Chinese grass turtle egg protein peptide extract with anti-oxidation effect as well as preparation method and application of Chinese grass turtle egg protein peptide extract
Chinese pond turtle egg protein peptides were prepared by ultrasound-assisted papain method and Sephadex G-25 dextran gel column chromatography, which solved the problem of unclear composition and structure of Chinese pond turtle egg protein peptides, and achieved efficient antioxidant function, which has broad application prospects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2026-01-19
- Publication Date
- 2026-04-10
AI Technical Summary
Existing research has focused little on protein peptides from Chinese pond turtle eggs, and there are no reports on the preparation of protein peptides from Chinese pond turtle eggs, clarifying their composition and structure, as well as their antioxidant activity, which limits their high-value utilization.
Chinese pond turtle egg protein peptides were prepared using an ultrasound-assisted papain method. Six peptide segments with high content were identified by LC-MS/MS. High-purity Chinese pond turtle egg protein peptide extracts were prepared by Sephadex G-25 dextran gel column chromatography, preserving their antioxidant activity.
The prepared Chinese pond turtle egg protein peptide extract has significant ABTS radical, DPPH radical, and hydroxyl radical scavenging ability and iron ion reducing ability, showing significant antioxidant effects and providing an antioxidant strategy.
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Figure CN121824680A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an extract of Chinese pond turtle egg protein peptides with antioxidant properties, its preparation method, and its application. It belongs to the field of bioprocessing technology. Background Technology
[0002] Bioactive peptides generally refer to a collective term for peptide segments containing two or more amino acids that possess functional activities such as antioxidation, antibacterial activity, and blood pressure reduction. Most bioactive peptides have a molecular weight of less than 3000 Da, and their biological activity is determined by the type, number, and sequence of amino acids. When proteins are enzymatically hydrolyzed or undergo physicochemical reactions, their spatial structure breaks down, exposing bioactive peptide segments that exhibit activities such as antioxidation, antibacterial activity, anti-inflammation, immunomodulation, and blood pressure reduction.
[0003] Natural aging occurs in all living organisms and is an extremely complex process. As aging progresses, excessive reactive oxygen species (ROS) are produced, weakening the body's antioxidant defense system. ROS formation causes cell membrane damage and oxidative stress on DNA. Oxidative damage leads to cell damage and apoptosis, exacerbating age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, heart disease, atherosclerosis, and diabetes. Antioxidants, as free radical scavengers, effectively reduce the level of free radicals in the body, thereby protecting biomolecules from damage and helping the body fight disease.
[0004] The Chinese pond turtle (Chinemys reevesii), also known as the grass turtle, mud turtle, or common pond turtle, lives in freshwater areas and is distributed throughout China. It is a nutritious and delicious aquatic product, and also a medicinal ingredient with significant medicinal value. Since 2017, my country's freshwater turtle production has consistently remained above 45,000 tons, with aquaculture production projected to reach approximately 55,000 tons in 2024. As a major edible turtle species in my country, the scale of turtle farming is expanding, and turtle eggs, as a byproduct, are also seeing increased production, presenting significant potential for development and utilization. Currently, most domestic and international research on the Chinese pond turtle focuses on the nutritional value of its eggs, incubation conditions, and factors influencing embryonic development, while research and development on turtle egg protein peptides are limited. Xu Wensi et al. (2022) analyzed the nutritional composition of Chinese pond turtle eggs and found that they are high in protein, low in fat, and low in sugar; they are also rich in vitamins and minerals, with high levels of essential amino acids and fatty acids, giving them high nutritional value. Furthermore, they are stable in oral and gastric digestive fluids and can be degraded into small oligopeptides in intestinal digestive fluids. Lu Xiaorong et al. (2023) also analyzed the nutritional composition of pond turtle eggs and found that they are rich in various amino acids, fatty acids, and minerals, with high levels of essential amino acids, unsaturated fatty acids, and vitamins B2 and E, and low cholesterol, indicating high nutritional value.
[0005] Chinese pond turtle eggs have the advantage of being developed into high-value-added products. However, existing research has paid little attention to the protein peptides in Chinese pond turtle eggs, and there are no reports on the preparation of protein peptides from Chinese pond turtle eggs. Clarifying their composition and structure, as well as their antioxidant activity, is the key to their high-value utilization. Summary of the Invention
[0006] In view of the above problems, firstly, the present invention provides a Chinese pond turtle egg protein peptide extract with antioxidant properties, wherein the protein peptides of the extract have high purity and the main components and structures are clearly defined.
