Anti-caries oral care composition and preparation method thereof

Abstract

The invention discloses an anti-caries oral care composition as well as a preparation method and application thereof, and belongs to the technical field of oral care drugs, the anti-caries oral care composition comprises probiotic bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptides and an acidic stabilizer; a tannic acid-calcium ion-polyvinylpyrrolidone coating and a casein phosphopeptide layer are sequentially arranged on the surface of the probiotic bifidobacterium infantis; the acid stabilizer is combined with the tannic acid-calcium ion-polyvinylpyrrolidone coating, so that the tannic acid-calcium ion-polyvinylpyrrolidone coating can be effectively stabilized. The anti-caries oral care composition can improve the chemical stability of BI (at) TA-Ca2 + (at) CPP in an oral environment, prevent excessive expansion after water absorption, and enhance the colonization stability of probiotics in the oral environment.

Description

Technical Field

[0001] The present invention relates to the technical field of oral care drugs, and particularly relates to an anti-caries oral care composition, a preparation method thereof and an application thereof. Background Art

[0002] Dental caries is a serious public health problem. The main cause of its occurrence is the formation of cariogenic plaque biofilm, in which the dynamic balance of oral bacteria is disrupted, and acid-producing bacteria play a dominant role, reducing the pH value of the biofilm. Therefore, the hard tissues of teeth may be eroded, resulting in demineralization and tooth decay. At present, the control and prevention of dental caries mainly adopt mechanical removal methods, local fluoride exposure and antibacterial agents. However, mechanical techniques cannot fundamentally reduce the number of cariogenic bacteria and only play a superficial role. The use of fluoride and antibacterial products is also limited by the difficult-to-enter tight structure of dental plaque biofilm, and high concentrations or long-term use may bring problems such as toxicity, tooth staining and drug resistance. In addition, the buffering effect of saliva makes it difficult for drugs to be continuously retained in the oral cavity. Therefore, anti-caries research fundamentally relies on safer and more effective strategies. Probiotics are members of the normal human microbiota. It has been reported that probiotics play an important role in maintaining the homeostasis of the internal environment through various biological functions. In recent years, the application of probiotics in clinical practice, especially in gastrointestinal diseases, has been increasingly emphasized and achieved good therapeutic effects. Currently, there are studies confirming that probiotics have a therapeutic effect on oral diseases such as dental caries, periodontitis, oral mucositis, and halitosis by regulating the balance of oral flora.

[0003] Nutrient acquisition, inhibition of the formation of cariogenic biofilm and immunomodulation. In addition, the continuously released antibacterial substances of probiotics are not affected by raw material consumption. However, there are some obstacles to the wide application of probiotics in anti-caries. Since most probiotics are not of oral origin, their chance of colonizing in the oral cavity is very low. In addition, most probiotics are Gram-positive strains and may be destroyed by lysozyme in saliva, further weakening their anti-caries effect. In addition, since tooth enamel is difficult to self-repair after tooth decay, combining the antibacterial film effect of probiotics with a remineralizing agent can achieve better results. In recent years, the probiotic coating strategy has provided a method to overcome the above obstacles. This strategy is mainly used in gastrointestinal diseases to enhance the resistance of probiotics to the harsh conditions of the gastrointestinal tract and improve their colonization ability, and its application in oral diseases has not been reported.

[0004] A study reported an effective layer-by-layer coating strategy of tannic acid (TA), calcium ions (Ca2+), and casein phosphopeptides (CPPs) to enhance the cariostatic effect mediated by probiotics through synergistic anti-biofilm and remineralization effects (Scheme 1). Tannic acid is a natural product found in green tea and has been approved by the US Food and Drug Administration (FDA) as a direct food additive with antioxidant, anti-inflammatory, antibacterial, and anticancer effects. Due to its unique polyphenol chemical structure, tannic acid has a strong adhesion ability to various substances such as metal ions, proteins, and polysaccharides. Tannic acid chelates with calcium ions (Ca2+) to form a metal-phenol network on probiotics. Ca2+ was selected to prepare the tannic acid-Ca2+ coating because the remineralization process is usually limited by the lack of Ca2+. Casein phosphopeptide is a bioactive polypeptide extracted from bovine milk casein. It complexes with the tannic acid-Ca2+ network to form the outermost layer, which is mainly constructed through electrostatic interactions between mineral ions and the inherent amino acid sequence of casein phosphopeptide (Cao M X, Qian Z Y, Liang Y J, et al. Layer-by-layer coated probiotics with tannic acid-Ca2+ and casein phosphopeptide complexes for caries prevention and enamel remineralization[J]. iScience, 2025, 28(1)). In addition to its well-known anti-tumor and remineralization effects, casein phosphopeptide also has acid resistance and protease inhibitory activities and has a high affinity for the tooth surface. Therefore, therapeutic probiotics were modified with functional coatings to enhance their cariostatic efficacy.

