Directional preparation method of intestinal immunity-enhanced sea cucumber peptide

Abstract

The invention relates to the technical field of biological deep processing and extraction, and discloses a directional preparation method of intestinal immunity-enhanced sea cucumber peptide, which comprises the following steps: (1) sea cucumber pretreatment; (2) carrying out deep penetration wall breaking on the supercritical CO2 / deep eutectic solvent; (3) performing enzymolysis treatment; (4) peptomics analysis; (5) performing molecular docking and virtual screening on the sea cucumber peptide and the TLR2 receptor; (6) membrane separation targeted enrichment; and (7) performing rotary evaporation, concentration and drying to obtain the sea cucumber peptide. Supercritical CO2 with strong permeation and diffusion characteristics is used as a carrier and cooperates with the deep-eutectic solvent to penetrate through a compact cuticle structure of sea cucumbers. The SC-CO2 acts on a stratum corneum lipid phase, a remarkable micropore expansion effect is initiated, and the pore density is greatly improved; functional ions in the deep eutectic solvent accurately destroy a salt bridge cross-linked network of cuticle key structure protein, and solvent components induce deep swelling of collagen through hydrogen-bond interaction, so that effective disintegration of the cuticle structure is realized.

Description

Technical Field

[0001] This invention relates to the field of deep processing and extraction technology of biotechnology, specifically to a method for the targeted preparation of intestinal immune-enhancing sea cucumber peptides. Background Technology

[0002] Sea cucumber peptides, as representative of marine-derived functional peptides, are rich in a variety of unique bioactive components, including collagen tripeptides with characteristic repeating sequences (Gly-Pro-Hyp), chondroitin sulfate with anti-inflammatory and immunomodulatory effects, and sea cucumber saponins with unique structures. These components endow sea cucumber peptides with significant physiological functions, particularly in immunomodulation (such as activating macrophages to secrete the anti-inflammatory factor IL-10), inhibiting tumor cell growth and metastasis, and repairing and enhancing the intestinal physical barrier (by regulating the expression of tight junction proteins such as Claudin and Occludin). Their low molecular weight (typically <2000 Da) not only ensures high bioavailability, making them easily absorbed by the intestine and exerting systemic effects, but also reduces the potential risk of immunogenicity. Therefore, sea cucumber peptides have broad application prospects in functional foods, special medical diets, nutritional supplements, and the biopharmaceutical field.

[0003] However, existing sea cucumber peptide preparation processes have many technical shortcomings, hindering their industrial application and precise functional development. These shortcomings are mainly reflected in the following aspects: (1) Inefficient pretreatment technology The unique cuticle structure of sea cucumber body walls, composed of cross-linked Alzheimer's proteins and chitin fiber networks, forms a dense barrier against enzymatic degradation. Traditional pretreatment methods have fundamental limitations: high-temperature cooking can lead to significant degradation of heat-sensitive functional components and is energy-intensive; while chemical permeation can improve enzymatic hydrolysis efficiency, it poses food safety risks due to chemical reagent residues; and ultrasonic-assisted treatment, due to uneven cavitation effects, can easily induce localized high temperatures, leading to the formation of off-odor substances and significantly reducing sensory quality.

[0004] (2) Poor controllability of enzymatic hydrolysis process Existing enzymatic hydrolysis systems face multiple challenges: First, due to insufficient removal of the stratum corneum barrier, the enzymatic hydrolysis efficiency is generally low, resulting in insufficient peptide yield; second, random enzymatic cleavage leads to a dispersed molecular weight distribution of peptides, with a high proportion of strongly hydrophobic peptides, causing persistent bitterness; finally, the shear force generated by traditional mechanical stirring easily disrupts the spatial conformation of immunologically active peptides, resulting in a significant reduction in their biological activity.

[0005] (3) Lagging functional peptide screening technology Current evaluation of sea cucumber peptide activity mainly relies on in vitro enzyme inhibition models, which is disconnected from in vivo immune regulation mechanisms. Specifically, this manifests in several ways: target selection is relatively limited, primarily focusing on antihypertensive / glycemic peptides, with insufficient attention paid to targets related to intestinal immunity; traditional separation and grading methods are based solely on molecular weight, making it difficult to accurately identify active epitopes that specifically bind to specific receptor domains; and the lack of quality control standards based on molecular interactions leads to significant batch-to-batch variations in immunostimulatory activity.

[0006] (4) Defects in separation and enrichment technology Existing separation processes struggle to balance efficiency and activity retention: chromatography has a long processing cycle and low target peptide recovery rate; traditional nanofiltration membranes suffer from severe membrane fouling and significant flux decay; and the drying process (especially the high-temperature conditions of spray drying) can easily lead to the inactivation of heat-sensitive peptides, resulting in loss of immune activity.

[0007] Therefore, there is an urgent need to develop an innovative process that integrates green pretreatment, precise receptor screening, and activity preservation to fundamentally overcome the technological bottlenecks in the industrialization of sea cucumber peptides. This invention fills a technological gap in this field through a systematic innovation of supercritical CO2-DES synergistic permeation, pulsed electric field time-controlled enzymatic hydrolysis, TLR2 receptor computer screening, and charged membrane enrichment. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for the targeted preparation of intestinal immune-enhancing sea cucumber peptides.

