A sea cucumber active peptide and its application

Abstract

The present invention discloses a sea cucumber active peptide and its application, belonging to the field of food science. The amino acid sequence of the sea cucumber active peptide is GYMLPK, and it has antioxidant, immunomodulatory and intestinal health regulating functions. The active peptide is extracted from sea cucumber protein using a biological enzymatic hydrolysis method, and can increase serum superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) content, and promote the growth of beneficial intestinal bacteria. The sea cucumber active peptide can be widely used in functional foods, health supplements and sports nutrition products.

Description

Technical Field

[0001] The present 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, due to their rich nutritional content and bioactive properties, have garnered widespread attention in the food and health product sectors. Research has shown that sea cucumber proteins contain numerous functional peptides, which may have antioxidant, immunomodulatory, and intestinal health-promoting effects in humans. They also have the potential to improve metabolism, combat fatigue, and protect cardiovascular health. Sea cucumber active peptides are rich in amino acids such as arginine, glycine, and glutamic acid, which play important roles in regulating the immune system and combating oxidative stress. Previous studies have shown that sea cucumber peptides can promote macrophage phagocytosis, increase immunoglobulin levels, and enhance natural killer (NK) cell activity. Furthermore, the low molecular weight of sea cucumber active peptides makes them easily absorbed by the body, enhancing their bioavailability. However, current sea cucumber products on the market primarily exist in the form of dried products or concentrated extracts. Developing effective extraction methods, optimizing purification processes, and improving the stability and functionality of sea cucumber active peptides warrants further research. Summary of the Invention

[0003] The present invention provides an active peptide extracted from sea cucumber, which has significant antioxidant, immune regulation and intestinal health regulation functions and can be widely used in the fields of food and health care products.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] A sea cucumber active peptide, the amino acid sequence of which is GYMLPK.

[0006] In order to ensure the quality and function of sea cucumber active peptides, the present invention adopts a biological enzymatic hydrolysis method, optimizes the enzymatic hydrolysis conditions, and ensures the efficient extraction of active peptides. The specific steps are as follows:

[0007] In step S1, fresh sea cucumbers are selected as raw material. Offal, sediment, and impurities are removed to ensure protein purity. Rinse with running water for 5-10 minutes, then soak in 0.9% NaCl solution for 10 minutes to remove residual salt. Add crushed sea cucumber powder to phosphate buffer (pH 7.4) at a ratio of 1:10 (g / mL) and stir thoroughly to fully dissolve the protein. Allow to stand at 4°C for 12 hours to allow the protein to fully swell, enhancing the efficiency of subsequent enzymatic hydrolysis.

[0008] In step S2, adjust the pH to 7.0-8.5 using NaOH (0.1M) or HCl (0.1M) to ensure optimal enzyme activity. Maintain the temperature between 40°C and 55°C. Temperatures below 40°C can lead to insufficient enzyme activity, resulting in inefficient protein hydrolysis. Temperatures above 55°C can cause enzyme inactivation, terminating the reaction. Maintain the enzymatic hydrolysis time for 4-6 hours, sampling every hour to monitor the degree of hydrolysis. Heat to 95°C for 10 minutes to inactivate the protease and prevent excessive hydrolysis.

[0009] In step S3, after enzymatic hydrolysis, the solution contains a large amount of protein fragments, polypeptides, and small peptides, requiring further purification to obtain the target functional peptides. Residues are removed by centrifugation (5000 rpm, 15 minutes) to obtain the supernatant of the enzymatic hydrolysis solution. The solution is then filtered through a 0.45 μm filter membrane to remove undissolved proteins and precipitates. Ultrafiltration is performed using a 10 kDa cut-off membrane. A 3 kDa dialysis bag is used to further remove small molecule impurities and improve the stability and functionality of the peptides.

