Toothpaste containing hydroxyapatite-silk fibroin oligopeptide mineralized material and preparation method thereof

Abstract

The present invention relates to a toothpaste comprising a hydroxyapatite-silk fibroin oligopeptide mineralized material and a preparation method thereof. Through extensive research, the present invention has subjected silk fibroin to treatments such as enzymatic hydrolysis, alkaline hydrolysis, and oxidative degradation of a specific process, which can significantly improve its specific surface area and dispersibility, making it easy to be evenly dispersed in an aqueous solution, and contributing to better remineralization with hydroxyapatite. Subsequently, a remineralization treatment is carried out with hydroxyapatite to obtain a mineralized material, and toothpaste is prepared on this basis, which can improve the promotion of enamel remineralization, increase the Vickers hardness of the enamel block after remineralization, reduce the degree of enamel demineralization, reduce the plaque index of patients with enamel demineralization chalky spots, and thus achieve an anti-caries effect. In addition, the present invention uses natural products such as silkworm cocoons as active ingredients, has good safety, low cost, and has great market prospects and social significance.

Description

Technical Field

[0001] The invention belongs to the field of natural product applications, and particularly relates to a toothpaste containing a hydroxyapatite-silk fibroin oligopeptide mineralized material and a preparation method thereof. Background Art

[0002] Mature human tooth enamel is a highly mineralized tissue composed primarily of inorganic minerals, primarily hydroxyapatite, which is arranged parallel and perpendicular to the enamel surface, forming a hydroxyapatite prismatic structure. Under normal circumstances, hydroxyapatite is a relatively stable structure, with demineralization and remineralization in a dynamic equilibrium. However, when this equilibrium is disrupted, enamel demineralization occurs and early-stage caries develop.

[0003] In clinical practice, fluoride is mainly used for remineralization treatment of enamel caries. However, excessive fluoride can lead to toxic side effects such as dental fluorosis, skeletal fluorosis, and acute fluorosis. Bionic enamel remineralization is a new treatment for early-stage caries. Hydroxyapatite has a mineral content similar to that of dental hard tissue and has high bioactivity and biocompatibility. The use of hydroxyapatite material alone for enamel remineralization mainly utilizes its nano-size effect to fill the demineralized areas, so the remineralization effect on enamel caries is limited. Therefore, it is particularly critical and important to develop a high-quality toothpaste that can efficiently repair enamel. Summary of the Invention

[0004] The purpose of the present invention is to address the toxic side effects such as dental fluorosis, skeletal fluorosis, and acute fluorosis caused by the use of fluoride for remineralization treatment in the prior art, and to develop a toothpaste that can effectively repair tooth enamel by processing silk fibroin through oxidative degradation and other processes to obtain silk fibroin oligopeptides, and then remineralizing them with hydroxyapatite.

[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 hydroxyapatite-silk fibroin oligopeptide mineralized material, comprising the following steps:

[0007] (1) preparing a silk fibroin solution from silkworm cocoons and adjusting the silk fibroin concentration to 10-100 mg / mL;

[0008] (2) adding neutral protease for the first enzymatic hydrolysis; then adding β-glucanase for the second enzymatic hydrolysis;

[0009] (3) adding NaOH to adjust the pH to alkaline, performing alkaline hydrolysis under shaking conditions, and then adjusting the pH to neutral;

[0010] (4) adding an oxidizing agent and carrying out an oxidative degradation reaction under stirring; then adding a reducing agent and continuing the stirring reaction;

[0011] (5) ultrafiltration of the reaction product to obtain a silk fibroin oligopeptide solution, and freeze-drying to obtain a silk fibroin oligopeptide solution;

[0012] (6) The silk fibroin oligopeptide obtained in step (5) 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.

[0013] Preferably, the silk fibroin solution in step (1) is prepared by the following steps:

[0014] (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;

[0015] (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;

[0016] (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.

[0017] Preferably, the concentration of the sodium carbonate solution in step (1.1) is 0.1-1.0 wt %; more preferably, the concentration of the sodium carbonate solution is 0.5 wt %.

