CN-121992058-A - Directional preparation method of intestinal immunity-enhanced sea cucumber peptide

CN121992058ACN 121992058 ACN121992058 ACN 121992058ACN-121992058-A

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 of (1) sea cucumber pretreatment; the method comprises the steps of (1) deep penetration wall breaking of supercritical CO 2 /eutectic solvent, (3) enzymolysis treatment, (4) peptide group analysis, (5) virtual screening of butt joint of sea cucumber peptide and TLR2 receptor molecules, (6) membrane separation targeting enrichment, and (7) rotary evaporation concentration and drying to obtain sea cucumber peptide. The invention uses the strong permeation and diffusion characteristic of supercritical CO 2 as a carrier, and cooperates with the eutectic solvent to penetrate the compact stratum corneum structure of sea cucumber. SC-CO 2 acts on the lipid phase of the stratum corneum to induce remarkable micropore expansion effect and greatly improve pore density, functional ions in the eutectic solvent precisely destroy a salt bridge crosslinked network of key structural proteins of the stratum corneum, and solvent components induce deep swelling of collagen through hydrogen bonding, so that the stratum corneum structure is effectively disintegrated.

Inventors

XIE WANCUI
LI HAOEN
LU KUAN
SUN YONGJUN
JU WENMING
GAO SHENG
YANG XIHONG

Assignees

青岛科技大学
好当家集团有限公司
先创数智(青岛)生物科技有限公司

Dates

Publication Date: 20260508
Application Date: 20251211

Claims (10)

