Preparation of silk fibroin oligopeptides based on high-pressure microfluidization technology and its application in toothpaste

Abstract

The present invention relates to a preparation of silk fibroin oligopeptide based on high-pressure microfluidization technology and its application in toothpaste. The present invention fully utilizes the advantages of excellent biocompatibility of silk fibroin, and by subjecting it to oxidative degradation, high-pressure microfluidization and other process treatments, and strictly controlling the homogenization pressure during the high-pressure microfluidization treatment, the particle size of the silk fibroin oligopeptide can be greatly reduced and its solubility performance can be improved. The toothpaste prepared using the mineralized material of the present invention can effectively reduce the degree of caries, increase the calcium / phosphorus molar ratio of the enamel surface, improve the repair effect on the enamel after demineralization, and help inhibit the progress of demineralization in patients with enamel caries and promote remineralization. In addition, the present invention uses silkworm cocoons from natural sources as the main preparation raw material, does not add any fluorine-containing chemical components, has low cost and good safety, can effectively avoid the various toxic and side effects caused by traditional fluorides in the treatment of dental caries, and has great market prospects and social significance.

Description

Technical Field

[0001] The present invention belongs to the field of application of natural products, and specifically relates to a preparation method of silk fibroin oligopeptide based on high-pressure microfluidization technology and its application in toothpaste, and especially relates to a preparation method of hydroxyl cellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidization technology and its application in toothpaste preparation. Background Art

[0002] Mature tooth enamel is known for its highly mineralized nature. Its primary component is inorganic minerals, the core of which is hydroxyapatite. Hydroxyapatite molecules are arranged in an orderly pattern within the enamel, parallel and perpendicular to its surface, forming a unique prismatic structure. In a healthy state, the demineralization and remineralization of hydroxyapatite maintain a dynamic equilibrium. However, if this balance is disrupted, tooth enamel undergoes demineralization, potentially leading to the development of early dental caries.

[0003] In the clinical practice of dental caries treatment, fluoride is often used as a traditional means for the remineralization treatment of enamel caries. However, it is worth noting that excessive use of fluoride may cause toxic side effects such as dental fluorosis, skeletal fluorosis, and even acute fluoride poisoning. To this end, researchers have explored an innovative early-stage dental caries treatment method - bionic enamel remineralization technology. The core of this method is to utilize hydroxyapatite materials, whose mineral composition is highly similar to that of dental hard tissue and has excellent bioactivity and biocompatibility. When hydroxyapatite is used alone for enamel remineralization, it mainly relies on its nano-scale tiny size characteristics to fill the demineralized areas. Although this method shows certain potential in promoting the remineralization of caries lesions, its remineralization effect under its sole action is still relatively limited. Summary of the Invention

[0004] The purpose of the present invention is to address the problems of a series of toxic side effects easily produced by the use of fluoride for remineralization treatment in the prior art and the low remineralization effect of hydroxyapatite. Based on the silk fibroin subjected to oxidative degradation and high-pressure microfluidization treatment to obtain silk fibroin oligopeptides, and then subjected to remineralization treatment with hydroxyapatite, a toothpaste with excellent solubility, good safety and effective anti-caries effect is developed.

[0005] To achieve the above object, the present invention is achieved by the following means:

[0006] The first aspect of the present invention provides a method for preparing a hydroxycellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidization technology, comprising the following steps:

[0007] (1) preparing a silk fibroin solution from silkworm cocoons and adjusting the silk fibroin concentration to 50-300 mg / mL;

[0008] (2) adding a reducing agent and continuing the stirring reaction, and then adding an oxidizing agent to carry out an oxidative degradation reaction under stirring to obtain a silk fibroin oligopeptide intermediate solution;

[0009] (3) placing the silk fibroin oligopeptide intermediate solution in a high-pressure microfluidizer for circulating homogenization;

[0010] (4) ultrafiltration of the circulated homogenized product to obtain a silk fibroin oligopeptide solution, which is then freeze-dried to obtain a silk fibroin oligopeptide;

[0011] (5) The silk fibroin oligopeptide obtained in step (4) is mixed with hydroxyapatite and dissolved in a saturated calcium nitrate solution, and a sodium phosphate solution is added dropwise under stirring to carry out a mineralization reaction; after the mineralization reaction is completed, the pH is adjusted to neutral, the precipitate is collected by centrifugation, dried, ultrafinely ground and sieved to obtain the product.