[0009] To achieve the above objectives, the technical solution of the present invention is: a method for the targeted preparation of intestinal immune-enhancing sea cucumber peptides, comprising the following steps: (1) Sea cucumber pretreatment: Sea cucumbers were quick-frozen with liquid nitrogen, pulverized, freeze-dried, and then degreased by subcritical propane extraction to obtain defatted sea cucumber powder; (2) Deep permeation and cell wall breaking of supercritical CO2 / eutectic solvent: Defatted sea cucumber powder is loaded into a high-pressure permeation vessel, and then supercritical CO2 and eutectic solvent are injected into the high-pressure permeation vessel simultaneously for synergistic cell wall breaking; (3) Enzymatic hydrolysis: The sea cucumber powder after cell wall disruption is added to phosphate buffer, mixed and then trypsin and subtilisin are added for enzymatic hydrolysis. During the enzymatic hydrolysis, a bipolar rectangular pulse electric field is used to assist the enzymatic hydrolysis. After enzymatic hydrolysis, the supernatant is obtained by centrifugation at 4~10℃. (4) Peptidomics analysis: The obtained supernatant was desalted and concentrated by ultrafiltration centrifuge tubes and then subjected to disulfide bond reduction, mercaptoalkylation protection and trypsin digestion. Then, high-throughput depth analysis was performed by LC-MS / MS to identify peptides and establish a molecular fingerprint of sea cucumber peptides. Characteristic peptides with molecular weight of 900~1300 Da, isoelectric point pI 3.5~5.0 and average hydrophobicity GRAVY value -1.5~0.5 were further screened. (5) Virtual screening of sea cucumber peptides and TLR2 receptor molecules: Based on the characteristic peptides obtained by peptidomics screening, the human TLR2-TLR1 heterodimer crystal structure was used as the receptor model. After optimization by 20ns molecular dynamics simulation, the key peptides in the characteristic peptides were precisely docked using AutoDock Vina, and 28 high-affinity candidate peptides were screened out. (6) Membrane separation and targeted enrichment: The supernatant obtained in step (3) is sequentially filtered through a 10000 Da molecular weight cutoff ultrafiltration membrane, a 3000 Da molecular weight cutoff nanofiltration membrane, a 1000 Da molecular weight cutoff nanofiltration membrane and a sulfonated polyether ketone charged reverse osmosis membrane to enrich the target peptide. (7) The enriched target peptides are concentrated by rotary evaporation, and then a freeze-drying protectant is added to the concentrate and vacuum belt drying is performed to obtain sea cucumber peptides.

[0010] Further; the subcritical propane extraction parameters in step (1) are: 30 minutes of cyclic extraction at 40±1℃ and 4.0±0.2MPa, with a propane solvent flow rate of 8 L / min·kg.

[0011] Further; the method for preparing the eutectic solvent in step (2) is as follows: choline chloride (ChCl) and glycerol (Gly) are mixed at a molar ratio of 1:2, and then 0.5% (w / w) citric acid is added. The mixture is stirred at 800 rpm at 60±1℃ until homogeneous and transparent; the temperature inside the high-pressure permeation reactor is 35.0±0.3℃, the pressure is 15.0±0.1 MPa, the CO2 flow rate is 3.5 L / min, and the eutectic solvent injection rate is 0.1 mL / g·min. -1 The synergistic cell wall disruption time is 30±1 min.

[0012] Further; the amount of trypsin added in step (3) is 2500±50U / g substrate, the amount of Bacillus subtilis protease added is 1800±30U / g substrate, and the parameters of the bipolar rectangular pulse electric field are: field strength: 12.5kV / cm±0.3kV / cm, pulse width: 18μs±0.5μs, frequency: 120Hz±5Hz; the enzymatic hydrolysis temperature is 40~60℃, and the enzymatic hydrolysis time is 100~200min; after centrifugation, the peptide solution is filtered through 1.2μm, 0.45μm and 0.22μm membranes in sequence to obtain the supernatant.

[0013] Further; the disulfide bond reduction, thiol alkylation protection, and trypsin digestion method in step (4) are as follows: add dithiothreitol to the supernatant after desalting and concentration at 56°C, with a final concentration of 10mM, and reduce for 30 minutes; then add iodoacetamide to a final concentration of 55mM in a dark room and alkylate for 30 minutes; finally add sequencing-grade trypsin at a trypsin / substrate mass ratio of 1:50 and digest at 37°C with shaking for 16 hours.

[0014] Further; in step (5), the center coordinates of the docking box in the precise docking are x=31.2, y=42.8, z=25.4, the box size is set to 26Å×24Å×20Å, the number of grid points is 60×58×50, the docking parameter exhaustion is set to 45, 30 binding conformations are generated, and the maximum energy difference threshold is 4.5kcal / mol.

[0015] Further; the 10000Da molecular weight cutoff ultrafiltration membrane mentioned in step (6) is a polyethersulfone hollow fiber ultrafiltration membrane with the following operating parameters: feed temperature 20-25℃, transmembrane pressure difference set to 0.15-0.25MPa, tangential flow velocity 2.5-5.0m / s, and volume concentration ratio of 3.0-5.0. The 3000Da molecular weight cutoff nanofiltration membrane is a zirconium oxide-titanium dioxide composite nanofiltration membrane. The operating parameters are: feed temperature 12-18℃, transmembrane pressure difference set at 0.40-0.50MPa, and tangential flow velocity 3.5-4.5 m / s. The 1000 Da molecular weight cutoff nanofiltration membrane is a polypiperazine amide nanofiltration membrane, operating at 0.65 MPa and 4–10 °C, with a membrane flux of 18 L / m³. 2 •h±5%.

[0016] Further; the rotary evaporation concentration temperature in step (7) is 10~45℃ and the pressure is 0.08~0.10MPa.

[0017] Further; the freeze-drying protectant in step (7) is trehalose and hydroxypropyl-β-cyclodextrin, wherein the trehalose is 0.2~0.8% of the concentrate mass and the hydroxypropyl-β-cyclodextrin is 0.1~0.5% of the concentrate mass.

[0018] Further; the vacuum belt drying in step (7) is a three-stage drying process: the first stage is pre-curing at 50~55℃ and 20kPa for 30~50min; the second stage is drying at 30~40℃ and 5 kPa for 180min; and the third stage is final equilibration at 20~25℃ and 1kPa for 60min.

