. The ultrasonic power is 135 W, and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse off time of 1 s).

[0070] C, Enzymolysis:

[0071] Adjust the pH of the hilsa egg solution after ultrasonic treatment to 7.0, add 1% flavor protease, and carry out enzymatic hydrolysis in a magnetic stirring water bath at 50 °C for 2.0 h to obtain an enzymolysis solution;

[0072] D, Enzyme inactivation and freeze-drying:

[0073] Boil the enzymolysis solution for 15 min to inactivate the enzyme. After the temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain hilsa egg-derived peptide powder.

[0074] Analysis of the experimental results shows that Figure 1It can be seen that when flavor protease is selected, the peptide content is (412.64 ± 4.56) mg / g, the oxygen radical absorbance capacity is (335.12 ± 8.11) μmol TE / g, and the ACE inhibitory capacity is (78.58 ± 0.64)%.

[0075] Example 5

[0076] A method for preparing hilsa shad egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0077] A. Freeze-drying and defatting of hilsa shad eggs:

[0078] First, wash and dehydrate the hilsa shad eggs with 10% brine until the water content of the eggs drops to 76%. Then, perform freeze-drying, pulverize to prepare freeze-dried hilsa shad egg powder, and obtain defatted hilsa shad eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0079] B. Ultrasonic treatment:

[0080] Add the defatted hilsa shad egg powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 135 W, and the ultrasonic time is 13 min (set as a pulse duration of 1 s and a pulse off-time of 1 s).

[0081] C. Enzymatic hydrolysis:

[0082] Adjust the pH of the hilsa shad egg solution after ultrasonic treatment to 8.0, add 1% compound protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 50 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0083] D. Inactivation of enzymes and freeze-drying:

[0084] Boil the enzymatic hydrolysate to inactivate the enzymes for 15 min. After the temperature drops to room temperature, centrifuge at 9000 r / min for 15 min, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain hilsa shad egg-derived peptides.

[0085] Analysis of the experimental results shows that Figure 2 It can be seen that when compound protease is selected, the peptide content is (216.25 ± 4.33) mg / g, the oxygen radical absorbance capacity is (288.83 ± 7.89) μmol TE / g, and the ACE inhibitory capacity is (70.60 ± 0.49)%.

[0086] Example 6

[0087] A method for preparing hilsa shad egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0088] A, Freeze-drying and defatting of shad eggs:

[0089] First, wash and dehydrate the shad eggs with 10% brine until the water content of the eggs drops to 76%. Then, conduct freeze-drying, pulverize them to prepare freeze-dried shad egg powder, and obtain defatted shad eggs after 30 minutes of extraction with a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40°C.

[0090] B, Ultrasonic treatment:

[0091] Add the defatted shad egg powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonication is below 40°C. The ultrasonic power is 135 W, and the ultrasonic time is 13 minutes (set with a pulse duration of 1 s and a pulse-off time of 1 s).

[0092] C, Enzymatic hydrolysis:

[0093] Adjust the pH of the shad egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and conduct enzymatic hydrolysis in a magnetic stirring water bath at 55°C for 2.0 h to obtain an enzymatic hydrolysate;

[0094] D, Inactivation of enzymes and freeze-drying:

[0095] Boil the enzymatic hydrolysate for 15 minutes to inactivate the enzymes. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 minutes, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain shad egg-derived peptides.

[0096] Analyze the experimental results as Figure 3 It can be seen that when the ultrasonic power is 135 W, the peptide content is (455.53 ± 6.32) mg / g, the oxygen radical absorbance capacity is (486.933 ± 6.88) μmol TE / g, and the ACE inhibition capacity is (92.56 ± 0.23)%.

[0097] Example 7

[0098] A method for preparing shad egg-derived peptides with both antioxidant activity and ACE inhibition activity, comprising the following steps:

[0099] A, Freeze-drying and defatting of shad eggs:

[0100] First, wash and dehydrate the shad eggs with 10% brine until the water content of the eggs drops to 76%. Then, conduct freeze-drying, pulverize them to prepare freeze-dried shad egg powder, and obtain defatted shad eggs after 30 minutes of extraction with a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40°C.

[0101] B. Ultrasonic treatment:

[0102] Add the defatted tenpounder egg powder into distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse-off time of 1 s).

[0103] C. Enzymatic hydrolysis:

[0104] Adjust the pH of the tenpounder egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate.

