Sea cucumber active peptide and application thereof

Abstract

The invention discloses sea cucumber active peptide and application thereof, and belongs to the field of food science. The amino acid sequence of the sea cucumber active peptide is GYMLPK, and the sea cucumber active peptide has the functions of oxidation resistance, immunoregulation and intestinal health regulation. The active peptide is extracted from sea cucumber protein by adopting a biological enzymolysis method, and can improve the activity of serum superoxide dismutase (SOD), reduce the content of malondialdehyde (MDA) and promote the growth of intestinal beneficial flora at the same time. The sea cucumber active peptide can be widely applied to functional foods, health supplements and sports nourishments.

Description

Technical Field

[0001] The invention relates to the field of food science and technology, and in particular to a sea cucumber active peptide and application thereof. Background Art

[0002] Sea cucumbers have received extensive attention in the field of food and health products due to their rich nutritional ingredients and biologically active functions. Studies have shown that sea cucumber proteins contain a large number of functional peptides, which may have antioxidant, immunomodulatory and intestinal health-promoting effects in the human body, and may also have the potential to improve metabolism, resist fatigue and protect cardiovascular health. Sea cucumber active peptides are rich in amino acids such as arginine, glycine and glutamic acid, which play an important role in regulating the immune system and resisting oxidative stress. Studies have shown that sea cucumber peptides can promote macrophage phagocytosis, increase immunoglobulin levels, and enhance the activity of natural killer cells (NK cells). In addition, the low molecular weight characteristics of sea cucumber active peptides make them easy to be absorbed by the human body, improving bioavailability. However, the sea cucumber products on the market are mainly in the form of dried products or concentrated extracts. How to effectively extract, optimize the purification process, and improve the stability and functionality of sea cucumber active peptides is worthy of in-depth study. Summary of the invention

[0003] The invention provides an active peptide extracted from sea cucumber, which has significant anti-oxidation, immunomodulatory and intestinal health regulating functions and can be widely used in the fields of food and health products.

[0004] To achieve the above object, the present invention provides the following technical solutions: A sea cucumber active peptide, whose amino acid sequence is GYMLPK.

[0005] In order to ensure the quality and function of sea cucumber active peptides, the present invention adopts biological enzymatic hydrolysis method, optimizes the enzymatic hydrolysis conditions, and ensures the efficient extraction of active peptides. The specific steps are as follows: Step S1, select fresh sea cucumber as raw material, remove viscera, mud and impurities to ensure the purity of protein extraction. Rinse with running water for 5-10 minutes, and then soak in 0.9% NaCl solution for 10 minutes to remove residual salt. Add the crushed sea cucumber powder to phosphate buffer (pH7.4) at a ratio of 1:10 (g / mL), stir evenly, and fully dissolve the protein. Let it stand at 4°C for 12 hours to allow the protein to fully swell and improve the efficiency of subsequent enzymatic hydrolysis.

[0006] Step S2, use NaOH (0.1M) or HCl (0.1M) to adjust the pH to 7.0-8.5 to ensure the optimal activity of the enzyme. The temperature is controlled between 40-55°C. If the temperature is lower than 40°C, the enzyme activity is insufficient, resulting in low protein hydrolysis efficiency. If the temperature is higher than 55°C, the enzyme may be inactivated, causing the reaction to terminate. The enzymatic hydrolysis time is maintained at 4-6 hours, and samples are taken every hour to detect the degree of enzymatic hydrolysis. Heat to 95°C for 10 minutes to inactivate the protease and avoid excessive hydrolysis.

[0007] Step S3: After the enzymatic hydrolysis is completed, the solution contains a large amount of protein fragments, polypeptides and small molecule peptides, which need to be further purified to obtain the target functional peptide segment. Remove the residue by centrifugation (5000rpm, 15min) to obtain the supernatant of the enzymatic hydrolyzate. Use a 0.45μm filter membrane to filter to remove undissolved proteins and precipitates. Use ultrafiltration technology and select a 10kDa cutoff membrane. Use a 3kDa dialysis bag to further remove small molecule impurities and improve the stability and functionality of the peptide.

