WO2018014841A1

WO2018014841A1 - Self-assembled collagen and preparation method therefor

Info

Publication number: WO2018014841A1
Application number: PCT/CN2017/093484
Authority: WO
Inventors: 裴雪涛; 南雪; 游子娟; 单紫筠; 魏红; 姚海雷; 贾茜媛; 龙冬莹
Original assignee: 华南生物医药研究院
Priority date: 2016-07-19
Filing date: 2017-07-19
Publication date: 2018-01-25
Also published as: CN107630060B; CN107630060A

Abstract

A self-assembled collagen and a preparation method therefor. The preparation method for the self-assembled collagen comprises the following steps: utilizing trypsin and papain in an enzymatic treatment of an animal-derived collagen so as to produce a hydrolysate containing small peptides; performing an ultrafiltration treatment on the hydrolysate so as to produce a collagen peptide solution; and mixing the collagen peptide solution with acetic acid and then performing a dialysis treatment so as to produce the self-assembled collagen, where the dialysis treatment employs a phosphate buffer as the dialysate. The method employs specific proteases for hydrolysis of collagen, not only produces the collagen peptide solution of low molecular weight, but also produces a specific "sticky end" at the distal end of the peptide, and forms, by means of self-assembly, a collagen fiber providing improved organ supporting and tissue protecting effects in the body.

Description

Self-assembling collagen and preparation method thereof

Priority information

The present application claims priority to and the benefit of the patent application No. 201610574236.5 filed on Jan. 19, 2016, the entire disclosure of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of protein engineering. Specifically, the present invention relates to the field of self-assembly of polypeptide molecules, and more particularly, the present invention relates to self-assembled collagen, a preparation method thereof and use thereof.

Background technique

Collagen is a natural protein produced by fibroblasts in animals. It is a structural protein of extracellular matrix, also known as collagen. It is widely found in connective tissues such as bones, skin and tendons in animals, and has tissues and organs. Good mechanical structure, which plays a role in supporting organs and protecting the body. The (Gly-XY)n repeating amino acid sequence unit is the molecular basis of the triple helix structure of the collagen, in which glycine (Gly) occupies the axial position of the triple helix, proline and hydroxyproline are high in the X and Y positions, respectively. Appears and plays an important role in the stability of the triple helix. Collagen and its degradation products are rich in natural moisturizing factors such as glycine, serine, alanine, aspartic acid and a large number of hydrophilic groups, so it has a moisturizing function. In addition, collagen has good permeability, can be filled between skin matrix and absorbed by the skin, and has the effect of repairing skin and whitening. Therefore, collagen and its degradation product collagen peptide are widely used in beauty care and cosmetics. .

Since the collagen extracted from the tissue has a relatively large molecular weight, it is generally about 200 kDa, and a substance having a molecular weight of more than 4 kDa is difficult to enter the dermis layer of human skin, resulting in absorption of collagen by the human body. The rate is low. Therefore, collagen oligopeptides having a molecular weight of about 1 kDa are produced by hydrolysis of proteases in vitro, and these small peptides can be absorbed by the skin, and can promote amino acid absorption and synthesis of body proteins. Collagen oligopeptide is obtained by hydrolyzing collagen with specific enzyme. When the collagen is hydrolyzed into a polypeptide, its triple helix structure is destroyed, and it can not support the organ and protect the body.

Polypeptide self-assembly technology refers to the spontaneous formation of stable aggregate structure by the interaction of non-covalent bonds under the thermodynamic equilibrium conditions of polypeptide molecules. The self-assembly mechanism utilizes non-covalent bonds of polypeptide molecules, such as intermolecular hydrogen bonds, ionic bonds, salt bonds, etc. to form polypeptide aggregates spontaneously. The self-assembly process of collagen is very complicated. Most of the current research focuses on the effects of collagen concentration, self-assembly time, solution pH, electrolytes and substrate on the self-assembly process of collagen.

Therefore, the preparation method of the collagen sample before self-assembly still needs to be improved.

Summary of the invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide a method capable of producing self-assembled collagen having a good supporting organ and protecting a biological function such as a body.

