CN115531239B - Nanoparticle, toothpaste and preparation method thereof - Google Patents

Abstract

The application provides a nanoparticle which is characterized by comprising keratin and chitosan. The nano particles have excellent effects on wound repair, hemostasis and anti-inflammation, have good biocompatibility, and can be added into oral care products, such as toothpaste, as an additive to quickly solve the oral care problems of oral ulcer and gingival bleeding.

Description

Nanoparticle, toothpaste and preparation method thereof

Technical Field

The application relates to the technical field of daily chemicals processing, in particular to a nanoparticle (especially a nanoparticle prepared from recombinant keratin), a preparation method of the nanoparticle and application thereof; a toothpaste and its preparation method are provided.

Background

With the increasing level of living, oral health issues are of great concern and consumer demand for functional toothpastes is increasing. The oral cavity is easy to grow bacteria because people need to ingest food through the oral cavity every day, so that various oral problems are caused, the oral problems are mainly caused by the fact that food residues are attached to the surfaces and the gaps of teeth, so that a large amount of bacteria are propagated, the bacteria corrode the teeth, and generated secretion forms hard tartar, so that the hard tartar is difficult to remove. Meanwhile, the bacteria decompose the gas generated by food to cause halitosis, and bacteria propagation or eating of irritant food and the like can cause swelling and pain of gum, gum bleeding, oral cavity ulceration and the like, so that the oral health and personal image of people are seriously affected, and the oral cavity environment is very necessary to clean teeth and oral cavity.

The toothpaste is a cleaner for daily cleaning teeth, can effectively clean the dental calculus adhered to the teeth, has the effects of strengthening teeth, protecting gingiva and keeping the oral cavity clean, and aims at various oral problems, various components are often added into the toothpaste to obtain the toothpaste with different functions so as to meet the daily use requirements of people. However, most of the toothpaste in the market at present mainly takes daily care, and some toothpaste with repairing function is only added with some antibacterial and anti-inflammatory components, so that the toothpaste is not only required to be used for a long time, but also has an unobvious repairing effect. Moreover, prolonged use of such toothpastes can lead to oral dependency. Meanwhile, the problems of dental ulcer, gingival bleeding and the like are repeatedly difficult to radically cure, and the oral cavity of a patient is puzzled.

The keratin is a middle silk structural protein, the amino acid composition is rich in a large amount of cysteine, a large amount of disulfide bonds are easily formed in molecules and among molecules, and the unique and stable space structure and excellent biological performance of the keratin are formed by combining the transverse connection of hydrogen bonds, salt bonds, van der Waals force and the like, so that the keratin material has good application prospect in the field of biological materials.

The most current processes for preparing keratin are hair chemical extraction, for example, the keratin in the hair is extracted by adopting a physical and chemical method, and the hair extraction keratin has the following problems: chemical extraction methods can break the spatial structure of proteins, resulting in poor stability of the extracted keratins with reduced activity; and the chemical agent is not effective in removing cytotoxicity that would cause keratin raw materials; the keratin extracted by the chemical method is a mixture of various keratin fragments, has a single component and poor quality control, and is not suitable for industrial mass production. Compared with the preparation process of the recombinant keratin and the traditional keratin, the preparation process of the recombinant keratin is more environment-friendly, and the yield and purity of the recombinant keratin are greatly improved.

Disclosure of Invention

The toothpaste is mainly prepared by adding Chinese herbal medicine components with anti-inflammatory and repairing functions in the market to solve the problems of dental ulcer, gingival bleeding and the like. However, there are problems in that the effect of treating the above oral problems is poor, and in addition to the repeatability and severity of the above oral problems, it is necessary to use such toothpaste for a long period of time, and there is a problem in that the use is dependent. Over time, the oral problems cannot be effectively and radically cured, and great trouble is brought to the oral cavity of the patient. Aiming at the defects of the prior art, the application provides the keratin-containing nano particles which have excellent effects on wound repair, hemostasis and anti-inflammation and have good biocompatibility, and the keratin-containing nano particles can be used as an additive to be added into oral care products, such as toothpaste, so that the oral care problems of oral ulcer and gingival bleeding can be rapidly solved. Especially for repeated canker sore, can effectively promote the repair of wound surface. Meanwhile, the chitosan and the keratin are used for preparing the composite nano particles, so that the mechanical property of the material is improved, the specific surface area and the hydrophilic property are increased, and the hemostatic effect is better.

It is an object of the present application to provide a nanoparticle comprising keratin and chitosan.

The technical scheme of the application is as follows:

1. a nanoparticle comprising keratin and chitosan.

2. The nanoparticle according to item 1,

the mass ratio of the chitosan to the keratin is (1-10): 1, preferably 4:1.

3. The nanoparticle of item 1 or 2,

the keratin is prepared by mechanical method, acid-base treatment method, reduction method, oxidation method, electrochemical reduction method, cuprammonium solution method, metal salt method, biological method or genetic engineering, preferably genetic engineering;

further preferably, the amino acid sequence of the keratin is shown as SEQ ID NO. 2; or the chitosan is carboxymethyl chitosan.

4. A method of preparing the nanoparticle of any one of claims 1-3, comprising the steps of:

dissolving keratin and chitosan in water to obtain an aqueous solution containing keratin and chitosan;

injecting the aqueous solution into hydrochloric acid solution to obtain nano suspension particles;

centrifuging and freeze-drying the nano suspension particles to obtain nano particles;

preferably, the method comprises the steps of,

the concentration of the keratin is 0.1-1mg/mL, and the concentration of the chitosan is 0.5-10mg/mL.

5. Use of the nanoparticle of any one of claims 1-3, or the nanoparticle prepared by the method of claim 4, in an oral care product;

Preferably, the oral care product is a toothpaste, mouthwash, tooth powder, dentifrice, floss or denture care product.

6. A toothpaste comprising the nanoparticle of any one of items 1-3, or the nanoparticle prepared by the method of item 4;

preferably, the nanoparticles are 1 to 10 parts by weight, preferably 1 to 3 parts by weight, relative to 100 parts by weight of toothpaste.

7. The toothpaste according to item 6,

the toothpaste also comprises one or more of bletilla striata extract, humectant, friction agent, stabilizer, foaming agent, pH stabilizer, thickener, sweetener and antiseptic.

8. The toothpaste according to item 7,

for 100 parts by weight of toothpaste, 0.01-1 part by weight of bletilla striata extract, 12-30 parts by weight of humectant, 10-25 parts by weight of friction agent, 0.1-2 parts by weight of stabilizer, 1-5 parts by weight of foaming agent, 0.01-0.5 part by weight of pH buffer, 1-8 parts by weight of thickening agent, 0.01-0.5 part by weight of sweetener and 0.01-0.5 part by weight of preservative.