[0007] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:
[0008] A Chinese pond turtle egg protein peptide extract with antioxidant properties; the peptide extract contains peptide segments with amino acid sequences as shown in SEQ ID NO. 1~6.
[0009] This invention extracts protein peptides from Chinese pond turtle eggs through a series of experimental steps. LC-MS / MS identified six peptide segments with high content, whose amino acid sequences are shown in SEQ ID NO. 1~6. Their relative molecular masses are less than 1100 Da and are, in descending order: 983.504, 387.248, 1094.515, 957.513, 414.248, and 458.274, with relative percentage contents of 9.71%, 7.26%, 6.87%, 3.74%, 3.01%, and 2.40%, respectively. The ABTS free radical scavenging ability, DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, and iron ion reducing ability of the prepared Chinese grass turtle egg protein peptide extract were 87.35±0.12%, 55.61±0.54%, 68.24±1.1%, and 60.12±0.76%, respectively. This indicates that the Chinese grass turtle egg protein peptide has significant antioxidant function, can effectively scavenge ABTS, DPPH, and hydroxyl free radicals, and enhance the reducing ability of ionic iron.
[0010] Secondly, the present invention provides a method for preparing an extract of Chinese pond turtle egg protein peptides with antioxidant properties.
[0011] The technical solution is as follows:
[0012] A method for preparing an extract of Chinese pond turtle egg protein peptides with antioxidant properties includes the following steps:
[0013] (1) Separation: Separate the egg white and yolk of the Chinese pond turtle egg, freeze and dry the egg white separately to obtain egg white protein;
[0014] (2) Enzymatic hydrolysis: Dissolve egg white protein in water, add protease, and use ultrasound-assisted enzymatic hydrolysis. After enzymatic hydrolysis, boil in a water bath to inactivate the enzyme, centrifuge and take the supernatant to obtain crude egg white protein peptide hydrolysate.
[0015] (3) Purification: The above-mentioned crude protein peptide hydrolysate of egg white was purified to obtain Chinese pond turtle egg protein peptide hydrolysate, which was then frozen and dried to obtain protein peptide powder.
[0016] Preferably, in the separation step, the egg white is placed separately in a sealed environment at -80°C and frozen for 3-4 hours.
[0017] Preferably, in the enzymatic hydrolysis step, the ultrasonic power is 200~300 W and the ultrasonic treatment time is 100~200 min.
[0018] Preferably, the enzyme used in the enzymatic hydrolysis step is papain, and the hydrolysis conditions are a temperature of 45-50 ℃ and a pH of 5-6; the enzyme inactivation is performed by a boiling water bath.
[0019] Preferably, in the enzymatic hydrolysis step, the amount of enzyme added is 8000 ~ 16000 U / g.
[0020] Preferably, the centrifugation conditions in the enzymatic hydrolysis step are: 5000 r / min, 4 ℃, 10 min.
[0021] As a preferred option, the specific experimental operation and conditions in the enzymatic hydrolysis step can be optimized as follows: dissolve the egg white of the Chinese pond turtle in water, adjust the pH to 5-6 with 0.1 mol / L HCl solution, add papain at a concentration of 8000-16000 U / g, then use ultrasonic power of 200-300W to enzymatically hydrolyze at 45-50 ℃ for 100-200 min, inactivate the enzyme with boiling water for 15 min, cool to room temperature, centrifuge (5000 r / min, 4 ℃, 10 min), and collect the supernatant to obtain the crude polypeptide hydrolysate of egg white.
[0022] Preferably, in the purification step, the crude polypeptide hydrolysate of egg white is filtered through a 0.45 μm aqueous membrane; the membrane-filtered protein peptide solution is passed through a Sephadex G-25 dextran gel column using an AKTA protein purifier, and then eluted with deionized water.
[0023] As a preferred option, the specific experimental operations and conditions in the purification step can be optimized as follows: filter the crude polypeptide hydrolysate of egg white using a 0.45 μm aqueous membrane; pass the membrane-filtered protein peptide solution through a Sephadex G-25 dextran gel column using an AKTA fully automated protein purification instrument, then elute with deionized water at a rate of 5.0 mL / min, collect in a 15 mL centrifuge tube, and freeze-dry to obtain Chinese pond turtle egg protein peptide powder.
[0024] Preferably, the drying process in steps (1) and (3) is freeze drying.
[0025] The freeze-drying method used here does not damage the activity of the extracted Chinese pond turtle egg protein peptides, and can well preserve the integrity of the structure and activity of the protein peptides.
