A dental composition

Abstract

This disclosure provides an oral composition comprising tourmaline, methyl salicylate, glycerin, and additives. The disclosure also provides the use of the oral composition for oral hygiene care, and further provides an oral hygiene product comprising the oral composition.

Description

Technical Field

[0001] This invention relates to the field of oral care, and more specifically to an oral composition. Background Technology

[0002] Tourmaline, also known as tourmaline stone, is a ring-structured silicate mineral powder characterized by boron content and containing aluminum, sodium, iron, magnesium, and lithium. It possesses unique properties such as piezoelectricity, pyroelectricity, conductivity, far-infrared radiation, and the release of negative ions. Through physical or chemical methods, it can be combined with other materials to create a variety of functional materials, which are used in environmental protection, electronics, medicine, chemical industry, light industry, and building materials. In oral care products, tourmaline-added functional toothpaste has a remarkable effect in quickly eliminating extrinsic oral odor (halitosis); it also has a significant relieving effect on patients with chronic gingivitis, gingival bleeding, and periodontitis (see "Pure Natural Mineral---Tourmaline Negative Ions and Their Efficacy in Oral Care Products," Chen Jianfen et al., Oral Care Products Industry, 2014, Issue 01).

[0003] In recent years, many oral care products using tourmaline have emerged. For example, patent application CN202211054638.4 describes a negative ion teeth whitening and cleaning preparation, which incorporates a negative ion agent, such as tourmaline. It is claimed that the addition of the negative ion agent can quickly remove tooth stains and odors, and eliminate oral bacteria. Patent application CN201911270174.9 describes a refreshing toothpaste that incorporates tourmaline and lactobacillus. The tourmaline continuously releases negative oxygen ions to remove bad breath, while the lactobacillus binds to and removes pathogens in the mouth through aggregation, preventing oral diseases (cavities, periodontal disease, and halitosis). Maintaining oral flora balance and freshening breath are also achieved through toothpaste containing tourmaline (CN201811181587.5), which adds tourmaline to remove odors and freshen breath. However, the high amount of tourmaline can negatively impact the added lactobacilli, resulting in limited improvement in oral hygiene. Toothpastes described in CN201911264097.6, CN201911270174.9, CN201810935590.5, CN201810646952.9, and CN201510199211.7 contain small amounts of tourmaline to remove odors. These toothpastes aim to achieve fresh breath and whiten teeth through the combined action of various ingredients, but the individual components rarely work synergistically. Therefore, in oral compositions containing tourmaline, it is difficult to fully utilize the individual components' effects. Summary of the Invention

[0004] The problem to be solved by the present invention is to address the problems existing in the prior art and to provide a heart-shaped oral composition.

[0005] In a first aspect of the invention, an oral composition is provided comprising the following components:

[0006] (1) Tourmaline, the proportion of tourmaline is 2-8 parts (by weight);

[0007] (2) Digestive enzymes, wherein the proportion of the protease is 2 parts (by weight), and the digestive enzymes include protease and lysozyme;

[0008] (3) Glycerin, wherein the proportion of glycerin is 15-30 parts (by weight);

[0009] (4) Additives, wherein the additives account for 1 part (by weight); the additives include licorice and Bletilla striata.

[0010] In some embodiments, the tourmaline is lithium tourmaline, comprising 3-6 parts (by weight). Many prior art oral compositions are prepared without considering the differences between different tourmalines. According to the website "The Mineral & Gemstone Kingdom" (http: / / www.minerals.net / tourmaline_chemical_formula.aspx), tourmaline is a group of isomorphous minerals with the same crystal lattice. Due to slight differences in their elemental distribution, each member of the tourmaline group has its own chemical formula. Different tourmalines do not function consistently, and their function is also related to the specific composition in which they are present.

[0011] In some implementations, the tourmaline comprises 5 parts (by weight).

[0012] In some embodiments, the protease is a subtilisin, the amino acid sequence of which contains more than 85% identity with the amino acid sequence shown in SEQ ID NO.1.

[0013] In some embodiments, the amino acid sequence of the subtilisin contains a mutation at the following positions relative to the amino acid sequence shown in SEQ ID NO.1: D40L or S101G.

[0014] In some embodiments, the subtilisin is divided into protease A and protease B, wherein the amino acid sequence of protease A has a mutation at the following position relative to the amino acid sequence shown in SEQ ID NO.1: D40L, and the amino acid sequence of protease B has a mutation at the following position relative to the amino acid sequence shown in SEQ ID NO.1: S101G.