1. A directional preparation method of intestinal immunity enhanced sea cucumber peptide is characterized by comprising the following steps: (1) Sea cucumber pretreatment, namely quick-freezing sea cucumber with liquid nitrogen, crushing, freeze-drying, and degreasing through subcritical propane extraction to obtain degreased sea cucumber powder; (2) Deep penetration wall breaking of supercritical CO 2 /eutectic solvent comprises loading defatted sea cucumber powder into high-pressure penetration kettle, and simultaneously injecting supercritical CO 2 and eutectic solvent into high-pressure penetration kettle for synergistic wall breaking; (3) Adding the wall-broken sea cucumber powder into phosphate buffer solution, mixing, adding trypsin and subtilisin for enzymolysis, adopting a bipolar rectangular pulse electric field to assist enzymolysis in the enzymolysis process, and centrifuging at 4-10 ℃ after enzymolysis to obtain supernatant; (4) Performing peptide group analysis, namely desalting and concentrating the obtained supernatant through an ultrafiltration centrifuge tube, performing disulfide bond reduction, sulfhydryl alkylation protection and trypsin digestion respectively, performing high-throughput deep analysis by adopting LC-MS/MS, performing peptide fragment identification, establishing a sea cucumber peptide molecular fingerprint, and further screening characteristic peptide fragments with molecular weight of 900-1300 Da, isoelectric point pI 3.5-5.0 and average hydrophobicity GRAVY value of-1.5-0.5; (5) Performing virtual butt joint screening on sea cucumber peptide and TLR2 receptor molecules, namely performing accurate butt joint on key peptide fragments in the characteristic peptide fragments by using AutoDock Vina to perform accurate butt joint on the key peptide fragments in a receptor model by using a humanized TLR2-TLR1 heterodimer crystal structure and performing 20ns molecular dynamics simulation optimization on the receptor model, and screening out 28 high-affinity candidate peptides; (6) Membrane separation targeted enrichment, namely filtering the supernatant obtained in the step (3) through a 10000Da cutoff molecular weight ultrafiltration membrane, a 3000Da cutoff molecular weight nanofiltration membrane, a 1000Da cutoff molecular weight nanofiltration membrane and a sulfonated polyether ketone charged reverse osmosis membrane in sequence to enrich target peptide; (7) And (3) carrying out rotary evaporation concentration on the enriched target peptide, then adding a freeze-drying protective agent into the concentrated solution, and carrying out vacuum belt drying to obtain the sea cucumber peptide.
2. The directional preparation method of the intestinal immunity-enhanced sea cucumber peptide, which is characterized in that the subcritical propane extraction parameter in the step (1) is 40+/-1 ℃ and 4.0+/-0.2 MPa, the circulation extraction is carried out for 30 minutes, and the flow rate of propane solvent is 8L/min.kg.
3. The directional preparation method of the intestinal immunity-enhanced sea cucumber peptide according to claim 1, wherein the preparation method of the eutectic solvent in the step (2) is characterized in that choline chloride (ChCl) and glycerin (Gly) are mixed according to a molar ratio of 1:2, then 0.5% (w/w) citric acid is added, stirring is carried out at 800rpm until the mixture is homogeneous and transparent at 60+/-1 ℃, the temperature in the high-pressure infiltration kettle is 35.0+/-0.3 ℃, the pressure is 15.0+/-0.1 MPa, the flow rate of CO 2 is 3.5L/min, the injection rate of the eutectic solvent is 0.1 mL/g.min -1 , and the synergistic wall breaking time is 30+/-1 min.
4. The directional preparation method of the intestinal immunity enhancement type sea cucumber peptide is characterized in that the trypsin addition amount in the step (3) is 2500+/-50U/g substrate, the subtilisin addition amount is 1800+/-30U/g substrate, the bipolar rectangular pulse electric field parameters comprise field intensity of 12.5 kV/cm+/-0.3 kV/cm, pulse width of 18 mu s+/-0.5 mu s, frequency of 120 Hz+/-5 Hz, enzymolysis temperature of 40-60 ℃ and enzymolysis time of 100-200 min, and supernatant fluid is obtained after the peptide liquid is filtered through membranes of 1.2 mu m, 0.45 mu m and 0.22 mu m in sequence after centrifugation.
5. The directional preparation method of the intestinal immunity enhancement type sea cucumber peptide, which is characterized by comprising the following steps of adding dithiothreitol into a desalted and concentrated supernatant at a final concentration of 10mM for 30 minutes under the condition of 56 ℃, adding iodoacetamide at a final concentration of 55mM for 30 minutes in a darkroom environment, alkylating, and finally adding sequencing-grade trypsin, wherein the mass ratio of trypsin to substrate is 1:50, and carrying out shaking enzymolysis for 16 hours at 37 ℃.
6. The method for preparing the intestinal immunopotentiating sea cucumber peptide according to claim 1, wherein in the step (5), the center coordinate of a butting box in the precise butting is x=31.2, y=42.8, z=25.4, the box size is set to be 26 a×24 a×20 a, the grid number is 60×58×50, the butting parameter is set to be 45, 30 binding conformations are generated, and the maximum energy difference threshold is 4.5kcal/mol.
7. The method for preparing the intestinal immunity enhanced sea cucumber peptide according to claim 1, wherein the 10000Da molecular weight cut-off ultrafiltration membrane in the step (6) is a polyethersulfone hollow fiber ultrafiltration membrane, the operation parameters are that the feeding temperature is 20-25 ℃, the transmembrane pressure difference is set to 0.15-0.25MPa, the tangential flow velocity is 2.5-5.0m/s, and the volume concentration ratio is 3.0-5.0; The 3000Da molecular weight cut-off nanofiltration membrane is a zirconia-titania composite nanofiltration membrane, the operation parameters are that the feeding temperature is 12-18 ℃, the transmembrane pressure difference is set to be 0.40-0.50MPa, and the tangential flow velocity is 3.5-4.5 m/s; the 1000Da molecular weight cut-off nanofiltration membrane is a polypiperazine amide nanofiltration membrane, and operates at 0.65MPa and 4-10 ℃, and the membrane flux is 18L/m 2 .h+/-5%.
8. The method for preparing the intestinal immunity-enhanced sea cucumber peptide according to claim 1, wherein the rotary evaporation concentration temperature in the step (7) is 10-45 ℃ and the pressure is 0.08-0.10 MPa.
9. The method for preparing the intestinal tract immunity enhancing sea cucumber peptide according to claim 1, wherein the freeze-drying protective agent in the step (7) is trehalose and hydroxypropyl-beta-cyclodextrin, the trehalose is 0.2-0.8% of the concentrated solution in mass, and the hydroxypropyl-beta-cyclodextrin is 0.1-0.5% of the concentrated solution in mass.
10. The directional preparation method of the intestinal immunity-enhanced sea cucumber peptide, which is characterized by comprising the steps of drying in a three-stage manner, wherein the vacuum belt drying in the step (7) is performed in a first stage of 50-55 ℃ and 20 kPa for 30-50 min, in a second stage of 30-40 ℃ and 5 kPa for 180min, and in a third stage of 20-25 ℃ and 1kPa for 60min.