[0019] The beneficial effects of this invention are: (1) This invention innovatively utilizes the strong permeation and diffusion characteristics of supercritical CO2 (SC-CO2) as a carrier to synergistically transport a specific ratio of eutectic solvent (DES) to penetrate the dense stratum corneum structure of sea cucumber. SC-CO2 acts on the lipid phase of the stratum corneum, inducing a significant micropore expansion effect (pore size can increase several times), greatly increasing pore density; at the same time, functional ions in DES (such as chloride ions) precisely disrupt the salt bridge cross-linking network of key structural proteins (Tg proteins) in the stratum corneum, while its solvent components (such as glycerol) induce deep swelling of collagen through hydrogen bonding (swelling rate as high as 85% or more). This dual synergistic mechanism achieves the effective disintegration of the stratum corneum structure.

[0020] (2) This invention constructs a computational biology screening platform based on a high-precision human TLR2 receptor model, and establishes strict multi-dimensional evaluation criteria such as binding energy strength, number of key hydrogen bonds, salt bridge formation, and spatial complementarity, realizing high-throughput virtual screening of sea cucumber enzymatic hydrolysis peptide libraries. A series of high-affinity peptides, such as DSSEEDR (binding energy -12.3 kcal / mol, approximately 26% better than the known ligand MDP), were successfully identified, which achieve ultra-stable binding with the receptor through multiple molecular forces. Based on this precise target, an innovatively designed four-level membrane coupling purification system (especially utilizing the specific electrostatic adsorption of sulfonated modified charged membranes) achieved efficient and highly selective enrichment of target immunomodulatory peptides, with a target peptide purity of 76.8% and a recovery rate as high as 89.2%. The enriched peptides obtained through this process exhibited superior immunostimulatory activity in cellular validation, demonstrating a 320% increase in the ability of THP-1 macrophages to secrete the key anti-inflammatory factor IL-10 (1850 pg / mL) compared to unenriched peptides, a 3.8-fold upregulation of RIP2 phosphorylation levels, and an effective dose (EC50) of [missing value]. 50 The concentration was significantly reduced to approximately 0.28 mg / mL (a reduction of approximately 77%), providing a strong guarantee of activity at the molecular level for functional foods.

[0021] (3) This invention utilizes pulsed electric field spatiotemporal control enzymatic hydrolysis technology to precisely regulate the enzymatic hydrolysis process at the molecular level. The optimized electric field parameters effectively reduced the Km value of the protease by approximately 40%, improving the hydrolysis efficiency. Combined with an intelligent two-stage temperature-controlled enzymatic hydrolysis strategy for selective shearing of hydrophobic bitter peptides, supplemented by real-time online monitoring with an electronic tongue, the proportion of strongly hydrophobic peptides (GRAVY>0) was successfully reduced to approximately 18%, resulting in a final product bitterness value of 1.8 (below the sensory threshold of 2.0), completely eliminating the additional chemical debittering step required by traditional processes. In the drying stage, an innovative vacuum belt drying process combined with a trehalose-hydroxypropyl β-cyclodextrin composite protection system was adopted to form a loose, brittle sheet structure with high porosity (approximately 82%) under mild low-temperature and low-pressure conditions. This process not only ensures excellent rehydration properties of the product (complete dissolution within 30 seconds), but more importantly, circular dichroism spectroscopy analysis confirmed that it has an extremely high retention rate of the higher spatial structures of active peptides (such as α-helices), with a loss of only 4.7%, far superior to traditional spray drying processes. At the same time, the energy consumption per unit of water evaporation is significantly reduced, requiring only about 36% of the energy consumption of traditional processes. This effectively solves the common problems of inactivation and high energy consumption that are prevalent in the industrial drying process of active peptides, ensuring the product's excellent bioactivity and long-term storage stability. Attached Figure Description

[0022] Figure 1 This is a diagram of the TLR2 receptor-sea cucumber peptide molecular docking model; Figure 2 This is a comparison chart of structure retention rate and energy consumption between vacuum belt drying and spray drying. Detailed Implementation

[0023] Example:

[0024] A method for the targeted preparation of intestinal immune-enhancing sea cucumber peptides, characterized by the following steps: (1) Sea cucumber pretreatment: Selected sea cucumbers (Apostichopus japonicus) from the Liaodong Peninsula waters, with a body wall thickness ≥8mm and a dry weight ≥200g / head. After removing the internal organs, the body wall was rinsed with flowing seawater for 30min, soaked in 3wt% NaCl-0.1wt% citric acid solution for 15min (to remove mucus), and rinsed with ultrapure water. The cleaned sea cucumber body wall was placed on a stainless steel tray and sent into a liquid nitrogen quick-freezing tunnel. Parameter settings: Pre-cooling zone: -80℃ strong wind circulation for 10min; Deep freezing zone: liquid nitrogen spray (-196℃) for 15min, core temperature ≤-50℃; Desorption zone: -40℃ equilibration for 20min. The quick-freezing rate reached 25℃ / min, and the ice crystal size was ≤20μm (Cryo-SEM verification), effectively inhibiting protein denaturation and cell structure damage.

[0025] After being quick-frozen with liquid nitrogen, the material is pulverized using a liquid nitrogen-protected ultra-micro vibration mill. Key parameters: liquid nitrogen injection volume 1.5L / kg raw material, maintaining a constant temperature environment of -45℃; vibration frequency 30Hz, amplitude 3mm, shear force dominant; pulverization time 15min, cycle × 3 times, intermittent cooling to prevent overheating.

[0026] After pulverization, the powder was sequentially passed through a vibrating sieve: primary sieve: 200 mesh (75μm) → retaining coarse particles (rework required if >10%); secondary sieve: 400 mesh (38μm) → collecting the main powder; tertiary sieve: 800 mesh (18μm) → recovering ultrafine components. The final product was a gradient-distributed powder with D10=42±3μm, D50=75±5μm, and D90=110±8μm (measured by Malvern laser particle size analyzer), and a specific surface area of ​​3.2±0.3m² / g (BET nitrogen adsorption method).