[0018] Preferably, the boiling time in step (1.1) is 30-90 min, the washing time is 5-20 min, and the boiling and washing processes are repeated 2-4 times; more preferably, the boiling time is 60 min, the washing time is 10 min, and the boiling and washing processes are repeated 3 times.

[0019] 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 40-70°C; more preferably, the concentration of the lithium bromide solution is 9.5 mol / L, and the temperature of the lithium bromide solution is 60°C.

[0020] 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 1:6.

[0021] Preferably, the centrifugal speed in step (1.2) is 5000-15000 rpm, and the time is 5-15 min; more preferably, the centrifugal speed is 9000 rpm, and the time is 10 min.

[0022] Preferably, the relative molecular mass retained by the dialysis bag in step (1.3) is 5-15 kDa, and the dialysis time is 1-3 days; more preferably, the relative molecular mass retained by the dialysis bag is 10 kDa, and the dialysis time is 2 days.

[0023] Preferably, the concentration of the polyethylene glycol solution in step (1.3) is 5-20%, and the concentration time is 5-20 hours; more preferably, the concentration of the polyethylene glycol solution is 10%, and the concentration time is 12 hours.

[0024] Preferably, the amount of the neutral protease in step (2) is 2-20% of the mass of the silk fibroin protein; the amount of the β-glucanase is 1-5% of the mass of the silk fibroin protein.

[0025] Preferably, the temperature of the first enzymatic hydrolysis in step (2) is 30-50°C, and the time is 30-90 min; more preferably, the temperature of the first enzymatic hydrolysis is 35-45°C, and the time is 45-75 min.

[0026] Preferably, the temperature of the second enzymatic hydrolysis in step (2) is 40-60° C., and the time is 10-60 min; more preferably, the temperature of the second enzymatic hydrolysis is 45-55° C., and the time is 20-40 min.

[0027] Preferably, in step (3), the pH is adjusted to 8.0-11.0; more preferably, the pH is adjusted to 8.5-9.5.

[0028] Preferably, the concentration of NaOH in step (3) is 0.5-3 mol / L; more preferably, the concentration of NaOH is 1 mol / L.

[0029] Preferably, the oscillation speed in step (3) is 30-100 rpm, the alkaline hydrolysis temperature is 40-60° C., and the time is 1-10 h; more preferably, the oscillation speed is 50-70 rpm, the alkaline hydrolysis temperature is 50-55° C., and the time is 3-8 h.

[0030] Preferably, the oxidant in step (4) is selected from hydrogen peroxide; more preferably, the oxidant is selected from hydrogen peroxide with a concentration of 2.5 mmol / L.

[0031] Preferably, the volume ratio of the oxidant to the silk fibroin solution is 0.0001-0.001:1.

[0032] Preferably, the temperature of the oxidative degradation reaction in step (4) is 30-60° C., and the time is 1-20 min; more preferably, the temperature of the oxidative degradation reaction is 40-50° C., and the time is 5-15 min.

[0033] Preferably, the reducing agent in step (4) is selected from vitamin C; more preferably, the reducing agent is selected from vitamin C with a concentration of 10 mmol / L.

[0034] Preferably, the volume ratio of the reducing agent to the silk fibroin solution is 0.0001-0.0005:1.

[0035] Preferably, the stirring reaction in step (4) is carried out at a temperature of 30-60°C and for a time of 10-60 min; more preferably, the stirring reaction is carried out at a temperature of 35-45°C and for a time of 20-40 min.

[0036] Preferably, the ultrafiltration in step (5) is specifically as follows: the reaction product is first separated by an ultrafiltration membrane with a molecular weight of 3 kDa, and the effluent is taken; then the product is separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate is taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution.

[0037] Preferably, the mass ratio of the silk fibroin oligopeptide to hydroxyapatite in step (6) is 1:1-3; more preferably, the mass ratio of the silk fibroin oligopeptide to hydroxyapatite is 1:1.2-2.0.

[0038] Preferably, in step (6), 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 0.02-0.06:1.

[0039] 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 0.05:1", if the amount of silk fibroin oligopeptide added is 5 g, the amount of saturated calcium nitrate solution added is 100 mL.