[0015] In some embodiments, the weight ratio of protease A to protease B is 1:2.

[0016] In some embodiments, the oral composition also contains 1 part by weight of an additive.

[0017] In some embodiments, the additive is licorice and Bletilla striata, wherein the weight ratio of licorice and Bletilla striata is 1:3-5.

[0018] In some embodiments, the weight ratio of Bletilla striata to licorice is 1:3.

[0019] In some embodiments, the amino acid sequence of the lysozyme is as shown in SEQ ID NO.1.

[0020] In a second aspect of the invention, the use of the said composition in oral hygiene care is provided.

[0021] In a third aspect of the invention, an oral hygiene product is provided, comprising the composition described in the first aspect.

[0022] The main advantages of this invention are:

[0023] 1. The oral composition of the present invention contains tourmaline, and a type of tourmaline with good odor-eliminating effect has been identified.

[0024] 2. The protease used in the existing technology was mutated, and a combination of two proteases was used. It was found that the protease has better stability and whitening effect compared with the existing technology.

[0025] 3. The addition of licorice and Bletilla striata to the oral composition significantly improves the taste of the oral composition. Detailed Implementation

[0026] To enable those skilled in the art to more clearly understand the purpose, technical solution, and advantages of the invention, the invention will be further described below in conjunction with embodiments.

[0027] Technical terms

[0028] The term "tourmaline," also known as jasper, refers to a silicate mineral, a multi-element natural mineral, primarily composed of magnesium, aluminum, iron, boron, and various trace elements. Tourmaline acquires an electrical charge when heated, a phenomenon known as pyroelectricity, hence its name. Each member of the tourmaline group has its own chemical formula due to slight differences in their elemental distribution. For example, in some embodiments, the tourmaline is black tourmaline, magnesium tourmaline, lithium tourmaline, calcium-lithium tourmaline, calcium-magnesium tourmaline, etc.

[0029] The term "protease" also includes peptidases, which are enzymes that hydrolyze peptide bonds. Proteases described herein include those derived from bacteria, fungi, plants, viruses, or animals, such as plant or microbial proteases, microbial proteases, and also chemically modified or protein-engineered mutants. In some embodiments, the protease is a serine protease, such as an S1 family protease (e.g., trypsin) or an S8 family protease (e.g., subtilisin). Serine proteases are a subgroup of proteases characterized by having a serine residue at the active site, which forms a covalent adduct with the substrate.

[0030] The term "subtilisin" refers to the S8 family of serine proteases, according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. Subtilisins can be divided into six subclasses: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanantibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0031] The term "wild-type protease" refers to a protease expressed by a naturally occurring organism (e.g., bacteria, archaea, yeast, fungi, plants, or animals found in nature). In some embodiments, the amino acid sequence of wild-type subtilisin is shown in SEQ ID NO.1.

[0032] The term "variant" refers to a polypeptide having protease activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at three or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that one or more amino acids, such as 1, 2, 3, 4, or 5 amino acids, are added adjacent to and immediately following an amino acid occupying a position. The term "subtilisase variant" refers to a variant of the parent subtilisase, i.e., a subtilisase variant that includes alterations (i.e., substitutions, insertions, and / or deletions) at three or more (e.g., several) positions compared to the parent subtilisase. In some embodiments, the amino acid sequence of the subtilisase variant has mutations at the following positions relative to the amino acid sequence shown in SEQ ID NO.1: D40L or S101G.

[0033] The mutant protein fragments, derivatives, or analogs of the present invention may be (i) mutant proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, wherein such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) mutant proteins having substituent groups in one or more amino acid residues; or (iii) mutant proteins formed by fusing a mature mutant protein with another compound (e.g., a compound that extends the half-life of the mutant protein, such as polyethylene glycol); or (iv) mutant proteins formed by fusing an additional amino acid sequence to the mutant protein sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify the mutant protein or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art. In some embodiments, the conserved substituted amino acids are preferably produced by amino acid substitution according to Table 1.

[0034] Table 1

[0035]

[0036] In some embodiments, other selected groups of amino acids that are considered to have mutually conserved substitutions (see, for example, Creighton, Proteins (1984)):

[0037] Table 2

[0038] Detailed Implementation

[0040] Example 1: Tourmaline Antibacterial Test

[0041] 1. Prepare black tourmaline powder, magnesium tourmaline powder, lithium tourmaline powder, calcium-lithium tourmaline powder, and calcium-magnesium tourmaline powder respectively, control their particle size (8000 mesh), and add the above tourmaline powders to the basic toothpaste components to obtain toothpastes containing different tourmaline powders, which are numbered 1A-1E respectively.