[0010] In step S4, the peptide solution is dispensed into freezing trays (less than 2 cm thick) to ensure uniform freezing. Pre-freeze at -40°C for 8-12 hours to allow the water to form fine ice crystals and minimize freeze-drying losses. Dry at -40°C under vacuum (0.1 mbar) for 24 hours to sublime the majority of the water. Raise the temperature to 20-25°C and maintain for 8 hours to remove any remaining bound water and improve powder stability. Grind the mixture into a 100-mesh fine powder using liquid nitrogen freeze-drying. Finally, seal and package the mixture and store it in a low-temperature, dry environment.

[0011] The sea cucumber active peptide can increase serum superoxide dismutase (SOD) activity, reduce malondialdehyde (MDA) content, and effectively reduce oxidative damage.

[0012] 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.

[0013] 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.

[0014] The sea cucumber active peptide can be used in functional foods, health supplements and sports nutrition products to enhance antioxidant capacity and immune function.

[0015] 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 bio-enzymatic hydrolysis method, has antioxidant, immune regulation and intestinal health regulation functions, can increase 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 at the same time enhance immune cell activity, increase the secretion levels of IgA, IgG and IL-10, and effectively improve the body's immunity. 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

[0016] Figure 1 The figure is a schematic diagram of the extraction process of sea cucumber active peptides of the present invention.

[0017] Figure 2 This is the test result of the antioxidant capacity of the sea cucumber active peptide of the present invention.

[0018] Figure 3 This is the effect of the sea cucumber active peptide of the present invention on the antioxidant indexes of mice.

[0019] Figure 4 This is the effect of the sea cucumber active peptide of the present invention on intestinal flora. DETAILED DESCRIPTION

[0020] The following is a clear and complete description of the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] 1. A method for preparing sea cucumber protein peptide ( Figure 1 ), the specific steps are as follows:

[0022] Step S1: Rinse fresh sea cucumbers with running water twice to remove surface sand and mucus. Soak in 0.5% NaCl solution for 5 minutes to remove attached microorganisms and salt. Rinse again with deionized water to ensure a clean surface. Use sterile scissors to cut the sea cucumber's abdomen open, remove the internal organs (including the intestines and gonads), and gently rinse the lumen with 0.1M phosphate buffered saline (PBS) to remove residual tissue. To prevent protein degradation and facilitate subsequent processing, freeze-dry the sea cucumbers. Pre-freeze the sea cucumber samples at -80°C for 12 hours to allow the water to form uniform ice crystals. A vacuum freeze dryer with a pressure of 0.01 Pa and a temperature of -50°C is used for 48 hours until the sea cucumbers are completely dry. The freeze-dried sea cucumbers have a porous structure, making them easier to grind. A high-speed grinder (12,000 rpm) is used to grind the freeze-dried sea cucumbers into a fine powder. Liquid nitrogen is used to cool the grinding process to prevent protein degradation due to frictional heating.

[0023] In step S2, a pH 7.4 phosphate buffer solution (PBS) is selected as the dissolution medium. The PBS formula is as follows: NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, and KH2PO4: 1.8 mM. The volume is adjusted to 1 L with deionized water, and the pH is adjusted to 7.4. Sea cucumber powder is weighed according to a ratio of 1:10 (g / mL) and added to the PBS solution. A magnetic stirrer (300 rpm) is used to stir at low speed for 30 minutes to gradually dissolve the protein. The solution is placed in a refrigerator at 4°C for 12 hours to ensure that the protein is fully dissolved and to reduce precipitation formation.

[0024] In step S3, to remove insoluble impurities and large precipitated particles, protein is separated using high-speed centrifugation. The centrifugation conditions are: 10,000 rpm, 4°C, and 10 min. The clear protein solution is collected after centrifugation, avoiding aspiration of the precipitate to reduce impurities. A double-layer absorbent cotton filter is used to remove larger particles. Filtering is performed using a 0.45 μm microporous membrane to remove fine impurities and bacteria. If the protein solution is viscous, 0.1% Triton X-100 can be added to reduce viscosity and improve filtration efficiency. After centrifugation and filtration, a transparent and clear sea cucumber protein solution is obtained. Protein concentration is measured using a UV-visible spectrophotometer (280 nm) to ensure that the protein concentration is within the range of 1-5 mg / mL.