[0040] Preferably, in step (6), the mass ratio of the silk fibroin oligopeptide to the sodium phosphate in the sodium phosphate solution is 0.5-2:1; more preferably, the mass ratio of the silk fibroin oligopeptide to the sodium phosphate in the sodium phosphate solution is 1-1.5:1.

[0041] Preferably, the temperature of the mineralization reaction in step (6) is 30-40° C., and the time is 24-96 h; more preferably, the temperature of the mineralization reaction is 35-38° C., and the time is 36-72 h.

[0042] The second aspect of the present invention provides a hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the above preparation method.

[0043] 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.

[0044] 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.

[0045] Preferably, the auxiliary materials include one or more of silicon dioxide, sorbitol, propylene glycol, xylitol, sodium carboxymethyl cellulose, sodium lauryl sulfate, carrageenan, sodium benzoate, and water.

[0046] Preferably, the toothpaste comprises the following ingredients in the following mass percentages:

[0047] 5-15% hydroxyapatite-silk fibroin oligopeptide mineralized material, 15-30% silicon dioxide, 5-15% sorbitol, 1-5% propylene glycol, 1-5% xylitol, 1-3% sodium carboxymethyl cellulose, 0.5-3% sodium lauryl sulfate, 0.5-2% carrageenan, 0.05-0.5% sodium benzoate, and the balance is water; the total mass percentage of each component is 100%.

[0048] Protein plays a key role in the inorganic-organic biomineralization of enamel mediated by ameloblasts. The remineralization of protein with hydroxyapatite imparts remarkable structural and mechanical properties to hydroxyapatite. This is because protein participates in the directional growth of hydroxyapatite crystals during the mineralization process. This hydroxyapatite-protein remineralization material can adsorb onto the surface of corroded enamel crystals, aligning bundles of enamel-like crystals and achieving the desired effect of repairing enamel.

[0049] Silk fibroin is a natural polymer extracted from silk fibers with excellent biocompatibility. However, due to the high molecular weight of silk fibroin, its specific surface area and dispersibility are relatively poor. In response to this, the present invention has carried out a large number of studies on silk fibroin, and has been subjected to treatments such as enzymolysis, alkaline hydrolysis and oxidative degradation of specific processes, which can significantly improve its specific surface area and dispersibility, making it easy to be evenly dispersed in an aqueous solution, and helping to better remineralize with hydroxyapatite. Subsequently, remineralization treatment is carried out with hydroxyapatite to obtain a mineralized material, and toothpaste is prepared with this, which can improve the promotion of enamel remineralization, increase the Vickers hardness of the enamel block after remineralization, reduce the degree of enamel demineralization, reduce the plaque index of patients with enamel demineralization chalky spots, and then achieve the effect of preventing caries. In addition, the present invention uses natural products such as silkworm cocoons as active ingredients, has good safety, low cost, and has great market prospects and social significance. DETAILED DESCRIPTION

[0050] 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.

[0051] Example 1

[0052] A hydroxyapatite-silk fibroin oligopeptide mineralized material, the preparation method of which comprises the following steps:

[0053] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0054] (2) Add 2 g of neutral protease (10,000 U / g) to 1 L of silk fibroin solution and perform enzymatic hydrolysis at 40°C for 1 h; then add 2.5 g of β-glucanase (3,000 U / g) and perform enzymatic hydrolysis at 50°C for 0.5 h.

[0055] (3) 1 mol / L NaOH was added to adjust the pH to 9.0, and the mixture was subjected to alkaline hydrolysis at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0056] (4) Add 0.5 mL of 2.5 mmol / L hydrogen peroxide and conduct an oxidative degradation reaction at 45°C with stirring for 10 minutes; then add 0.3 mL of 10 mmol / L vitamin C and conduct a stirring reaction at 40°C for 0.5 hours.

[0057] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0058] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0059] 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: 10% hydroxyapatite-silk fibroin oligopeptide mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0060] Example 2

[0061] A hydroxyapatite-silk fibroin oligopeptide mineralized material, the preparation method of which comprises the following steps:

[0062] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0063] (2) 1 L of silk fibroin solution was added with 4 g of neutral protease (10,000 U / g) and enzymatically hydrolyzed at 35°C for 75 min; then 1 g of β-glucanase (3,000 U / g) was added and enzymatically hydrolyzed at 45°C for 40 min.