[0042] Table 3

[0043]

[0044] 2. *Actinomyces actinomycete* ATCC 29523, *Streptococcus mutans* CGMCC 12499, and *Fusobacterium nucleatum* ATCC 25586 were used as test strains. They were cultured according to standard methods (at 37℃) to obtain bacterial suspensions of the above three strains (the final bacterial suspension concentration was adjusted to 1×10⁻⁶). 9(CFU / mL). Take 1 mL of each toothpaste composition from 1A to 1E and mix thoroughly to prepare toothpaste solution. Then, use serially diluted toothpaste solutions to test the minimum inhibitory concentration (MIC) of each group. The test results are as follows:

[0045] Table 4

[0046]

[0047] As shown in Table 4, compared with experimental groups 1A, 1B, 1D, and 1E, experimental group 1C had the lowest MIC value (MIC values ​​for *Actinomyces actinomycete* ATCC 29523, *Streptococcus mutans* CGMCC 12499, and *Fusobacterium nucleatum* ATCC25586 were as low as 1 / 82 mg / L, 1 / 135 mg / L, and 1 / 98 mg / L, respectively). This indicates that when the amount of tourmaline powder added is 2 parts, the addition of lithium tourmaline powder to the toothpaste composition has the best antibacterial effect, while the use of other tourmalines has a poor antibacterial effect.

[0048] 3. Experiments on the amount of tourmaline added.

[0049] To investigate the effect of different tourmaline addition amounts on the antibacterial effect, the inventors designed a comparative experiment to determine the optimal amount of tourmaline powder for achieving the best antibacterial effect. The following experimental design was conducted (the toothpaste base ingredients and their contents used in the experiment were the same as those in Table 3, and the MIC values ​​were as follows):

[0050] Table 5

[0051]

[0052] As shown in Table 5, compared with other experimental groups, experimental group 2C showed better antibacterial effect, and its antibacterial effect was significantly better than other cross-sectional experimental groups when the amount of lithium tourmaline powder added was in the range of 2-8 parts. *Actinomyces actinomycetes* is a Gram-negative short bacillus, a facultative anaerobic bacterium with pili, no spores, and no motility. In recent years, it has been recognized as closely related to periodontitis, especially invasive periodontitis. *Streptococcus mutans* is a Gram-positive coccus, one of the most prevalent streptococci in the oral microbiota, and a major component of dental plaque. *Fusobacterium nucleatum* is a Gram-negative obligate anaerobic bacterium, one of the oral commensal bacteria, closely related to periodontal pockets, and clinical studies have shown its association with gingivitis and periodontitis. Selecting lithium tourmaline as a toothpaste ingredient, with an addition amount of 2-8 parts, showed a good inhibitory effect on the above three oral microbiota.

[0053] Example 2 Construction and activity detection of protease variants

[0054] 1. Construction of protease mutant expression vector

[0055] The Bacillus subtilis enzyme sequence described in CN201811556321.4 (sequence number SEQ ID NO:3 in that patent application, and sequence number SEQ ID NO:1 in this invention) was further mutated. The inventors predicted key amino acid sites that might affect its biological function using bioinformatics and mutated these sites, resulting in new Bacillus subtilis enzyme A and Bacillus subtilis enzyme B.

[0056] First, based on the sequence of wild-type Bacillus subtilis enzyme (hereinafter referred to as WT) SEQ ID NO.1, the inventors constructed five single-point mutants between positions 30 and 200, and constructed the nucleotide sequences encoding WT and the mutants into the pET32a expression vector. Then, the obtained expression vectors were transformed into BL21(DE3) Escherichia coli (NEB) for expression. After purification and activity detection, the following mutant proteases were obtained.