The present invention has been completed based on the following findings of the inventors: since collagen is rich in charged amino acids such as glutamic acid, lysine and aspartic acid, peptides having a specific amino acid end can be obtained by protease hydrolysis, for example, the terminal is The positively charged glutamic acid and the negatively charged lysine form a "sticky end" which promotes self-assembly of the collagen polypeptide to form collagen fibers.

Thus, in accordance with one aspect of the invention, the invention provides a method of making self-assembling collagen. According to an embodiment of the present invention, the method comprises the steps of: enzymatically treating animal-derived collagen with trypsin and papain to obtain an enzymatic hydrolysate containing a small molecule peptide; and ultrafiltration treatment of the enzymatic hydrolysate In order to obtain a collagen polypeptide solution; the collagen polypeptide solution is mixed with acetic acid and subjected to dialysis treatment to obtain the self-assembled collagen, wherein the dialysis treatment uses a phosphate buffer as a dialysate external solution.

The inventors have surprisingly found that using the method of the present invention for preparing self-assembled collagen, specific Protease hydrolyzes collagen to obtain not only a low molecular weight collagen polypeptide solution, but also a specific "sticky end" at the end of the peptide to form collagen fibers by self-assembly. The protease is directly hydrolyzed into a small peptide, which can be effectively absorbed by the human body, but it cannot be self-assembled into collagen in the body, and cannot function as a supporting organ and protect the body. Therefore, collagen is hydrolyzed with a specific protease to produce a "sticky end", and collagen fibers are formed by self-assembly, so that its biological function can be better exerted.

The animal-derived collagen may be selected from animal collagen, such as bovine collagen, porcine collagen or horse collagen, and may also be selected from fish collagen. There are many types of collagen, and the common types are type I, type II, type III, type V and type XI. Type I collagen has been extensively studied for its richest content in nature, and the biomaterials made therefrom have the advantages of good biocompatibility, easy degradation, low toxicity and high structural strength. In the examples of the present invention, the method of the present invention is mainly described by taking type I collagen as an example, but the method of the present invention is not limited to the self-assembly of type I collagen oligopeptide.

In addition, the method of preparing self-assembled collagen according to an embodiment of the present invention may further have the following additional technical features:

According to an embodiment of the invention, the enzymatic treatment further comprises:

Performing the first hydrolysis treatment with trypsin for 2 to 6 hours;

Performing a second hydrolysis treatment on the first hydrolyzed product using papain for 2 to 6 hours;

The product of the second hydrolysis treatment is centrifuged, and the supernatant is collected, and the supernatant constitutes the hydrolyzed product. Thereby, the hydrolysis efficiency is high and the specificity is good.

According to an embodiment of the present invention, a step of boiling water bath inactivation activity is included after both the first hydrolysis treatment and the second hydrolysis treatment.

According to some specific examples of the present invention, the animal-derived collagen is firstly hydrolyzed with trypsin for 2 to 6 hours, the enzyme is inactivated by a boiling water bath, and further hydrolyzed with papain for 2 to 6 hours, inactivated by boiling water bath, and centrifuged. The precipitate was discarded and the supernatant was collected. The collagen is sequentially hydrolyzed by trypsin and papain, and the hydrolysis efficiency is high, the specificity is good, and the production cost is low. The boiling water bath inactivation refers to the enzyme The reaction tube was placed in a boiling water bath for 20 minutes to inactivate papain and trypsin.

According to an embodiment of the present invention, the enzymatically hydrolyzed product is ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 1 to 5 kDa, preferably, ultrafiltered with an ultrafiltration membrane having a molecular weight cut off of 1 kDa, and is suitable for screening of small molecular peptides.

According to an embodiment of the present invention, in the step (2), the collagen polypeptide solution is mixed with 0.05 to 1.0 mol/L of acetic acid, wherein the final concentration of the collagen polypeptide is 0.5 to 5 mg/mL. That is, the collagen polypeptide solution is sufficiently dissolved in acetic acid at a concentration of 0.05 to 1.0 mol/L to prepare an acid solution having a final concentration of the collagen polypeptide of 0.5 to 5 mg/mL, preferably the concentration of the collagen polypeptide solution. It was sufficiently dissolved in 0.5 mol/L of acetic acid to prepare an acid solution having a final concentration of collagen polypeptide of 3 mg/mL.