[0005] The tannic acid molecule contains multiple phenolic hydroxyl groups, and these hydroxyl groups can form stable chelates with metal ions (such as Ca 2+ ). When tannic acid chelates with calcium ions, a certain degree of swelling may occur, which is mainly due to the increase in the intermolecular distance caused by the introduction of metal ions. Effects on casein phosphopeptide: If tannic acid swells, it may change its interaction with casein phosphopeptide. The swollen tannic acid may occupy more space, thus affecting the distribution and stability of casein phosphopeptide in tannic acid.

[0006] When tannic acid chelates with calcium ions (Ca 2+) When the chelated metal - phenol network expands, it may cause some calcium ions to be released from the chelation structure. Since casein phosphopeptide has a strong calcium - ion chelating ability, the released calcium ions may bind to casein phosphopeptide. This process may change the distribution and function of casein phosphopeptide in the composite material, which can be understood from the following aspects: 1. Redistribution of calcium ions. Release mechanism: When tannic acid expands, the coordination bonds in its molecular structure may become loose, causing calcium ions to be released from the chelation ring of tannic acid. The released calcium ions will move freely in the composite material. Binding ability: Casein phosphopeptide has multiple phosphoserine residues, which can form stable chelates with calcium ions. Therefore, the released calcium ions may preferentially bind to casein phosphopeptide. Change in spatial distribution: The redistribution of calcium ions will cause a change in the binding position of casein phosphopeptide and calcium ions. The calcium ions that were originally bound to tannic acid are now bound to casein phosphopeptide, which may change the spatial distribution of casein phosphopeptide in the composite material. Aggregation phenomenon: Due to the strong calcium - ion chelating ability of casein phosphopeptide, the released calcium ions may cause casein phosphopeptide to aggregate in local areas, forming a high - concentration calcium - casein phosphopeptide complex. This aggregation may change the overall structure and uniformity of the composite material. Summary of the Invention

[0007] As described in the above - mentioned prior art, one of the purposes of the present invention is to provide an anti - caries oral care composition, which can improve the stability of BI@tannic acid - Ca2 +@casein phosphopeptide in the oral environment and enhance the stability of probiotic colonization in the oral environment.

[0008] Another purpose of the present invention is to provide a manufacturing method of the anti - caries oral care composition. The method has simple steps and can industrially produce the anti - caries oral care composition.

[0009] Another purpose of the present invention is to provide the application of the anti - caries oral care composition in the preparation of anti - caries oral care toothpaste.

[0010] One of the purposes of the present invention is achieved by the following technical solution:

[0011] An anti - caries oral care composition, comprising: probiotic Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, and an acid stabilizer; the surface of the probiotic Bifidobacterium infantis has a tannic acid - calcium ion - polyvinylpyrrolidone coating and a casein phosphopeptide layer in sequence; the acid stabilizer binds to the tannic acid - calcium ion - polyvinylpyrrolidone coating and can effectively stabilize the tannic acid - calcium ion - polyvinylpyrrolidone coating.

[0012] Further, the concentration of the probiotic Bifidobacterium infantis (BI) is 1×107 CFU / mL.

[0013] Further, the acidic stabilizer is an organic compound containing a carboxyl group or a phosphate group.

[0014] Further, the organic compound containing a carboxyl group or a phosphate group includes citric acid and / or triethylamine phosphate.

[0015] Further, it also includes chitosan, and the casein phosphopeptide layer coats the chitosan coating.