[0057] Table 6

[0058]

[0059] Determining the whitening effects of various proteases

[0060] The teeth whitening effect was measured using the same method as in Example 1 of CN201811556321.4:

[0061] (1) Staining of dense hydroxyapatite:

[0062] Seven standard dense hydroxyapatite sheets, each 30 mm long, 20 mm wide, and 4 mm thick, were prepared for use in a toothbrush. After cutting and grinding, the hydroxyapatite sheets were washed with clean water, soaked in softened water, and air-dried before being fixed on a staining machine. The staining machine then used a mixture of artificial saliva (Solarbio, A7990), tea extract, and coffee extract to stain the hydroxyapatite sheets. The sheets were subjected to a cycle of staining solution immersion, heating aging, and staining solution immersion on the staining machine. After 12 hours, the staining was completed, and the sheets were air-dried for use.

[0063] (2) The toothpaste composition of the experimental group was applied to the surface of all 7 standard dense hydroxyapatite tablets. The basic toothpaste composition consisted of the following components: 40 parts calcium carbonate, 20 parts sorbitol, 15 parts glycerin, 1 part cellulose gum, 1 part sodium lauryl sulfate, 0.3 parts fragrance, and 2 parts lithium tourmaline powder. Then, each protease mutant and wild-type protease (WT) were added to the basic toothpaste composition at a weight ratio of 2 parts. After brushing with a toothbrush for 20 minutes, the tablets were quickly removed, rinsed with water, and dried before use. A whiteness meter was then used to measure the whiteness of the experimental group that had not undergone toothbrush cleaning (this group was only stained without toothpaste composition application and toothbrush cleaning), and then the whiteness after the toothbrush cleaning experiment was measured. The difference was the effect of the whitening substance.

[0064] Table 7

[0065]

[0066] As shown in Table 7, compared with toothpaste compositions containing wild-type protease (WT) and other mutant toothpaste compositions, toothpaste compositions containing protease variants (D40L, S101G) shown in SEQ ID NO:1 have significantly improved teeth whitening efficacy.

[0067] The inventors then conducted an experiment on the ratio of the two mutant proteins. The experimental method was the same as steps (1) and (2), and the total amount of mutant protease added was still 2 parts by weight. The experimental results are shown in Table 8.

[0068] Table 8

[0069]

[0070] Example 3: Taste Test of Toothpaste Composition

[0071] Through the experiments in Examples 1 and 2, the inventors obtained toothpaste components with good antibacterial and whitening effects. To improve the taste of the toothpaste, the inventors added 1 part by weight of additives (licorice and Bletilla striata) to the toothpaste composition. Since Bletilla striata has a high viscosity, the inventors tested the brushing comfort of different viscosities and found that the tooth comfort was higher when the amount of licorice exceeded that of Bletilla striata. Therefore, the inventors set the amount of licorice added to be more than that of Bletilla striata.

[0072] In this embodiment, the toothpaste base composition (by weight) is as follows: 40 parts calcium carbonate, 20 parts sorbitol, 15 parts glycerin, 1 part cellulose gum, 1 part sodium lauryl sulfate, 0.3 parts fragrance, 2 parts lithium tourmaline powder, and 2 parts Bacillus subtilis protease (of which protease A is 0.66 parts and protease B is 1.34 parts). Then, the ratio of licorice and Bletilla striata was set to 1:1-1:10 respectively. Ten ordinary consumers (all adults, aged 20-45 years) were selected, and toothpastes with different proportions of additives were evaluated using a scale of 1-10. The following results were obtained.

[0073] Table 9

[0074]

[0075] As shown in Table 9, the taste evaluation is better when the weight ratio of Bletilla striata to licorice is between 1:3 and 5, especially when the weight ratio is 1:4.

[0076] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

[0077] sequence list

[0078]

Claims

1. An oral composition comprising the following components: (1) tourmaline, the proportion of tourmaline being 2-8 parts by weight; (2) digestive enzyme, the digestive enzyme being protease, the proportion of the protease being 2 parts by weight; (3) glycerol, the proportion of the glycerol being 15-30 parts by weight; (4) additive, the proportion of the additive being 1 part by weight; the additive comprising liquorice and bletilla striata; the protease being subtilisin, the subtilisin comprising protease A and protease B, protease A and protease B respectively having mutations at the following positions in the amino acid sequence shown in SEQ ID NO. 1: D40L, S101G; the weight ratio of protease A to protease B being 1:

2.

2. The composition according to claim 1, the tourmaline being lithium tourmaline, the proportion of the tourmaline being 3-6 parts by weight.

3. The composition according to claim 2, the proportion of the tourmaline being 5 parts by weight.

4. The composition according to claim 3, the weight ratio of bletilla striata to liquorice being 1:3-5.

5. An oral hygiene product comprising the composition according to any one of claims 1-4.

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