[0012] Preferably, the silk fibroin solution in step (1) is prepared by the following steps:

[0013] (1.1) Cut the cocoons into pieces and add them to a sodium carbonate solution for boiling, followed by soaking and washing with water; repeat the boiling and washing process 1-5 times, and dry them to obtain degummed silk;

[0014] (1.2) Dissolving the degummed silk in lithium bromide solution, filtering to remove large particles of impurities, and then centrifuging to obtain the supernatant to obtain a crude silk fibroin extract;

[0015] (1.3) The crude silk fibroin extract is transferred to a dialysis bag for dialysis; when the water level in the dialysis bag no longer changes, the dialysis bag containing the crude silk fibroin extract is placed in a polyethylene glycol solution for concentration to obtain the product.

[0016] Preferably, the concentration of the sodium carbonate solution in step (1.1) is 0.1-5.0 wt %; more preferably, the concentration of the sodium carbonate solution is 3 wt %.

[0017] Preferably, the boiling time in step (1.1) is 10-50 min, the washing time is 10-30 min, and the boiling and washing processes are repeated 2-4 times; more preferably, the boiling time is 30 min, the washing time is 20 min, and the boiling and washing processes are repeated 3 times.

[0018] Preferably, the concentration of the lithium bromide solution in step (1.2) is 5-15 mol / L, and the temperature of the lithium bromide solution is 60-90°C; more preferably, the concentration of the lithium bromide solution is 9 mol / L, and the temperature of the lithium bromide solution is 70°C.

[0019] Preferably, in step (1.2), the mass ratio of the degummed silk to the lithium bromide solution is 1:3-10; more preferably, the mass ratio of the degummed silk to the lithium bromide solution is 1:5.

[0020] Preferably, the centrifugal speed in step (1.2) is 5000-15000 rpm, and the time is 10-30 min; more preferably, the centrifugal speed is 8000 rpm, and the time is 20 min.

[0021] Preferably, the relative molecular mass retained by the dialysis bag in step (1.3) is 5-15 kDa, and the dialysis time is 1-5 days; more preferably, the relative molecular mass retained by the dialysis bag is 8 kDa, and the dialysis time is 3 days.

[0022] Preferably, the concentration of the polyethylene glycol solution in step (1.3) is 5-20%, and the concentration time is 10-30 hours; more preferably, the concentration of the polyethylene glycol solution is 15%, and the concentration time is 24 hours.

[0023] Preferably, the reducing agent in step (2) is selected from vitamin C; more preferably, the reducing agent is selected from vitamin C with a concentration of 5 mmol / L.

[0024] Preferably, the volume ratio of the reducing agent to the silk fibroin solution is 0.0005-0.003:1.

[0025] Preferably, the oxidant in step (2) is selected from hydrogen peroxide; more preferably, the oxidant is selected from hydrogen peroxide with a concentration of 5 mmol / L.

[0026] Preferably, the volume ratio of the oxidant to the silk fibroin solution is 0.001-0.01:1.

[0027] Preferably, the stirring reaction in step (2) is carried out at a temperature of 40-60° C. and for a time of 20-60 min; more preferably, the stirring reaction is carried out at a temperature of 45-55° C. and for a time of 40-50 min.

[0028] Preferably, the temperature of the oxidative degradation reaction in step (2) is 30-60° C., and the time is 10-60 min; more preferably, the temperature of the oxidative degradation reaction is 40-50° C., and the time is 20-40 min.

[0029] Preferably, the pressure of the cyclic homogenization in step (3) is 70-120 MPa, and the number of times is 1-5 times; more preferably, the pressure of the cyclic homogenization is 80-110 MPa, and the number of times is 2-4 times.

[0030] Preferably, the ultrafiltration in step (4) is specifically as follows: the reaction product is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa, and the effluent is taken; then the product is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution.