[0016] The second object of the present invention is achieved by the following technical solution:

[0017] A manufacturing method of an anti-caries oral care composition includes the following steps:

[0018] S1. Suspend Bifidobacterium infantis (BI) in deionized water at a concentration of 1×107 CFU / mL, and its optical density at 600 nm is 0.4;

[0019] S2. Add 150 mL of a tannic acid solution with a concentration of 40 mg / mL to 150 mL of a calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of a polyvinylpyrrolidone solution with a mass percentage of 0.5 - 2.0 wt% and 5 mL of glucono-δ-lactone with a mass percentage of 0.1% - 2%, stir evenly, and then add the bacterial solution from step S1. Centrifuge at 8000 rpm for 5 min to form the tannic acid-calcium ion-polyvinylpyrrolidone coating on the surface of the Bifidobacterium infantis (BI);

[0020] S3. Resuspend the bacterial solution from step S2 in deionized water at a bacterial density of 1×107 CFU / mL, then add 200 mL of casein phosphopeptide (casein phosphopeptide) with a concentration of 40 mg / mL, and mix at a constant rotation speed of 80 rpm for 2 h to obtain Bifidobacterium infantis @ tannic acid-calcium ion-polyvinylpyrrolidone coating @ casein phosphopeptide, which is the anti-caries oral care composition described above.

[0021] Further, after step S3, resuspend the Bifidobacterium infantis @ tannic acid-calcium ion-polyvinylpyrrolidone coating @ casein phosphopeptide in deionized water at a bacterial density of 1×107 CFU / mL, add chitosan with a mass percentage of 1.0 - 3.0 wt%, and mix at a constant rotation speed of 80 rpm for 2 h to obtain the anti-caries oral care composition described above.

[0022] The third object of the present invention is to provide an application of an anti-caries oral care composition in the preparation of anti-caries oral care toothpaste, which also includes a pH slow-release regulator, a flavor, and a sweetener.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) For an anti-caries oral care composition provided by the present invention, the prior art is as follows: First, tannic acid and CaCl2 are successively mixed with BL to prepare BI@tannic acid-Ca2+. With the assistance of calcium ions, tannic acid crosslinks on the surface of BL to form a clear and dense film. Transmission electron microscopy (TEM) observation shows that there is a film with a thickness of 100 nm around BL cells. Next, casein phosphopeptide is encapsulated with BI@tannic acid-Ca2+ through electrostatic interaction and hydrogen bonding to obtain BI@tannic acid-Ca2+@casein phosphopeptide, and its outermost layer is obvious with a thickness of 200 nm. This diverse combination can prevent dental caries from multiple angles and improve the anti-caries effect. In the present invention, there is a coating of tannic acid-calcium ion-acidic stabilizer-polyvinylpyrrolidone on the surface of the probiotic Bifidobacterium infantis in sequence. Polyvinylpyrrolidone can prevent tannic acid from swelling excessively after absorbing moisture. Since hydrogen bonds can be formed between the phenolic hydroxyl and carboxyl groups in tannic acid and the pyrrolidone ring of PVP, the formation of hydrogen bonds makes the interaction between tannic acid and polyvinylpyrrolidone closer, forming a stable network structure; and the molecular chain structure of polyvinylpyrrolidone is relatively compact and can form a close arrangement in the tannic acid-Ca2+ coating. This close arrangement not only provides mechanical support but also restricts the swelling of tannic acid, improving the stability of BI@tannic acid-Ca2+@casein phosphopeptide in the oral environment; in the tannic acid-calcium ion-acidic stabilizer-polyvinylpyrrolidone coating, the acidic stabilizer can form a stable complex with calcium ions to prevent the precipitation of calcium ions. This chelation effect can effectively prevent the dissociation and loss of calcium ions, reduce the coating structure change and dissolution caused by ion migration, improve the stability of BI@tannic acid-Ca2+@casein phosphopeptide in the oral environment, and strengthen the colonization stability of probiotics in the oral environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0026] Figure 1 It is a graph showing the colonization amount result of the probiotic Bifidobacterium infantis in the saliva sample in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0027] Next, in combination with specific embodiments, the present invention will be further described. It should be noted that, on the premise of no conflict, any combination can be formed among the following-described embodiments or technical features to form a new embodiment.

[0028] Example 1

[0029] This example provides an anti-caries oral care composition, including: probiotic Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, and acidic stabilizer citric acid; there are tannic acid-calcium ion-citric acid-polyvinylpyrrolidone coatings and casein phosphopeptide layers on the surface of probiotic Bifidobacterium infantis in sequence; the acidic stabilizer combines with the tannic acid-calcium ion-polyvinylpyrrolidone coating, and can effectively stabilize the tannic acid-calcium ion-polyvinylpyrrolidone coating.

[0030] In this example, the concentration of probiotic Bifidobacterium infantis (BI) is 1×10 7 CFU / mL.