[0026] Thirdly, the present invention provides the application of the Chinese pond turtle egg protein peptide extract, specifically its application in antioxidant applications.
[0027] The Chinese scorpion turtle egg protein peptide extract has significant effects on ABTS free radical scavenging rate, DPPH free radical scavenging rate, hydroxyl free radical scavenging rate and enhanced iron ion reducing ability.
[0028] By measuring the ABTS free radical scavenging rate, DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, and iron ion reducing capacity, it was found that the Chinese pond turtle egg protein peptide extract can effectively scavenge ABTS free radicals, DPPH free radicals, and hydroxyl free radicals and enhance iron ion reducing capacity. This indicates that the extracted Chinese pond turtle egg protein peptide extract has significant antioxidant effects and can provide a strategy for the antioxidant properties of Chinese pond turtle egg protein peptide extract.
[0029] The present invention has the following beneficial effects:
[0030] (1) The protein peptide extract of Chinese grass turtle egg was prepared by ultrasound-assisted papain method. The preparation process is simple, the obtained peptides have high purity, the main components and structures are clear, and the activity of the protein peptide extract is well maintained.
[0031] (2) The preparation process does not involve any organic solvents and the operating conditions are mild, which ensures the safety of the product;
[0032] (3) In the separation and purification process, chromatography column separation was used, which is not only faster than chromatography but also simpler to operate;
[0033] (4) Protein peptide extract has the ability to scavenge ABTS free radicals, DPPH free radicals, hydroxyl free radicals and enhance the reduction ability of iron ions, and has significant antioxidant effects. It can provide a strategy for the anti-oxidation of Chinese grass turtle egg protein peptides and has broad application prospects. Attached Figure Description
[0034] Figure 1 This is the Basepeak image of the purified Chinese pond turtle egg protein peptide identified by LC-MS / MS.
[0035] Figure 2 The scavenging rate of ABTS free radicals by purified extracts of Chinese pond turtle egg protein peptides at different concentrations;
[0036] Figure 3 The scavenging rate of DPPH free radicals by purified extracts of Chinese pond turtle egg protein peptides at different concentrations;
[0037] Figure 4 The scavenging rate of hydroxyl radicals by purified extracts of Chinese pond turtle egg protein peptides at different concentrations;
[0038] Figure 5 The reduction capacity of iron ions by purified extracts of Chinese pond turtle egg protein peptides at different concentrations. Detailed Implementation
[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0040] Example 1: Preparation of an extract of Chinese pond turtle egg protein peptides with antioxidant properties
[0041] An extract of protein peptides from the egg of the Chinese pond turtle with antioxidant properties was prepared by the following method:
[0042] (1) Separation: Peel the shell off the Chinese pond turtle eggs, and use an egg white separator to separate the egg white and yolk. Take the egg white separately and freeze it at -80℃ for 3-4 hours, and then freeze-dry it to obtain egg white protein;
[0043] (2) Enzymatic hydrolysis: Dissolve 0.5g of Chinese pond turtle egg white in 50mL of deionized water, adjust the pH to 5-6 with 0.1 mol / L HCl solution, add papain, the amount of enzyme added is 8000-16000U / g, then use ultrasonic power 200-300W, enzymatic hydrolysis at 45-50℃ for 100-200min, then boil water to inactivate the enzyme for 15min, cool to room temperature, centrifuge (5000 r / min, 4℃, 10min) to obtain supernatant, freeze dry to obtain crude protein peptides of Chinese pond turtle egg;
[0044] (3) Purification: The crude protein peptides were dissolved in 50 mL of deionized water to prepare a solution with a concentration of 2 mg / mL to 5 mg / mL, and filtered through a 0.45 μm aqueous membrane. The membrane-filtered protein peptide solution was passed through a Sephadex G-25 dextran gel column using an AKTA fully automated protein purification system, and then eluted with deionized water at a rate of 5.0 mL / min. The elution was collected in a 15 mL centrifuge tube and freeze-dried to obtain purified Chinese pond turtle egg protein peptide powder.
[0045] Analysis and testing showed that the purity of the Chinese pond turtle egg protein peptide obtained in this embodiment was 96.77%.
[0046] Example 2: Peptide composition and structural identification of Chinese pond turtle egg protein peptides
[0047] The peptide composition and structure of Chinese pond turtle egg protein peptides were identified using LC-MS / MS technology. The mass spectrometry information was compared with PDB and NCBI databases, revealing six peptides with high content, as shown in Table 1.