9. The toothpaste according to item 8,

the humectant is one or more than two of sorbitol, glycerol and polyethylene glycol;

The friction agent is hydrated silica;

the stabilizer is polyethylene glycol-400;

the foaming agent is sodium lauryl sulfate;

the pH buffering agent is sodium pyrophosphate;

the thickener is one or more than two selected from cellulose gum, xanthan gum, carrageenan, deer pectin, guar gum and carrageenan;

the sweetener is sodium cyclamate and/or saccharin sodium;

the preservative is one or more than two selected from sodium benzoate, methylisothiazolinone and potassium sorbate.

10. A method for preparing a toothpaste according to any one of claims 6 to 9,

dissolving a thickening agent and a stabilizing agent in a humectant, and stirring to form a solution A;

dissolving sweetener, pH buffer and preservative in water, and stirring to form solution B;

adding water and humectant into the solution B, and then adding the solution A to form glue; then adding nano particles, a foaming agent, a bletilla striata extract and a friction agent; stirring to obtain a solution C;

and (3) introducing the solution C into a paste making machine to make paste, thereby obtaining the toothpaste.

The application has the following beneficial technical effects:

the nano particles of the application have excellent hydrophilic performance and water absorption performance, good solubility and sealing performance, and when the nano particles are used for oral care, oral problems such as gingival bleeding occur, the nano particles have good water absorption performance, and can quickly absorb water in blood, thereby achieving the effects of enriching platelets and coagulation factors and realizing quick hemostasis.

When the nano particles contain keratin, especially recombinant keratin, the adsorption capacity and mechanical property of the material can be enhanced after the nano particles are crosslinked with chitosan, bleeding points are closed, and the physical hemostatic effect is improved.

The nano particles can promote proliferation of epithelial cells and accelerate healing of wounds.

The toothpaste provided by the application can be used for rapidly promoting wound healing, promoting wound repair and has the functions of stopping bleeding and diminishing inflammation.

Drawings

FIG. 1 is a graph of particle size of nanoparticles of example 2;

fig. 2 is a graph showing the wound repair hemostatic effect of experimental example 1;

FIG. 3 is a graph showing the effect of hemostasis time of wound surface repair in Experimental example 1;

FIG. 4 is a graph showing the effect of wound surface repair on blood loss in experimental example 1;

FIG. 5 is a graph showing the effect of hemostasis in wound surface repair in Experimental example 2;

FIG. 6 is a graph showing the effect of healing rate of wound surface repair in Experimental example 2;

FIG. 7 shows the results of the hydrophilicity test in Experimental example 3;

FIG. 8 is a graph showing the effect of evaluating cell proliferation in Experimental example 4.

Detailed Description

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The description and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As used throughout the specification and claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description proceeds with reference to the general principles of the description. The scope of the application is defined by the appended claims.

The present application provides a nanoparticle comprising keratin and chitosan.

Keratin is a fibrous natural protein, which is the main constituent of wool, human hair, mammalian hooves, horns, nails, and poultry feathers. Generally, keratins are classified according to secondary structure, and can be classified into alpha-keratins (molecular weight 40-60 kDa) and beta-keratins (molecular weight 10-25 kDa).

The keratin preparation methods disclosed at present are mainly mechanical methods, acid-base treatment methods, reduction methods, oxidation methods, electrochemical reduction methods, cuprammonium solution methods, metal salt methods and biological methods.

The mechanical extraction method is generally to apply pressure and heat to break disulfide bonds between keratin molecules in hair, but the mechanical extraction method can only obtain proteins with lower molecular weight, even polypeptide mixture.

The oxidation method is to break disulfide bond in keratin to oxidize into sulfonic group with oxidant to obtain keratin. The oxidizing agent is generally peroxygen such as peroxyformic acid, peracetic acid, hydrogen peroxide, etc. However, the oxidation cleavage of peptide bonds is easy to occur, and the average molecular weight of the obtained keratin is not high.

The reduction method is to reduce disulfide bonds in the keratin into sulfhydryl groups by using a reducing agent to obtain the keratin. The reducing agent used is generally a mercapto compound such as sodium thioglycolate, thioglycollic acid, etc. However, the whole process is complex, the keratin is unstable, and the prepared solution is oxidized and reduced again to insoluble substances.

The acid-base treatment method is to make the soluble keratin by swelling hair with strong acid and alkali agent and hydrolyzing at a certain temperature, and is usually used together with reducer. However, hydrolysis of peptide bonds and decomposition of disulfide bonds of protein macromolecules inevitably occur in the acid-base treatment process. The extraction process can produce alkaline waste water and steam of waste acid, and pollute the environment.

The biological method mainly utilizes keratinase produced by microorganisms to induce decomposition of keratin, and the enzymolysis of the higher structure of the keratin into secondary structures such as amino acid, polypeptide and the like. However, this method is generally time-consuming and the products are mostly polypeptides with lower molecular weight.

In the process of preparing keratin by genetic engineering, genetic engineering bacteria are adopted to strengthen gene transcription and translation, so that efficient expression and active secretion are achieved, and the production intensity of keratin can be effectively improved. Recombinant keratin generally has single extracellular protein activity through performance improvement, and simplifies purification work downstream of fermentation. Novel keratins are sought by genetic engineering techniques.

Chitosan is both a major disinfecting component and a natural biological hemostatic. Chitosan is a chitosan deacetylated product, is an amorphous semitransparent substance, is nontoxic, harmless and easy to biodegrade, has excellent properties such as biofunctionality, compatibility, adsorptivity, film forming property, hygroscopicity, blood compatibility, safety, microbial degradability and the like, is widely focused by various industries, and has made great progress in application research in various fields such as medicine, food, chemical industry, cosmetics, water treatment, metal extraction and recovery, biochemical and biomedical engineering and the like. However, chitosan is insoluble in water and organic solvents and only in dilute acid, so that the application of chitosan is limited to a certain extent.

Carboxymethyl chitosan (CMC) is a type of chitosan derivative obtained by reacting chitosan with monochloroacetic acid under alkaline conditions. Carboxymethyl chitosan is an amphoteric polyelectrolyte, and can be effectively complexed with metal ions.

In some embodiments of the application, the keratin is a keratin prepared by mechanical, acid-base treatment, reduction, oxidation, electrochemical reduction, cuprammonium solution, metal salt, biological or genetic engineering, preferably genetic engineering.

In some embodiments of the application, the keratin has the amino acid sequence shown in SEQ ID NO. 2.