[0031] In this example, the function of the acidic stabilizer is as follows: the acidic stabilizer can combine with calcium ions to prevent the aggregation of Ca 2+ and PO4 3- . For example, using organic compounds containing carboxyl or phosphate groups, such as citric acid and triethylamine phosphate, can effectively stabilize the tannic acid-Ca 2 + coating.

[0032] Citric acid: Citric acid is a common acidic stabilizer and contains multiple carboxyl groups. Citric acid can form a stable complex with calcium ions to prevent the precipitation of calcium ions. This chelation effect can effectively prevent the dissociation and loss of calcium ions, reduce the change and dissolution of the coating structure caused by ion migration, thereby enhancing the stability of the coating, preventing it from quickly dissolving in a humid environment, and can significantly improve the stability of the tannic acid-Ca 2 + coating.

[0033] Triethylamine phosphate: Triethylamine phosphate contains phosphate groups and can form a stable complex with calcium ions to prevent the precipitation of calcium ions.

[0034] Microstructural analysis: Through techniques such as scanning electron microscopy (SEM) and X-ray diffraction (XRD), it can be observed that the microstructure of the coating is more uniform and there is no obvious precipitate formation.

[0035] In this example, the function of polyvinylpyrrolidone is as follows:

[0036] Polyvinylpyrrolidone is a polymer material with excellent hydrophilicity. Its hydrophilicity mainly stems from the relatively polar lactam groups in its molecular structure. These polar groups can form stable hydrated structures with water molecules through hydrogen bonding, enabling polyvinylpyrrolidone to exhibit good solubility and hydrophilicity in water. Specifically, when polyvinylpyrrolidone encounters water, it quickly absorbs water, and water molecules form a hydrogel through hydrogen bonding complexation, thus demonstrating good hydrophilic properties.

[0037] 1. Enhanced hydrophilicity: Hydrogen bonds can form between the phenolic hydroxyl groups and carboxyl groups in tannic acid and the pyrrolidone ring of polyvinylpyrrolidone. The formation of hydrogen bonds makes the interaction between tannic acid and polyvinylpyrrolidone closer, forming a stable network structure. The oxygen atoms in the phenolic hydroxyl groups and carboxyl groups of tannic acid can form hydrogen bonds with the hydrogen atoms in water molecules, and the oxygen atoms in the pyrrolidone ring can also form a hydrogen bond network with the hydrogen atoms in water molecules, thereby enhancing the hydrophilicity of the material. This network can absorb water, but at the same time restricts the free movement of water molecules, thus reducing the excessive swelling of tannic acid. This composite structure can absorb more water, but at the same time, due to the presence of polyvinylpyrrolidone, it can limit the excessive swelling of the material, thereby controlling the swelling rate, preventing tannic acid from swelling excessively after absorbing water, and the molecular chain structure of polyvinylpyrrolidone can provide certain mechanical support to limit the swelling of the material.

[0038] Mechanical support: The molecular chain structure of polyvinylpyrrolidone is relatively compact and can form a close arrangement in the composite material. This close arrangement provides mechanical support and enhances the overall strength of the material.

[0039] Swelling property: The hydrophilicity of polyvinylpyrrolidone enables it to absorb water, but the compactness of its molecular chain structure limits the excessive swelling of the material. Therefore, polyvinylpyrrolidone can maintain a relatively stable size after absorbing water and will not swell significantly like tannic acid.

[0040] In this embodiment, the pH slow-release regulator (such as D-(+)-glucono-δ-lactone, GDL) can regulate the pH value inside the tannic acid-Ca2+ coating, buffer the external acidic environment, and reduce the influence of the acidic environment on the crosslinked network. If the external environment itself is acidic, the slow-release acidification characteristic of the pH slow-release regulator can form a dynamic equilibrium with the external acidic environment. Although the pH slow-release regulator will reduce the pH value inside the hydrogel, its slow-release characteristic can prevent the internal pH value from dropping significantly instantaneously, thereby buffering the influence of the external acidic environment on the hydrogel to a certain extent.

[0041] In this embodiment, due to the high-affinity binding of casein phosphopeptide and hydroxyapatite, after the double-coated probiotics are colonized on the tooth surface, they can not only continuously secrete various antibacterial substances, but also shed tannic acid and casein phosphopeptide during the proliferation process. Tannic acid and casein phosphopeptide play an anti-biofilm role by inhibiting the adhesion of dental plaque bacteria to the acquired pellicle and reducing the formation of glucan on tooth enamel (the main component of the extracellular matrix of the biofilm).