[0008] Step S4, pack the peptide solution into freezing trays (within 2 cm thickness) to ensure uniform freezing, pre-freeze at -40°C for 8-12 hours to form fine ice crystals and reduce freeze-drying losses. Dry at -40°C under vacuum (0.1 mbar) for 24 hours to sublimate most of the water. Raise the temperature to 20-25°C and maintain for 8 hours to remove the remaining bound water and improve the stability of the powder. Then freeze-grind it with liquid nitrogen into 100 mesh fine powder, finally seal and package it, and place it in a low-temperature drying environment.

[0009] The sea cucumber active peptide can increase serum superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) content, and effectively reduce oxidative damage.

[0010] The sea cucumber active peptide can promote the growth of beneficial intestinal flora, regulate the balance of intestinal microecology, and increase the level of short-chain fatty acids (SCFAs), such as acetic acid and butyric acid, thereby enhancing the intestinal barrier function.

[0011] The sea cucumber active peptide can promote the activity of immune cells, increase the secretion levels of IgA, IgG and IL-10, and improve the body's immunity.

[0012] The sea cucumber active peptide can be used in functional foods, health supplements and sports nutrition products to enhance antioxidant capacity and immune function.

[0013] Technical effects and advantages of the present invention: The present invention provides an active peptide GYMLPK (SEQID NO: 6) extracted from sea cucumber. The active peptide is prepared by an optimized biological enzymatic hydrolysis method, has antioxidant, immune regulation and intestinal health regulation functions, can improve serum superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) content, reduce oxidative damage, promote the growth of beneficial intestinal flora, increase short-chain fatty acids (SCFAs) levels, and enhance immune cell activity, increase IgA, IgG and IL-10 secretion levels, effectively improve the body's immunity, and the active peptide has a small molecular weight and high bioavailability, is suitable for functional foods, health supplements and sports nutrition products, can be widely used in the fields of antioxidant, intestinal regulation and immune enhancement, and has good market prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a schematic diagram of the extraction process of the sea cucumber active peptides of the present invention.

[0015] Figure 2 This is the test result of the antioxidant capacity of the sea cucumber active peptide of the present invention.

[0016] Figure 3 The present invention shows the influence of the sea cucumber active peptide on the antioxidant indexes of mice.

[0017] Figure 4 This is the effect of the sea cucumber active peptide of the present invention on intestinal flora. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] 1. A method for preparing sea cucumber protein peptide ( Figure 1 ), the specific steps are as follows: Step S1, rinse the fresh sea cucumber with running water for several times to remove the sea sand and mucus attached to the surface, soak it in 0.5% NaCl solution for 5 minutes to remove the attached microorganisms and salt, and wash it again with deionized water to ensure that the surface is clean; cut the abdomen of the sea cucumber with sterile scissors, remove the internal organs (including intestines and gonads), and gently rinse the inner cavity with 0.1M phosphate buffer (PBS) to remove residual tissue; in order to prevent protein degradation and facilitate subsequent processing, the sea cucumber needs to be freeze-dried, and the sea cucumber sample is placed in a -80℃ refrigerator for pre-freezing for 12 hours to allow the water to form uniform ice crystals. A vacuum freeze dryer with a pressure of 0.01Pa and a temperature of -50℃ is used for 48 hours until the sea cucumber is completely dry. The freeze-dried sea cucumber has a porous structure, which is convenient for subsequent grinding; a high-speed grinder (speed 12,000rpm) is used to grind the freeze-dried sea cucumber into fine powder. During the grinding process, liquid nitrogen is used for cooling to prevent the protein from being degraded due to frictional heat.

[0020] Step S2, using pH 7.4 phosphate buffer (PBS) as the dissolution medium, the PBS formula is: NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 1.8mM, dilute to 1L with deionized water, and adjust the pH to 7.4; weigh sea cucumber powder according to a ratio of 1:10 (g / mL), add PBS solution; use a magnetic stirrer (300rpm) to stir at a low speed for 30 minutes to gradually dissolve the protein, and place the solution in a 4°C refrigerator for 12 hours to ensure that the protein is fully dissolved and reduce precipitation formation.