[0031] Preferably, the mass ratio of the silk fibroin oligopeptide to hydroxyapatite in step (5) is 1:1-3; more preferably, the mass ratio of the silk fibroin oligopeptide to hydroxyapatite is 1:2-2.5.

[0032] Preferably, in step (5), the mass volume ratio of the silk fibroin oligopeptide to the saturated calcium nitrate solution is 0.01-0.1:1; more preferably, the mass volume ratio of the silk fibroin oligopeptide to the saturated calcium nitrate solution is 0.02-0.05:1.

[0033] It should be understood that, unless otherwise specified, the "mass-to-volume ratio" in the context of the present invention should be understood in a conventional manner in the art, that is, the ratio of the mass of the solid substance (in g) to the liquid volume (in mL). For example, when the "mass-to-volume ratio of silk fibroin oligopeptide to saturated calcium nitrate solution is 0.04:1", if the amount of silk fibroin oligopeptide added is 4 g, the amount of saturated calcium nitrate solution added is 100 mL.

[0034] Preferably, in step (5), the mass ratio of the silk fibroin oligopeptide to the sodium phosphate in the sodium phosphate solution is 0.1-1:1; more preferably, the mass ratio of the silk fibroin oligopeptide to the sodium phosphate in the sodium phosphate solution is 0.2-0.5:1.

[0035] Preferably, the temperature of the mineralization reaction in step (5) is 30-50°C, and the time is 24-120h; more preferably, the temperature of the mineralization reaction is 35-45°C, and the time is 48-96h.

[0036] The second aspect of the present invention provides a hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the above preparation method.

[0037] The third aspect of the present invention provides the use of the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the above preparation method in the preparation of toothpaste.

[0038] A fourth aspect of the present invention provides a toothpaste comprising the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the above preparation method, and auxiliary materials.

[0039] Preferably, the auxiliary materials include one or more of calcium carbonate, sorbitol, glycerin, hydrated silica, saccharin sodium, xanthan gum, sodium lauryl sulfate, cellulose gum, parahydroxybenzoate, and water.

[0040] Preferably, the toothpaste comprises the following ingredients in the following mass percentages:

[0041] 2.5-10% hydroxyapatite-silk fibroin oligopeptide mineralized material, 10-20% calcium carbonate, 10-20% sorbitol, 5-10% glycerol, 1-5% hydrated silica, 1-5% saccharin sodium, 1-3% xanthan gum, 0.5-3% sodium lauryl sulfate, 0.5-2% cellulose gum, 0.05-0.5% parahydroxybenzoate, and the balance is water; the total mass percentage of each component is 100%.

[0042] Proteins play a central role in the ameloblast-guided biomineralization of enamel. They are closely linked to the remineralization of hydroxyapatite, significantly enhancing its structural and mechanical properties. Key to this process lies in the ability of proteins to precisely regulate the directional growth of hydroxyapatite crystals, thereby forming a unique hydroxyapatite-protein composite remineralization material. Silk fibroin, a natural polymer extracted from silk fibers, exhibits excellent biocompatibility, but its high molecular weight results in poor solubility.

[0043] The present invention utilizes specific oxidative degradation and high-pressure microfluidization technology to treat silk fibroin; wherein high-pressure microfluidization is a good physical modification method, which sprays silk fibroin at high speed and collides it on an impact ring, generating shearing, cavitation and other effects to break it up. During the sample processing process, silk fibroin is simultaneously subjected to multiple mechanical forces, thereby inducing changes in its macromolecular structure and properties. By subjecting silk fibroin to high-pressure microfluidization technology, its particle size can be significantly reduced, thereby greatly improving the solubility of the protein. This improvement greatly promotes the effective remineralization process of silk fibroin and hydroxyapatite. As a result, the mineralized material has excellent adsorption capacity, can fit tightly to the surface of damaged enamel crystals, guide and arrange into bundles of enamel-like crystal layers, and effectively achieve the repair and regeneration of tooth enamel.