In some embodiments of the application, the mass ratio of the chitosan to the keratin is (1-10): 1, preferably 4:1;

for example, the mass ratio of the chitosan to the keratin is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any range therebetween.

The application also provides a preparation method of the nano-particles, wherein keratin and chitosan are dissolved in water to obtain an aqueous solution containing the keratin and the chitosan; injecting the aqueous solution into hydrochloric acid solution to obtain nano suspension particles; and centrifuging and freeze-drying the nano-suspension particles to obtain the nano-particles.

In some embodiments of the application, the concentration of keratin is 0.1-1mg/mL and the concentration of chitosan is 0.5-10mg/mL;

for example, the concentration of the keratin may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mg/mL or any range therebetween;

the chitosan concentration may be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/mL or any range therebetween.

The present application provides the use of the above nanoparticles in an oral care product; preferably, the oral care product is a toothpaste, mouthwash, tooth powder, dentifrice, floss or denture care product.

The application provides toothpaste comprising the above nanoparticles.

When the recombinant keratin disclosed by the application is used for hemostasis, the water absorption speed is high, a colloid is formed after water absorption, and a wound can be closed. The wound can stop bleeding rapidly, and gel blocks are removed after hemostasis, so that secondary bleeding is avoided. When commercially available carboxymethyl chitosan is applied to the wound to stop bleeding, the carboxymethyl chitosan has better water absorption speed, forms blocky crusts to block the wound, but is fragile; the wound is fast in hemostasis, and the blood clot is removed after hemostasis, so that secondary bleeding is avoided.

When the nanoparticle (i.e. recombinant keratin and carboxymethyl chitosan nanoparticle) disclosed by the application is applied to a wound to stop bleeding, the nanoparticle rapidly absorbs water to form blocky crusts to block the wound, the strength is high, the nanoparticle is not easy to break up by arterial hemorrhage, the required nanoparticle amount is small, the bleeding stopping speed is high, femoral artery hemorrhage can be stopped about 1-2min, blood clots are removed after bleeding stopping, and secondary bleeding is avoided.

Compared to the commercially available keratin nanoparticles (i.e., nanoparticles of comparative example 3) and the carboxymethyl chitosan nanoparticles of comparative example 2, the recombinant keratin nanoparticles of comparative example 1, when applied to the same wound, the nanoparticles prepared using the chitosan and recombinant keratin recovered the best, the fastest and highest wound healing rate for the same wound recovery period with the same particles. When the nano particles contain keratin, especially recombinant keratin, the adsorption capacity and mechanical property of the material can be enhanced after the nano particles are crosslinked with chitosan, so that the effect of closing wound surfaces can be better achieved, and the hemostatic effect on a physical layer is better.

The nano particles prepared from chitosan and recombinant keratin have the advantages of high water absorption and swelling speed, good sealing performance and no liquid outflow after inversion. The material has excellent hydrophilic performance and water absorption performance, and good solubility and sealing performance, is used for oral care, when oral problems such as gingival bleeding occur, the material has good water absorption performance, can quickly absorb water in blood, and achieves the effect of enriching platelets and coagulation factors, thereby realizing quick hemostasis.

The nano particles prepared from chitosan and recombinant keratin have better effect of promoting epithelial cell proliferation, so that wound healing is better promoted and wound healing is accelerated.

In some embodiments of the application, the nanoparticles are 1 to 10 parts by weight, preferably 1 to 3 parts by weight, relative to 100 parts by weight of toothpaste;

for example, the nanoparticles may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 parts by weight or any range therebetween, relative to 100 parts by weight of the toothpaste.

In some embodiments of the present application, the toothpaste further comprises one or more of bletilla striata extract, humectant, abrasive, stabilizer, foaming agent, pH stabilizer, thickener, sweetener, and antiseptic.

In some embodiments of the present application, the bletilla striata extract is 0.01-1 part by weight, the humectant is 12-30 parts by weight, the abrasive is 10-25 parts by weight, the stabilizer is 0.1-2 parts by weight, the foaming agent is 1-5 parts by weight, the pH buffer is 0.01-0.5 parts by weight, the thickener is 1-8 parts by weight, the sweetener is 0.01-0.5 parts by weight, and the preservative is 0.01-0.5 parts by weight, relative to 100 parts by weight of the toothpaste;

For example, the bletilla striata extract may be 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 part by weight or any range therebetween, relative to 100 parts by weight of the toothpaste;

the humectant may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 parts by weight or any range therebetween, relative to 100 parts by weight of the toothpaste;

the abrasive may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 parts by weight or any range therebetween, relative to 100 parts by weight of the toothpaste;

the stabilizing agent may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 parts by weight or any range therebetween, relative to 100 parts by weight of the toothpaste;

the foaming agent may be 1, 2, 3, 4, 5 parts by weight or any range therebetween with respect to 100 parts by weight of the toothpaste;

the pH buffer may be 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 parts by weight or any range therebetween, relative to 100 parts by weight of the toothpaste;

The thickener may be 1, 2, 3, 4, 5, 6, 7, 8 parts by weight or any range therebetween with respect to 100 parts by weight of the toothpaste;

the sweetener may be 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 parts by weight or any range therebetween with respect to 100 parts by weight of the toothpaste;

the preservative may be 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 parts by weight or any range therebetween with respect to 100 parts by weight of the toothpaste.

In some embodiments of the application, the humectant is one or more selected from sorbitol, glycerin, polyethylene glycol.

In some embodiments of the application, the abrasive is hydrated silica.

In some embodiments of the application, the stabilizer is polyethylene glycol-400.

In some embodiments of the application, the foaming agent is sodium lauryl sulfate.

In some embodiments of the application, the pH buffer is sodium pyrophosphate.

In some embodiments of the application, the thickener is selected from one or more of cellulose gum, xanthan gum, carrageenan, deer pectin, guar gum and carrageenan.

In some embodiments of the application, the sweetener is sodium cyclamate and/or sodium saccharin.

In some embodiments of the application, the preservative is selected from one or more of sodium benzoate, methylisothiazolinone, potassium sorbate.

The application provides a preparation method of toothpaste, wherein a thickening agent and a stabilizing agent are dissolved in a humectant and stirred to form a solution A; dissolving sweetener, pH buffer and preservative in water, and stirring to form solution B; adding water and humectant into the solution B, and then adding the solution A to form glue; then adding nano particles, a foaming agent, a bletilla striata extract and a friction agent; stirring to obtain a solution C; and (3) introducing the solution C into a paste making machine to make paste, thereby obtaining the toothpaste.