[0042] In this embodiment, casein phosphopeptide: Casein phosphopeptide is a bioactive peptide rich in phosphoserine residues extracted from milk casein, which has the ability to chelate calcium ions and other minerals and is commonly used to improve the bioavailability of minerals. Due to the large number of phosphate groups in its structure, these phosphate groups will dissociate into negative charges under physiological conditions, so casein phosphopeptide is a negatively charged peptide.

[0043] Chitosan is a natural cationic polysaccharide whose molecular structure contains amino groups (—NH2). Under acidic conditions (pH < 6.5), these amino groups can be protonated to form —NH3 + and thus carry a positive charge, while casein phosphopeptide is a negatively charged peptide, and chitosan and casein phosphopeptide can bind through electrostatic interaction.

[0044] pH value range: Research shows that under acidic conditions (such as when the pH value is below 6.0), the degree of protonation of the amino groups of chitosan is higher and the ability to carry a positive charge is stronger. The isoelectric point of casein phosphopeptide is 4.6. When the pH value is higher than 4.6, casein phosphopeptide carries a negative charge, and casein phosphopeptide is also more likely to carry a negative charge in an acidic environment, which is conducive to electrostatic interaction with positively charged chitosan. Therefore, the pH value between 4.5 and 6.0 may be the optimal range for realizing the electrostatic interaction between chitosan and casein phosphopeptide.

[0045] In this embodiment, the mechanism by which tannic acid inhibits the activity of GTF (glucosyltransferase) and reduces the formation of glucan.

[0046] Tannic acid inhibits the activity of glucosyltransferase (GTF) through various mechanisms, thereby reducing the formation of glucan and playing an anti-biofilm role. The following is a specific explanation:

[0047] 1. Inhibiting the activity of glucosyltransferase

[0048] Glucosyltransferase is a key enzyme that can catalyze the decomposition of sucrose and synthesize glucan. Glucan is the main component of dental plaque biofilm, providing an attachment point for bacteria and enhancing the stability of the biofilm.

[0049] Inhibitory mechanism of tannic acid: Tannic acid binds to the active site of glucosyltransferase through its polyphenol structure, interfering with the catalytic process of the enzyme, thereby inhibiting the activity of glucosyltransferase. This inhibition reduces the synthesis of glucan, meaning that bacteria lose their attachment points, thus reducing the formation and stability of biofilms and weakening the formation of dental plaque biofilms.

[0050] Functions of tannic acid and casein phosphopeptide: Tannic acid can inhibit the activity of GTF, thereby reducing the formation of glucan. Casein phosphopeptide interferes with the synthesis process of glucan by releasing calcium ions and phosphate ions. These mechanisms work together to reduce the accumulation of glucan on tooth enamel, thus weakening the formation of biofilms.

[0051] This example also provides a preparation method of an anti-caries oral care composition, including the following steps:

[0052] S1. Suspend Bifidobacterium infantis (BI) in deionized water at a concentration of 1×107 CFU / mL, and its optical density at 600 nm is 0.4;

[0053] S2. Add 150 mL of tannic acid solution with a concentration of 40 mg / mL to 150 mL of calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of polyvinylpyrrolidone solution with a mass percentage of 0.5 wt% and 5 mL of citric acid with a mass percentage of 0.1%, stir evenly, and then add the bacterial solution in step S1. Centrifuge at 8000 rpm for 5 min to form a tannic acid-calcium ion-polyvinylpyrrolidone coating on the surface of Bifidobacterium infantis (BI);

[0054] S3. Resuspend the bacterial solution in step S2 in deionized water at a bacterial density of 1×107 CFU / mL, then add 200 mL of casein phosphopeptide with a concentration of 40 mg / mL, and mix at a constant rotation speed of 80 rpm for 2 h to obtain Bifidobacterium infantis @ tannic acid-calcium ion-citric acid-polyvinylpyrrolidone coating @ casein phosphopeptide, which is an anti-caries oral care composition.

[0055] Example 2

[0056] This example provides an anti-caries oral care composition, including: probiotic Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, and acidic stabilizer triethyl phosphate; there are a tannic acid-calcium ion-polyvinylpyrrolidone coating and a casein phosphopeptide layer on the surface of probiotic Bifidobacterium infantis in sequence; the acidic stabilizer binds to the tannic acid-calcium ion-polyvinylpyrrolidone coating and can effectively stabilize the tannic acid-calcium ion-polyvinylpyrrolidone coating.