[0044] The present invention makes full use of the advantages of the excellent biocompatibility of silk fibroin. By subjecting it to oxidative degradation, high-pressure microfluidization and other process treatments, and strictly controlling the homogenization pressure during the high-pressure microfluidization treatment, the particle size of the silk fibroin oligopeptide can be greatly reduced, its solubility performance can be improved, and it can help promote the mineralization fusion with hydroxyapatite. The toothpaste prepared using the mineralized material of the present invention can effectively reduce the degree of caries, improve the calcium / phosphorus molar ratio of the enamel surface, improve the repair effect on the enamel after demineralization, and at the same time help to inhibit the progress of demineralization in patients with enamel caries and promote remineralization. In addition, the present invention uses silkworm cocoons from natural sources as the main preparation raw materials, does not add any fluorine-containing chemical components, has low cost and good safety, can effectively avoid the various toxic and side effects caused by traditional fluorides in the process of treating dental caries, and has great market prospects and social significance. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0046] Example 1

[0047] A hydroxycellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidics technology, wherein the preparation method comprises the following steps:

[0048] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0049] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 50°C for 45 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 45°C with stirring for 30 min to obtain a silk fibroin oligopeptide intermediate solution.

[0050] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and homogenized three times at 95 MPa.

[0051] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0052] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0053] Furthermore, the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following ingredients in the following mass percentages: 5% hydroxyapatite-silk fibroin oligopeptide mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0054] Example 2

[0055] A hydroxycellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidics technology, wherein the preparation method comprises the following steps:

[0056] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0057] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 45°C for 50 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 40°C with stirring for 40 min to obtain a silk fibroin oligopeptide intermediate solution.

[0058] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and circulated and homogenized for 4 times at 80 MPa.

[0059] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0060] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0061] Furthermore, the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following ingredients in the following mass percentages: 5% hydroxyapatite-silk fibroin oligopeptide mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0062] Example 3

[0063] A hydroxycellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidics technology, wherein the preparation method comprises the following steps:

[0064] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0065] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 55°C for 40 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 50°C with stirring for 20 min to obtain a silk fibroin oligopeptide intermediate solution.

[0066] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and homogenized twice at 110 MPa.

[0067] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0068] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0069] Furthermore, the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following ingredients in the following mass percentages: 5% hydroxyapatite-silk fibroin oligopeptide mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0070] Comparative Example 1

[0071] A mineralized material, the preparation method of which comprises the following steps:

[0072] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0073] (2) Take 1 L of silk fibroin solution, place it in a high-pressure microfluidizer, and circulate homogenization for 3 times at 95 MPa.

[0074] (3) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0075] (4) 3 g of the silk fibroin oligopeptide obtained in step (3) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0076] Furthermore, the mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following ingredients in the following mass percentages: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% saccharin sodium, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% parahydroxybenzoate, and the balance is water.

[0077] Comparative Example 2

[0078] A mineralized material, the preparation method of which comprises the following steps:

[0079] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0080] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 50°C for 45 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 45°C with stirring for 30 min to obtain a silk fibroin oligopeptide intermediate solution.

[0081] (3) The silk fibroin oligopeptide intermediate solution is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then the solution is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain the silk fibroin oligopeptide.

[0082] (4) 3 g of the silk fibroin oligopeptide obtained in step (3) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0083] Furthermore, the mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following ingredients in the following mass percentages: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% saccharin sodium, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% parahydroxybenzoate, and the balance is water.

[0084] Comparative Example 3

[0085] A mineralized material, the preparation method of which comprises the following steps:

[0086] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0087] (2) 1 L of silk fibroin solution was placed in a high-pressure microfluidizer and homogenized three times at 95 MPa.

[0088] (3) After the cyclic homogenization treatment is completed, 2 mL of 5 mmol / L vitamin C is added and stirred at 50°C for 45 minutes; then 5 mL of 5 mmol / L hydrogen peroxide is added and oxidative degradation reaction is carried out at 45°C with stirring for 30 minutes to obtain a silk fibroin oligopeptide intermediate solution.

[0089] (4) The supernatant of the silk fibroin oligopeptide intermediate solution was first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate was taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain the silk fibroin oligopeptide.