Compared with the common toothpaste on the market, the toothpaste of the application has obvious effect on treating gingival bleeding and canker sore under the same condition, and symptoms and pain feel are basically disappeared within one week. When the nanoparticle containing recombinant keratin and chitosan is used in toothpaste, the nanoparticle has the effects of rapidly promoting wound healing and wound repair. The toothpaste provided by the application has the functions of rapidly promoting wound healing, promoting wound repair, stopping bleeding and diminishing inflammation.

EXAMPLE 1 preparation of recombinant keratins

1.1 Synthesis and screening of recombinant Keratin amino acid sequences

The keratin amino acid sequence is shown in SEQ ID NO.1 (marked as K31), and the total length of the protein is 416 amino acids. In the structural region of K31, the domain formed by the first amino acid has strong hydrophobicity, and the active structure of keratin is mainly an intermediate alpha-helix structure, and the application further modifies the amino acid sequence shown in SEQ ID NO.1 on the basis of the structure: a large amount of cysteines exist at the front end of the original sequence, and a large amount of disulfide bonds and mismatches are easy to form in the process of escherichia coli expression and purification, so that the efficiency is reduced. After modification, the keratin amino acid sequence of the application is shown as SEQ ID NO.2 (marked as K31-modification), and the recombinant keratin of the application simultaneously maintains the active region of the amino acid sequence shown as SEQ ID NO. 1. The modified keratin sequence is shown in SEQ ID NO.2, and amino acids 56-367 of K31 are reserved.

SEQ ID NO.2 is as follows:

KETMQFLNDRLASYLEKVRQLERDNAELENLIRERSQQQEPLLCPSYQSYFKTIEELQQKILCTKSENARLVVQIDNAKLAADDFRTKYQTELSLRQLVESDINGLRRILDELTLCKSDLEAQVESLKEELLCLKSNHEQEVNTLRCQLGDRLNVEVDAAPTVDLNRVLNETRSQYEALVETNRREVEQWFTTQTEELNKQVVSSSEQLQSYQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESEARYSSQLSQVQSLITNVESQLAEIRSDLERQNQEYQVLLDVRARLECEINTYRSLL ESEDCNL

1.2 preparation of recombinant keratins

1.2.1 amplification of fragments of interest

1) Synthesis of target Gene

The coding region sequence of the recombinant keratin subjected to the preliminary screening is optimized according to the codon usage preference of the escherichia coli in an escherichia coli codon usage preference data table, and the codon which has lower usage frequency and can influence ribosome passing efficiency in the translation process is replaced by the codon with higher usage frequency on the premise of ensuring that the protein sequence of the recombinant keratin is unchanged and only utilizing the degeneracy of the codon, so that the nucleic acid sequence with optimized codon is obtained, and the obtained sequence is shown as SEQ ID NO.3 in a sequence table.

According to the nucleic acid sequence of the target gene, the template gene is obtained through complete gene synthesis and sequencing verification, and is shown as SEQ ID NO. 3.

SEQ ID NO.3 is as follows:

GAAAAAGAAACCATGCAGTTTCTGAATGATCGTCTGGCGAGCTACCTGGAGAAAGTACGCCAGCTGGAACGCGATAATGCCGAACTGGAAAATCTGATTCGCGAACGCAGCCAGCAGCAGGAACCGCTGCTGTGCCCGAGCTACCAGAGCTATTTTAAAACCATTGAAGAACTGCAGCAGAAAATTCTGTGCACCAAAAGCGAAAACGCGCGCCTGGTTGTACAGATTGATAACGCCAAACTGGCGGCCGATGATTTCCGCACCAAATATCAGACCGAACTGAGCCTGCGCCAGCTGGTGGAAAGCGATATTAACGGTCTGCGCCGTATCCTGGATGAACTGACCCTGTGCAAATCCGATCTGGAAGCGCAGGTGGAAAGCCTGAAAGAAGAACTGCTGTGCCTGAAAAGCAACCATGAACAGGAAGTGAACACCCTGCGCTGCCAGCTGGGCGATCGTCTGAATGTGGAGGTGGATGCGGCCCCGACGGTGGATCTGAACCGCGTGCTGAACGAAACCCGTAGCCAATATGAAGCGCTGGTGGAAACCAACCGTCGTGAAGTGGAACAGTGGTTTACGACTCAGACCGAAGAACTGAATAAACAGGTGGTGAGTAGCTCAGAACAGCTGCAGTCATATCAGGCCGAAATCATTGAACTGCGCCGCACCGTGAACGCGCTGGAAATTGAACTGCAGGCCCAGCACAATCTGCGTGATAGCCTGGAAAATACCCTGACCGAAAGCGAAGCGCGCTATAGCAGCCAGCTGAGCCAGGTACAGAGCCTGATCACCAACGTGGAAAGCCAGCTGGCCGAAATTCGCAGCGATCTGGAACGCCAGAACCAGGAATATCAGGTGCTGCTGGATGTGCGCGCGCGCCTGGAATGCGAAATTAACACCTATCGCAGTCTGCTGGAAAGCGAAGACTGCAACCTG

2) Primers are designed according to the nucleic acid sequence of the target gene. PCR amplification was performed using the synthesized template gene as a template and F-KRT31: CCCATATGGAAAATCTGTATTTTCAGGGTGA (SEQ ID NO. 4) and R-KRT31: CGGGATCCCAGGTTGCAGTCTTCGCTTTCCAG (SEQ ID NO. 5) as primers.

PCR reaction system: 10 mu mol/L primer 1 mu L,1 mu L target gene or linearized pET28a-His-TEV vector gene, dNTP (2.5 mM each) 4 mu L,10 XBuffer (Mg-containing) ²⁺ ) 5. Mu.L, 1. Mu. L Pfu DNA Polymerase, water was added to make up to a total volume of 50. Mu.L.

PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 98℃for 10s, annealing at 55℃for 5s, elongation at 72℃for 30s,30 cycles; finally, the extension is carried out for 5min at 72 ℃.

3) And (5) carrying out agarose gel electrophoresis detection on the PCR amplified product. The amplified target fragment (about 1.0 kb) has the same size as the expected fragment, and the target gene fragment and the pET28a-His-TEV vector gene fragment are obtained.

1.2.2 construction of recombinant plasmids

1) Purified target gene fragment 4. Mu.L and pET28a-His-TEV linearized vector 6. Mu.L, 10XCloneEZ Buffer 2. Mu.L, cloneEZ Enzyme 2. Mu.L, deionized water make up 20. Mu.L, were mixed, kept at 22℃for 30 minutes, and then kept on ice for 5 minutes.