[0057] In this example, the concentration of the probiotic Bifidobacterium infantis (BI) is 1×10⁷ CFU / mL.

[0058] This example also provides a method for preparing an anti-caries oral care composition, which includes the following steps:

[0059] S1. Suspend Bifidobacterium infantis (BI) in deionized water at a concentration of 1×10⁷ CFU / mL, and its optical density at 600 nm is 0.4;

[0060] S2. Add 150 mL of tannic acid solution with a concentration of 40 mg / mL to 150 mL of calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of polyvinylpyrrolidone solution with a mass percentage of 2 wt% and 5 mL of triethylamine phosphate with a mass percentage of 2% as an acidic stabilizer, stir evenly, and then add the bacterial solution from step S1. Centrifuge at 8000 rpm for 5 min to form a tannic acid-calcium ion-triethylamine phosphate-polyvinylpyrrolidone coating on the surface of Bifidobacterium infantis (BI);

[0061] S3. Resuspend the bacterial solution from step S2 in deionized water at a bacterial density of 1×10⁷ CFU / mL, then add 200 mL of casein phosphopeptide with a concentration of 40 mg / mL, and mix at a constant rotation speed of 80 rpm for 2 h to obtain Bifidobacterium infantis @ tannic acid-calcium ion-polyvinylpyrrolidone coating @ casein phosphopeptide, which is an anti-caries oral care composition.

[0062] Example 3

[0063] This example provides an anti-caries oral care composition, including: the probiotic Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, and the acidic stabilizer triethylamine phosphate; there are a tannic acid-calcium ion-polyvinylpyrrolidone coating and a casein phosphopeptide layer on the surface of the probiotic Bifidobacterium infantis in sequence; the acidic stabilizer combines with the tannic acid-calcium ion-polyvinylpyrrolidone coating, which can effectively stabilize the tannic acid-calcium ion-polyvinylpyrrolidone coating.

[0064] In this example, the concentration of the probiotic Bifidobacterium infantis (BI) is 1×10⁷ CFU / mL.

[0065] This example also provides a method for preparing an anti-caries oral care composition, which includes the following steps:

[0066] S1. Suspend Bifidobacterium infantis (BI) in deionized water at a concentration of 1×10⁷ CFU / mL, and its optical density at 600 nm is 0.4;

[0067] S2. Add 150 mL of tannic acid solution with a concentration of 40 mg / mL to 150 mL of calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of polyvinylpyrrolidone solution with a mass percentage of 1 wt% and 5 mL of triethylamine phosphate as an acidic stabilizer with a mass percentage of 0.5%. Stir evenly, and then add the bacterial solution from step S1. Centrifuge at 8000 rpm for 5 min to form a tannic acid-calcium ion-polyvinylpyrrolidone coating on the surface of Bifidobacterium infantis (BI).

[0068] S3. Resuspend the bacterial solution from step S2 in deionized water at a bacterial density of 1×10⁷ CFU / mL, and then add 200 mL of casein phosphopeptide with a concentration of 40 mg / mL. Mix at a constant rotation speed of 80 rpm for 2 h to obtain Bifidobacterium infantis @ tannic acid-calcium ion-triethylamine phosphate-polyvinylpyrrolidone coating @ casein phosphopeptide, which is a kind of anti-caries oral care composition.

[0069] Example 4

[0070] This example provides an anti-caries oral care composition, including: probiotic Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, acidic stabilizer citric acid, chitosan; there are a tannic acid-calcium ion-polyvinylpyrrolidone coating and a casein phosphopeptide layer on the surface of probiotic Bifidobacterium infantis in sequence; the casein phosphopeptide layer coats the chitosan coating.

[0071] In this example, the concentration of probiotic Bifidobacterium infantis (BI) is 1×10⁷ CFU / mL.

[0072] This example also provides a preparation method of an anti-caries oral care composition, including the following steps:

[0073] S1. Suspend Bifidobacterium infantis (BI) in deionized water at a concentration of 1×10⁷ CFU / mL, and its optical density at 600 nm is 0.4.

[0074] S2. Add 150 mL of tannic acid solution with a concentration of 40 mg / mL to 150 mL of calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of polyvinylpyrrolidone solution with a mass percentage of 0.5 wt% and 5 mL of citric acid as an acidic stabilizer with a mass percentage of 0.1%. Stir evenly, and then add the bacterial solution from step S1. Centrifuge at 8000 rpm for 5 min to form a tannic acid-calcium ion-polyvinylpyrrolidone coating on the surface of Bifidobacterium infantis (BI).