[0105] D. Enzyme inactivation and freeze-drying:

[0106] Boil the enzymatic hydrolysate for 15 min to inactivate the enzyme. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0107] Analyze the experimental results as Figure 3 It can be seen that when the ultrasonic power is 170 W, the peptide content is (483.74 ± 6.88) mg / g, the oxygen radical absorbance capacity is (555.76 ± 4.88) μmol TE / g, and the ACE inhibition capacity is (93.29 ± 0.42)%.

[0108] Example 8

[0109] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibition activity, comprising the following steps:

[0110] A. Freeze-drying and defatting of tenpounder eggs:

[0111] First, wash and dehydrate the tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then perform freeze-drying, pulverize them to prepare freeze-dried tenpounder egg powder, and obtain defatted tenpounder eggs after extracting for 30 min with a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0112] B. Ultrasonic treatment:

[0113] Add the defatted tenpounder egg powder into distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 205 W, and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse-off time of 1 s).

[0114] C. Enzymatic hydrolysis:

[0115] Adjust the pH of the ultrasonicated tenpounder egg solution to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate.

[0116] D. Inactivate enzymes and freeze-dry:

[0117] Boil the enzymatic hydrolysate to inactivate enzymes for 15 min. After its temperature drops to room temperature, centrifuge at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0118] Analyze the experimental results as Figure 3 It can be seen that when the ultrasonic power is 205 W, the peptide content is (462.30 ± 7.33) mg / g, the oxygen radical absorbance capacity is (538.59 ± 5.99) μmol TE / g, and the ACE inhibition capacity is (92.91 ± 0.19)%.

[0119] Example 9

[0120] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibition activity, comprising the following steps:

[0121] A. Freeze-dry and defat tenpounder eggs:

[0122] First, wash and dehydrate tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then perform freeze-drying, pulverize to prepare freeze-dried tenpounder egg powder, and obtain defatted tenpounder eggs after extraction with a supercritical CO2 extractor for 30 min under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0123] B. Ultrasonic treatment:

[0124] Add the defatted tenpounder egg powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasound is lower than 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 7 min (set as a pulse duration of 1 s and a pulse off time of 1 s).

[0125] C. Enzymatic hydrolysis:

[0126] Adjust the pH of the ultrasonicated tenpounder egg solution to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate.

[0127] D. Inactivate enzymes and freeze-dry:

[0128] Boil the enzymatic hydrolysate to inactivate the enzyme for 15 min. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibitory rate. Freeze-dry the supernatant to obtain the hilsa fish egg-derived peptide.

[0129] Analyze the experimental results as Figure 4 It can be seen that when the ultrasonic time is 7 min, the peptide content is (461.33 ± 6.15) mg / g, the oxygen radical absorbance capacity is (460.24 ± 13.73) μmol TE / g, and the ACE inhibitory capacity is (93.23 ± 0.32)%.

[0130] Example 10

[0131] A method for preparing hilsa fish egg-derived peptide with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0132] A. Freeze-drying and defatting of hilsa fish eggs:

[0133] First, wash and dehydrate the hilsa fish eggs with 10% brine until the water content of the fish eggs drops to 76%. Then carry out freeze-drying, pulverize to prepare freeze-dried hilsa fish egg powder, and obtain defatted hilsa fish eggs after extraction with a supercritical CO2 extractor for 30 min under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0134] B. Ultrasonic treatment:

[0135] Add the defatted hilsa fish egg powder to distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 13 min (set with a pulse duration of 1 s and a pulse off time of 1 s).

[0136] C. Enzymatic hydrolysis:

[0137] Adjust the pH of the hilsa fish egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and carry out enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0138] D. Enzyme inactivation and freeze-drying:

[0139] Boil the enzymatic hydrolysate to inactivate the enzyme for 15 min. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibitory rate. Freeze-dry the supernatant to obtain the hilsa fish egg-derived peptide.

[0140] Analyze the experimental results as Figure 4It can be seen that when the ultrasonic time is 13 min, the peptide content is (488.42 ± 4.77) mg / g, the oxygen radical absorbance capacity is (564.76 ± 5.16) μmol TE / g, and the ACE inhibitory capacity is (94.49 ± 0.51)%.