2) Taking 100 mu L of DH5 alpha or Top10 escherichia coli competent cells, adding the mixed solution, placing on ice for incubation for 30 minutes under the condition of flick number; heat shock in a 42 ℃ water bath for 90 seconds and incubation on ice for 5 minutes; 1mL of SOC culture medium is added into the cells, and the cells are gently shaken for 1 hour at 37 ℃ with the rotating speed of 200rpm; the cells were collected by centrifugation at 5000rpm for 5 minutes, and resuspended in 100. Mu.L of SOC liquid medium.

3) The cells were spread evenly on antibiotic-containing plates and incubated overnight at 37 ℃.3 positive clones were individually picked and grown overnight at 37℃and 220rpm/min in 5mL LB medium containing 50. Mu.g/mL kanamycin (Kan) antibiotics.

4) 3mL of the extracted plasmid (Tian Gen plasmid extraction kit) is taken from the bacterial liquid of each sample, and the sequence is carried out by sending Nanjing Jinsri, and the plasmid which is correctly sequenced is named as pET28a-His-TEV-KERATIN plasmid.

1.2.3 construction of E.coli genetically engineered bacteria

1) BL21 (DE 3) competent cells were dissolved in ice, and 1. Mu.L of recombinant plasmid was added to BL21 (DE 3) competent cells and gently mixed. After standing in ice for 30min, heat shock was applied at 42℃for 60s and the ice was returned rapidly. Then, 450. Mu.L of LB medium at room temperature was added to the competent cells and the mixture was shaken at 220rpm for 1 hour at 37℃on a shaker.

2) Then, 100. Mu.L of the bacterial liquid was applied to LB plate containing Kan-type antimicrobial property from the tube, and cultured overnight at 37 ℃.2 positive clones were picked from the plate and inoculated into 5mL LB medium containing 50. Mu.g/mL Kan antibiotics, cultured at 37℃and 220rpm for about 3 hours, and the bacterial liquid was taken for seed preservation, with OD 600=0.6. Thus obtaining the escherichia coli genetic engineering bacteria.

1.2.4 Induction of recombinant protein expression

1) 20g of the thus-collected precipitated bacterial cells were resuspended in 100mL of Tris buffer, 50mM Tris (pH 8.0), 500mM NaCl,5%wt Glycerol. The homogenizer was crushed 2 times, mixed well, centrifuged at 4000rpm for 0.5h at 4℃and the precipitate was collected for use as the target protein was present in the inclusion bodies.

2) The collected pellet was dissolved by resuspension with 40mL denaturing buffer (50 mM Tris (pH 8.0), 500mM NaCl,5% glycerol, 20mM beta-mercaptoethanol, 8M urea, balance deionized water). After sufficient lysis, the supernatant was centrifuged at 12000rpm for 1h and transferred to a 5mL HisFF affinity column (equilibrated with denaturing Buffer).

3) The column was washed with 10-fold denaturing buffer. The eluted protein was then collected by stage elution with denaturing buffers containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM and 500mM imidazole, followed by desalting buffer with dialysate (25 mM Tris pH=8.0, 10mM imidazole, 20mM beta-mercaptoethanol, 20mM cysteine, balance deionized water).

4) Adding TEV enzyme into the protein solution obtained in the step 3), carrying out enzymolysis for 2 hours at 25 ℃, removing fusion HIS tag at the N-terminal of the protein, and centrifuging to collect supernatant. Transfer to a 5mL HisFF affinity column (equilibrated with dialysate), rinse with 3-fold dialysate, and collect the flow-through.

5) Dialyzing with dialysate (20 mM beta-mercaptoethanol, 20mM cysteine, and deionized water for 12 hr), and lyophilizing to obtain target protein.

Wherein the recombinant keratin after modification is denoted as K31-modified (or YL 2), and the keratin after no modification is denoted as K31-original (or YL1, i.e. keratin in CN 111202868B).

Example 2 preparation of nanoparticles

Preparing the recombinant keratin YL2 freeze-dried powder in the embodiment 1 into an aqueous solution, wherein the concentration of the recombinant keratin freeze-dried powder is 0.5mg/mL, then adding carboxymethyl chitosan (from Zhejiang Australia biotechnology Co., ltd.) into the aqueous solution, wherein the concentration of the carboxymethyl chitosan is 2mg/mL, the mass ratio of the carboxymethyl chitosan to the recombinant keratin is 4:1, and slightly shaking and dissolving the aqueous solution to obtain the solution of the recombinant keratin and the carboxymethyl chitosan. The solution was aspirated with a syringe and fixed to a syringe pump. 2000mL of hydrochloric acid at pH 3.0 was then pipetted into a 2500mL beaker of ice bath. The solution was injected into the hydrochloric acid solution at a rate of 0.1mL/min by a syringe and a syringe pump under ultrasonic (ultrasonic cytoclasis apparatus with power of 350 w) to obtain nano-suspended particles. And then removing the supernatant by centrifugation (centrifugation parameters: 14000rpm,4 ℃ C., 10 min), and freeze-drying the obtained precipitate by a freeze dryer to obtain nano particles, namely nano particle freeze-dried powder.

The surface morphology of the nanoparticles was observed by scanning electron microscopy. The nanoparticle suspension was first smeared onto tinfoil paper in a petri dish and dried in a freeze dryer. Then spraying a layer of gold on the surface of the dried nano particles, detecting by adopting a scanning electron microscope with 20kv accelerating voltage under the vacuum condition, and collecting scanning images of keratin nano suspended particles, wherein the experimental operation is as follows:

closing the test cover, starting the instrument and waiting for 30min to stabilize the laser;

starting Zetasizer software to prepare a sample;

selecting a sample cell to conform to the sample and test type;

filling the sample cell with the prepared sample;

creating an SOP measurement, selecting 'test-start SOP' from Zetasizer software, selecting the necessary SOP and selecting 'open', then displaying instructions on the screen, and 'measurement show' will be displayed immediately.

The sample is inserted into the sample cell, the 'start' is clicked, the temperature of the sample is waited to be stable, the measurement is executed, the test result is displayed, and after the test is completed, the test result is stored in a file.

The scanning electron microscope picture of the nano particles is shown in figure 1.

As can be seen from fig. 1, the nanoparticles are spherical and have an average size between 150 and 250 nm.

Example 3

Example 3 differs from example 2 only in that: the concentration of the recombinant keratin freeze-dried powder is 0.5mg/mL, then carboxymethyl chitosan is added, the concentration of the carboxymethyl chitosan is 0.5mg/mL, the mass ratio of the carboxymethyl chitosan to the recombinant keratin is 1:1, and the rest conditions are the same.

Example 4

Example 4 differs from example 2 only in that: the concentration of the recombinant keratin freeze-dried powder is 0.5mg/mL, then carboxymethyl chitosan is added, the concentration of the carboxymethyl chitosan is 5mg/mL, the mass ratio of the carboxymethyl chitosan to the recombinant keratin is 10:1, and the rest conditions are the same.

