Sea cucumber intestine antioxidant and pancreatic lipase inhibitory activity polypeptide

Abstract

A novel polypeptide with double biological activities is obtained from sea cucumber intestines, and the sequence of the polypeptide is KADANNQWVDVK through high performance liquid chromatography purification and mass spectrometry analysis and identification. The in-vitro activity evaluation of a system shows that the polypeptide not only shows remarkable antioxidant capacity and can effectively remove ABTS free radicals, but also shows relatively strong pancreatic lipase inhibitory activity, and the IC50 value of the polypeptide reaches a remarkable level. The marine active peptide with double functions of oxidation resistance and metabolism regulation shows a good application prospect in the field of functional food and medicine development. The sea cucumber intestines are treated through an enzymolysis technology, low-molecular-weight polypeptides are separated through dialysis, de novo sequencing is carried out, and high-throughput polypeptides are synthesized. The polypeptide KADANNQWVDVK (lysine-alanine-aspartic acid-alanine-asparagine-asparagine-glutamine-tryptophan-valine-asparagine-valine-lysine) with the activity of resisting oxidation and inhibiting pancreatic lipase is screened, and the polypeptide KADANNQWVDVK (lysine-alanine-aspartic acid-alanine-asparagine-asparagine-asparagine-glutamine-tryptophan-valine-asparagine-valine-lysine) with the activity of resisting oxidation and inhibiting pancreatic lipase is verified. According to the method, sea cucumber processing byproducts are fully utilized, the resource utilization rate is increased, a scientific basis is provided for high-value utilization of sea cucumber intestines, and meanwhile, a potential material basis is provided for developing natural antioxidants and peptides with lipid-lowering functions.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and natural product development, specifically to the development of active peptides with antioxidant and lipid-lowering functions using marine biological resources. Background Technology

[0002] Sea cucumber intestines are a byproduct of sea cucumber processing, rich in protein and bioactive substances, but are usually discarded or used at low value, resulting in resource waste. In recent years, bioactive peptides have become a research hotspot, but traditional peptide preparation methods suffer from low efficiency, high cost, and environmental pollution. Therefore, developing efficient and green sea cucumber intestine bioactive peptide preparation technology is of great significance for increasing the added value of sea cucumber byproducts and promoting the development of functional foods. Sea cucumber intestines are rich in high-quality protein, and after enzymatic hydrolysis, they can release low-molecular-weight peptides with biological functions such as anti-oxidation and inhibition of pancreatic lipase activity.

[0003] In existing technologies, the preparation of bioactive peptides from sea cucumber intestinal protein typically employs an enzymatic hydrolysis process that simulates gastrointestinal digestion. Specifically, freeze-dried and pulverized sea cucumber intestinal raw materials are first subjected to enzymatic hydrolysis using pepsin and trypsin under simulated physiological conditions to obtain protein hydrolysates. Subsequently, ultrafiltration is used to retain small peptides with molecular weights below 3 kDa, followed by further fine separation and purification using dextran gel chromatography (e.g., G-25 packing material). For structural identification, de novo sequencing combined with the PEAKS Studio 10.6 bioinformatics analysis platform is used to accurately resolve the bioactive peptide sequences. To meet the demands of high-throughput preparation, this technology employs a solid-phase parallel synthesis system based on a cartridge tip, guided by an Fmoc protection strategy, using a stepwise extension method from the C-terminus to the N-terminus to complete the chemical synthesis of the target peptide. By establishing an in vitro evaluation system including ABTS free radical scavenging ability and pancreatic lipase inhibitory activity, bioactive peptides with both antioxidant and lipid-lowering effects were successfully screened.

[0004] However, existing technologies still suffer from insufficient utilization of sea cucumber processing byproducts and low efficiency in screening bioactive peptides. This invention, by optimizing the extraction process, significantly improves the yield and activity of functional polypeptides in sea cucumber intestines, achieving value-added utilization of processing waste. The obtained polypeptide products not only exhibit excellent free radical scavenging capabilities but also significant pancreatic lipase inhibition, providing high-quality raw materials for developing novel naturally derived antioxidants and lipid-lowering functional factors. This technology not only improves the resource utilization efficiency of sea cucumber processing waste but also opens up new avenues for the development of marine bioactive substances. Summary of the Invention

[0005] To address the shortcomings of traditional peptide synthesis techniques, this study innovatively developed a novel integrated green synthesis strategy based on a paper carrier. This method offers significant advantages: simple operation, high synthesis efficiency, strong environmental friendliness, and good versatility, making it particularly suitable for the rapid construction and screening of large-scale peptide libraries.

