CN117398295A - Oral composition - Google Patents

Abstract

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

Description

An oral composition

Technical Field

The invention relates to the field of oral care, in particular to an oral composition.

Background

Tourmaline, also called tourmaline or tourmaline, is a silicate mineral powder with a cyclic structure, characterized by boron, of aluminum, sodium, iron, magnesium, lithium, has unique properties of piezoelectricity, pyroelectric property, conductivity, far infrared radiation, anion release and the like, can be compounded with other materials by a physical or chemical method to prepare various functional materials, and is applied to the fields of environmental protection, electronics, medicine, chemical industry, light industry, building materials and the like. In the aspect of oral products, the tourmaline-added functional toothpaste has the outstanding effect of rapidly removing exogenous oral odor (halitosis); has obvious relieving effect on patients with chronic gingivitis, gingival bleeding and periodontitis (see 'pure natural minerals-tourmaline negative ions and efficacy research thereof in oral products', chen Jianfen and the like, oral care products industry, 2014 and 01).

In recent years, a number of oral products using tourmaline have appeared, for example, in patent application CN202211054638.4, an anionic tooth whitening cleaning preparation is described, in which an anionic agent is added, and the anionic agent can be used as tourmaline, and after the anionic agent is added, the oral products can be used for rapidly removing stains and peculiar smell and removing oral bacteria; patent application CN201911270174.9 describes a fresh toothpaste, in which tourmaline (i.e. tourmaline) and lactobacillus are added into the toothpaste, the added tourmaline component continuously releases negative oxygen ions and can remove bad breath, the lactobacillus is combined with pathogenic bacteria in the oral cavity through agglutination and removes the pathogenic bacteria to prevent oral diseases (tooth decay, periodontal disease and halitosis) from happening, the oral flora balance is maintained, and the breath is freshened, and also CN201811181587.5 is added into the toothpaste to remove bad breath, but the added tourmaline has a large amount and affects the added lactobacillus, but the effect of improving oral hygiene is limited; a small amount of tourmaline is added into the toothpaste described in CN201911264097.6, CN201911270174.9, CN201810935590.5, CN201810646952.9 and CN201510199211.7 to remove peculiar smell, and the effects of refreshing breath and whitening teeth are achieved through the effects of the components, but the synergistic effects of the components are difficult to achieve in practice. Therefore, it is difficult to sufficiently exert the functions of the respective components in the oral composition containing tourmaline.

Disclosure of Invention

The invention aims to solve the problems in the background art and provides a heart-shaped oral composition.

In a first aspect of the present invention, there is provided an oral composition comprising the following components:

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

(2) 2 parts by weight of digestive enzymes including protease and lysozyme;

(3) Glycerol, the proportion of which is 15-30 parts by weight;

(4) An additive in a proportion of 1 part by weight; the additive comprises licorice and bletilla striata.

In some embodiments, the tourmaline is a lithium tourmaline, and the proportion of the tourmaline is 3-6 parts (by weight). In The prior art, when many oral compositions are prepared, the difference of different tourmaline is not considered, and according to The content recorded in The website of The Mineral & Gemstone Kingdom (http:// www.minerals.net/tourmaline_chemical_formula. Aspx), the tourmaline is a group of isomorphous ores with The same crystal lattice. Each member of the tourmaline group has its own chemical formula due to a small difference in element distribution thereof. Different tourmaline is not consistent in action, and the action of the tourmaline is also related to the specific condition of the composition in which the tourmaline is positioned.

In some embodiments, the tourmaline is present in a proportion of 5 parts by weight.

In some embodiments, the protease is a subtilisin, the subtilisin amino acid sequence comprising an amino acid sequence having greater than 85% identity to the amino acid sequence set forth in SEQ ID No. 1.

In some embodiments, the amino acid sequence of the subtilisin has a mutation at the following positions relative to the amino acid sequence set forth in SEQ ID No. 1: D40L or S101G.

In some embodiments, the subtilisin is divided into protease a and protease B, the amino acid sequence of which protease a has a mutation at the following positions relative to the amino acid sequence set forth in SEQ ID No. 1: d40L, the amino acid sequence of protease B having a mutation at the following position relative to the amino acid sequence shown in SEQ ID No. 1: S101G.

In some embodiments, the weight ratio of protease a to protease B is 1:2.

In some embodiments, the oral composition further comprises 1 part by weight of an additive.

In some embodiments, the additive is licorice and bletilla striata, wherein the weight ratio of the licorice to the bletilla striata is 1:3-5.

In some embodiments, the weight ratio of bletilla striata to licorice is 1:3.

In some embodiments, the lysozyme amino acid sequence is as shown in SEQ ID NO. 2.