[0027] The sea cucumber powder was then evenly spread on a freeze-drying tray (thickness ≤ 1 cm) and subjected to the following processes in a vacuum freeze dryer: ① pre-freezing at -45℃ for 4 hours; ② main drying stage at -25℃ / 10Pa for 16 hours; ③ desorption drying stage at 25℃ / 5Pa for 8 hours. The final moisture content was ≤ 3.0% (Karl Fischer method). Subsequently, a subcritical propane extraction system was used: circulated extraction was performed at 40℃ and 4.0 MPa for 30 minutes, with a propane solvent flow rate of 8 L / min·kg. The defatting rate was > 98.5%, and the residual lipids were < 0.5% (verified by Soxhlet extraction). Propane extraction can eliminate the interference of lipid oxidation on subsequent processes and avoid collagen denaturation caused by high temperatures.

[0028] (2) Deep permeation and cell wall disruption by supercritical CO2 / eutectic solvent: Choline chloride (ChCl) and glycerol (Gly) were mixed at a molar ratio of 1:2, and then 0.5% (w / w) citric acid was added as a permeation enhancer. The mixture was stirred magnetically at 800 rpm for 2 hours in a constant temperature reactor at 60℃ until it was homogeneous and transparent, and the eutectic solvent (DES) was obtained. The viscosity was measured to be 115 mPa·s (25℃) by rheometer, the conductivity was 12.5 mS / cm, and the pH was 4.8.

[0029] The defatted sea cucumber powder obtained in step (1) was processed at a concentration of 0.45 g / cm³. 3 The high-pressure permeation reactor was filled with supercritical CO2 and a eutectic solvent simultaneously for synergistic cell disruption. The reactor parameters were: temperature 35.0℃, pressure 15.0 MPa, CO2 flow rate 3.5 L / min, and DES injection rate 0.1 mL / g·min. -1The DES injection and supercritical CO2 synergistic cell disruption process lasted for 30 minutes. CO2 molecules carried DES nanodroplets through the stratum corneum, disrupting the cell wall through a dual mechanism: 1) Micropore expansion effect: CO2 dissolves in the lipid phase of the stratum corneum, expanding the micropore diameter from 0.1-0.3 μm to 1.2-1.8 μm; 2) Protein swelling and depolymerization: Chloride ions in DES disrupt the Arg-Glu salt bridges of Tg proteins, and glycerol hydroxyl groups form hydrogen bond networks with collagen peptide chains, resulting in a swelling degree of 85.7% in the cross-linked structure. Real-time monitoring showed pressure fluctuations ≤ ±0.15% and temperature gradients ≤ ±0.5℃. The penetration endpoint was determined by an online weighing system (DES absorption ≥ 0.22 g / g matrix), with a total time of 30 minutes and a unit energy consumption of 0.8 kWh / kg. After the cell wall disruption process, the pressure in the high-pressure permeation reactor is slowly reduced to atmospheric pressure. The resulting material is a loose, powdery solid uniformly impregnated with DES, with an angle of repose of 38°±2°, still exhibiting good solid flow characteristics, and can be directly used in subsequent enzymatic hydrolysis steps. According to the "NY / T 4471-2025 Technical Specification for Steam Explosion of Corn Stalk Feed," the typical energy consumption range of steam explosion technology for processing similar dense biomass (corn stalks) corresponds to a theoretical energy consumption of approximately 2.4 kWh / kg under the conditions described in this invention. Based on this, the supercritical CO2 / eutectic solvent deep permeation cell wall disruption of this invention saves approximately 67.2% of energy.

[0030] (3) Enzymatic hydrolysis: The solid material obtained after supercritical CO2 / DES cell disruption pretreatment was mixed with 0.1M phosphate buffer (pH 7.4) at a material-to-liquid ratio of 1:12 (w / v), and the solid concentration was adjusted to 15%. Trypsin (Sigma T6567, activity 2500 U / g substrate) and subtilisin (Sigma P5380, activity 1800 U / g substrate) were added. Trypsin specifically cleaves the carboxyl-terminal peptide bonds of arginine (Arg) and lysine (Lys); subtilisin has broad-spectrum endopeptide hydrophobicity and aromatic amino acid (such as leucine and phenylalanine) peptide bonds.

[0031] A bipolar rectangular pulse generator was used for assisted enzymatic hydrolysis with an electric field strength of 12.5 kV / cm, a pulse width of 18 μs, and a frequency of 120 Hz. Analysis using the Lineweaver-Burk double reciprocal plot method showed that under these pulsed electric field parameters, the apparent Michaelis constants (Km) of trypsin and subtilisin decreased to 0.48 mM and 0.52 mM, respectively. Compared to the conventional enzymatic hydrolysis conditions without electric field assistance (Km values ​​of 0.80 mM and 0.87 mM, respectively), this represents an average reduction of approximately 40%, indicating a significant improvement in enzyme-substrate affinity. A two-stage temperature-controlled enzymatic hydrolysis method was employed: The first stage, the macropeptide release stage (0-90 min): the temperature was kept constant at 45.0℃, and the viscosity of the hydrolysate was dynamically monitored online using a rotational viscometer: the initial value was 1250 cP, and when the viscosity dropped to 450 cP (a decrease of 64%), the process transitioned to the next stage (preferentially hydrolyzing the collagen cross-linked region to release peptides with a molecular weight >5 kDa); The second stage, the bitter peptide shearing stage (90-150 min): the temperature was raised to 55.0℃, and the bitterness value was monitored in real time using an electronic tongue (Insent TS-5000Z): the initial value was 4.5, and the reaction was terminated when it dropped to 1.8 (below the sensory threshold of 2.0) (the aminopeptidase activity was enhanced, selectively cleaving the N-terminal amino acids of hydrophobic peptides).

[0032] Immediately inactivate the enzymatic hydrolysate: first, treat it in a 90℃ hot water bath for 12 min; then, cool it to 25℃ using a plate heat exchanger at a rate of 8℃ / min; finally, add 0.1% (w / w) ε-polylysine (food-grade antibacterial agent) to ensure biological stability.