According to the presently illustrated embodiment, the phosphate buffer solution has a concentration of 50 mM, a pH of 6.5 to 8.5, and a pH of 7.0.

According to another aspect of the present invention, there is provided a self-assembling collagen obtained by the method of one or more of the above-described embodiments of the present invention. The foregoing description of the technical features and advantages of the method for preparing self-assembled collagen of one or both of the present invention is equally applicable to the self-assembled collagen of this aspect of the present invention, and will not be described herein.

This aspect of the invention provides a self-assembling collagen gel, which is determined by the inventors' repeated adjustment test, including the hydrolysis of collagen by a specific enzyme to obtain a specific concentration of collagen acid solution, and the enzymatic hydrolysate is subjected to ultrafiltration. It is fully dissolved in acetic acid to prepare an acid solution, and is dialyzed in a dialysis bag, so that the self-assembled collagen can be obtained simply and conveniently.

It should be noted that, as far as the inventors are aware, there has not been any report on the self-assembly of collagen polypeptide after hydrolysis of collagen by a specific enzyme. The preparation method provided by the inventors allows self-assembly of polypeptide molecules by simple collagenase digestion to obtain self-assembled collagen which can better support organs and protect the body in vivo.

According to still another aspect of the present invention, there is provided the use of the self-assembled collagen of one aspect of the present invention described above in the preparation of a food, a health care product, a skin care product, and a pharmaceutical. Self-assembled collagen has the toughness, strength and thermal stability of collagen, and has the easy absorption of collagen, and It has the characteristics of supporting organs and protecting the body that collagen oligopeptides do not have. It has a huge application space in the food industry, biomedical and beauty skin care products.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 shows an SDS-PAGE electropherogram of collagen prior to self-assembly in one embodiment of the present invention;

2 shows an SDS-PAGE electrophoresis pattern of collagen self-assembly in one embodiment of the present invention;

3 shows a scanning electron micrograph (SEM) of a self-assembled anterior collagen film in one embodiment of the present invention;

4 shows a scanning electron micrograph (SEM) of a self-assembled procollagen film in one embodiment of the present invention.

detailed description

The invention is described in detail below with reference to the preferred embodiments of the invention.

Unless otherwise stated, the reagents and instruments not specifically addressed in the following examples may be derived from conventional commercially available products.

Example 1

Self-assembled collagen was prepared to prepare self-assembled bovine type I collagen as an example.

Self-assembled collagen was prepared by using 5 mg/ml bovine type I collagen aqueous solution as a raw material.

(1) The concentration of collagen was added to trypsin (Trypsin) at E/S of 8000 U/g, and after hydrolysis at 37 °C for 3 h, the pH was adjusted with 0.1 mol/L NaOH during the hydrolysis to maintain the pH of the reaction system at 8.0, boiling water bath. The enzyme was inactivated for 20 min, and further hydrolyzed with papain (Papain) at 50 ° C for 3 hours. During the hydrolysis, the pH was adjusted with 0.1 mol/L NaOH to maintain the pH of the reaction system at 7.0. The enzyme was boiled in a boiling water bath for 20 min and cooled. Finally, centrifuge at 5000 g/min for 10 min, discard the precipitate, and collect the supernatant.

(2) The hydrolyzate obtained in the step (1) was subjected to ultrafiltration using a 1 kDa ultrafiltration membrane to obtain a collagen polypeptide solution, which was placed in a refrigerator at 4 ° C for use.

(3) The collagen polypeptide solution obtained in the step (2) is sufficiently dissolved in 0.05 mol/L acetic acid to prepare an acid solution of 1 mg/mL, and dialyzed against a dialysis bag at 4 ° C for 12 hours at a concentration of 50 mM and a pH of 7.0. Phosphate buffered solution (PBS) is a dialyzed external solution to obtain self-assembled bovine type I collagen.

(4) The self-assembled procollagen polypeptide solution and the self-packed collagen polypeptide solution obtained in the step (3) were respectively dissolved in a 0.5 mol/L acetic acid solution, and thoroughly stirred and dissolved for 12 hours to prepare a 5 mg/mL membrane solution. A plate having a diameter of 3.5 cm/well was used as a mold, and a 2 ml sample was added. The refrigerator was chilled overnight at -20 ° C, and lyophilized under vacuum at -40 ° C, 1 Pa for 24 h. The surface morphology of the film was observed by scanning electron microscopy (SEM) after drying.