[0025] 2. Peptide sequence screening of sea cucumber active peptides

[0026] To further screen and identify the polypeptide sequences of sea cucumber active peptides, this study employed liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and data analysis using specialized mass spectrometry analysis software to obtain the amino acid sequence information of the target active peptides. LC-MS / MS assay conditions include both liquid chromatography and mass spectrometry conditions. The liquid chromatography conditions used a C18 reverse-phase column (3 μm, 250 mm × 75 μm, Waters) for separation, with water (0.1% formic acid) as phase A and acetonitrile (0.1% formic acid) as phase B. Gradient elution was used to optimize the separation effect, wherein phase A gradually decreased from 95% to 55% within 0-50 min, further decreased to 25% from 50-58 min, and decreased to 5% from 58-60 min and maintained for 2 min. Then, phase A returned to 95% within 60-65 min to balance the system and ensure 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 and improve detection sensitivity.

[0027] Mass spectrometry analysis was performed in positive ion detection mode. The parameters for the primary mass spectrometry scan were set to a resolution of 120,000, a mass range of m / z 200–1800, and an AGC target value of 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 The collision energy was 35eV, and the selective fragmentation mode ensured precise fragmentation of peptides, improving the accuracy of peptide sequence analysis. In addition, to further optimize peptide detection, 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.

[0028] 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 criteria were screened out, see Table 1.

[0029] Table 1 Screening results of sea cucumber active peptides

[0030] 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 VYAPKG 676.2 3.5 6 QFTGRP 725.4 3.8 6 TKLMPG 643.7 2.7 6 RGYPKV 665.5 3.3 6 LRYFGP 702.3 3.6 6 VMPKLY 658.2 2.8 6 QRLFAG 735.6 3.4 6 TMKLVG 678.4 2.5 6 VRYMGP 702.2 3.7 6 FKLTMP 689.5 2.9 6 APVGKM 644.3 3.0 6 LMPKVG 677.5 2.6 6 KMPGVL 655.7 3.2 6 FYLRGP 693.8 3.1 6 GYPMLK 691.2 2.8 6

[0031] The present study further analyzed the antioxidant and immunomodulatory abilities of these peptides and used Discovery Studio molecular docking software to simulate their interactions with key proteins, such as superoxide dismutase (SOD), to assess their biological activities. To assess antioxidant capacity, the free radical scavenging abilities of the active peptides were first determined using a DPPH free radical scavenging assay, using vitamin C (VC, 50 μg / mL) as a positive control. The results showed that GYMLPK, LRFTAP, and QFTGRP exhibited the highest DPPH scavenging rates, reaching 85.3%, 84.1%, and 82.9%, respectively, significantly exceeding those of the other peptides (P < 0.05). Furthermore, in an ABTS+ scavenging assay, GYMLPK and LRFTAP achieved scavenging rates of 78.5% and 76.2%, respectively, at a concentration of 50 μg / mL, also significantly exceeding those of the other active peptides (P < 0.01), demonstrating their strong antioxidant potential. To assess immunomodulatory capacity, the present invention used a macrophage phagocytosis assay to observe the effects of the active peptides on the phagocytic activity of RAW264.7 cells, and an ELISA assay to measure changes in the levels of the 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 increased by 41.5% and 38.9% compared to the control group (P < 0.01), and IL-10 levels increased by 56.4% and 52.7%, respectively (P < 0.001), demonstrating that these two active peptides have significant immunoenhancing effects. Furthermore, IL-6 levels in the cell culture medium of the FYLRGP and GYPMLK groups decreased significantly (by 39.2% and 35.8%), indicating their effective regulatory effects on inflammatory factors. GYMLPK, LRFTAP, and QFTGRP demonstrated the highest DPPH and ABTS+ scavenging abilities (85.3%, 84.1%, and 82.9%, respectively). Other peptides exhibited moderate antioxidant activity, but lower than that of GYMLPK. GYMLPK and LRFTAP promoted macrophage phagocytosis and significantly increased IL-10 levels (by 56.4%). FYLRGP and GYPMLK significantly regulated IL-6 levels. Based on the antioxidant and immunomodulatory evaluation results, GYMLPK was identified as the target active peptide for further research and application development.