[0090] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0091] Furthermore, the prepared mineralized material is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following components in percentage by weight: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0092] Comparative Example 4

[0093] A mineralized material, the preparation method of which comprises the following steps:

[0094] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0095] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 50°C for 45 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 45°C with stirring for 30 min to obtain a silk fibroin oligopeptide intermediate solution.

[0096] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and circulated and homogenized three times at 65 MPa.

[0097] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0098] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0099] Furthermore, the prepared mineralized material is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following components in percentage by weight: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0100] Comparative Example 5

[0101] A mineralized material, the preparation method of which comprises the following steps:

[0102] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0103] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 50°C for 45 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 45°C with stirring for 30 min to obtain a silk fibroin oligopeptide intermediate solution.

[0104] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and circulated and homogenized three times at 125 MPa.

[0105] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0106] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0107] Furthermore, the prepared mineralized material is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following components in percentage by weight: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0108] Comparative Example 6

[0109] A mineralized material, the preparation method of which comprises the following steps:

[0110] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 3wt% sodium carbonate solution, boil for 30 minutes, and then soak and wash them with distilled water for 20 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 70℃, 9.0mol / L lithium bromide solution (the mass ratio of degummed silk to lithium bromide solution is 1:5), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 8000rpm for 20 minutes to obtain a crude silk fibroin extract; transfer the crude silk fibroin extract to a dialysis bag with a cutoff relative molecular mass of 8kDa, and dialyze with ultrapure water for 3 days until the water level in the dialysis bag no longer changes; further, place the dialysis bag containing the crude silk fibroin extract in a 15% polyethylene glycol solution and concentrate it for 24 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 100mg / mL.

[0111] (2) 1 L of silk fibroin solution was added with 2 mL of 5 mmol / L vitamin C and stirred at 50°C for 45 min; then 5 mL of 5 mmol / L hydrogen peroxide was added and oxidative degradation reaction was carried out at 45°C with stirring for 30 min to obtain a silk fibroin oligopeptide intermediate solution.

[0112] (3) The concentration of the silk fibroin oligopeptide intermediate solution was adjusted to 100 mg / mL, and the solution was placed in a high-pressure microfluidizer and circulated and homogenized three times at 155 MPa.

[0113] (4) The supernatant of the product after the circulation homogenization is first separated by an ultrafiltration membrane with a molecular weight of 2 kDa to remove macromolecular substances, and the effluent is taken; then it is separated by an ultrafiltration membrane with a molecular weight of 0.75 kDa, and the retentate is taken to obtain a 0.75-2 kDa silk fibroin oligopeptide solution, which is then freeze-dried to obtain silk fibroin oligopeptide.

[0114] (5) 3 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 7 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 20 mL of 3 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring. The mixture was subjected to a mineralization reaction at 40° C. for 72 h. After the mineralization reaction, the pH was adjusted to 7.0, the precipitate was collected by centrifugation, dried, and ultrafinely ground to pass through a 20,000 mesh sieve.

[0115] Furthermore, the prepared mineralized material is used to prepare toothpaste according to conventional methods in the art, and the toothpaste comprises the following components in percentage by weight: 5% mineralized material, 15% calcium carbonate, 15% sorbitol, 7% glycerol, 3% hydrated silica, 2% sodium saccharin, 2% xanthan gum, 2% sodium lauryl sulfate, 1% cellulose gum, 0.2% paraben, and the balance is water.

[0116] Verification Example 1

[0117] Solubility is the most important functional property of protein oligopeptide. When silk fibroin oligopeptide is treated with high-pressure microfluidization, its particle size becomes smaller, solubility increases, and the corresponding specific surface area increases, which is beneficial to the subsequent mineralization step with hydroxyapatite. To this end, the silk fibroin oligopeptide samples in Examples 1-3 and Comparative Examples 1-6 were taken for solubility performance testing, and the specific steps were as follows: 0.2g silk fibroin oligopeptide sample was dissolved in 20mL deionized water, stirred for 30min, adjusted to neutral pH, stirred for 30min, centrifuged at 5000rpm for 10min, and an appropriate amount of supernatant was diluted. The protein content in the supernatant was determined by the Folin phenol method, with dioctanoic acid (BCA) as the standard protein. Wherein the solubility was calculated according to the following formula:

[0118] Solubility = protein content in supernatant / total protein content in sample × 100%.