[0075] S3. Resuspend the bacterial solution from step S2 in deionized water at a bacterial density of 1×107 CFU / mL, then add 200 mL of casein phosphopeptide with a concentration of 40 mg / mL, and mix for 2 h at a constant rotation speed of 80 rpm to obtain Bifidobacterium infantis @ tannic acid-calcium ion-citric acid-polyvinylpyrrolidone coating @ casein phosphopeptide;

[0076] S4. Resuspend Bifidobacterium infantis @ tannic acid-calcium ion-citric acid-polyvinylpyrrolidone coating @ casein phosphopeptide in deionized water at a bacterial density of 1×107 CFU / mL, add 5 mL of chitosan with a mass percentage of 1.0 - 3.0 wt%, and mix for 2 h at a constant rotation speed of 80 rpm to obtain an anti-caries oral care composition.

[0077] Example 5

[0078] Provide an application of an anti-caries oral care composition in the preparation of an anti-caries oral care toothpaste, which also includes a pH slow-release regulator, a flavor, and a sweetener.

[0079] In this example, the pH slow-release regulator (such as D-(+)-glucono-δ-lactone, GDL) can regulate the pH value inside the tannic acid-Ca2+ coating, buffer the external acidic environment, and reduce the impact of the acidic environment on the cross-linked network. If the external environment itself is acidic, the slow-release acidification characteristic of the pH slow-release regulator can form a dynamic balance with the external acidic environment. Although the pH slow-release regulator will decrease the pH value inside the hydrogel, its slow-release characteristic can prevent the internal pH value from dropping significantly instantaneously, thereby buffering the impact of the external acidic environment on the hydrogel to a certain extent. This method not only improves the mechanical properties of the hydrogel but also endows it with antibacterial function.

[0080] Comparative Example 1

[0081] Different from Example 1, this comparative example provides an anti-caries oral care composition, including: probiotic Bifidobacterium infantis, tannic acid, calcium ion, casein phosphopeptide, acidic stabilizer citric acid; there are tannic acid-calcium ion-citric acid coating and casein phosphopeptide layer on the surface of probiotic Bifidobacterium infantis in sequence.

[0082] Comparative Example 2

[0083] Different from Example 1, this comparative example provides an anti-caries oral care composition, including: probiotic Bifidobacterium infantis, tannic acid, calcium ion, casein phosphopeptide; there are tannic acid-calcium ion coating and casein phosphopeptide layer on the surface of probiotic Bifidobacterium infantis in sequence.

[0084] Experimental Example

[0085] Three-week-old Sprague-Dawley rats were prepared for in vivo anti-caries experiments. To create a caries environment, except for the blank group, rats in other groups were treated with 2.4 mg / mL ampicillin and 0.5 mg / mL streptomycin for 4 days to eliminate the original bacteria in the oral cavity. Then, the diet was replaced with a cariogenic diet supplemented with 5% sucrose solution, and all rats were locally inoculated with Streptococcus mutans at a concentration of 1×108 CFU / mL for 4 days, and infection occurred 2 days later. Then the rats were randomly divided into 8 groups, with 5 rats in each group, and were respectively given the anti-caries oral care composition of Example 1, in which the concentration of the probiotic Bifidobacterium infantis (BI) was 1×107 CFU / mL, the probiotic Bifidobacterium infantis (negative control group), or a commercial cetylpyridinium chloride mouthwash containing 0.01% sodium fluoride (positive control group). All rats received treatment once in the morning and once every night for 6 consecutive weeks. Saliva samples of the mice were collected regularly (weekly) for analyzing the colonization of probiotics and oral health indicators.

[0086] The plate counting method was used to detect the colonization amount of the probiotic Bifidobacterium infantis in the saliva samples, with the unit of CFU / mL. Specifically, the saliva samples were diluted 10 times, 100 μL of the samples were taken and inoculated on the corresponding culture medium, and cultured at 37 °C for 24 hours, and the colony-forming units (CFU / mL) were counted. The results are as Figure 1 shown.

[0087] Probiotic colonization: 16S rDNA sequencing was used to analyze the microbial community in the saliva samples to evaluate the colonization of probiotics.