Example 5

Example 5 differs from example 2 only in that: the recombinant keratin lyophilized powder was replaced with 0.5mg/mL of commercially available keratin, with the remaining conditions being the same. Wherein the commercially available keratin is commercially available hydrolyzed keratin peptide (purchased from Hubei's Jian peptide biotechnology Co., ltd.), specifically chicken duck hair hydrolyzed keratin, which is a mixture of keratin amino acid fragments of different molecular weights, with an average molecular weight of 2000 daltons.

Comparative example 1

Comparative example 1 differs from example 2 only in that the concentration of recombinant keratin lyophilized powder is 0.5mg/mL, no carboxymethyl chitosan is added, and the remaining conditions are the same.

Comparative example 2

Comparative example 1 differs from example 2 only in that no recombinant keratin lyophilized powder was added, the carboxymethyl chitosan concentration was 2mg/mL, and the remaining conditions were the same.

Comparative example 3

Comparative example 3 differs from comparative example 1 only in that the recombinant keratin lyophilized powder was replaced with 0.5mg/mL of commercially available keratin, with the remaining conditions being the same. Wherein the commercially available keratin is commercially available hydrolyzed keratin peptide (purchased from Hubei's Jian peptide biotechnology Co., ltd.), specifically chicken duck hair hydrolyzed keratin, which is a mixture of keratin amino acid fragments of different molecular weights, with an average molecular weight of 2000 daltons.

Table 1 parameters of examples 2-5 and comparative examples 1-3 are as follows (nanoparticle preparation)

The commercially available keratins in table 1 are commercially available hydrolyzed keratins (purchased from Hubei's Jian peptide biotechnology Co., ltd.) and are specifically chicken duck hair hydrolyzed keratins, which are a mixture of keratin amino acid fragments of different molecular weights with an average molecular weight of 2000 daltons.

TABLE 2 comparison of parameters for example 6 (preparation of toothpaste)

EXAMPLE 6 preparation of toothpaste

5g of thickener (carrageenan 1g, xanthan gum 1g, cellulose sodium 3 g) and polyethylene glycol-4001 g are dissolved in 15g of glycerin, and flat-permeation stirring is adopted in a premix pot to fully disperse the materials until no caking phenomenon exists, so that a solution A is formed;

Dissolving 0.3g of saccharin sodium, 0.5g of sodium pyrophosphate and 0.3g of sodium benzoate in water in a pre-dissolving pot, stirring to form a solution B, and then conveying the solution B into a glue making water pot to be uniformly stirred;

adding water and 5g of sorbitol into the solution B in a glue making pot, and then adding the solution A to form glue; then adding 2g of the nano particles in the example 2, 3g of sodium lauryl sulfate, 0.5g of bletilla striata extract, 15g of hydrated silica and stirring to obtain a solution C, wherein the total weight is 100g;

and (3) introducing the solution C into a glue weighing pot for weighing, starting a vacuum device of the paste making machine, and controlling the vacuum degree to be-0.05 to-0.03 MPa. Opening a valve to allow the solution C to uniformly enter the paste maker, and opening a scraping plate (the rotating speed is 40-60 r/min) until the solution C is completely entered; when the vacuum degree is rapidly reduced due to the completion of the feeding of the solution C, the glue feeding valve is closed timely, so that the paste is prepared and the toothpaste is formed.

Experimental example

Experimental example 1 wound repair

SD rat femoral artery hemorrhage model was established. The experimental method is as follows:

the experimental SD rats were 25, randomly divided into 5 groups of 5. Control groups (compression hemostasis with medical cotton balls only); applying commercially available keratin at the wound site for hemostasis; applying the recombinant keratin of example 1 to a wound site for hemostasis; applying commercially available carboxymethyl chitosan to the wound site for hemostasis; the nanoparticles of example 2 (i.e., recombinant keratin + carboxymethyl chitosan nanoparticles) were applied to the wound site to stop bleeding (the nanoparticles of example 2 were sprinkled over the wound).

The experiments were conducted after 7 days of feeding in an SPF environment, free diet. Anesthesia was performed using 10% chloral hydrate at the time of the experiment.

After the rats are completely anesthetized, dehairing and skin preparation are carried out in the tested area, the skin is incised layer by layer, the femoral artery is separated, ophthalmic scissors are used for shearing the femoral artery, after the rats naturally bleed for 5 seconds, cotton balls are used for absorbing the bled blood, and then five groups of rats are respectively hemostatic according to groups. The hemostatic time was recorded and the exuded blood was absorbed with a cotton ball weighed in advance and the blood loss was calculated by weighing. The results are shown in FIGS. 2-4.

As can be seen from fig. 2 to 4, when the recombinant keratin of example 1 is applied to a wound site to stop bleeding, the water absorption speed is high, and a gel is formed after the water absorption, so that the wound can be closed. The wound can stop bleeding after 2-3min, and gel blocks are removed after hemostasis, and secondary bleeding is avoided.

When commercially available keratin is applied to a wound to stop bleeding, the water absorption performance is poor, the water absorption is almost free, the bleeding is easy to disperse by arterial massive hemorrhage, the bleeding amount is large, and the bleeding is basically free of a hemostatic effect; after major bleeding, the wound surface was still oozed by observation for 5 min. .

When commercially available carboxymethyl chitosan is applied to the wound to stop bleeding, the carboxymethyl chitosan has better water absorption speed, forms blocky crusts to block the wound, but is fragile; the wound can stop bleeding for 3min, and the blood clot is removed after stopping bleeding, so that secondary bleeding is avoided.

When the nanoparticles of the example 2 (i.e. the recombinant keratin and carboxymethyl chitosan nanoparticles) are applied to the wound to stop bleeding, the nanoparticles rapidly absorb water to form blocky crusts to block the wound, the strength is high, the nanoparticles are not easy to be dispersed by arterial hemorrhage, the required amount of the nanoparticles is small, the bleeding speed is high, femoral artery hemorrhage can be stopped about 1-2min, blood clots are removed after bleeding stopping, and secondary bleeding is avoided.

As can be seen from fig. 2 to 4, the nanoparticles prepared from the recombinant keratin of the present application are remarkable in hemostatic effect.

The hemostatic time of the nanoparticle prepared by the chitosan and the recombinant keratin is greatly shortened, compared with a control group (medical cotton ball) and the commercially available keratin, the hemostatic time can be reduced from 300 seconds to less than 100 seconds, the hemostatic time is one third of the hemostatic time before, and compared with the recombinant keratin and the commercially available carboxymethyl chitosan, the hemostatic time is also improved by about 50 percent. In addition, the hemostatic time corresponds to the amount of blood loss, and the longer the hemostatic time is, the more the amount of blood loss is. When the hemostatic time is shortened, the blood loss is also greatly reduced, and the blood loss is reduced to less than about 2g from 6g in the control group.