The SEM results are shown in Figure 2. The self-assembly of collagen is better than that before self-assembly. The self-assembly collagen membrane is loose and has a large pore size (82.0±21.6) μm. However, after self-assembly, the collagen membrane is arranged more closely, and the pore diameter becomes smaller (52.7±15.9) μm, which is more flexible.

The method described in Example 1 is also applicable to the self-assembly of collagen oligopeptides of type I, type II, type III, type V and type XI such as porcine collagen, horse collagen, and fish collagen, which will not be described herein.

Example 2 electrophoresis analysis of the obtained collagen polypeptide and self-assembled collagen polypeptide

The collagen polypeptide solution obtained in Example 1 and the self-installed collagen polypeptide solution were subjected to electrophoresis analysis, as follows:

SDS-PAGE gel electrophoresis analysis

The bovine type I collagen polypeptide obtained before and after self-assembly obtained in Example 1 was identified by SDS-PAGE gel electrophoresis using a concentration of 5% concentrated gel, 20% and 12% separation gel.

The results are shown in Fig. 1. Comparing the collagen with the standard protein Maker, it can be seen that the molecular weight of the bovine collagen polypeptide after hydrolysis becomes smaller, and most of them are concentrated below 10 kDa, and S represents the example 1 in the figure. Bovine type I collagen polypeptide after protease hydrolysis.

As a result, as shown in Fig. 2, after the self-assembly of the collagen polypeptide, the molecular weight was significantly increased, mainly focusing on 22.5 kDa, 10 kDa and 12 kDa, and S in the figure indicates the self-assembled bovine type I collagen polypeptide in Example 1. It is indicated that the collagen polypeptide has reconstituted the triple helix structure of collagen after undergoing a certain degree of self-assembly. In the figure, M denotes Maker, and S denotes a sample.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A material, or feature, is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

A method for preparing self-assembled collagen, comprising:

(1) enzymatically treating animal-derived collagen with trypsin and papain to obtain an enzymatic hydrolysate containing a small molecule peptide;

(2) subjecting the enzymatically hydrolyzed product to ultrafiltration treatment to obtain a collagen polypeptide solution;

(3) The collagen polypeptide solution is mixed with acetic acid and subjected to dialysis treatment to obtain the self-assembled collagen, wherein the dialysis treatment uses a phosphate buffer solution as a dialysate external solution.
The method according to claim 1, wherein the animal-derived collagen is animal collagen or fish collagen.
The method according to claim 2, wherein the animal-derived collagen is bovine collagen, porcine collagen or equine collagen.
The method of claim 1 wherein said enzymatic treatment further comprises:

Performing the first hydrolysis treatment with trypsin for 2 to 6 hours;

Performing a second hydrolysis treatment on the first hydrolyzed product using papain for 2 to 6 hours;

The product of the second hydrolysis treatment is centrifuged, and the supernatant is collected, and the supernatant constitutes the hydrolyzed product.
The method according to claim 4, wherein the step of boiling water bath inactivation is included after both the first hydrolysis treatment and the second hydrolysis treatment.
The method according to claim 1, wherein the enzymatic product is subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 1 to 5 kDa.
The method according to claim 6, wherein the enzymatic product is subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 1 kDa.
The method according to claim 1, wherein in the step (2), the collagen polypeptide solution is mixed with 0.05 to 1.0 mol/L of acetic acid, wherein the final concentration of the collagen polypeptide is 0.5 to 5 mg. /mL,

Preferably, the collagen polypeptide solution is mixed with 0.5 mol/L of acetic acid, wherein the final concentration of the collagen polypeptide is 3 mg/mL.
The method according to claim 1, wherein the phosphate buffer solution has a concentration of 50 mM, a pH of 6.5 to 8.5, and preferably a pH of 7.0.
A self-assembling collagen prepared by the method of any one of claims 1-10.
Use of the self-assembled collagen of claim 10 for the preparation of a food, a health care product, a cosmetic or a skin care product.