[0119] The test results are shown in Table 1 below. The results show that under the conditions of Comparative Examples 1-3, the solubility of silk fibroin oligopeptide is lower than that of Examples 1-3. The silk fibroin oligopeptide treated with high-pressure microjet can change the aggregation of the oligopeptide, refine the particle size, and significantly improve the solubility. In Comparative Example 1, the silk fibroin was not subjected to oxidative degradation and was directly treated with high-pressure microjet, and the solubility was low. The reason is that the silk fibroin is a macromolecule and is difficult to be converted into silk fibroin oligopeptide without oxidative degradation, and the solubility of macromolecule silk fibroin is poor. Since Comparative Example 2 was not treated with high-pressure microjet, its solubility was significantly lower than that of Examples 1-3. In Comparative Example 3, the silk fibroin was first treated with high-pressure microjet and then subjected to oxidative degradation of the silk fibroin oligopeptide, and the solubility was low. The reason is that the silk fibroin is a macromolecule and is limited by the molecular weight of the material. The high-pressure microjet treatment has poor processing ability for macromolecules and it is difficult to evenly disperse and dissolve the macromolecules in water. First, oxidative degradation, then high-pressure micro-jet treatment, after the protein can be fully oxidatively degraded into small molecule oligopeptides, high-pressure micro-jet treatment is then used to change the degree of refinement of the peptide, so that the oligopeptide molecules are more evenly and delicately dispersed in water, thereby improving solubility. And when high-pressure micro-jet treatment, homogenization pressure also has a key impact on the solubility of silk fibroin. Specifically, in Comparative Example 4, under a relatively low homogenization pressure of 65MPa, the silk fibroin oligopeptide has a smaller particle size under the dual action of shear force and impact force, but its solubility is not high, which is not enough to disperse and dissolve well in the water system. When the homogenization pressure is too large and increases to 125MPa (Comparative Example 5) and 155MPa (Comparative Example 6), the solubility of the silk fibroin oligopeptide decreases. This is because the excessive expansion of the protein molecules in the unfolding process causes the hydrophobic interaction and electrostatic attraction between molecules to enhance and produce aggregates, thereby reducing its solubility. It is clear through a large number of studies that when the homogenization pressure reaches 95MPa, the solubility of the silk fibroin oligopeptide is the best.

[0120] Table 1 Solubility of silk fibroin oligopeptides in Examples 1-3 and Comparative Examples 1-6

[0121]

[0122]

[0123] Verification Example 2

[0124] The mineralized materials prepared in Examples 1-3 and Comparative Examples 1-6 were respectively used to study their effects on the remineralization of tooth enamel.

[0125] The enamel samples were prepared as follows: fresh bovine teeth were obtained, soft tissue removed, the crown and root separated using a hard tissue slicer, the crown surface polished to a smooth surface using a grinder and polisher, ultrasonically cleaned for 30 minutes, rinsed with deionized water, placed in a 0.9% sodium chloride solution, and stored at 4°C until ready for use. The enamel samples were observed under a microscope, and intact enamel samples free of cracks, caries, or fluorosis were selected, rinsed with deionized water, and air-dried. The samples were then randomly divided into a negative group (no demineralization treatment), a demineralization group, an example group, and a comparative example group.

[0126] The demineralization and remineralization experimental procedures were as follows: all enamel samples were immersed in demineralization solution (50 mmol / L acetic acid, 2.2 mmol / L potassium dihydrogen phosphate solution, 2.2 mmol / L calcium chloride dihydrate solution, 0.5 mol / L sodium bicarbonate solution, pH 4.5), placed in a 37°C shaker at 100 rpm for demineralization for 72 h, and rinsed with deionized water to form artificial caries.