[0033] (4) Centrifugation: After enzymatic hydrolysis, the mixed system was first rapidly cooled to 25°C using a plate heat exchanger, and then entered a three-stage gradient centrifugation purification process: the primary centrifugation was carried out at 8,000g and 10°C for 15 minutes to effectively settle cell wall fragments with a particle size >50μm and undigested tissue blocks. The supernatant was collected and the solid loss rate was measured to be <2%. The secondary centrifugation treated the supernatant at 15,000g and 4°C for 30 minutes to specifically remove denatured protein aggregates of 1-50μm. The removal rate of impurity proteins with a molecular weight >20kDa was verified by SDS-PAGE electrophoresis to be >95%. The tertiary centrifugation was carried out at 30,000g and 4°C for 45 minutes to completely separate colloidal particles of 0.1-1μm, reducing the turbidity to below 25 NTU, and obtaining a clear amber peptide solution. The entire centrifugation process was carried out under the protection of a cooling jacket to ensure that the temperature fluctuation of the feed solution was ≤±1°C. The interface changes were monitored by an online density meter to achieve dynamic optimization of centrifugation parameters.

[0034] The centrifuged peptide solution underwent deep purification through a three-stage terminal filtration system: first, residual particles were removed by a 1.2 μm glass fiber pre-filtration membrane, with the pressure difference controlled within 0.05 MPa; then, hydrophobic impurities were adsorbed by a 0.45 μm nylon membrane, with the membrane flux maintained at 150 L / m³. 2 • h; Finally, microorganisms were removed through a 0.22 μm polyvinylidene fluoride (PVDF) sterilization membrane, with the operating temperature kept constant at 20℃ and the transmembrane pressure difference ≤0.1MPa. The supernatant obtained was measured by an online UV-Vis spectrophotometer (600nm wavelength) and the transmittance was ≥97.5%, and HPLC analysis showed no macromolecular polymer residues.

[0035] (5) Peptidomics analysis: 1.0 mL of the supernatant obtained in step (4) was desalted and concentrated in a 3 kDa ultrafiltration centrifuge tube, and then the following treatment was performed: First, dithiothreitol (DTT, final concentration 10 mM) was added at 56℃ for 30 minutes to open the disulfide bonds; then iodoacetamide (IAA, final concentration 55 mM) was added in a dark room for 30 minutes to alkylate and block the free thiol groups; finally, sequencing-grade trypsin (enzyme / substrate mass ratio 1:50) was used to enzymatically digest the peptides at 37℃ for 16 hours with shaking to cut the peptides into fragments suitable for mass spectrometry analysis. After the enzymatic digestion was terminated, the sample was separated by a nanoliter ultra-high performance liquid chromatography system, and nonlinear gradient elution was performed at a constant temperature of 40℃ and a flow rate of 300 nL / min: the initial acetonitrile phase (containing 0.1% formic acid) ratio was 5%, which increased to 35% within 100 minutes, jumped to 80% within 5 minutes and maintained for 5 minutes, and finally equilibrated to the initial conditions in 10 minutes. Mass spectrometry acquisition was performed using the QExactive HF-X platform. In positive ion mode, the spray voltage was 2.4 kV, the capillary temperature was 280 °C, the first-stage full scan resolution was 120,000 (m / z 350-1600), and the first 20 precursor ions were automatically selected for HCD fragmentation based on intensity (normalized collision energy 28%). The second-stage resolution was 17,500, and the dynamic exclusion time was 30 seconds.

[0036] Raw data were processed using MaxQuant software (v2.1.0), with the UniProt sea cucumber protein database (2023 version) as the benchmark. Precursor ion mass tolerance was set to ±10 ppm, and fragment ion tolerance to ±0.02 Da. Cysteine ​​alkylation was the fixed modification, while variable modifications included methionine oxidation and N-terminal acetylation. The false detection rate (FDR) for both peptide and protein identification was controlled below 1%. Ultimately, over 1200 non-redundant peptides were identified, and a detailed sea cucumber peptide molecular fingerprint was constructed. Then, a three-dimensional screening algorithm was used to identify target peptides: precise calculation of molecular weight (900-1300 Da range), isoelectric point (pI 3.5-5.0 range), and average hydrophobicity (GRAVY value -1.5 to 0.5) were performed, resulting in the selection of 112 characteristic peptides, including the high-frequency sequences DSSEEDR (molecular weight 1052.3 Da, pI 4.2, with strong negative charge), EGDSPASSK (molecular weight 987.1 Da, pI 4.7, containing characteristic phosphorylation sites), and ADGQDSLPR (molecular weight 1218.4 Da, pI 4.5, C-terminal arginine enhances receptor binding). This analysis not only constructed a molecular fingerprint of sea cucumber peptides but also provided a structurally well-defined candidate library for subsequent receptor docking, achieving a leap from empirical enzymatic digestion to rational design. Based on the statistical analysis of the GRAVY values ​​of all 1200+ peptides identified by peptidomics, the proportion of strongly hydrophobic peptides (GRAVY > 0) was 17.9%, confirming that the enzymatic hydrolysis process effectively controlled the formation of bitter peptides.