[0064] (3) 1 mol / L NaOH was added to adjust the pH to 9.5, and the mixture was subjected to alkaline hydrolysis at 70 rpm and 50°C for 3 h, and then the pH was adjusted to 7.0.

[0065] (4) Add 1 mL of 2.5 mmol / L hydrogen peroxide and allow to react at 40°C for 15 minutes under stirring; then add 0.5 mL of 10 mmol / L vitamin C and allow to react at 35°C for 40 minutes under stirring.

[0066] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0067] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0068] 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: 10% hydroxyapatite-silk fibroin oligopeptide mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0069] Example 3

[0070] A hydroxyapatite-silk fibroin oligopeptide mineralized material, the preparation method of which comprises the following steps:

[0071] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0072] (2) Take 1L of silk fibroin solution, add 1g of neutral protease (10000U / g) and enzymatically hydrolyze at 45℃ for 45min; then add 0.5g of β-glucanase (3000U / g) and enzymatically hydrolyze at 55℃ for 20min.

[0073] (3) 1 mol / L NaOH was added to adjust the pH to 8.5, and the mixture was subjected to alkaline hydrolysis at 50 rpm and 55°C for 8 h, and then the pH was adjusted to 7.0.

[0074] (4) Add 0.2 mL of 2.5 mmol / L hydrogen peroxide and allow to react at 50°C with stirring for 5 minutes; then add 0.1 mL of 10 mmol / L vitamin C and allow to react at 45°C with stirring for 20 minutes.

[0075] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0076] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0077] 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: 10% hydroxyapatite-silk fibroin oligopeptide mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0078] Comparative Example 1

[0079] A mineralized material, the preparation method of which comprises the following steps:

[0080] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0081] (2) Take 1 L of silk fibroin solution, add 2.5 g of β-glucanase (3000 U / g) and enzymatically hydrolyze at 50°C for 0.5 h.

[0082] (3) 1 mol / L NaOH was added to adjust the pH to 9.0, and the mixture was subjected to alkaline hydrolysis at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0083] (4) Add 0.5 mL of 2.5 mmol / L hydrogen peroxide and conduct an oxidative degradation reaction at 45°C with stirring for 10 minutes; then add 0.3 mL of 10 mmol / L vitamin C and conduct a stirring reaction at 40°C for 0.5 hours.

[0084] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0085] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0086] Furthermore, the mineralized material prepared above is used to prepare toothpaste according to conventional methods in the art. The toothpaste comprises the following components in percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0087] Comparative Example 2

[0088] A mineralized material, the preparation method of which comprises the following steps:

[0089] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0090] (2) Take 1L of silk fibroin solution, add 2.5g of β-glucanase (3000U / g) and enzymatically hydrolyze at 50℃ for 0.5h; then add 2g of neutral protease (10000U / g) and enzymatically hydrolyze at 40℃ for 1h.

[0091] (3) 1 mol / L NaOH was added to adjust the pH to 9.0, and the mixture was subjected to alkaline hydrolysis at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0092] (4) Add 0.5 mL of 2.5 mmol / L hydrogen peroxide and conduct an oxidative degradation reaction at 45°C with stirring for 10 minutes; then add 0.3 mL of 10 mmol / L vitamin C and conduct a stirring reaction at 40°C for 0.5 hours.

[0093] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0094] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0095] 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 percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0096] Comparative Example 3

[0097] A mineralized material, the preparation method of which comprises the following steps:

[0098] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0099] (2) Add 2 g of neutral protease (10,000 U / g) to 1 L of silk fibroin solution and perform enzymatic hydrolysis at 40°C for 1 h; then add 2.5 g of β-glucanase (3,000 U / g) and perform enzymatic hydrolysis at 50°C for 0.5 h.

[0100] (3) Add 0.5 mL of 2.5 mmol / L hydrogen peroxide and conduct an oxidative degradation reaction at 45°C with stirring for 10 minutes; then add 0.3 mL of 10 mmol / L vitamin C and conduct a stirring reaction at 40°C for 0.5 hours.

