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A kind of elastase inhibitory peptide and its preparation method and application

A technology of elastase and inhibitory peptides, applied in the field of protein polypeptides, can solve problems such as protease inhibitory peptides that have not yet been seen, and achieve good acid-base environment stability, improved stability, and high economic benefits

Active Publication Date: 2022-04-26
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there is no protease inhibitory peptide product prepared from walnut meal

Method used

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  • A kind of elastase inhibitory peptide and its preparation method and application
  • A kind of elastase inhibitory peptide and its preparation method and application
  • A kind of elastase inhibitory peptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Extraction of protease inhibitory peptide-polypeptide solution

[0041] The walnut pulp used in this example is a by-product obtained after oil extraction by hydraulic pressing (cold pressing).

[0042] (1) Degreasing

[0043] The walnut meal is ground by a grinder and passed through a 60-mesh sieve for later use. Mix walnut meal powder and petroleum ether at a ratio of 1:5 (w / v), and stir in a magnetic stirrer at room temperature, replace petroleum ether after 3 hours, and repeat three times. The processed walnut meal powder was placed in a fume hood to volatilize organic reagents to obtain degreased powder. The degreased walnut meal powder was stored in a sealed environment at 4°C for future use.

[0044] (2) Protein extraction

[0045] Following the method of Mao and Hua (2012) with slight modifications. Accurately weigh 40.0g of walnut dregs defatted powder, mix it with deionized water at a ratio of 1:10 (w / v), adjust its pH value to 11.0 with 1mo...

Embodiment 2

[0059] Embodiment 2: Separation and purification of protease inhibitory peptide

[0060] The enzymatic hydrolyzate of walnut meal obtained by ultrasonic-alkaline protease hydrolysis contains free amino acids, polypeptides of different lengths, and proteins with a small degree of hydrolysis.

[0061] In this example, walnut meal polypeptides were purified by means of ultrafiltration, using elastase inhibitory ability as an indicator, and the polypeptide sequences of the purified components were identified by LC-MS / MS. Through PyRx virtual screening combined with Autodock Vina molecular docking and amino acid sequencing combined with literature research, the peptides that may have strong elastase inhibitory ability were screened out.

[0062] (1) ultrafiltration

[0063] The enzymatic hydrolysis product of walnut meal prepared in Example 1 was subjected to ultrafiltration to purify and enrich the active ingredients. The enzymolysis solution was separated by ultrafiltration usi...

Embodiment 3

[0087] Example 3 Verification of polypeptide activity

[0088] (1) Artificial synthesis of peptides

[0089] The synthesis of peptides adopts the Fmoc solid-phase synthesis method, and peptides are synthesized according to the amino acid sequence, and powdered peptides are obtained after cleavage, precipitation, and purification. The synthesis process was entrusted to Nanjing Yuanpeptide Biotechnology Co., Ltd. The purity of the peptide segment FFVPF and the peptide segment NSLNLPIL were both greater than 95%.

[0090] (2) Activity verification of artificially synthesized peptides

[0091] According to the method described in Example 1, the inhibitory rate of peptides of different concentrations to elastase was measured, and the half-inhibitory concentration (IC) of peptides to elastase was calculated according to the fitting of the experimental results. 50 ). IC of peptide FFVPF 50 The value is 0.469±0.010mg / mL, the IC of NSLNLPIL 50 The value is 0.635±0.030, which has ...

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Abstract

The invention belongs to the technical field of protein polypeptides, and in particular relates to an elastase inhibitory peptide extracted from walnut dregs, a preparation method and application thereof. The amino acid sequence from the N-terminal to the C-terminal of the inhibitory peptide is: Phe-Phe-Val-Pro-Phe (FFVPF) or Asn-Ser-Leu-Asn-Leu-Pro-Ile-Leu (NSLNLPIL). The half-inhibitory concentration (IC) of FFVPF to elastase 50 ) reaches 0.469 ± 0.010mg / mL, and has the characteristics of partial decomposition after gastric digestion and complete decomposition in the intestinal tract; the half-inhibitory concentration (IC) of NSLNLPIL to elastase 50 ) reached 0.635±0.030mg / mL. The short peptidase provided by the invention has obvious inhibitory effect, uses walnut meal as raw material, has low production cost and high economic benefit. The present invention also prepares nanoparticles loaded with short peptides. The encapsulation efficiency of CS-TPP to short peptides can reach 94.58±0.23%, the particle size is between 140nm and 180nm, and the zeta potential is +33mV to +36mV, and has a relatively high Good environmental stability.

Description

Technical field: [0001] The invention belongs to the technical field of protein polypeptides, and in particular relates to an elastase inhibitory peptide extracted from walnut dregs, a preparation method and application thereof. Background technique: [0002] Elastin, a fibrous protein found in the extracellular matrix, is essential for the elasticity of the lungs, arteries, ligaments and skin. As the body matures and ages, the dissolution of elastin accelerates the development of diseases of aging. Elastase is a proteolytic enzyme characterized by degrading elastin, which exists in microorganisms, snake venom, and many mammalian cells and tissues, including pancreas, leukocytes, and macrophages. In recent years, many studies have shown that elastase is associated with skin photoaging, lung diseases, rheumatoid arthritis and so on. [0003] The skin is mainly composed of supporting and connecting proteins, among which collagen and elastin are particularly important for mai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K17/10C07K17/02A61K38/08A61K8/64A61K9/51A61K47/36A61K47/02A61P11/00A61P17/18A61P19/02A61P29/00A61Q19/08
CPCC07K7/06C07K17/10C07K17/02A61K9/5161A61K9/5115A61P17/18A61P11/00A61P19/02A61P29/00A61K8/64A61Q19/08A61K38/00A61K36/52A61K2800/82A61K2800/805
Inventor 任迪峰王潇漪陈诗佳彭芃
Owner BEIJING FORESTRY UNIVERSITY
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