[0037] (6) Virtual screening of sea cucumber peptide docking with TLR2 receptor molecules: The TLR2-TLR1 heterodimer crystal structure (PDB:6NIG) was used as the receptor model. The TLR2 subunit 425-445 deletion loop region (sequence Gly-Ser-Lys-Arg-Val-Pro-Glu) was completed using Modeller 10.4 software. Systematic molecular dynamics optimization was performed using the AMBER18 force field: Initially, 5,000 steps of the steepest descent method were performed to minimize energy (using ff14SB force field parameters to eliminate atomic spatial conflicts). Subsequently, under the NVT ensemble, the temperature was increased from 0K to 310K physiological temperature in 100ps over a time step of 2fs (using a Langevin thermostat, collision frequency 1.0ps). -1The model was then maintained at a constant pressure of 1 bar (Berendsen pressure coupling algorithm) for 1 ns in the NPT ensemble (SHAKE constraint hydrogen bonding enabled); finally, the production stage was performed in a TIP3P explicit water molecule environment for 20 ns trajectory simulation (one conformation frame was saved every 10 ps). The optimized model was validated by PROCHECK: the Ramachandran conformation diagram showed that 96.3% of the residues were located in the allowed region (of which 92.7% of the LRR domains of the TLR2 subunit were in the optimal φ / ψ angle space), the root mean square deviation (RMSD) of Cα atoms at the TLR2-TLR1 dimer interface was 0.17 ± 0.02 nm, and the bond length fluctuation of key hydrogen bond networks (such as TLR2 / Arg334-TLR1 / Glu388) was ≤ 0.08 Å.

[0038] For the target peptide DSSEEDR (Asp-Ser-Ser-Glu-Glu-Asp-Arg), the lowest energy conformation was predicted using the PEP-FOLD4 server (50,000 simulated annealing iterations to form a characteristic β-turn structure). Preprocessing was performed using AutoDockTools 1.5.6: polar hydrogen atoms were added to the receptor and Gasteiger charges were assigned. Precise docking was performed using AutoDockVina, with the center coordinates of the docking box defined as x=31.2, y=42.8, z=25.4 (covering the positively charged pocket Lys331-Arg334 cluster of the TLR2 subunit and the adjacent interface of the TLR1 subunit). The box size was set to 26 Å (x-axis) × 24 Å (y-axis) × 20 Å (z-axis), and the number of grid points was 60 × 58 × 50. The exhaustiveness of the docking parameters was set to 45, generating 30 binding conformations with a maximum energy difference threshold of 4.5 kcal / mol. The docking results were rigorously screened using a four-tier standard: the first tier was based on a binding energy of ≤-10.5 kcal / mol calculated using Vina (this threshold was calibrated using a TLR2 / NF-κB luciferase reporter gene assay, corresponding to HEK293 cell EC50). 50 =0.23±0.03mg / mL); the second level requires the formation of ≥5 hydrogen bonds (using HBPLUS software analysis, focusing on verifying the interaction with TLR2 / Lys331 NZ atoms (bond length ≤3.0Å), TLR2 / Arg334NH1 atoms (bond angle ≥130°), and TLR1 / Tyr365OH groups (for receptor bifunctionality); the third level requires the presence of ≥2 salt bridges (the distance between the carboxyl oxygen of peptide Glu4 / Asp6 and the nitrogen atom of TLR2 / Arg334 / Lys331 is ≤3.5Å as measured by PyMOL); the fourth level uses PDBePISA to calculate the solvent-accessible surface area of ​​the peptide-receptor complex dimer interface to be ≥850Ų (the original structure Pam3CSK4 has a buried area of ​​926Ų). Figure 1 A docking model of TLR2 receptor-sea cucumber peptide molecule (DSSEEDR) was demonstrated.

[0039] In-depth analysis of the mechanism of action of the DSSEEDR peptide: Its Glu4 Oε1 atom forms a 2.78±0.03Å salt bridge with TLR2 / Arg334 NH1 (bond angle 156.8°±2.1°); Asp6 Oδ1 atom establishes a 2.85±0.05Å hydrogen bond with TLR2 / Lys331 NZ, while Asp6 Oδ2 atom forms a 3.02±0.07Å hydrogen bond with the carbonyl oxygen of the Lys331 main chain; Ser2 Oγ atom generates a 2.91±0.04Å hydrogen bond with the carbonyl oxygen of the TLR1 / Ser346 main chain (bond angle 142.5°); the peptide's net charge -4 (pI 4.2) forms a strong electrostatic complementarity with the positive potential region (+18kT / e) of the receptor pocket. 20 ns molecular dynamics simulations showed that the root mean square fluctuation (RMSF) of the Cα atom in the peptide was ≤0.32 Å (with a fluctuation of only 0.10 Å between residues in the TLR2 binding critical region from 330 to 350 Å), and the binding free energy calculated by MM-GBSA was -27.9 ± 1.1 kcal / mol. Finally, 28 high-affinity candidate peptides were screened from 112 peptides, with a binding energy distribution of -11.1 ± 1.3 kcal / mol. The key peptide DSSEEDR accounted for 3.2% of the total peptide content, and its binding energy of -12.8 kcal / mol was significantly better than that of the control ligand Pam3CSK4 (-9.6 kcal / mol), providing a precise target for the quaternary membrane enrichment system.

[0040] (7) Membrane separation and targeted enrichment: The supernatant obtained in step (4) was subjected to targeted enrichment using a four-stage tandem membrane system: The first stage used a polyethersulfone (PES) hollow fiber ultrafiltration membrane (molecular weight cutoff 10kDa), which was operated under the conditions of transmembrane pressure difference 0.25MPa, tangential flow rate 5m / s and temperature 25℃ to concentrate the initial volume to 1 / 4, effectively removing large molecular impurity proteins >10kDa. HPLC verification showed a rejection rate of 99.3%; The second stage used a zirconium oxide-titanium dioxide composite nanofiltration membrane (molecular weight cutoff 3kDa), with operating parameters set as transmembrane pressure difference 0.45MPa, flow rate 4m / s and temperature 15℃. Small and medium molecular peptides and inorganic salts of 500Da-3kDa were removed through the "concentration-dilution-reconcentration" mode (dilution ratio 1:1). The sodium ion removal rate is >98%, and the conductivity decreases from 12.5 mS / cm to 0.8 mS / cm. The third-stage polypiperazine amide nanofiltration membrane (molecular weight cutoff 1 kDa) operates at 0.65 MPa high pressure and 10℃ low temperature, retaining 1-3 kDa peptides while allowing the target peptides (900-1300 Da) to permeate efficiently, with a membrane flux stable at 18 L / m²·h ± 5%. The fourth-stage core is a sulfonated polyetherketone (PEK-C) charged reverse osmosis membrane (Guiyang Times Wharton Technology Co., Ltd.), whose surface zeta potential reaches -38 mV (pH 4.0) after sulfonation modification. Under a transmembrane pressure difference of 0.8 MPa, it specifically captures negatively charged target peptides (pI 3.5-5.0) through electrostatic adsorption, and uses a binary dialysis concentration process (at pH 6.0). PBS was used as the dialysis buffer, and the mixture was cyclically repeated three times at a volume replacement ratio of 1:1. The target peptide was eventually enriched in the concentrate to 8.7 mg / mL with a purity of 76.8% and a recovery rate of 89.2%.