[0141] Example 11

[0142] A method for preparing hilsa shad egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0143] A. Freeze-drying and defatting of hilsa shad eggs:

[0144] First, wash and dehydrate the hilsa shad eggs with 10% brine until the water content of the eggs is reduced to 76%. Then, perform freeze-drying, pulverize to obtain freeze-dried hilsa shad egg powder, and obtain defatted hilsa shad eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0145] B. Ultrasonic treatment:

[0146] Add the defatted hilsa shad egg powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 19 min (set with a pulse duration of 1 s and a pulse off time of 1 s).

[0147] C. Enzymolysis:

[0148] Adjust the pH of the hilsa shad egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0149] D. Inactivating the enzyme and freeze-drying:

[0150] Boil the enzymatic hydrolysate for 15 min to inactivate the enzyme. After the temperature drops to room temperature, centrifuge at 9000 r / min for 15 min, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain hilsa shad egg-derived peptides.

[0151] Analyze the experimental results as Figure 4 It can be seen that when the ultrasonic time is 19 min, the peptide content is (484.88 ± 6.79) mg / g, the oxygen radical absorbance capacity is (545.93 ± 10.58) μmol TE / g, and the ACE inhibitory capacity is (94.42 ± 0.25)%.

[0152] Example 12

[0153] A method for preparing hilsa fish egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0154] A. Freeze-drying and defatting of hilsa fish eggs:

[0155] First, wash and dehydrate the hilsa fish eggs with 10% brine until the water content of the fish eggs is reduced to 76%. Then, perform freeze-drying, pulverize them to prepare freeze-dried hilsa fish egg powder, and obtain defatted hilsa fish eggs after extraction for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0156] B. Ultrasonic treatment:

[0157] Add the defatted hilsa fish egg powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is below 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse off time of 1 s).

[0158] C. Enzymatic hydrolysis:

[0159] Adjust the pH of the hilsa fish egg solution after ultrasonic treatment to 8.0, add 1% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0160] D. Inactivation of enzymes and freeze-drying:

[0161] Boil the enzymatic hydrolysate for 15 min to inactivate the enzymes. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain hilsa fish egg-derived peptides.

[0162] Analyze the experimental results as Figure 5 It can be seen that when 1% alkaline protease is selected, the peptide content is (484.88 ± 5.45) mg / g, the oxygen radical absorbance capacity is (564.76 ± 6.31) μmol TE / g, and the ACE inhibition capacity is (94.38 ± 0.32)%.

[0163] Example 13

[0164] A method for preparing hilsa fish egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0165] A. Freeze-drying and defatting of hilsa fish eggs:

[0166] First, clean and dehydrate the tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then, perform freeze-drying, crush them and prepare freeze-dried tenpounder egg powder. After extraction with a supercritical CO2 extractor for 30 minutes under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C, defatted tenpounder eggs are obtained.

[0167] B. Ultrasonic treatment:

[0168] Add the defatted tenpounder egg powder to distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is below 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 13 minutes (set with a pulse duration of 1 s and a pulse off time of 1 s).

[0169] C. Enzymatic hydrolysis:

[0170] Adjust the pH of the tenpounder egg solution after ultrasonic treatment to 8.0, add 3% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate.

[0171] D. Inactivation of enzymes and freeze-drying:

[0172] Boil the enzymatic hydrolysate to inactivate the enzymes for 15 minutes. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 minutes, take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0173] Analysis of the experimental results shows that Figure 5 when 3% alkaline protease is selected, the peptide content is (541.77 ± 6.18) mg / g, the oxygen radical absorbance capacity is (590.81 ± 7.13) μmol TE / g, and the ACE inhibition capacity is (96.31 ± 0.27)%.

[0174] Example 14

[0175] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibition activity, comprising the following steps:

[0176] A. Freeze-drying and defatting of tenpounder eggs:

[0177] First, clean and dehydrate the tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then, perform freeze-drying, crush them and prepare freeze-dried tenpounder egg powder. After extraction with a supercritical CO2 extractor for 30 minutes under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C, defatted tenpounder eggs are obtained.

[0178] B. Ultrasonic treatment:

[0179] Add the defatted tenpounder egg powder to distilled water at a ratio of 1:70 (mg / mL). Immerse the beaker in an ice-water bath to ensure that the temperature of all samples after sonication is below 40 °C. The sonication power is 170 W and the sonication time is 13 min (set with a pulse duration of 1 s and a pulse-off time of 1 s).