[0006] In this invention, some commonly used abbreviations have the following meanings:

[0007] ABTS: 2,2′-Aza-bis(3-ethylbenzothiazoline-6-sulfonic acid)

[0008] Fmoc: fluorenemethyloxycarbonyl;

[0009] DIEA: N,N'-Diisopropylethylamine;

[0010] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate;

[0011] TFA: Trifluoroacetic acid;

[0012] DMF: N,N-dimethylformamide;

[0013] TIBS: Triisopropylsilane

[0014] (A) Enzymatic hydrolysis: The freeze-dried and pulverized sea cucumber intestine raw material was subjected to stepwise enzymatic hydrolysis using pepsin and trypsin under simulated gastrointestinal digestion conditions to obtain protein hydrolysate.

[0015] (B) Fractional purification: First, the enzymatic hydrolysate is initially separated by ultrafiltration to collect components with a molecular weight of less than 3 kDa; then, it is further purified by dextran G-25 gel chromatography column to collect the active components with the maximum ultraviolet absorption at a wavelength of 214 nm.

[0016] (C) Sequence analysis: The purified peptide components were sequenced using de novo sequencing technology, and the mass spectrometry data were processed using PEAKSStudio 10.6 software to predict the complete peptide sequence;

[0017] (D) Peptide synthesis: Selecting potential active sequences, using solid-phase synthesis based on the Fmoc protection strategy, the target peptide molecule is constructed stepwise from the C-terminus to the N-terminus;

[0018] (E) Activity screening: The bioactivity of the synthesized peptides was evaluated through in vitro experiments, with a focus on their ability to scavenge ABTS free radicals and their inhibitory effect on pancreatic lipase, so as to screen out target peptides with significant antioxidant and lipid-lowering activities. Attached Figure Description

[0019] Figure 1HPLC chromatogram of crude peptide KADANNQWVDVK (lysine-alanine-aspartic acid-alanine-asparagine-asparagine-glutamine-tryptophan-valine-aspartic acid-lysine) prepared by the synthesis method implemented according to the present invention.

[0020] Figure 2 Mass spectrum of crude peptide KADANNQWVDVK (lysine-alanine-aspartic acid-alanine-asparagine-asparagine-glutamine-tryptophan-valine-aspartic acid-lysine) prepared by the synthesis method implemented according to the present invention.

[0021] Figure 3 Secondary mass spectrometry fragment sequences of the crude peptide KADANNQWVDVK (lysine-alanine-aspartic acid-alanine-asparagine-asparagine-glutamine-tryptophan-valine-aspartic acid-lysine) prepared by the synthesis method implemented according to the present invention. Detailed Implementation

[0022] This section describes in detail the specific embodiments of the present invention. It should be noted that the embodiments listed herein are merely representative examples of the present invention and are not exhaustive of all possible embodiments. Any technical solutions derived by those skilled in the art based on these embodiments without inventive effort, according to the technical solutions disclosed in this specification, fall within the protection scope of this patent.

[0023] In this invention, the terms "peptide product" and "crude peptide" specifically refer to primary products prepared by solid-phase synthesis that have not undergone any purification treatment. These initially synthesized peptide mixtures can be purified using various separation and purification techniques (such as high-performance liquid chromatography) to ultimately obtain high-purity target peptide products.