[0048] Table 1. Analysis of protein peptide composition in Chinese pond turtle eggs.
[0049] Serial Number sequence length Relative molecular mass (Da) relative percentage content (%) SEQ ID NO.1 NPEQRIVE 8 983.504 9.71 SEQ ID NO.2 VAKA 4 387.248 7.26 SEQ ID NO.3 LDGGHIFEAH 10 1094.515 6.87 SEQ ID NO.4 KDNIDILQ 8 957.513 3.74 SEQ ID NO.5 VTVP 4 414.248 3.01 SEQ ID NO.6 VEVL 4 458.274 2.40
[0050] Example 3: Determination of ABTS free radical scavenging rate of protein peptides from Chinese pond turtle eggs
[0051] Step 1: Dissolve the purified extract of Chinese pond turtle egg protein peptides in ultrapure water to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.
[0052] ABTS free radical scavenging rate determination: After step 1, accurately weigh 38.4 mg of ABTS and 67.5 mg of K₂S₂O₈ and dissolve them in 10.0 mL and 100.0 mL of ultrapure water, respectively. Take 2.0 mL of each solution, mix well, and incubate at room temperature in the dark for 12 h. Dilute with 5.0 mM phosphate buffer (pH 7.4) to a absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS working solution. Add 100.0 μL of Chinese pond turtle egg protein peptide solution (or PBS buffer) and 100.0 μL of ABTS working solution to a 96-well plate and incubate in the dark for 10 min. After the reaction, measure the absorbance at 734 nm and take the average of three experiments. The formula is:
[0053] ABTS clearance rate = (A1-A0) / A1×100%
[0054] Where: A1 is the absorbance of the sample, and A0 is the absorbance of the reference standard.
[0055] Test results as follows Figure 2 As shown, within the range of 0-0.8 mg / mL, the ABTS free radical scavenging rate increased with the increase of the concentration of Chinese turtle protein peptide, and reached a maximum of 87.35±0.12% at 0.8 mg / mL.
[0056] Example 4: Determination of DPPH free radical scavenging rate of Chinese pond turtle egg protein peptides
[0057] Step 1: Dissolve the purified extract of Chinese pond turtle egg protein peptides in ultrapure water to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.
[0058] DPPH free radical scavenging rate determination: After step 1, accurately weigh 0.002 g of DPPH and dissolve it in 50.0 ml of anhydrous ethanol. Store in the dark and prepare fresh before use. Add 100.0 μL of Huacao turtle egg protein peptide solution to a 96-well plate, and add 100.0 μL of 0.1 mmol / L DPPH anhydrous ethanol solution to each well. React in the dark for 30 min. Using anhydrous ethanol and ultrapure water as references, measure the absorbance at 517 nm and take the average of three experiments. The formula is:
[0059] DPPH removal rate = (A0 - A1 + A2) / A0 × 100%
[0060] Where: A0 is the absorbance of DPPH anhydrous ethanol solution + ultrapure water at 517 nm, A1 is the absorbance of DPPH anhydrous ethanol solution + Chinese pond turtle egg protein peptide solution at 517 nm, and A2 is the absorbance of anhydrous ethanol + Chinese pond turtle egg protein peptide solution at 517 nm.
[0061] Test results as follows Figure 3 As shown, when the concentration of Chinese pond turtle protein peptide is 0-1.0 mg / mL, the DPPH free radical scavenging rate increases in a concentration-dependent manner, reaching a maximum of 55.61±0.54% at 1.0 mg / mL.
[0062] Example 5: Determination of hydroxyl radical scavenging rate of protein peptides from Chinese pond turtle eggs
[0063] Step 1: Dissolve the purified extract of Chinese pond turtle egg protein peptides in ultrapure water to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.
[0064] Hydroxyl radical scavenging rate determination: After step 1, prepare 8.8 mmol / L H2O2 solution, 9.0 mmol / L salicylic acid-ethanol solution, and 9.0 mmol / L ferrous sulfate solution. Add 100.0 μL each of the Chinese pond turtle egg protein peptide solution and the above three solutions sequentially. After reacting at 37 ℃ for 1 h, measure the absorbance at 510 nm using a microplate reader and calculate the scavenging rate. Take the average of three experiments. The formula for the free radical scavenging rate is:
[0065] Clearance rate (%) = (A0 - (A0)) i -A j )) / A0×100%
[0066] Where: A0 is the absorbance value of the blank control; A i A represents the absorbance of the sample. j The absorbance value is the value without the addition of the colorimetric reagent H2O2.