In a second aspect of the invention there is provided the use of the composition in oral hygiene care.

In a third aspect of the present invention there is provided an oral hygiene product comprising the composition of the first aspect.

The invention has the main advantages that:

1. the oral composition contains tourmaline and finds out the tourmaline type with better effect of eliminating peculiar smell.

2. The protease adopted in the prior art is mutated, and the combination of the two proteases is adopted, so that the protease stability and the whitening effect are better compared with the prior art.

3. The liquorice and the bletilla striata are added into the oral composition, so that the taste of the oral composition is obviously improved.

Detailed Description

The invention is further described below in connection with examples in order to more clearly understand the objects, technical solutions and advantages of the invention by those skilled in the art.

Technical terminology

The term tourmaline is also called tourmaline, which is a silicate mineral and is a multi-element natural mineral, and the main component of the tourmaline is magnesium, aluminum, iron, boron and other trace elements. Tourmaline will charge when heated, this phenomenon is called pyroelectric effect, so the name tourmaline is obtained. Each member of the tourmaline group has its own chemical formula due to a small difference in the distribution of its elements. For example, in some embodiments, the tourmaline is a schorl, a magnesian tourmaline, a lithium tourmaline, a callith tourmaline, a calomel tourmaline, or the like.

The term "protease" also includes peptidases, meaning enzymes that hydrolyze peptide bonds, and proteases described herein include proteases of bacterial, fungal, plant, viral or animal origin, e.g., plant or microbial, microbial origin, and chemically modified or protein engineered mutants. In some embodiments, the protease is a serine protease, e.g., an S1 family protease (e.g., trypsin) or an S8 family protease (e.g., subtilisin). Serine proteases are a subgroup of proteases characterized by serine at the active site, which forms a covalent adduct with a substrate.

The term "subtilisin" refers to the S8 family protease of serine proteases, according to Siezen et al, proteinEngng.4 (1991) 719-737 and Siezen et al, protein Science6 (1997) 501-523. Subtilisins can be divided into 6 sub-categories, namely the Subtilisin (Subtilisin) family, the thermophilic proteinase (thermotase) family, the proteinase K family, the lanthiolysin peptidase (Lantibiotic peptidase) family, the Kexin family and the Pyrolysin family.

The term "wild-type protease" means 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 the wild-type subtilase is set forth in SEQ ID NO. 1.

The term "variant" means a polypeptide having protease activity that comprises alterations (i.e., substitutions, insertions, and/or deletions) at three or more (e.g., several) positions. Substitution means that an amino acid occupying one position is replaced by a different amino acid; deletion means removal of an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids, such as 1, 2, 3, 4 or 5 amino acids, adjacent to and immediately following the amino acid occupying a position. The term subtilase variant refers to a variant of a subtilase parent, i.e. a subtilase variant, which is a subtilase comprising alterations (i.e. substitutions, insertions and/or deletions) at three or more (e.g. several) positions compared to the parent subtilase. In some embodiments, the amino acid sequence of the variant of subtilase is mutated with respect to the amino acid sequence set forth in SEQ ID No.1 at the following positions: G40L or a101G.

The mutein fragment, derivative or analogue of the present invention may be (i) a mutein having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mutein formed by fusion of a mature mutein with another compound, such as a compound that extends the half-life of the mutein, e.g. polyethylene glycol, or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence, such as a leader or secretory sequence or a sequence used to purify the mutein or a pro-protein sequence, or a fusion protein formed with an antigen IgG fragment. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein. In certain embodiments, conservatively substituted amino acids are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

In certain embodiments, other selected groups of amino acids that are considered conservative substitutions for one another (see, e.g., cright on, proteins (1984)):

TABLE 2

Group 1	Ala(A)、Gly(G)
		Group 2	Asp(D)、Glu(E)
Group 3	Asn(N)、Gln(Q)
		Group 4	Arg(R)、Lys(K)
Group 5	Ile(I)、Leu(L)、Met(M)、Val(V)
		Group 6	Phe(F)、Tyr(Y)、Trp(W)
Group 7	Ser(S)、Thr(T)
		Group 8	Cys(C)、Met(M)

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1 tourmaline antibacterial test

1. Preparing black tourmaline powder, magnesium tourmaline powder, lithium tourmaline powder, calcium lithium tourmaline powder and calcium magnesium tourmaline powder respectively, controlling particle diameter (8000 mesh), and adding the above various tourmaline powder into basic toothpaste components to obtain toothpaste containing different tourmaline powder with numbers of 1A-1E respectively.