[0021] Step S3, in order to remove insoluble impurities and large particle precipitation, high-speed centrifugation is used to separate protein. The centrifugal conditions are: speed 10000rpm, temperature 4°C, time 10min, take the clear protein solution after centrifugation, avoid absorbing the precipitation layer to reduce impurities. Use a double-layer absorbent cotton filter cloth to remove larger particles, and use a 0.45μm microporous filter membrane to filter to remove fine impurities and bacteria. If the protein solution is viscous, 0.1% Triton X-100 can be appropriately added to reduce viscosity and improve filtration efficiency. After centrifugation and filtration, a transparent and clear sea cucumber protein solution is obtained. The protein concentration is determined by a UV-visible spectrophotometer (280nm) to ensure that the protein concentration is within the range of 1-5mg / mL.

[0022] 2. Peptide sequence screening of sea cucumber active peptides In order to further screen and identify the polypeptide sequence of sea cucumber active peptides, the present invention uses liquid chromatography-tandem mass spectrometry (LC-MS / MS) for analysis and uses professional mass spectrometry analysis software for data analysis to obtain the amino acid sequence information of the target active peptide. The LC-MS / MS determination conditions include liquid chromatography conditions and mass spectrometry conditions. In the liquid chromatography conditions, a C18 reverse phase column (3 μm, 250 mm×75 μm, Waters) was used for separation, water (0.1% formic acid) was used as phase A, acetonitrile (0.1% formic acid) was used as phase B, and gradient elution was used to optimize the separation effect, wherein phase A gradually decreased from 95% to 55% within 0-50 min, phase A continued to decrease to 25% from 50-58 min, phase A decreased to 5% from 58-60 min and maintained for 2 min, and then phase A returned to 95% within 60-65 min to balance the system, ensuring the stability and repeatability of subsequent analysis; the injection volume was set to 4 μL, and the flow rate was controlled at 300 nL / min to ensure efficient separation of the target peptide segment and improve the detection sensitivity.

[0023] Mass spectrometry analysis was performed in positive ion detection mode. The parameters of the primary mass spectrometry scan were set as resolution 120,000, mass range m / z 200-1800, and AGC target value 2×10 5 To ensure high sensitivity and high resolution of peptide detection. The secondary mass spectrometry scanning used HCD (high energy collision dissociation) mode for fragmentation analysis. The specific parameters included a resolution of 30,000, a mass range of m / z 100-1600, and an AGC target value of 1×10 4 , collision energy 35eV, and selective fragmentation mode ensure accurate cleavage of peptides and improve the accuracy of peptide sequence analysis. In addition, in order to further optimize the detection of peptides, the experiment selected a valence range of 1-5 and a separation window of 1.6m / z to ensure efficient separation and accurate identification of target active peptides in complex samples.

[0024] Through LC-MS / MS data analysis, the sequences of multiple sea cucumber active peptides were obtained and screened based on the following criteria: 1. Peak area ≥ 2×10 7 ; 2. Number of amino acids ≤ 6; 3. Molecular weight ≤ 1000Da. Finally, 18 short peptide sequences that met the standards were screened out, see Table 1.

[0025] Table 1 Screening results of sea cucumber active peptides Amino acid sequence Molecular weight (Da) <![CDATA[Peak area (×10 7 ).]]> Number of amino acids GYMLPK 694.4 3.2 6 LRFTAP 717.6 3.1 6 NMLKVP 689.3 2.9 6 VYAPK 676.2 3.5 6 QFTGRP 725.4 3.8 6 TKLMPG 643.7 2.7 6 RGYPK 665.5 3.3 6 LRYF 702.3 3.6 6 VMPKL 658.2 2.8 6 QRLFAG 735.6 3.4 6 TMKLVG 678.4 2.5 6 VRYMGP 702.2 3.7 6 FKL 689.5 2.9 6 APVGKM 644.3 3.0 6 LMPVG 677.5 2.6 6 KMPVL 655.7 3.2 6 FYLRGP 693.8 3.1 6 GJK 691.2 2.8 6