[0032] 3. Analysis of the antioxidant capacity of sea cucumber active peptides

[0033] To investigate the antioxidant capacity of sea cucumber active peptides, a DPPH free radical scavenging assay was performed. Different concentrations (10-100 μg / mL) of sea cucumber active peptides were dissolved in DPPH reagent and allowed to react for 30 minutes in the dark to ensure sufficient reaction. Subsequently, the absorbance of the solution was measured at a wavelength of 517 nm, 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.

[0034] 4. Verification of the in vivo antioxidant effect of sea cucumber active peptides

[0035] The study was conducted using an ICR male mouse model. Eight-week-old healthy mice were randomly divided into a control group (administered with normal saline by gavage) and an experimental group (administered with sea cucumber active peptides at a dose of 100 mg / kg by gavage). The mice were gavaged for four consecutive weeks. After the experiment, the serum of the mice was collected to measure superoxide dismutase (SOD) activity and malondialdehyde (MDA) content to evaluate antioxidant capacity. Figure 3 The experimental results showed that compared with the control group, the SOD activity of the experimental group 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.

[0036] 5. Analysis of the effects of sea cucumber active peptides on intestinal health

[0037] To investigate the effects of sea cucumber active peptides on intestinal flora, this study employed 16S rRNA high-throughput sequencing technology. Eight-week-old ICR mice were randomly divided into a control group (gavage with normal saline) and an experimental group (gavage with 100 mg / kg of sea cucumber active peptides). These treatments were administered for four consecutive weeks to simulate the regulatory effects of long-term sea cucumber active peptide intake on intestinal flora. Following the experiment, feces were collected and total DNA extracted. High-throughput sequencing was used to analyze the fecal flora and determine alpha diversity (the richness of the microbial population) and beta diversity (the variability in the composition of the microbial population). 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.

[0038] 6. Short-chain fatty acid analysis

[0039] Short-chain fatty acids (SCFAs) are key metabolic products of the intestinal microbiota and play a crucial role in maintaining intestinal barrier function, regulating intestinal microecological balance, and enhancing immune function. To evaluate the effects of sea cucumber active peptides on SCFAs, this study used gas chromatography-mass spectrometry (GC-MS) to determine the levels of acetic acid, propionic acid, and butyric acid in mouse feces. During the experiment, fecal samples were homogenized, acidified, extracted with solvents, and ultrafiltered before GC-MS analysis to quantitatively determine the abundance of different SCFAs. Results showed that compared with the control group, the fecal levels of acetic acid and butyric acid in the experimental group (gavage with 100 mg / kg sea cucumber active peptides) were significantly increased (p < 0.01), while propionic acid levels did not change significantly (p > 0.05). Acetic acid and butyric acid, as major SCFAs, not only provide energy and promote intestinal epithelial cell proliferation, but also improve intestinal barrier integrity, reduce inflammatory responses, and thus enhance immunity. This result shows that sea cucumber active peptides can promote the metabolism of beneficial bacteria and increase the synthesis levels of acetic acid and butyric acid, thereby optimizing the intestinal environment and improving the host's immune defense capabilities. It has good intestinal health-promoting effects and application value.

[0040] 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 principles of the present invention should be included in the scope of protection 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. An application of sea cucumber active peptide, characterized in that , Use of the sea cucumber active peptide described in claim 1 in the preparation of food, health products and sports nutrition products.

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