[0180] C, Enzymatic hydrolysis:

[0181] Adjust the pH of the sonicated tenpounder egg solution to 8.0, add 5% alkaline protease, and perform enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 2.0 h to obtain an enzymatic hydrolysate;

[0182] D, Inactivation of enzymes and freeze-drying:

[0183] Boil the enzymatic hydrolysate for 15 min to inactivate the enzymes. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0184] Analyze the experimental results as Figure 5 It can be seen that when 5% alkaline protease is selected, the peptide content is (538.31 ± 5.16) mg / g, the oxygen radical absorbance capacity is (543.03 ± 6.99) μmol TE / g, and the ACE inhibition capacity is (95.55 ± 0.36)%.

[0185] Example 15

[0186] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0187] A, Freeze-drying and defatting of tenpounder eggs:

[0188] First, wash and dehydrate tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then perform freeze-drying, pulverize to prepare freeze-dried tenpounder egg powder, and obtain defatted tenpounder eggs after extraction with a supercritical CO2 extractor for 30 min under extraction pressure of 12 MPa and extraction temperature of 40 °C.

[0189] B, Sonication:

[0190] Add the defatted tenpounder egg powder to distilled water at a ratio of 1:70 (mg / mL). Immerse the beaker in an ice-water bath to ensure that the temperature of all samples after sonication is below 40 °C. The sonication power is 170 W and the sonication time is 13 min (set with a pulse duration of 1 s and a pulse-off time of 1 s).

[0191] C, Enzymatic hydrolysis:

[0192] Adjust the pH of the ultrasonicated tenpounder egg solution to 8.0, add 3% alkaline protease, and enzymatically hydrolyze it in a magnetic stirring water bath at 55°C for 1.0 h to obtain a hydrolyzate;

[0193] D, Inactivation of enzymes and freeze-drying:

[0194] Boil the hydrolyzate to inactivate the enzymes for 15 min. After the temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain tenpounder egg-derived peptides.

[0195] Analyze the experimental results as Figure 6 It can be seen that when the enzymatic hydrolysis time is 1.0 h, the peptide content is (492.91 ± 5.35) mg / g, the oxygen radical absorbance capacity is (513.77 ± 7.67) μmol TE / g, and the ACE inhibition capacity is (94.57 ± 0.47)%.

[0196] Example 16

[0197] A method for preparing tenpounder egg-derived peptides with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0198] A, Freeze-drying and defatting of tenpounder eggs:

[0199] First, wash and dehydrate tenpounder eggs with 10% brine until the water content of the eggs drops to 76%. Then, perform freeze-drying, pulverize them to prepare freeze-dried tenpounder egg powder, and obtain defatted tenpounder eggs after extracting for 30 min with a supercritical CO2 extractor at an extraction pressure of 12 MPa and an extraction temperature of 40°C.

[0200] B, Ultrasonic treatment:

[0201] Add the defatted tenpounder egg powder to distilled water at a ratio of 1:70 (mg / mL), and immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonication is below 40°C. The ultrasonic power is 170 W, and the ultrasonic time is 13 min (set to a pulse duration of 1 s and a pulse off time of 1 s).

[0202] C, Enzymatic hydrolysis:

[0203] Adjust the pH of the ultrasonicated tenpounder egg solution to 8.0, add 3% alkaline protease, and enzymatically hydrolyze it in a magnetic stirring water bath at 55°C for 2.0 h to obtain a hydrolyzate;

[0204] D, Inactivation of enzymes and freeze-drying:

[0205] Boil the enzymatic hydrolysate to inactivate the enzyme for 15 min. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain the hilsa-herring-roe-derived peptide.

[0206] Analyze the experimental results as Figure 6 It can be seen that when the enzymatic hydrolysis time is 2.0 h, the peptide content is (543.57 ± 4.56) mg / g, the oxygen radical absorbance capacity is (595.90 ± 5.13) μmol TE / g, and the ACE inhibition capacity is (95.93 ± 0.55)%.

[0207] Example 17

[0208] A method for preparing hilsa-herring-roe-derived peptide with both antioxidant activity and ACE inhibitory activity, comprising the following steps:

[0209] A. Freeze-drying and defatting of hilsa-herring roe:

[0210] First, wash and dehydrate the hilsa-herring roe with 10% brine until the water content of the roe drops to 76%. Then carry out freeze-drying, pulverize it to prepare freeze-dried hilsa-herring-roe powder, and obtain defatted hilsa-herring roe after extracting for 30 min by a supercritical CO2 extractor under the conditions of an extraction pressure of 12 MPa and an extraction temperature of 40 °C.