[0041] Compared to traditional gel chromatography, membrane separation targeted enrichment reduces processing time from 8 hours to 2.5 hours, lowers energy consumption by 65%, and leaves no organic solvent residue. The selectivity coefficient (K0) of the sulfonated PEK-C membrane for the target peptide is significantly higher. + / Na + =5.3) is very ideal.

[0042] (8) Rotary evaporation concentration: The enriched liquid obtained in step (7) is transferred to a rotary evaporator for gradient concentration. First, the free water is evaporated at 40℃ / 0.10MPa until the volume is reduced by 50%. Then, it is continuously concentrated at 45℃ / 0.08MPa until the original volume is 1 / 3. The solid content is monitored by an online refractometer and is 35%. Finally, a dark amber viscous liquid is obtained, the proportion of target peptide is increased to 68.3%, and the bitterness value of electronic tongue is 1.5.

[0043] (9) Vacuum belt drying: Trehalose and hydroxypropyl-β-cyclodextrin are added to the concentrate obtained in step (8), wherein the trehalose is 0.5% of the concentrate mass and the hydroxypropyl-β-cyclodextrin is 0.1% of the concentrate mass. After mixing, the mixture is coated on a Teflon conveyor belt with a thickness of 0.5 mm and subjected to three-stage drying: pre-curing at 50℃ / 20 kPa for 40 min to form a porous skeleton; main drying at 35℃ / 5 kPa for 180 min to achieve internal moisture diffusion control (effective diffusion coefficient 2.1×10). -10 m 2 / s); 25℃ / 1kPa final equilibration for 60 min reduced the water activity to 0.15. Porous flakes of sea cucumber peptide (porosity 82%) were obtained, with an energy consumption of 0.65 kWh / kg per unit of water evaporation. Complete dissolution occurred in water at 25℃ for 30 seconds. Circular dichroism spectroscopy confirmed that the α-helical mass content loss was only 4.7%. See [link to relevant documentation]. Figure 2 It reduces emissions by 61% compared to traditional spray drying.

[0044] The conventional spray drying method is as follows: the concentrated liquid obtained in step (8) is dried in a centrifugal spray dryer without adding a freeze-drying protectant. The drying parameters are: inlet air temperature 180℃, outlet air temperature 85℃, atomizing disc speed 18,000 rpm, and feed rate 8 mL / min to obtain sea cucumber peptide powder product for energy consumption and α-helical content comparison analysis.

[0045] Validation of intestinal immune activity: The dried sea cucumber peptide product obtained in this invention was dissolved in sterile PBS and filtered for sterilization using a sterile polyethersulfone (PES) needle filter with a pore size of 0.22 micrometers. A co-culture model was used to simulate the intestinal immune environment by co-culturing human colon adenocarcinoma cell line Caco-2 with macrophages derived from peripheral blood mononuclear cells (both provided by the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Caco-2 cells were seeded onto polycarbonate membranes of Transwell inserts and cultured for 21 days to form a complete monolayer intestinal epithelial barrier. Peripheral blood mononuclear cells were induced to differentiate into macrophages and then seeded into the lower chamber of a Transwell. Different concentrations of sea cucumber peptide samples (0, 50, 100, 200 μg / mL) were added to the upper chamber and co-incubated with Caco-2 cells for 24 hours. The cell culture supernatant from the lower chamber was collected, and the secretion level of the anti-inflammatory factor interleukin-10 (IL-10) was detected by enzyme-linked immunosorbent assay (ELISA). Simultaneously, macrophages were collected, and the phosphorylation level of RIP2 protein was analyzed by Western blotting. The results showed that, compared with the unenriched raw enzymatically hydrolyzed peptides, the enriched peptides prepared in this invention significantly enhanced the immunomodulatory function of macrophages. Simultaneous detection and analysis at a concentration of 200 μg / mL induced macrophages to secrete high levels of the anti-inflammatory factor interleukin-10 (IL-10), reaching 1850 pg / mL, an increase of approximately 320% compared to the unenriched peptide group. Furthermore, Western blotting analysis of the same experimental sample confirmed a 3.8-fold upregulation of the phosphorylation level of the key signaling pathway protein RIP2. These data clearly demonstrate that the sea cucumber peptide product prepared in this invention can effectively regulate intestinal immunity through the TLR2 pathway, exhibiting significant anti-inflammatory and immune-balanced functions. The effective dose (EC50) of the sea cucumber peptide of the present invention inducing IL-10 secretion in a macrophage model was determined by dose-response curve analysis to be approximately 0.28 mg / mL, which is about 77% lower than that of the unenriched peptide (EC50 of approximately 1.22 mg / mL).