TABLE 3 Table 3

2. Actinobacillus ATCC 29523, streptococcus mutans CGMCC 1.2499 and clostridium nucleatum ATCC25586 are adopted as test bacteriaA strain. Culturing according to conventional method (37deg.C) to obtain bacterial solutions of the above three strains (final concentration of bacterial solution is adjusted to 1×10) ⁹ CFU/mL). Taking 1mL of each toothpaste composition 1A-1E, and fully mixing to prepare toothpaste liquid. The Minimum Inhibitory Concentration (MIC) of each group will then be tested using a gradient diluted toothpaste. The test results were as follows:

TABLE 4 Table 4

As can be seen from Table 4, the MIC values of experiment group 1C were smallest (MIC values for Actinobacillus actinomycetemcomitans ATCC 29523, streptococcus mutans CGMCC 1.2499, fusobacterium nucleatum ATCC25586 were as low as 1/82mg/L, 1/135mg/L and 1/98mg/L, respectively) compared with experiment groups 1A, 1B, 1D and 1E, which indicates that the antibacterial effect of adding lithium tourmaline powder to the toothpaste composition was optimal when the tourmaline powder was added in 2 portions, whereas the antibacterial effect was poor when other tourmaline was used.

3. Aiming at tourmaline addition experiments.

In order to detect the influence of different tourmaline addition amounts on the antibacterial effect, the inventor designs a comparison experiment to find out the most suitable tourmaline powder addition amount when the antibacterial effect is best, and designs the following determination experiment (the basic components and the content of toothpaste adopted in the experiment are the same as those in Table 3, MIC values):

TABLE 5

As shown in Table 5, the antibacterial effect of the 2C experimental group was better than that of the other experimental groups, and the antibacterial effect was significantly better than that of the other transverse experimental groups when the addition amount of the lithium tourmaline powder was in the range of 2-8 parts. Actinobacillus actinomycetemcomitans is a gram-negative short bacillus, is a pilus, spore-free and unpowered facultative anaerobe, and is recently recognized as being closely related to periodontitis, particularly invasive periodontitis; the streptococcus mutans is gram-positive coccus, is one of streptococcus with the largest proportion in the natural bacterial group of the oral cavity, and is one of main components of dental plaque; fusobacterium nucleatum is a gram-negative obligate anaerobic bacterium, is one of oral symbiotic bacteria, is closely related to a tooth Zhou Shen bag, and is clinically researched to be related to gingivitis and periodontitis. The additive amount of the lithium tourmaline is 2-8 parts, which has good inhibition effect on the three oral bacteria groups.

EXAMPLE 2 construction of protease variants and Activity detection

1. Construction of protease mutant expression vector

Further mutation was performed according to the subtilase sequence described in CN201811556321.4 (SEQ ID NO:3 in this patent application, SEQ ID NO:1 in the present invention). The inventor predicts key amino acid sites which possibly affect the biological functions of the bacillus subtilis through bioinformatics, and mutates the amino acid sites to obtain novel bacillus subtilis A and bacillus subtilis B.

First, the inventors constructed 5 single site mutants between positions 30-200 according to the condition of wild type subtilase (hereinafter referred to as WT) SEQ ID NO:3 sequence, and constructed nucleotide sequences encoding WT and mutants onto pET32a expression vectors, and then transformed each obtained expression vector into BL21 (DE 3) E.coli (NEB) for expression, and purified and activity tested to obtain the following mutant proteases.

TABLE 6

Mutant numbering	Mutation mode
		3A	D40L
3B	A101G
		3C	T37V
3D	G46R
		3E	G186T

2. Determination of whitening efficacy of each protease

Tooth whitening effect determination was performed by the same method as in CN201811556321.4 example 1:

(1) Staining of dense hydroxyapatite:

7 standard dense hydroxyapatite sheets 30mm long, 20mm wide and 4mm thick were prepared for use in a brushing machine. And (3) after the cutting and grinding are finished, cleaning the hydroxyapatite sheet with clear water, soaking the sheet in softened water, naturally airing the sheet, fixing the sheet on a dyeing machine, then dyeing the hydroxyapatite sheet by adopting a mixture of artificial saliva (Solarbio, A7990), tea extract and coffee extract on the dyeing machine, and after 12 hours, finishing dyeing and airing the sheet for later use in a dyeing liquid soaking-heating aging-dyeing liquid soaking cycle.