[0211] B. Ultrasonic treatment:

[0212] Add the defatted hilsa-herring-roe powder to distilled water at a ratio of 1:70 (mg / mL), immerse the beaker in an ice-water bath to ensure that the temperature of all samples after ultrasonic treatment is lower than 40 °C. The ultrasonic power is 170 W, and the ultrasonic time is 13 min (set as the pulse duration of 1 s and the pulse-off time of 1 s).

[0213] C. Enzymatic hydrolysis:

[0214] Adjust the pH of the hilsa-herring-roe solution after ultrasonic treatment to 8.0, add 3% alkaline protease, and carry out enzymatic hydrolysis in a magnetic stirring water bath at 55 °C for 3.0 h to obtain an enzymatic hydrolysate;

[0215] D. Enzyme inactivation and freeze-drying:

[0216] Boil the enzymatic hydrolysate to inactivate the enzyme for 15 min. After its temperature drops to room temperature, centrifuge it at 9000 r / min for 15 min, and take the supernatant to measure the peptide content, oxygen radical absorbance capacity, and ACE inhibition rate. Freeze-dry the supernatant to obtain the hilsa-herring-roe-derived peptide.

[0217] Analyze the experimental results as Figure 6It can be seen that when the enzymatic hydrolysis time is 3.0 h, the peptide content is (531.88 ± 3.71) mg / g, the oxygen radical absorbance capacity is (554.65 ± 12.79) μmol TE / g, and the ACE inhibition capacity is (93.67 ± 0.28)%.

[0218] It can be seen from Examples 1 - 5 that the preferred protease for enzymatic hydrolysis of tenpounder eggs is alkaline protease, which can result in the highest peptide content, ORAC, and ACE inhibition rate in the obtained enzymatic hydrolysate. It can be seen from Examples 6 - 8 that the optimal extraction ultrasonic power is 170 W, and through response surface experiments, 166 W is the best, which can result in the highest peptide content, ORAC, and ACE inhibition rate in the obtained enzymatic hydrolysate. It can be illustrated by Examples 9 - 11 that the optimal extraction ultrasonic time is 13 min, and through response surface experiments, 13 min is the best, which can result in the highest peptide content, ORAC, and ACE inhibition rate in the obtained enzymatic hydrolysate. It can be seen from Examples 12 - 14 that the preferred enzyme addition amount is 3%, and through response surface experiments, 3.4% is the best, which can result in the highest peptide content, ORAC, and ACE inhibition rate in the obtained enzymatic hydrolysate. It can be seen from Examples 15 - 17 that the optimal extraction enzymatic hydrolysis time is 2.0 h, and through response surface experiments, 1.8 h is the best, which can result in the highest peptide content, ORAC, and ACE inhibition rate in the obtained enzymatic hydrolysate.

[0219] Therefore, according to the above examples and related data, the optimal process conditions are as follows: select 3% alkaline protease, ultrasonic power of 166 W, ultrasonic time of 13 min, enzyme addition amount of 3.4%, and enzymatic hydrolysis time of 1.8 h.

[0220] Example 18

[0221] In the present invention, alkaline protease, ultrasonic power of 166 W, ultrasonic time of 13 min, enzyme addition amount of 3.4%, and enzymatic hydrolysis time of 1.8 h are selected. Other preparation process conditions are the same as those in Example 1. Under these process conditions, the peptide content of the tenpounder egg peptide prepared is (552.84 ± 2.77) mg / g, the oxygen radical absorbance capacity is (603.99 ± 7.32) μmol TE / g, the ACE inhibition capacity is (96.93 ± 0.35)%, and the IC 50 is 0.48 mg / mL. The tenpounder egg-derived peptide prepared by the present invention has both antioxidant activity and ACE inhibition activity.