[0024] Example 1: Enzymatic hydrolysis of sea cucumber intestinal protein

[0025] First, fresh sea cucumber intestines were freeze-dried and ground into a fine powder, which was then sieved through a 90-mesh sieve to ensure uniform particle size. Subsequently, simulating the human gastrointestinal digestive environment, hydrolysis was performed stepwise using pepsin and trypsin. The reaction conditions were based on literature methods and further optimized. The specific experimental procedure is as follows:

[0026] Sample pretreatment: Disperse an appropriate amount of sea cucumber intestine powder in 0.1M HCl buffer (pH 1.5) and stir to fully suspend the powder;

[0027] Pepsin hydrolysis: Add pepsin and react at a constant temperature of 37°C for 3 hours;

[0028] pH adjustment and trypsin hydrolysis: After adjusting the pH of the system to 6.4, trypsin was added and hydrolysis was continued at 37°C for 3.2 hours.

[0029] Reaction termination and product collection: Heat in a 95℃ water bath for 10 minutes to inactivate the enzyme, cool and centrifuge (10 minutes), collect the supernatant to obtain the sea cucumber intestinal protein hydrolysate.

[0030] Example 2: Isolation and purification of polypeptides

[0031] First, ultrafiltration (with a molecular weight cutoff of 3 kDa) was used for preliminary fractionation of the enzymatic hydrolysis products, retaining fractions with molecular weights less than 3 kDa. Then, fine separation was performed using a Sephadex G-25 gel chromatography column (1.6 cm × 80 cm) with deionized water as the eluent, at a flow rate controlled at 1.0 mL / min. Target peaks were detected by UV light (214 nm), and the corresponding fractions were collected. Finally, the purified peptide solution was freeze-dried and stored at -20°C for later use.

[0032] Example 3: Sequence Identification and Chemical Synthesis of Polypeptides

[0033] First, the low molecular weight peptide was characterized using liquid chromatography-tandem mass spectrometry (LC-MS / MS) combined with de novo sequencing. The obtained mass spectrometry data were processed using PEAKS Studio 10.6 bioinformatics software, successfully deriving the sequence of the target peptide (e.g., KADANNQWVDVK). In the synthesis stage, a solid-phase synthesis technique based on the Fmoc protecting group was employed. Through a C-terminal-initiated stepwise condensation reaction and periodic deprotection processes, the target peptide was efficiently prepared, and the final product achieved the required purity.

[0034] Example 4: In vitro activity detection

[0035] ABTS free radical scavenging experiment

[0036] The ABTS+ stock solution was prepared by mixing equal volumes of ABTS solution (8 mg / mL) and potassium persulfate (1.32 mg / mL). Before use, it was stored at room temperature protected from light for 12 hours. Then, the ABTS+ stock solution was diluted with potassium phosphate-K3PO4 buffer (PBS) (100 mM, pH 7.4) to prepare the ABTS+ working solution, with an absorbance range of 1.000–1.099 at 734 nm.

[0037] Reaction system: ABTS + working solution + peptide samples (different concentrations), incubated in the dark for 30 min;

[0038] The absorbance at 734 nm was measured, and the free radical scavenging rate was calculated.

[0039] Pancreatic lipase inhibition experiment

[0040] The specific steps are as follows:

[0041] 50 μL of enzyme (1 mg / ml) and 20 μL of peptide samples at different concentrations were pre-incubated in 96-well microplates at 37 °C for 10 min. The absorbance at 405 nm was measured after incubation for 15 min by adding 80 μL of 10 mM p-nitrophenyl butyrate to initiate the enzymatic reaction.

[0042] Calculate the inhibition rate of the peptide on pancreatic lipase.

[0043] Experimental results

[0044] Through the above experiments, the polypeptide KADANNQWVDVK, which has good antioxidant and pancreatic lipase inhibitory effects, was screened out.

Claims

1. A sea cucumber intestinal antioxidant and pancreatic lipase inhibitory polypeptide, characterized in that... The sequence of the novel bioactive peptide is KADANNQWVDVK, and the molecular formula of the novel bioactive peptide is C. 60 H 94 N 18 O 20 The structural formula of the new polypeptide is 2. The active peptide of KADANNQWVDVK according to claim 1, characterized in that: KADANNQWVDVK exhibits 60% inhibition of pancreatic lipase at a concentration of 0.013 mg / mL; furthermore, the aforementioned polypeptide possesses antioxidant activity, demonstrating an 80% antioxidant scavenging rate against ABTS free radicals at a concentration of 0.1 mg / mL.

Images