[0127] Artificially demineralized enamel samples were immersed in a 10 mg / mL solution of the mineralizing material prepared in each group for 20 minutes. The samples were then rinsed with deionized water and placed in an acidic buffer solution (50 mmol / L acetic acid, 2.25 mmol / L calcium chloride dihydrate solution, 0.5 mol / L sodium bicarbonate solution, 1.5 mmol / L potassium dihydrogen phosphate solution, 130 mmol / L potassium chloride solution, pH 5.0) for 30 minutes. The samples were then rinsed with deionized water and dried with filter paper. Finally, the samples were immersed in a neutral buffer solution (20 mmol / L HEPES buffer, 2.25 mmol / L calcium chloride dihydrate, 1.5 mmol / L potassium dihydrogen phosphate solution, 130 mmol / L sodium chloride solution, pH 7.0) for 10 minutes. The samples were rinsed with deionized water and dried with filter paper. This process was repeated six times daily for 10 days, with the samples remaining in the neutral buffer solution overnight. The reagents were changed daily. This was done to remineralize artificial caries.

[0128] After remineralization, enamel samples were embedded and cut longitudinally along the center of the fenestration under a steady water flow using a hard tissue cutter. Thin slices of 110 μm were obtained, resulting in a complete, flat surface. The sections were then stained with 0.1 mmol / L rhodamine B fluorescent dye for 1 hour. Excess dye was rinsed with distilled water, dried, and sealed with glycerol. The sections were then placed under a laser scanning confocal microscope and scanned at an excitation wavelength of 529 nm. Rhodamine B penetration was observed at 10x magnification. Scanned images were processed and analyzed using Image J v1.8.0 software and expressed as total fluorescence. Laser scanning confocal microscopy is an advanced method for observing demineralization and remineralization of dental hard tissues. Measuring total fluorescence in carious lesions using laser scanning confocal microscopy provides insights into the demineralization and remineralization status of the specimen. Tooth enamel itself does not fluoresce; therefore, it must be stained with a dye such as rhodamine B. Fluorescence emission is then generated by laser scanning confocal microscopy, which is then quantitatively analyzed using Image J software. Fluorescent dye molecules can penetrate into the micropores of demineralized enamel, and the demineralization degree of the demineralized area is proportional to the total fluorescence amount.

[0129] The results are shown in Table 2 below. The results demonstrate that the crystal structures formed during the mineralization process of the samples prepared in Examples 1-3 of the present invention effectively hindered further penetration of fluorescent dye molecules into the micropores of demineralized enamel in tooth tissue, a phenomenon that indicates a reduction in the severity of caries lesions. This not only confirms the positive effect of the present examples in promoting enamel remineralization, but also strongly demonstrates their effectiveness and potential as a caries prevention strategy.

[0130] Oxidative degradation of silk fibroin can degrade the macromolecular silk fibroin into small molecular silk fibroin oligopeptides, which is convenient for subsequent mineralization with hydroxyapatite. Comparative Example 1 is poorer than Examples 1-3 in the functional effect of toothpaste prepared with the silk fibroin oligopeptides without oxidative degradation. Silk fibroin is first oxidatively degraded and then subjected to high-pressure microfluidization treatment. The purpose is to first degrade the macromolecular silk fibroin into small molecular silk fibroin oligopeptides. Since the peptide segments have spontaneous aggregation, after being subjected to high-pressure microfluidization treatment, the aggregation of the oligopeptides can be changed and more loosely distributed in the solution, which is convenient for subsequent mineralization with hydroxyapatite. If silk fibroin is first subjected to high-pressure microfluidization treatment, due to its too large molecular weight, the protein particle size is difficult to refine (Comparative Example 3), and the functional effect of toothpaste prepared with it is poorer than Examples 1-3.

[0131] Table 2 Effects of Examples 1-3 and Comparative Examples 1-6 on Enamel Remineralization

[0132]

[0133] Furthermore, the surface elemental composition of remineralized enamel was characterized using an FEI Nova Nano SEM 450 field emission scanning electron microscope. The calcium-to-phosphorus ratio was calculated based on the calcium-to-phosphorus content obtained through energy spectrum analysis to determine the degree of demineralization. The lower the calcium-to-phosphorus ratio, the more pronounced the demineralization and the more severe the acid erosion of the enamel. The test results are shown in Table 3 below. The results show that after repair with the toothpastes of Examples 1-3, the calcium / phosphorus molar ratio on the enamel surface was increased, demonstrating that the embodiments of the present invention have a restorative effect on demineralized enamel.