[0222] Experimental Example Application of the tenpounder egg-derived peptide prepared in Example 18 of the present invention

[0223] The present invention selects the shad egg peptide prepared in Example 18 to conduct an experiment on the lifespan of Caenorhabditis elegans. For the shad egg peptide treatment groups, the selected concentrations are: 0.05, 0.10, 0.15, 0.20 mg / mL. The blank group is treated in parallel with distilled water, and the positive control group selects the anti-aging drug D+Q star combination developed in the United States, that is, the positive control group with a mass ratio of dasatinib (Dasatinib BMS 354825) + quercetin (Quercetin HY18085) of 1:10, and the total concentration of D+Q is: 0.15 mg / mL or 0.1 mg / mL. For the above-mentioned shad egg peptide treatment groups, blank group and positive control group, the E. coli OP50 bacterial solution (kindly provided by Caenorhabditis Geneties Center (University of Minnesota, CGC)) is mixed and diluted to the corresponding concentration and then evenly spread on the solid medium for the growth of Caenorhabditis elegans (NGM) medium. The larvae hatched synchronously are added to the loaded NGM medium for drug treatment (that is, treated with shad egg peptide at different concentrations, distilled water treatment, and the American anti-aging drug D+Q combination treatment respectively), and the drug treatment time is 48 h. The first day of drug treatment is recorded as day 0.

[0224] After the drug treatment is completed, the nematodes corresponding to each sample concentration are picked onto the NGM plate coated with E. coli OP50 bacterial solution. 10 nematodes are picked for each plate, and five parallel samples are made. The nematodes are transferred to a new NGM plate coated with OP50 every two days and the number of dead nematodes is counted. In the later stage of the experiment, since the activity of the nematodes is low and no offspring are produced, which affects the counting results, the dead nematodes are selected for counting to prevent damage to the aging nematodes caused by picking worms and affecting the experimental results. The criterion for judging nematode death is that there is no response when the picking needle gently touches the nematode.

[0225] The specific experimental protocol is as follows:

[0226] (1) Preparation of drug-containing culture plates:

[0227] 1) Preparation of bacterial solution: Use an inoculation loop to pick up the OP50 bacterial solution and streak a single colony on the LB plate without antibiotics. Incubate at 37 °C for 12 h. Take out the LB plate containing the OP50 single colony, pick a single colony with a sterile pipette tip and transfer it to 50 mL of LB medium, and incubate it on a shaker at 37 °C for 12 h with shaking.

[0228] (2) Preparation of NGM plates containing OP50 bacterial solution and drugs: Take 2 mL of the prepared bacterial solution in a laminar flow hood, centrifuge at 15,000×g for 2 min, and discard the supernatant. Resuspend the pellet in 160 μL of dd H2O, and use a pipette to aspirate 80 μL of the bacterial solution and add it to the center of a blank NGM plate. Gently spread the bacterial solution evenly to form a bacterial plaque. Place the NGM plate coated with the bacterial solution under a closed laminar flow hood to dry, and sterilize it by ultraviolet irradiation for 1 h. Preparation of culture plates containing SPs: Filter the drug solution through a 0.22 μm microporous filter membrane to remove bacteria. Use a pipette to aspirate 80 μL of the prepared drug solution and evenly spread it on a blank NGM plate to cover the bacterial solution.

[0229] (2) C. elegans culture and synchronization

[0230] C. elegans culture: Use a pick to transfer C. elegans to an NGM plate containing Escherichia coli, and incubate it in a 20 °C incubator. 0.2 mL of OP50 is spread on the NGM as food for C. elegans.

[0231] C. elegans synchronization:

[0232] 1) In a laminar flow hood, rinse the NGM plate several times with sterile M9 buffer to wash off C. elegans and eggs. Collect a large number of gravid C. elegans in a centrifuge tube (1.5 mL centrifuge tube) with M9 buffer, and rinse 4 times with M9 buffer to remove impurities and food. During this period, centrifuge at a low speed of 2000 rpm for 1 min to sediment C. elegans. Do not disrupt the sedimented C. elegans mass when removing the supernatant. After the last wash, discard the supernatant, leaving about 500 μL of worm solution (the egg content washed off in a 90 mm diameter culture medium is about 300 per time). The egg concentration in the remaining worm solution after removing the supernatant is about 600 eggs / mL;

[0233] 2) Add 1 mL of C. elegans lysis solution (a 1:1 mixture of 10% sodium hypochlorite and 1 M sodium hydroxide) to the centrifuge tube containing the worm solution at a volume ratio of 2:1.

[0234] 3) Vortex until the worms are lysed, for no more than 5 min.

[0235] 4) Centrifuge the tube in a tabletop centrifuge at 1300×g, with a centrifugal radius of 10 cm, at 20 °C for 2 min to precipitate the eggs.

[0236] 5) After discarding the supernatant until 0.1 mL remains, add sterile water to 5 mL, shake well, repeat steps 4) and 5). Use a pipette to transfer the eggs in the remaining 0.1 mL of liquid to the edge of a clean NGM plate inoculated with an Escherichia coli OP50 lawn, and incubate at 20 °C for 48 h to obtain L4-stage C. elegans.