[0026] The present invention further conducted an in-depth analysis of its antioxidant capacity and immunomodulatory capacity, and used Discovery Studio molecular docking software to simulate its interaction with key proteins such as superoxide dismutase (SOD) to evaluate its biological activity. In terms of antioxidant capacity evaluation, the DPPH free radical scavenging experiment was first used to determine the free radical scavenging capacity of each active peptide, and vitamin C (VC, 50μg / mL) was used as a positive control. The results showed that GYMLPK, LRFTAP and QFTGRP had the highest DPPH scavenging rates, reaching 85.3%, 84.1% and 82.9%, respectively, which were significantly higher than other peptides (P<0.05). In addition, in the ABTS+ scavenging experiment, the scavenging rates of GYMLPK and LRFTAP reached 78.5% and 76.2%, respectively, at a concentration of 50μg / mL, which were also significantly better than other active peptides (P<0.01), indicating that these peptides have strong antioxidant capacity. In terms of immune regulation ability assessment, the present invention uses macrophage phagocytosis experiment to observe the effect of active peptides on the phagocytic activity of RAW264.7 cells, and ELISA experiment to determine the level changes of inflammatory factors IL-6, TNF-α and IL-10. The results showed that at a concentration of 100 μg / mL, the phagocytic index of RAW264.7 cells in the GYMLPK and LRFTAP groups was increased by 41.5% and 38.9% compared with the control group (P<0.01), and the IL-10 level increased by 56.4% and 52.7%, respectively (P<0.001), indicating that these two active peptides have significant immune enhancement effects. In addition, the IL-6 level in the cell culture fluid of the FYLRGP and GYPMLK groups decreased significantly (reduced by 39.2% and 35.8%), indicating that it has a good regulatory effect on inflammatory factors. GYMLPK, LRFTAP and QFTGRP had the highest DPPH and ABTS+ scavenging abilities (the clearance rates were 85.3%, 84.1% and 82.9%, respectively). Other peptides showed moderate antioxidant activity, but lower than GYMLPK. GYMLPK and LRFTAP promoted macrophage phagocytosis and significantly increased IL-10 levels (increased by 56.4%). FYLRGP and GYPMLK significantly regulated IL-6 levels. Based on the antioxidant and immunomodulatory evaluation results, GYMLPK was finally identified as the target active peptide for further research and application development.

[0027] 3. Analysis of the antioxidant capacity of sea cucumber active peptides In order to study the antioxidant capacity of sea cucumber active peptides, DPPH free radical scavenging experiment was used for detection. During the experiment, sea cucumber active peptides of different concentrations (10-100μg / mL) were dissolved in DPPH reagent and reacted for 30 minutes under light-proof conditions to ensure that the reaction was fully carried out. Subsequently, the absorbance change of the solution was measured at a wavelength of 517nm, and the free radical scavenging rate was calculated. Figure 2 The results showed that sea cucumber active peptides can effectively scavenge DPPH free radicals, and their scavenging rate significantly increases with increasing concentration, showing a concentration-dependent trend. At high concentrations, their antioxidant capacity is close to or reaches the level of vitamin C, which indicates that sea cucumber active peptides have good antioxidant activity and can be used as natural antioxidants in the field of food and health products.

[0028] 4. Verify the antioxidant effect of sea cucumber active peptides in vivo The ICR male mouse model was used for the study. 8-week-old healthy mice were randomly divided into a control group (gavage with normal saline) and an experimental group (gavage with sea cucumber active peptides at a dose of 100 mg / kg). The gavage was continued for 4 weeks. After the experiment, the mouse serum was collected to detect superoxide dismutase (SOD) activity and malondialdehyde (MDA) content to evaluate the antioxidant capacity. Figure 3 The experimental results showed that compared with the control group, the SOD activity of the experimental group of mice was significantly increased, indicating that sea cucumber active peptides can enhance the body's ability to scavenge free radicals. At the same time, the MDA content was significantly reduced, reflecting a decrease in lipid peroxidation levels, which helps to reduce oxidative damage and thus exert an antioxidant effect.