[0101] (4) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0102] (5) 4 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0103] 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 percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0104] Comparative Example 4

[0105] A mineralized material, the preparation method of which comprises the following steps:

[0106] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0107] (2) 1 L of silk fibroin solution was added with 1 mol / L NaOH to adjust the pH to 9.0, and then alkaline hydrolysis was performed at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0108] (3) Add 2 g of neutral protease (10,000 U / g) and enzymatically hydrolyze at 40°C for 1 h; then add 2.5 g of β-glucanase (3,000 U / g) and enzymatically hydrolyze at 50°C for 0.5 h.

[0109] (4) Add 0.5 mL of 2.5 mmol / L hydrogen peroxide and conduct an oxidative degradation reaction at 45°C with stirring for 10 minutes; then add 0.3 mL of 10 mmol / L vitamin C and conduct a stirring reaction at 40°C for 0.5 hours.

[0110] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0111] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0112] 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 percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0113] Comparative Example 5

[0114] A mineralized material, the preparation method of which comprises the following steps:

[0115] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0116] (2) Add 2 g of neutral protease (10,000 U / g) to 1 L of silk fibroin solution and perform enzymatic hydrolysis at 40°C for 1 h; then add 2.5 g of β-glucanase (3,000 U / g) and perform enzymatic hydrolysis at 50°C for 0.5 h.

[0117] (3) 1 mol / L NaOH was added to adjust the pH to 9.0, and the mixture was subjected to alkaline hydrolysis at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0118] (4) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0119] (5) 4 g of the silk fibroin oligopeptide obtained in step (4) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0120] 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 percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0121] Comparative Example 6

[0122] A mineralized material, the preparation method of which comprises the following steps:

[0123] (1) Select high-quality clean silk cocoons, cut them into pieces, add them to a boiling 0.5 wt% sodium carbonate solution, boil for 60 minutes, and then soak and wash them with distilled water for 10 minutes; repeat the above boiling and washing process 3 times, and dry them to obtain degummed silk; dissolve the degummed silk in a 9.5 mol / L lithium bromide solution at 60°C (the mass ratio of degummed silk to lithium bromide solution is 1:6), and after cooling to room temperature, use a filter to remove large particles of impurities, centrifuge at 9000 rpm for 10 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 10 kDa, and dialyze with ultrapure water for 2 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 10% polyethylene glycol solution and concentrate it for 12 hours to obtain a silk fibroin solution, and adjust the silk fibroin concentration to 50 mg / mL.

[0124] (2) Take 1L of silk fibroin solution, add 0.5mL of 2.5mmol / L hydrogen peroxide, and perform oxidative degradation reaction at 45℃ with stirring for 10min; then add 0.3mL of 10mmol / L vitamin C and perform stirring reaction at 40℃ for 0.5h.

[0125] (3) Add 2 g of neutral protease (10,000 U / g) and enzymatically hydrolyze at 40°C for 1 h; then add 2.5 g of β-glucanase (3,000 U / g) and enzymatically hydrolyze at 50°C for 0.5 h.

[0126] (4) 1 mol / L NaOH was added to adjust the pH to 9.0, and the mixture was subjected to alkaline hydrolysis at 60 rpm and 55°C for 5 h, and then the pH was adjusted to 7.0.

[0127] (5) The supernatant of the reaction product was first separated by an ultrafiltration membrane with a molecular weight of 3 kDa to remove macromolecular substances, and the effluent was taken; then the supernatant was separated by an ultrafiltration membrane with a molecular weight of 0.5 kDa, and the retentate was taken to obtain a 0.5-3 kDa silk fibroin oligopeptide solution, which was freeze-dried to obtain silk fibroin oligopeptide.

[0128] (6) 4 g of the silk fibroin oligopeptide obtained in step (5) was mixed with 6 g of hydroxyapatite and dissolved in 100 mL of saturated calcium nitrate solution. 10 mL of 2 mol / L sodium phosphate solution was added dropwise at 37° C. with stirring, and the mixture was subjected to mineralization reaction at 37° C. for 48 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 10,000 mesh sieve.