Experimental example 2 evaluation of acute wound repair model of rat

The model building method comprises the following steps: 32 SD rats were randomly divided into 4 groups of 8, and anesthetized (0.5 mL/100 g) by intraperitoneal injection of chloral hydrate (10 wt.%). After shaving and conventional sterilization of the back under sterile conditions, 1.5X1.5 cm was created with the spine as midline ² The wound surface is excised from the skin of the size, and the skin around the wound is fixed by using a surgical suture.

The SD four groups of rats were treated as follows: applying the keratin nanoparticles of comparative example 3 at the wound site for hemostasis; applying the recombinant keratin nanoparticles of comparative example 1 at the wound site for hemostasis; applying the carboxymethyl chitosan nanoparticle of comparative example 2 at the wound site to stop bleeding; the nanoparticles of example 2 (i.e., recombinant keratin + carboxymethyl chitosan nanoparticles) were applied at the wound site for hemostasis.

Mice of the experimental group were kept in a sterile environment and the healing process of the wound surface was observed. On days 3, 6, 9, 12, 15 and 18 post-wound, the area of the wound was measured by a method of combining a transparent film with image processing software, and the following formula:

the wound healing rate was calculated. Wherein A is ₀ For the initial area of wound surface A _t Is the wound area on day "t" after the wound. The statistical results are shown in fig. 5 and 6 below.

The rat acute wound repair model proves that: the composite material has obviously better wound healing speed than other groups. As can be seen from fig. 5, the nanoparticles prepared using the recombinant keratin and chitosan (i.e., the nanoparticles of example 2) were best repaired within the same wound restoration period in the case of applying the same particles to the same wound. The minimal wound area was seen very clearly compared to the control group and commercially available keratin. That is, wound recovery was fastest using nanoparticles prepared comprising chitosan and recombinant keratin (i.e., nanoparticles of example 2) during the same period of time. From the healing rate data of fig. 6, it can be seen that the wound healing rate was highest using the nanoparticles prepared with chitosan and recombinant keratin (i.e., the nanoparticles of example 2). The recombinant keratin nanoparticle of comparative example 1 has better wound healing effect and the nanoparticle of example 2 has the best wound healing effect compared to the commercially available keratin nanoparticle (i.e., the nanoparticle of comparative example 3) and the carboxymethyl chitosan nanoparticle of comparative example 2.

Experimental example 3 hydrophilicity test

Taking 3 15mL centrifuge tubes, respectively adding 5g of deionized water into the centrifuge tubes for standby;

taking 0.5g of freeze-dried nano particles in example 2 (namely recombinant keratin and carboxymethyl chitosan nano particle freeze-dried powder), comparative example 1 (recombinant keratin nano particle freeze-dried powder) and comparative example 2 (carboxymethyl chitosan nano particle freeze-dried powder), respectively placing the freeze-dried nano particles into a centrifuge tube, compacting, and enabling the lower layer of the freeze-dried nano particles to just contact with the upper layer of the water surface;

after 1min, obliquely placing the centrifuge tube with a tube orifice downwards, and observing whether water flows out or not, so as to judge the sealing property of the material;

after the sealing test is completed, the freeze-dried nano particles are completely immersed in water, after 1min, the residual water in the tube is poured out, the tube is weighed, and the water absorption capacity is obtained by calculating the difference between the front water weight and the rear water weight. The results are shown in FIG. 7 and Table 3.

As can be seen from FIG. 7, the nanoparticles of comparative example 1, namely, the nanoparticles of recombinant keratin lyophilized powder alone, have a slow water absorption swelling rate, a good sealing property, and a small amount of liquid flows out after inversion. The nanoparticles of comparative example 2, namely the nanoparticles of the chitosan lyophilized powder alone, had poor swelling properties, were rapidly dispersed in water, had poor sealing properties, and had liquid flow out after inversion. The nanoparticle freeze-dried powder of the embodiment 2 contains the nanoparticle of the recombinant keratin and the carboxymethyl chitosan, has high water absorption and swelling speed and good sealing performance, and does not flow out after inversion. The nanoparticle of example 2 has excellent hydrophilic performance and water absorption performance, good solubility and sealing performance, and when used for oral care, when oral problems such as gingival bleeding occur, the material has good water absorption performance, and can quickly absorb water in blood, thereby achieving the effect of enriching platelets and coagulation factors, and further achieving quick hemostasis.

TABLE 3 Table 3

Note that: since the water was directly poured out after the inversion of comparative example 2, the water absorption could not be counted.

As can be seen from fig. 7 and table 3, the nanoparticle lyophilized powder is excellent in hydrophilic property and water absorption property, and the mechanical properties of the composite material are due to a single raw material.

Experimental example 4 evaluation of cell proliferation

The detection is carried out by CCK-8 method. CCK-8 is a rapid and efficient kit for testing cell proliferation. The kit contains WST-8, which is reduced into yellow formazan product with high water solubility by the deoxidizing hydrogenase in the cells under the action of the electron carrier 1-Methoxy PMS. The amount of formazan product produced is proportional to the number of living cells. Thus, this property can be used to directly conduct cell proliferation analysis. The faster the cell proliferates, the darker the color. For the same cells, the shade of color and the number of cells are linear.

The specific method comprises the following steps: cell digestion was performed when the HOEC oral epithelial cells grew to 95%, the cells were centrifuged and collected, and cell suspensions were prepared with serum-containing DMEM medium and cell counts were performed. In a 96-well plate according to 5X 10 ³ cell density seeding of cells/well, volume of cell culture solution was 100 μl/well. Comparative example 3 (commercially available keratin), comparative example 2 (only chitosan), comparative example 1 (only recombinant keratin), example 2 (recombinant keratin) White + chitosan), the four groups of particles are respectively provided with concentration gradients: 0.005, 0.01, 0.05, 0.1, 0.14, 0.16, 0.2mg/mL. After the cells to be cultured adhere to the wall, HOEC cells are treated according to the set groups and concentrations. The data acquisition time was set to 0h, 24h, 48h, after the incubation time was reached, the liquid was changed, 10. Mu.L of CCK-8 reagent and DMEM medium were added to each well, and incubation was continued for 2 hours. The absorbance of each hole at 450nm is detected, the data are counted and analyzed, and a cell growth curve is drawn. The results are shown in FIG. 8.