[0088] Oral health index evaluation:

[0089] Caries score: Micro-computed tomography was used to observe the caries situation of the mice's teeth for caries scoring. High-resolution micro-computed tomography was used, with the source voltage of 70 kV and the source current of 200 mA. The mandible was dissected to evaluate its anti-caries effect. The original images were reconstructed into three-dimensional images using CTVox software, and the volume of the residual enamel was quantified by C tannic acid n software.

[0090] It can be seen from Figure 1 that compared with Comparative Example 1 and Comparative Example 2, the colonization rate of the probiotic Bifidobacterium infantis in the rats of the groups of Examples 1-4 was significantly higher than that of Comparative Example 1 and Comparative Example 2, and Comparative Example 2 was the worst, indicating that the anti-caries oral care compositions of the groups of Examples 1-4 could successfully colonize in the oral cavity of the rats.

[0091] Compared with Comparative Example 1 and Comparative Example 2, the results showed that the caries score of the rats in the group of Example 1 was significantly lower than that of Comparative Example 1 and Comparative Example 2, indicating that the anti-caries oral care composition of Example 1 could effectively prevent caries.

[0092] Microbial community analysis: The results showed that, compared with Comparative Example 1 and Comparative Example 2, in the oral microbial community of the rats in Example 1 group, the abundance of the probiotic Bifidobacterium infantis increased significantly, while the abundance of cariogenic bacteria (such as Streptococcus mutans) decreased significantly.

[0093] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection required by the present invention.

Claims

1. A caries-preventive oral care composition, characterized in that, Comprising: Bifidobacterium infantis, tannic acid, calcium ions, polyvinylpyrrolidone, casein phosphopeptide, acidic stabilizer; The surface of the probiotic Bifidobacterium infantis is successively coated with tannic acid - calcium ion - acidic stabilizer - polyvinylpyrrolidone coating and casein phosphopeptide layer.

2. The anti-caries oral care composition according to claim 1, wherein, The concentration of the Bifidobacterium infantis is 1×10 7 CFU / mL.

3. The anti-caries oral care composition according to claim 1, wherein, The acidic stabilizer is an organic compound containing a carboxyl group or a phosphate group.

4. The anti-caries oral care composition according to claim 3, wherein The organic compound containing a carboxyl group or a phosphate group includes citric acid and / or triethylamine phosphate.

5. The anti-caries oral care composition according to claim 1, wherein It also includes chitosan, and the casein phosphopeptide layer coats the chitosan coating.

6. A method for preparing an anti-caries oral care composition according to any one of claims 1 to 5, characterized in that, Including the following steps: S1. Suspend Bifidobacterium infantis in deionized water at a concentration of 1×10 7 CFU / mL, and its optical density at 600 nm is 0.4; S2. Add 150 mL of tannic acid solution with a concentration of 40 mg / mL to 150 mL of calcium chloride solution with a concentration of 10 mg / mL, then add 30 mL of polyvinylpyrrolidone solution with a mass percentage of 0.5 - 2.0 wt% and 5 mL of glucono - δ - lactone with a mass percentage of 0.1% - 2%, stir evenly, and then add the bacterial solution from step S1. Centrifuge at 8000 rpm for 5 min to form the tannic acid - calcium ion - polyvinylpyrrolidone coating on the surface of the Bifidobacterium infantis (BI); S3. Resuspend the bacterial solution from step S2 at a bacterial density of 1×10 7 CFU / mL in deionized water, then add 200 mL of casein phosphopeptide (CPP) with a concentration of 40 mg / mL, and mix at a constant rotation speed of 80 rpm for 2 h to obtain Bifidobacterium infantis@tannic acid-calcium ion-polyvinylpyrrolidone coating@casein phosphopeptide, which is the described anti-caries oral care composition.

7. The preparation method of an anti-caries oral care composition according to claim 6, characterized in that, After step S3, the Bifidobacterium infantis @ tannic acid - calcium ion - polyvinylpyrrolidone coating @ casein phosphopeptide is resuspended in deionized water at a bacterial density of 1×10 7 CFU / mL, and 1.0 - 3.0 wt% of chitosan is added, and the mixture is stirred at a constant speed of 80 rpm for 2 h to obtain the anti - caries oral care composition.

8. Use of a caries-preventive oral care composition according to any one of claims 1 to 5 in the preparation of a caries-preventive oral care toothpaste, characterized in that, It also includes a pH slow - release regulator, fragrance, and sweetener.

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