[0067] Test results as follows Figure 4 As shown, the protein peptides of Chinese pond turtle eggs have significant hydroxyl radical scavenging ability in the concentration range of 0-1.0 mg / mL, with a maximum of 68.24±1.1%.
[0068] Example 6: Determination of the iron reducing power of Chinese pond turtle egg protein peptides
[0069] Step 1: Dissolve the purified extract of Chinese pond turtle egg protein peptides in ultrapure water to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.
[0070] Determination of iron ion reducing power: After step 1, take 0.25 mL of 1% potassium ferricyanide, 0.25 mL of Chinese pond turtle egg protein peptide solution, and 0.25 mL of pH 6.6 phosphate buffer into a 1.5 mL centrifuge tube, mix well, heat in a 50 ℃ water bath for 20 min, then remove and cool to room temperature. Add 0.25 mL of 10% trichloroacetic acid solution, mix well, and centrifuge at 3000 r / min for 10 min. Take 100 μL of the supernatant, add 20 μL of 1% FeCl3 solution and 100 μL of deionized water into a 96-well plate, mix well, let stand for 20 min, and measure the absorbance at 700 nm (A). Take 100 μL of the supernatant, add 120 μL of deionized water into a 96-well plate, mix well, let stand for 20 min, and measure the absorbance at 700 nm (B). The formula for calculating iron ion reducing power is as follows:
[0071] Iron ion reducing power = (AB) / A × 100%
[0072] Where: A is the sample absorbance value; B is the sample background absorbance value (deionized water replaces FeCl3).
[0073] Test results as follows Figure 5 As shown, the protein peptides extracted from Chinese pond turtle eggs in this experiment have good iron ion reducing ability, and the iron ion reducing ability gradually increases with increasing concentration, reaching a maximum value of 60.12±0.76% at 1.0 mg / mL.
[0074] In summary, this invention utilizes an ultrasound-assisted enzymatic hydrolysis method combined with Sephadex G-25 dextran gel column chromatography to prepare a purified extract of Chinese pond turtle egg protein peptides. This extract exhibits significant ABTS radical scavenging, DPPH radical scavenging, and hydroxyl radical scavenging abilities, as well as enhanced iron ion reducing capacity, providing a strategy for the antioxidant effects of Chinese pond turtle egg protein peptides.
Claims
1. A Chinese pond turtle egg protein peptide extract, characterized in that, The protein peptide extract contains peptides with amino acid sequences as shown in SEQ ID NO.1~6.
2. A method for preparing the Chinese pond turtle egg protein peptide extract as described in claim 1, comprising the following steps: (1) Separation: Separate the egg white and yolk of the Chinese pond turtle egg, freeze and dry the egg white separately to obtain egg white protein; (2) Enzymatic hydrolysis: Dissolve egg white protein in water, add protease, and use ultrasound-assisted enzymatic hydrolysis. After enzymatic hydrolysis, boil in a water bath to inactivate the enzyme, centrifuge and take the supernatant to obtain crude egg white protein peptide hydrolysate. (3) Purification: The above-mentioned crude protein peptide hydrolysate of egg white was purified to obtain Chinese pond turtle egg protein peptide hydrolysate, which was then frozen and dried to obtain protein peptide powder.
3. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: In the separation step, the egg whites are placed separately in a sealed environment at -80°C and frozen for 3-4 hours.
4. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: In the enzymatic hydrolysis step, the ultrasonic power is 200~300 W and the ultrasonic treatment time is 100~200 min.
5. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: In the enzymatic hydrolysis step, the enzyme used is papain, and the hydrolysis conditions are a temperature of 45-50 ℃ and a pH of 5-6; the enzyme inactivation is performed by a boiling water bath.
6. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 5, characterized in that: In the enzymatic hydrolysis step, the amount of enzyme added is 8000 ~ 16000 U / g.
7. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: In the enzymatic hydrolysis step, the centrifugation conditions are: 5000 r / min, 4 ℃, 10 min.
8. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: In the purification step, the crude polypeptide hydrolysate of egg white is filtered through a 0.45 μm aqueous membrane; the membrane-filtered protein peptide solution is passed through a Sephadex G-25 dextran gel column using an AKTA fully automated protein purification instrument, and then eluted with deionized water.
9. The method for preparing a Chinese pond turtle egg protein peptide extract according to claim 2, characterized in that: The drying process in the separation and purification steps employs freeze drying.
10. The application of the Chinese pond turtle egg protein peptide extract according to claim 1 in antioxidant pharmaceuticals and food.