Description

Directional preparation method of intestinal immunity-enhanced sea cucumber peptide Technical Field The invention relates to the technical field of biological deep processing and extraction, in particular to a directional preparation method of intestinal immunity enhancement type sea cucumber peptide. Background Sea cucumber peptide is used as the representative of ocean source functional peptide, and is rich in various unique bioactive components including collagen tripeptide with characteristic repeated sequence (Gly-Pro-Hyp), chondroitin sulfate with anti-inflammatory and immunoregulatory effects, sea cucumber saponin with special structure, etc. These components confer a remarkable physiological function to the sea cucumber peptide, particularly in terms of immunomodulation (e.g., activating macrophage secretion of anti-inflammatory factor IL-10), inhibition of tumor cell growth and metastasis, and repair and enhancement of the physical barrier of the intestinal tract (by modulating expression of tight junction proteins such as Claudin and Occludin). Its low molecular weight nature (typically <2000 Da) not only ensures high bioavailability, making it easier for the intestinal tract to absorb and exert systemic effects, but also reduces the potential risk of immunogenicity. Therefore, the sea cucumber peptide has wide application prospect in the fields of functional foods, special medical diet, nutritional supplements and biological medicines. However, the existing sea cucumber peptide preparation process has more technical defects, and the industrialization application and functional precise development of the existing sea cucumber peptide preparation process are restricted. The main aspects are as follows: (1) Inefficiency of pretreatment technique The unique cuticle structure of the sea cucumber body wall is composed of a crosslinked Alzheimer protein and a chitin fiber network, so that a compact enzymolysis-resistant barrier is formed. The traditional pretreatment method has the fundamental limitations that the high-temperature stewing method can lead to obvious degradation of thermosensitive functional components and has higher energy consumption, the chemical permeation method can improve enzymolysis efficiency, but has the risk of food safety caused by chemical reagent residues, and the ultrasonic auxiliary treatment is uneven due to cavitation effect, so that peculiar smell substances are easily generated due to local high temperature, and the sensory quality is obviously reduced. (2) The controllability of the enzymolysis process is poor The conventional enzymolysis system has multiple challenges, namely firstly, the enzymolysis efficiency is generally low due to insufficient breaking of stratum corneum barrier, the peptide yield is insufficient, secondly, the random digestion leads to the dispersion of the molecular weight distribution of peptide fragments, wherein the peptide fragments with strong hydrophobicity occupy higher proportion, and the sustained bitter taste is caused, and finally, the shearing force generated by the traditional mechanical stirring is easy to destroy the space conformation of the immunocompetent peptide, so that the biological activity of the immunocompetent peptide is obviously reduced. (3) Functional peptide screening technique hysteresis The current sea cucumber peptide activity evaluation mainly depends on an in-vitro enzyme inhibition model, and is disjointed with an in-vivo immune regulation mechanism. The method is characterized in that screening targets are single, the screening targets are mainly concentrated on antihypertensive/hypoglycemic peptides, attention on related targets of intestinal immunity is insufficient, the traditional separation and classification method is carried out only according to molecular weight, active epitopes specifically combined with specific receptor domains are difficult to accurately identify, and meanwhile, quality control standards based on molecular interaction layers are lacked, so that the difference of immunostimulation activities among batches is large. (4) Defects of separation and enrichment technology The existing separation process has the problems of long treatment period of chromatography and low recovery rate of target peptide, serious membrane pollution of the traditional nanofiltration membrane, remarkable flux attenuation, and easy inactivation of heat-sensitive peptide fragments in the drying process (particularly in the high-temperature condition of spray drying) to cause immune activity loss. Therefore, an innovative process integrating green pretreatment, receptor precise screening and activity preservation is urgently required to be developed, and the technical bottleneck of sea cucumber peptide industrialization is fundamentally broken through. The invention fills the technical blank in the field through systematic innovation of supercritical CO 2 -DES collaborative permeation, pulse electric field time