The results show that: the ability of the nanoparticles of example 2 to promote epithelial cell proliferation is significantly better than comparative examples 1-3.

As can be seen from fig. 8, the nanoparticle containing recombinant keratin and chitosan of example 2 has a better effect of promoting epithelial cell proliferation and thus wound healing than that of comparative example 3 (nanoparticle of commercially available keratin), comparative example 2 (nanoparticle containing only chitosan), and comparative example 1 (nanoparticle containing only recombinant keratin). The re-epithelialization of the wound surface comprises three stages of proliferation, migration and differentiation of epithelial cells, wherein the proliferation of the epithelial cells is a precondition of migration and differentiation, and newly proliferated cells continuously migrate to the wound surface and pave wound bed until the epidermization is completed. Example 4 the pro-healing efficacy of materials was demonstrated from the cellular level by comparing the pro-epithelial cell proliferation capacity of different materials. After 24 hours of culture of the epithelial cells in the cell culture medium containing the recombinant keratin-and chitosan-containing nanoparticles of example 2, the OD was significantly higher than in the other treated groups, indicating that the cell numbers were significantly higher than in the other groups under these conditions, and the faster the cell division proliferation was. Is more beneficial to the subsequent migration and differentiation of cells and accelerates the healing of wound surfaces.

Experimental example 5 toothpaste repairing hemostatic Effect

50 subjects with gingival bleeding and 50 subjects with dental ulcers were selected for the test, and the oral gums of the subjects were photographed. Dividing gingival bleeding subjects into two groups, one group of 25 persons; the subjects with canker sore were equally divided into two groups, one group of 25 persons, one group using the toothpaste prepared in example 6 and the control group using a common commercially available toothpaste. The subjects brushed their teeth 1 time, 3 minutes/time each in the morning and evening, and were not spicy during use for one week. Other oral cavity cleaning products cannot be used in the test period of each group of subjects, and after one week of use, the oral gums of the subjects are photographed, and the effects before and after use are compared. The results are shown in Table 4, where the clinical observations herein are statistics of the effectiveness of the patient after use.

TABLE 4 Table 4

Wherein:

the effect is obvious: the symptoms and pain completely disappear within one week;

the effect is improved: the symptoms and pain are obviously relieved within one week;

the effect is not obvious: within a week, the symptoms and pain are not relieved or aggravated.

As is clear from Table 4, the toothpaste of example 6 of the present application was remarkable in the treatment of gingival bleeding and oral ulcer under the same conditions as the commercially available common toothpaste, and the symptoms and pain sensation were substantially disappeared within one week. Illustrating that the nanoparticle of example 2 containing recombinant keratin and chitosan shows an effect of rapidly promoting wound healing and wound repair when applied to toothpaste. The toothpaste of the embodiment 6 of the application can quickly promote wound healing, promote wound repair and has the functions of stopping bleeding and diminishing inflammation.

Sequence listing

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Claims (15)

1. A nanoparticle for an oral care product comprising keratin and chitosan; the amino acid sequence of the keratin is shown as SEQ ID NO. 2 or SEQ ID NO. 1;

the mass ratio of the chitosan to the keratin is (1-10): 1, wherein the chitosan is carboxymethyl chitosan.

2. The nanoparticle according to claim 1, wherein,

the mass ratio of the chitosan to the keratin is 4:1.

3. The nanoparticle according to claim 1 or 2, wherein,

the keratin is prepared by a mechanical method, an acid-base treatment method, a reduction method, an oxidation method, an electrochemical reduction method, a cuprammonium solution method, a metal salt method, a biological method or genetic engineering.

4. The nanoparticle according to claim 3, wherein,

the keratin is genetically prepared keratin.

5. A method of preparing the nanoparticle of any one of claims 1-4, comprising the steps of:

dissolving keratin and chitosan in water to obtain an aqueous solution containing keratin and chitosan;

injecting the aqueous solution into hydrochloric acid solution to obtain nano suspension particles;

and centrifuging and freeze-drying the nano-suspension particles to obtain the nano-particles.

6. The method according to claim 5, wherein,

the concentration of the keratin is 0.1-1mg/mL, and the concentration of the chitosan is 0.5-10mg/mL.

7. Use of a nanoparticle according to any one of claims 1 to 4, or a nanoparticle prepared by a method according to claim 5 or 6, in an oral care product.

8. The use according to claim 7, wherein,

the oral care product is a toothpaste, mouthwash, tooth powder, dentifrice, floss or denture care product.

9. A toothpaste comprising the nanoparticle of any one of claims 1 to 4, or the nanoparticle prepared by the method of claim 5 or 6.

10. The toothpaste according to claim 9, wherein,

the nano particles are 1 to 10 parts by weight with respect to 100 parts by weight of the toothpaste.

11. The toothpaste according to claim 9, wherein,

the nano particles are 1 to 3 parts by weight with respect to 100 parts by weight of the toothpaste.

12. The toothpaste according to claim 9, wherein,

the toothpaste also comprises one or more of bletilla striata extract, humectant, friction agent, stabilizer, foaming agent, pH buffer, thickener, sweetener and antiseptic.

13. The toothpaste according to claim 12, wherein,

for 100 parts by weight of toothpaste, 0.01-1 part by weight of bletilla striata extract, 12-30 parts by weight of humectant, 10-25 parts by weight of friction agent, 0.1-2 parts by weight of stabilizer, 1-5 parts by weight of foaming agent, 0.01-0.5 part by weight of pH buffer, 1-8 parts by weight of thickening agent, 0.01-0.5 part by weight of sweetener and 0.01-0.5 part by weight of preservative.

14. The toothpaste according to claim 13, wherein,

the humectant is one or more than two of sorbitol, glycerol and polyethylene glycol;

The friction agent is hydrated silica;

the stabilizer is polyethylene glycol-400;

the foaming agent is sodium lauryl sulfate;

the pH buffering agent is sodium pyrophosphate;

the thickener is one or more than two selected from cellulose gum, xanthan gum, carrageenan, deer pectin, guar gum and carrageenan;

the sweetener is sodium cyclamate and/or saccharin sodium;

the preservative is one or more than two selected from sodium benzoate, methylisothiazolinone and potassium sorbate.

15. A method of preparing a toothpaste according to any one of claim 9 to 14,

dissolving a thickening agent and a stabilizing agent in a humectant, and stirring to form a solution A;

dissolving sweetener, pH buffer and preservative in water, and stirring to form solution B;

adding water and humectant into the solution B, and then adding the solution A to form glue; then adding nano particles, a foaming agent, a bletilla striata extract and a friction agent; stirring to obtain a solution C;

and (3) introducing the solution C into a paste making machine to make paste, thereby obtaining the toothpaste.