[0029] 5. Analysis of the effects of sea cucumber active peptides on intestinal health In order to study the effect of sea cucumber active peptides on intestinal flora, this study used 16SrRNA high-throughput sequencing technology for analysis. In the experiment, 8-week-old ICR mice were randomly divided into a control group (oral administration of normal saline) and an experimental group (oral administration of 100 mg / kg sea cucumber active peptides), and the treatment was given for 4 consecutive weeks to simulate the regulatory effect of long-term intake of sea cucumber active peptides on intestinal flora. After the experiment, the mouse feces were collected and total DNA was extracted. The fecal flora was analyzed by high-throughput sequencing technology to determine the α diversity (richness of microbial populations) and β diversity (difference in flora composition). Figure 4 The experimental results showed that sea cucumber active peptides significantly increased the abundance of beneficial bacteria in the intestine (such as bifidobacteria and lactic acid bacteria), and reduced the proportion of conditional pathogens (such as Escherichia coli and Clostridium perfringens), while promoting the production of intestinal short-chain fatty acids (SCFAs, such as acetic acid and butyric acid), further improving the intestinal environment and enhancing intestinal barrier function.

[0030] 6. Short-chain fatty acid analysis Short-chain fatty acids (SCFAs) are important products of intestinal flora metabolism and play an important role in maintaining intestinal barrier function, regulating intestinal microecological balance and enhancing immune function. In order to evaluate the effect of sea cucumber active peptides on SCFAs, this study used gas chromatography-mass spectrometry (GC-MS) to determine the content of acetic acid, propionic acid and butyric acid in mouse feces. During the experiment, the mouse fecal samples were homogenized and subjected to GC-MS analysis after acidification, solvent extraction and ultrafiltration steps to quantitatively determine the abundance of different short-chain fatty acids. The experimental results showed that compared with the control group, the content of acetic acid and butyric acid in the feces of mice in the experimental group (100 mg / kg gavage of sea cucumber active peptides) increased significantly (p<0.01), while the content of propionic acid did not change significantly (p>0.05). As the main short-chain fatty acids, acetic acid and butyric acid can not only provide energy and promote the proliferation of intestinal epithelial cells, but also improve the integrity of the intestinal barrier and reduce inflammatory response, thereby enhancing the body's immunity. This result shows that sea cucumber active peptides can optimize the intestinal environment and enhance the host's immune defense capabilities by promoting the metabolism of beneficial bacteria and increasing the synthesis levels of acetic acid and butyric acid. It has good intestinal health-promoting effects and application value.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A sea cucumber active peptide, characterized in that: The amino acid sequence of the sea cucumber active peptide is GYMLPK.

2. A sea cucumber active peptide according to claim 1, characterized in that: The preparation method of the sea cucumber active peptide comprises the following steps: Step S1, taking fresh sea cucumber as raw material, pretreatment; Step S2, enzymolyzing the sea cucumber protein with protease, controlling the enzymolysis temperature at 40-55° C. and the pH at 7.0-8.5; Step S3, purifying by ultrafiltration technology to obtain peptides within the target molecular weight range; Step S4, preparing sea cucumber active peptide powder by freeze-drying technology.

3. A sea cucumber active peptide according to claim 2, characterized in that: The protease used is selected from trypsin, neutral protease, flavor protease or a combination thereof.

4. A sea cucumber active peptide according to claim 1, characterized in that , the application of the sea cucumber active peptide in food, health products and sports nutrition products.

5. A sea cucumber active peptide according to claim 1, characterized in that , the application of the sea cucumber active peptide in regulating intestinal flora, improving oxidative stress status and enhancing immune function.

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