[0237] (3) Drug intervention

[0238] Experimental grouping: control group (CK), positive group (treated with anti-aging drugs D+Q at 0.15 mg / mL or 0.1 mg / mL), and macrura reevesii egg peptide at 0.05, 0.10, 0.15, 0.20 mg / mL. The control group used ddH2O as a solvent control.

[0239] (4) Lifespan determination: Pick L4-stage nematodes onto NGM plates containing drugs and OP50, with 30 nematodes in each group, and three plates in parallel for each concentration. Culture at 20°C; perform drug treatment, and mark the age of the nematodes as 0 d from the day of drug administration. During the experiment, pick the drug-treated nematodes onto new plates every 2 d to avoid interference from offspring. Observe the status of the nematodes every day, record the number of surviving and dead nematodes, and observe the growth status of the nematodes. The criterion for nematode death is that the body does not bend or the head does not swing after being touched, and the body is rigid. Record the daily death number of the nematodes and make a growth curve.

[0240] Table 1. Comparison table of total lifespan, average lifespan, median lifespan, maximum lifespan, and improvement rate of each sample group

[0241]

[0242] Analysis of the experimental results shows that Figure 7 as can be seen, at concentrations of 50, 100, 150, 200 (μg / mL), the average lifespan of nematodes treated with macrura reevesii egg peptide was significantly increased (P < 0.001) compared with the blank group by: 23.38 ± 0.578%, 24.38 ± 1.192%, 33.33 ± 1.081%, 31.34 ± 0.708%; when the intervention dose (150 μg / mL) was the same, the improvement rates of the average lifespan of nematodes treated with macrura reevesii egg peptide and the D+Q anti-aging combination (150 μg / mL) were 33.33% and 30.34% respectively. This experiment shows that the macrura reevesii egg-derived peptide prepared in the present invention is superior to the D+Q anti-aging star combination developed in the United States and has significant anti-aging activity.

Claims

1. A green preparation method of macrura reevesii egg-derived peptide, characterized in that, It includes the following steps: Firstly, clean and dehydrate the hilsa shad eggs, then perform freeze-drying, and after pulverization, prepare freeze-dried powder of hilsa shad eggs; after extracting oil by supercritical CO2, obtain defatted powder of hilsa shad eggs; add distilled water and perform ultrasonic treatment to obtain a hilsa shad egg solution, then add protease for enzymatic hydrolysis, perform enzyme inactivation and centrifugation on the enzymatic hydrolysate, take the supernatant and perform freeze-drying to obtain hilsa shad egg-derived peptides.

2. The preparation method of the hilsa herring egg-derived peptide according to claim 1, wherein The conditions for the supercritical CO2 extraction are: extraction pressure is 12 MPa, extraction temperature is 40 °C, and extraction time is 30 min.

3. The preparation method of the macrura reevesii egg-derived peptide according to claim 1, wherein The ratio of the hilsa shad egg protein powder to distilled water is 1:70 (mg / mL).

4. The preparation method of the macrura reevesii egg-derived peptide according to claim 1, wherein The ultrasonic conditions are: ultrasonic power is 135 - 205 W, and ultrasonic treatment time is 7 - 19 min.

5. The preparation method of the hilsa herring egg-derived peptide according to claim 1, characterized in that, After ultrasonic treatment, it also includes adjusting the pH of the hilsa shad egg solution to 7.0 - 8.0 and then performing enzymatic hydrolysis.

6. The preparation method of the hilsa herring egg-derived peptide according to claim 1, characterized in that, The protease includes one or several of alkaline protease, trypsin, papain, flavor protease or compound protease.

7. The preparation method of the macrura reevesii egg-derived peptide according to claim 1, characterized in that, The addition amount of the protease is 1% - 5%.

8. The preparation method of the macrura reevesii egg-derived peptide according to claim 1, characterized in that, The enzyme inactivation temperature is a water bath at 90 - 100 °C for 15 min, the centrifugation speed is 9000 r / min, and the centrifugation time is 15 min.

9. Hilsa shad egg-derived peptides prepared by the method according to any one of claims 1 - 8.

10. Use of the hilsa shad egg-derived peptides according to claim 9 in the preparation of antioxidant, blood pressure-lowering, life-prolonging and / or anti-aging products.

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