[0129] 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 percentage by mass: 10% mineralized material, 20% silicon dioxide, 10% sorbitol, 2% propylene glycol, 2% xylitol, 2% sodium carboxymethyl cellulose, 1% sodium lauryl sulfate, 1% carrageenan, 0.1% sodium benzoate, and the balance is water.

[0130] Verification Example 1

[0131] The mineralized materials prepared in the examples and comparative examples were respectively used to study their effects on the remineralization of tooth enamel.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] The results are shown in Table 1 below. These results demonstrate that the crystals generated by the remineralization of the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared in accordance with the present invention reduced the amount of fluorescent dye entering the micropores of demineralized enamel in tooth tissue, reducing the extent of caries lesions and demonstrating a remineralizing effect. This also further demonstrates that the present invention promotes the remineralization of tooth enamel, thereby achieving a caries-preventing effect.

[0137] Table 1 Effects of Examples and Comparative Examples on Enamel Remineralization

[0138]

[0139] Furthermore, all the enamel blocks were placed under a Vickers hardness tester before demineralization, after demineralization and after the remineralization experiment, and the Vickers hardness (VHN) was measured. The hardness of the artificial caries after remineralization was analyzed using the embodiments and comparative examples. The test results are shown in Table 2 below. The results show that the change in dentin microhardness is directly related to its mineral content, and the Vickers hardness of the enamel decreases significantly after demineralization. The Vickers hardness of the enamel block after remineralization in the embodiment of the present invention is greatly improved compared with that after demineralization, indicating that the Vickers hardness value can be better improved after treatment with the embodiment, and the degree of enamel demineralization can be reduced.

[0140] Table 2 Vickers hardness of tooth enamel before and after remineralization of the samples of the embodiment and comparative example

[0141]

[0142] Verification Example 2

[0143] The toothpastes prepared in the examples and comparative examples were used to study their repairing effects on enamel demineralization and chalky spots. The specific steps were as follows: 200 subjects were randomly divided into 10 groups after informed consent, namely a control group (using ordinary toothpaste not containing hydroxyapatite), an example group and a comparative example group (n=20 in each group). The subjects cleaned their teeth with the example and comparative example toothpaste every morning and evening for half a year.

[0144] The enamel demineralization index (EDI) is the main indicator reflecting the degree of enamel demineralization. It is measured according to the following method: the EDI value is calculated based on whether there are chalky spots on the surfaces of the 8 upper teeth. The tooth surface is divided into four areas: gingival, maxillary, mesial and distal, with the crown as the center. The degree of demineralization is divided into 4 levels, where the absence of chalky spots on the enamel surface is scored as 0, enamel surface spots <50% is scored as 1, 100%> enamel surface spots >50% is scored as 2, and surface spots occupying the entire area or the presence of cavities is scored as 3. The EDI value = the sum of the demineralization scores of each part of the observed tooth accounted for the number of observed tooth areas.

[0145] The test results are shown in Table 3. The results show that the EDI value of the toothpaste prepared using the examples of the present invention is significantly lower than that of the control groups, indicating that the toothpaste prepared in the examples can effectively inhibit demineralization when applied to subjects with enamel demineralization and chalky spots.

[0146] Table 3 Comparison of enamel demineralization index (EDI) of subjects in Example and Comparative Example

[0147]

[0148]

[0149] Furthermore, the plaque index of each group was measured: after drying the tooth surface, the amount of plaque on the tooth surface near the gingival margin was directly observed with the naked eye in combination with probing. A score of 0 indicates no plaque near the gingival margin, 1 indicates plaque visible only after probing, 2 indicates moderate plaque at the gingival margin or tooth surface, and 3 indicates extensive plaque in the gingival sulcus, gingival margin, and tooth surface.

[0150] The test results are shown in Table 4. The results show that after six months of treatment with the toothpaste prepared in the examples of the present invention, the plaque index of the subjects was significantly lower than that of the control group, indicating that the toothpaste prepared in the examples is beneficial for the subjects to better control plaque, which may be because the preparation can inhibit the adhesion of oral bacteria to hydroxyapatite.