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A method for the targeted preparation of intestinal immune-enhancing sea cucumber peptides, characterized in that, The steps are as follows: (1) Sea cucumber pretreatment: Sea cucumbers were quick-frozen with liquid nitrogen, pulverized, freeze-dried, and then degreased by subcritical propane extraction to obtain defatted sea cucumber powder; (2) Deep permeation and cell wall breaking of supercritical CO2 / eutectic solvent: Defatted sea cucumber powder is loaded into a high-pressure permeation vessel, and then supercritical CO2 and eutectic solvent are injected into the high-pressure permeation vessel simultaneously for synergistic cell wall breaking; (3) Enzymatic hydrolysis: The sea cucumber powder after cell wall disruption is added to phosphate buffer, mixed and then trypsin and subtilisin are added for enzymatic hydrolysis. During the enzymatic hydrolysis, a bipolar rectangular pulse electric field is used to assist the enzymatic hydrolysis. After enzymatic hydrolysis, the supernatant is obtained by centrifugation at 4~10℃. (4) Peptidomics analysis: The obtained supernatant was desalted and concentrated by ultrafiltration centrifuge tubes and then subjected to disulfide bond reduction, mercaptoalkylation protection and trypsin digestion. Then, high-throughput depth analysis was performed by LC-MS / MS to identify peptides and establish a molecular fingerprint of sea cucumber peptides. Characteristic peptides with molecular weight of 900~1300 Da, isoelectric point pI 3.5~5.0 and average hydrophobicity GRAVY value -1.5~0.5 were further screened. (5) Virtual screening of sea cucumber peptides and TLR2 receptor molecules: Based on the characteristic peptides obtained by peptidomics screening, the human TLR2-TLR1 heterodimer crystal structure was used as the receptor model. After optimization by 20ns molecular dynamics simulation, the key peptides in the characteristic peptides were precisely docked using AutoDock Vina, and 28 high-affinity candidate peptides were screened out. (6) Membrane separation and targeted enrichment: The supernatant obtained in step (3) is sequentially filtered through a 10000 Da molecular weight cutoff ultrafiltration membrane, a 3000 Da molecular weight cutoff nanofiltration membrane, a 1000 Da molecular weight cutoff nanofiltration membrane and a sulfonated polyether ketone charged reverse osmosis membrane to enrich the target peptide. (7) The enriched target peptides are concentrated by rotary evaporation, and then a freeze-drying protectant is added to the concentrate and vacuum belt drying is performed to obtain sea cucumber peptides.

2. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The subcritical propane extraction parameters in step (1) are: 30 minutes of cyclic extraction at 40±1℃ and 4.0±0.2MPa, with a propane solvent flow rate of 8 L / min·kg.

3. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The method for preparing the eutectic solvent in step (2) is as follows: choline chloride (ChCl) and glycerol (Gly) are mixed at a molar ratio of 1:2, and then 0.5% (w / w) citric acid is added. The mixture is stirred at 800 rpm at 60±1℃ until homogeneous and transparent. The high-pressure permeation reactor is maintained at a temperature of 35.0±0.3℃, a pressure of 15.0±0.1 MPa, a CO2 flow rate of 3.5 L / min, and an eutectic solvent injection rate of 0.1 mL / g·min. -1 The synergistic cell wall disruption time is 30±1 min.

4. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: In step (3), the amount of trypsin added is 2500±50 U / g substrate, and the amount of subtilisin added is 1800±30 U / g substrate. The parameters of the bipolar rectangular pulse electric field are: field strength: 12.5kV / cm±0.3kV / cm, pulse width: 18μs±0.5μs, frequency: 120Hz±5Hz; the enzymatic hydrolysis temperature is 40~60℃, and the enzymatic hydrolysis time is 100~200min; after centrifugation, the peptide solution is filtered through 1.2μm, 0.45μm and 0.22μm membranes to obtain the supernatant.

5. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The disulfide bond reduction, thiol alkylation protection, and trypsin digestion methods in step (4) are as follows: Dithiothreitol is added to the desalted and concentrated supernatant at 56°C to a final concentration of 10 mM, and the reduction is carried out for 30 minutes; then iodoacetamide is added in a dark room to a final concentration of 55 mM, and alkylation is carried out for 30 minutes; finally, sequencing-grade trypsin is added at a trypsin / substrate mass ratio of 1:50, and the mixture is shaken at 37°C for 16 hours.

6. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: In step (5), the center coordinates of the docking box in the precise docking are x=31.2, y=42.8, z=25.

4. The box size is set to 26Å×24Å×20Å, the number of grid points is 60×58×50, the exhaustiveness of the docking parameters is set to 45, 30 binding conformations are generated, and the maximum energy difference threshold is 4.5kcal / mol.

7. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The 10000Da molecular weight cutoff ultrafiltration membrane mentioned in step (6) is a polyethersulfone hollow fiber ultrafiltration membrane. The operating parameters are: feed temperature 20-25℃, transmembrane pressure difference set to 0.15-0.25MPa, tangential flow velocity 2.5-5.0m / s, and volume concentration ratio 3.0-5.

0. The 3000Da molecular weight cutoff nanofiltration membrane is a zirconium oxide-titanium dioxide composite nanofiltration membrane. The operating parameters are: feed temperature 12-18℃, transmembrane pressure difference set at 0.40-0.50MPa, and tangential flow velocity 3.5-4.5 m / s. The 1000 Da molecular weight cutoff nanofiltration membrane is a polypiperazine amide nanofiltration membrane, operating at 0.65 MPa and 4–10 °C, with a membrane flux of 18 L / m³. 2 •h±5%.

8. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The rotary evaporation concentration temperature in step (7) is 10~45℃ and the pressure is 0.08~0.10MPa.

9. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The freeze-drying protectant in step (7) is trehalose and hydroxypropyl-β-cyclodextrin, wherein the trehalose is 0.2-0.8% of the concentrate mass and the hydroxypropyl-β-cyclodextrin is 0.1-0.5% of the concentrate mass.

10. The method for targeted preparation of intestinal immune-enhancing sea cucumber peptides according to claim 1, characterized in that: The vacuum belt drying described in step (7) is a three-stage drying process: the first stage is pre-curing at 50~55℃ and 20 kPa for 30~50 min; the second stage is drying at 30~40℃ and 5 kPa for 180 min; and the third stage is final equilibration at 20~25℃ and 1 kPa for 60 min.

Images