[0134] Table 3 Calcium / P molar ratio of tooth enamel surface in Examples 1-3 and Comparative Examples 1-6

[0135]

[0136] Verification Example 3

[0137] The toothpastes prepared in Examples 1-3 and Comparative Examples 1-6 were used to study their repairing effects on demineralized chalky plaques on tooth enamel. The specific steps were as follows: 100 subjects were randomly divided into 10 groups, namely a control group (using ordinary toothpaste without hydroxyapatite), an example group, and a comparative example group (n=10 in each group). The subjects cleaned their teeth with the example and comparative example toothpastes every morning and evening for 3 months. The inclusion criteria for the subjects were as follows: (1) informed and voluntary participation in this study; (2) clear chalky plaques were visible on the tooth enamel surface; (3) early enamel caries were determined based on the patient's complaints and imaging examination results.

[0138] The enamel demineralization rate of each group of subjects was tested before treatment and 6 months and 12 months after treatment according to the following formula:

[0139] Enamel demineralization rate = number of demineralized teeth / total number of teeth × 100%.

[0140] The test results are shown in Table 4. The results show that the demineralization rate of the toothpaste prepared using Examples 1-3 of the present invention was significantly lower than that of the control group 6 and 12 months after treatment, indicating that the toothpaste prepared using the process of the examples can effectively inhibit demineralization and promote remineralization.

[0141] Table 4 Comparison of the incidence of demineralization in patients with Examples 1-3 and Comparative Examples 1-6

[0142]

[0143] The above detailed description of the analytical methods involved in the present invention provides a detailed introduction. It should be noted that the above description is intended solely to help those skilled in the art better understand the methods and concepts of the present invention, and is not intended to limit the relevant content. Without departing from the principles of the present invention, those skilled in the art may make appropriate adjustments or modifications to the present invention, and such adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. A method for preparing a hydroxycellulose-silk fibroin oligopeptide mineralized material based on high-pressure microfluidics technology, characterized in that: The steps include: (1) Prepare silk fibroin solution from silk cocoons and adjust the silk fibroin concentration to 50-300 mg / mL; (2) adding a reducing agent and continuing the stirring reaction, and then adding an oxidizing agent and performing an oxidative degradation reaction under stirring to obtain a silk fibroin oligopeptide intermediate solution; the reducing agent is selected from vitamin C; the oxidizing agent is selected from hydrogen peroxide; (3) placing the silk fibroin oligopeptide intermediate solution in a high-pressure microfluidizer for cyclic homogenization; the cyclic homogenization pressure is 70-120 MPa, and the number of times is 1-5 times; (4) ultrafiltration of the product after cyclic homogenization to obtain a silk fibroin oligopeptide solution, which is then freeze-dried to obtain a silk fibroin oligopeptide solution; the ultrafiltration step specifically comprises the following steps: first separating the product after the reaction through an ultrafiltration membrane with a molecular weight of 2 kDa, and taking the effluent; then separating the product through an ultrafiltration membrane with a molecular weight of 0.75 kDa, and taking the retentate to obtain a 0.75-2 kDa silk fibroin oligopeptide solution; (5) The silk fibroin oligopeptide obtained in step (4) is mixed with hydroxyapatite and dissolved in a saturated calcium nitrate solution, and a sodium phosphate solution is added dropwise under stirring to carry out a mineralization reaction; after the mineralization reaction is completed, the pH is adjusted to neutral, the precipitate is collected by centrifugation, dried, ultrafinely ground and sieved to obtain the product.

2. The preparation method according to claim 1, characterized in that The stirring reaction in step (2) is carried out at a temperature of 40-60° C. and for a time of 20-60 min.

3. The preparation method according to claim 1, characterized in that The temperature of the oxidative degradation reaction in step (2) is 30-60° C. and the time is 10-60 min.

4. The hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the preparation method according to any one of claims 1 to 3.

5. Use of the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the preparation method according to any one of claims 1 to 3 in the preparation of toothpaste.

6. A toothpaste, characterized in that The invention comprises a hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the preparation method according to any one of claims 1 to 3, and auxiliary materials.

Images