[0151] Table 4 Comparison of plaque index values ​​of subjects in Example and Comparative Example

[0152]

[0153]

[0154] It can be clearly seen from the above that the toothpaste prepared by the method of the present invention is significantly better than the comparative examples in terms of enamel mineralization, improvement of enamel hardness, improvement of enamel demineralization and inhibition of dental plaque. The main reason is that, since silk fibroin is neutral, it can be moderately degraded into smaller molecular silk fibroin oligopeptides under neutral conditions by using neutral protease hydrolysis, exposing more enzyme cleavage sites and preparing for subsequent β-glucanase hydrolysis. In Comparative Example 1, silk fibroin was directly hydrolyzed by β-glucanase without being hydrolyzed by neutral protease, and the order of silk fibroin enzyme hydrolysis was changed in Comparative Example 2, both of which resulted in a lack of β-glucanase cleavage sites on the surface of silk fibroin, making it difficult to obtain active small molecule oligopeptides by enzymatic hydrolysis, thereby significantly reducing the performance of the toothpaste finally obtained.

[0155] In Comparative Example 3, the mineralized material prepared by silk fibroin without alkaline hydrolysis was used to make toothpaste, and the larger molecular weight silk fibroin oligopeptides could not be fully and thoroughly degraded into silk fibroin oligopeptides; in Comparative Example 4, the mineralized material prepared by silk fibroin first undergoing alkaline hydrolysis and then enzymatic hydrolysis was used to make toothpaste, and alkaline hydrolysis would cause the silk fibroin to be severely denatured, and the enzyme cleavage sites would be wrapped inside the protein, making it difficult to be enzymatically degraded by biological enzymes; in Comparative Example 5, the silk fibroin was not subjected to oxidative degradation treatment, and the larger molecular weight silk fibroin oligopeptides could not be fully and thoroughly degraded into silk fibroin oligopeptides, and it was difficult to mineralize with hydroxyapatite later; although Comparative Example 6 was subjected to oxidative degradation treatment, since it was carried out before enzymatic hydrolysis and alkaline hydrolysis, the silk fibroin was severely chelated and folded after oxidative degradation, and the enzyme cleavage sites were wrapped inside the protein, making it difficult to be degraded by biological enzymes. Therefore, the performance of the toothpaste prepared according to Comparative Examples 3-6 was also far inferior to that of the present invention.

[0156] 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 hydroxyapatite-silk fibroin oligopeptide mineralized material, characterized in that: The steps include: (1) Prepare silk fibroin solution from silk cocoons and adjust the silk fibroin concentration to 10-100 mg / mL; (2) adding neutral protease for the first enzymatic hydrolysis; then adding β-glucanase for the second enzymatic hydrolysis; (3) Add NaOH to adjust the pH to alkaline, perform alkaline hydrolysis under oscillation conditions, and then adjust the pH to neutral; (4) adding an oxidizing agent and carrying out an oxidative degradation reaction under stirring; then adding a reducing agent and continuing the stirring reaction; the oxidizing agent is selected from hydrogen peroxide, and the reducing agent is selected from vitamin C; (5) ultrafiltration of the reaction product to obtain a silk fibroin oligopeptide solution, which is then freeze-dried to obtain a silk fibroin oligopeptide solution; (6) The silk fibroin oligopeptide obtained in step (5) 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 temperature of the first enzymatic hydrolysis in step (2) is 30-50°C and the time is 30-90 minutes.

3. The preparation method according to claim 1, characterized in that The temperature of the second enzymatic hydrolysis in step (2) is 40-60°C and the time is 10-60 minutes.

4. The preparation method according to claim 1, characterized in that In step (3), the pH is adjusted to 8.0-11.

0.

5. The preparation method according to claim 1, characterized in that The temperature of the oxidative degradation reaction in step (4) is 30-60° C. and the time is 1-20 min.

6. The preparation method according to claim 1, characterized in that The stirring reaction in step (4) is carried out at a temperature of 30-60° C. and for a time of 10-60 min.

7. The preparation method according to claim 1, characterized in that The temperature of the mineralization reaction in step (6) is 30-40°C.

8. The hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the hydroxyapatite-silk fibroin oligopeptide mineralized material prepared according to the preparation method according to any one of claims 1 to 7 in the preparation of toothpaste.

10. 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 7, and auxiliary materials.

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