(2) The toothpaste composition of the experimental group was applied to the surface of all 7 standard dense hydroxyapatite tablets, the base toothpaste composition ingredients were as follows: 40 parts of calcium carbonate, 20 parts of sorbitol, 15 parts of glycerin, 1 part of cellulose gum, 1 part of sodium lauryl sulfate, 0.3 part of essence and 2 parts of lithium tourmaline powder. Then adding each protease mutant and wild protease (WT) into the basic toothpaste composition according to 2 weight parts, brushing for 20 minutes by using a tooth brushing machine, rapidly taking out, and washing with clear water and airing for later use. And then adopting a whiteness meter to firstly measure the whiteness of a set of experiments which are not subjected to decontamination by a tooth brushing machine (the set is an experiment set which is only subjected to dyeing and is not subjected to toothpaste composition coating and tooth brushing by the tooth brushing machine), and then measuring the whiteness after the experiment of decontamination by tooth brushing, wherein the difference value is the effect of the whitening substance.

TABLE 7

As can be seen from Table 7, the toothpaste composition comprising the protease variant shown in SEQ ID NO:1 (D40L, A101G) showed significantly improved whitening efficacy relative to teeth as compared to the wild-type protease (WT) toothpaste composition and other mutant toothpaste compositions.

The inventors also carried out experiments on the mixture ratio of the two mutant proteins in the same manner as in the steps (1) and (2), the total addition amount of the mutant protease was still 2 parts by weight, and the experimental results are shown in table 8.

TABLE 8

D40L mutant and A101G ratio	Testing whiteness
		1:1	11
1:2	27
		1:3	12
1:4	19
		1:5	12
1:6	17
		1:7	16
2:1	17
		3:1	18
4:1	16
		5:1	15
6:1	12
		7:1	20
D40L alone	19
		Individual A101G	20

Example 3 taste experiment of toothpaste composition

Through experiments of examples 1 and 2, the inventor obtains toothpaste components with good antibacterial and whitening effects, and in order to improve the taste of the toothpaste, the inventor adds 1 part by weight of additive (liquorice and bletilla striata) into the toothpaste composition, and the inventor sets the liquorice addition to be more than the bletilla striata because the bletilla striata has higher viscosity and the comfort level of teeth is higher when the liquorice amount exceeds the bletilla striata through testing the comfort level of teeth of different viscosities.

In this example, the toothpaste base composition (in parts by weight) was as follows: 40 parts of calcium carbonate, 20 parts of sorbitol, 15 parts of glycerin, 1 part of cellulose gum, 1 part of sodium lauryl sulfate, 0.3 part of essence, 2 parts of lithium tourmaline powder and 2 parts of subtilisin (wherein protease A is 0.66 part and protease B is 1.34 parts), then respectively setting the ratio of liquorice to bletilla striata to be 1:1-1:10, selecting 10 common consumers (both adults, the ages of 20-45 years), and evaluating the toothpaste with the additives in different ratios by adopting 1-10 minutes, and obtaining the following results.

TABLE 9

Experimental group	Weight ratio of bletilla striata to liquorice	Taste scoring
			4A	1:1	2
4B	1:2	4
			4C	1:3	8
4D	1:4	9
			4E	1:5	8
4F	1:6	1
			4G	1:7	4
4H	1:8	3

As shown in the test results of Table 9, the taste was better when the weight ratio of bletilla striata to licorice was 1:3-5, and the taste was especially best when the weight ratio of bletilla striata to licorice was 1:4.

Variations and modifications to the above would be obvious to persons skilled in the art to which the invention pertains from the foregoing description and teachings. Therefore, the invention is not limited to the specific embodiments disclosed and described above, but some modifications and changes of the invention should be also included in the scope of the claims of the invention. In addition, although specific terms are used in the present specification, these terms are for convenience of description only and do not limit the present invention in any way.

Claims (10)

1. An oral composition comprising the following components:

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

(2) A digestive enzyme, which is a protease, in a proportion of 2 parts by weight;

(3) Glycerol, the proportion of which is 15-30 parts by weight;

(4) An additive in a proportion of 1 part by weight; the additive comprises licorice and bletilla striata.

2. The composition of claim 1, wherein the tourmaline is lithium tourmaline and the proportion of the tourmaline is 3-6 parts by weight.

3. The composition according to claim 2, wherein the tourmaline is present in an amount of 5 parts by weight.

4. The composition of claim 1, wherein the protease is a subtilisin.

5. The subtilisin according to claim 5 having a mutation in the amino acid sequence set forth in SEQ ID No.1 at the position: D40L or S101G.

6. The composition of any one of claims 5, wherein the protease comprises protease a and protease B.

7. The composition according to claim 6, wherein protease A and protease B are mutated with respect to the amino acid sequence shown in SEQ ID NO.1 at the following positions: d40L, S101G.

8. The composition of claim 7, wherein the weight ratio of protease a to protease B is 1:2; the weight ratio of the bletilla striata to the liquorice is 1:3-5.

9. Use of the composition of any one of claims 1-8 in oral hygiene care.

10. An oral hygiene product comprising the composition of any one of claims 1-8.