CN109481336B - Oral care compositions and uses thereof - Google Patents

Abstract

The invention relates to the field of articles for daily use, and provides an oral care composition and application thereof. An oral care composition comprising a protease variant, which is a variant of a parent protease, which parent protease is a subtilisin, which protease variant comprises at least the following amino acid substitutions: G97D + A98R + S99N, wherein the positions correspond to the amino acid positions of the polypeptide of SEQ ID NO: 1. The invention also provides an application of the oral care composition.

Description

Oral care compositions and uses thereof

Technical Field

The invention relates to the field of articles for daily use, in particular to an oral care composition and application thereof.

Background

In the oral cavity of a human, a large amount of protein substances which are unfavorable for teeth exist. A large amount of protein substances exist in food residues in human oral cavity, various functional proteins support the survival and growth of oral flora, and salivary proteins form an acquired film on the surface of teeth, and browning of the film is an important reason for tooth yellowing. Since protease is a biological enzyme capable of hydrolyzing proteins, methods of removing food residues, inhibiting oral flora, and whitening teeth using protease in oral care products have been used.

Unfortunately, since protease is also a protein per se, in aqueous oral care products such as toothpastes, mouthwashes and mouth sprays, proteases have been shown to be unstable during effective use due to their tendency to self-degrade. Meanwhile, natural protease is greatly influenced by conditions such as temperature and pH value, and although the oral care composition added with the protease at present is optimized correspondingly, the expected effect is still difficult to achieve.

Disclosure of Invention

The present invention aims to overcome the above-mentioned disadvantages of the prior art and to provide an oral care composition and its use.

In order to solve the technical problems, the invention adopts the following technical scheme. An oral care composition comprising a protease variant, which is a variant of a parent protease, which is a subtilisin,

the protease variant comprises at least the following amino acid substitutions: G97D + A98R + S99N, wherein the positions correspond to the amino acid positions of the polypeptide of SEQ ID NO: 1.

Preferably, the protease variant further comprises the substitution L124M.

Preferably, the protease variant further comprises one or more alterations selected from the group consisting of: Q12K, S101R, a151G, N184G, a194P, N218S, N155, a 161R.

Preferably, the protease variant comprises at least 3 site substitutions.

Preferably, the parent protease is identical to SEQ ID NO:2 with at least 95% sequence identity.

Preferably, the protease variant has an amino acid sequence with at least 96.6% sequence identity to SEQ ID NO 3.

Preferably, the protease variant has an amino acid sequence represented by SEQ ID NO 3.

Preferably, the oral care composition further comprises one or more of catalase, lipase, protease, laccase, dextranase, lysozyme.

Use of an oral care composition as described above in a toothpaste or a liquid oral care composition.

The invention has the beneficial effects that:

the protease variants contained in the oral care compositions provided herein have improved stability or tooth whitening properties compared to the parent subtilase, and are therefore more suitable for use in toothpaste and liquid oral care compositions. .

Drawings

FIG. 1 is an alignment of the amino acid sequences of subtilisin BPN' (SEQ ID: 1) with subtilisin 309(SEQ ID: 2) and a subtilisin variant (SEQ ID: 3).

Detailed Description

For those skilled in the art to more clearly understand the objects, technical solutions and advantages of the present invention, the following description will be further provided in conjunction with the accompanying drawings and examples.

Definition of

A liquid oral care composition is one in which the composition is liquid at ambient temperature (e.g., 25 ℃).

The term "improved property" refers to an improved characteristic relative to a parent protease in relation to a protease variant as defined herein. Such improved properties include, but are not limited to, improved tooth whitening performance, protease activity, improved thermal stability, improved pH activity profile, improved pH stability, improved stability under storage conditions, and chemical stability.

"improved stability" or "increased stability" is defined herein as the relative increase in protease activity after aging of an oral care composition comprising a variant protease relative to the stability of the composition comprising the parent protease when tested under the same conditions. Protease stability is determined by comparing the residual protease activity measured after incubation at a specific temperature for a specific period of time in a liquid oral care composition comprising the protease of interest. Protease stability can be measured using an assay as described in example 5.

The term "tooth whitening properties" is used as the ability of an oral care composition to remove tooth staining materials, for example, during brushing and rinsing. Tooth whitening performance can be measured as shown in example 4.

The term "improved tooth whitening performance" is defined herein as the tooth whitening performance of a protease variant according to the invention relative to a parent protease, which is expressed herein as "equivalent" if there is no significant or meaningful difference between two or more oral care compositions of interest.

The term "residual activity" or "residual activity" as used herein means remaining after storage, in particular in a toothpaste or liquid oral care compositionOr residual protease activity. When residual activity is measured after adding protease to an oral care composition, at t₁The activity of the protease is measured. Then after a certain amount of storage time, typically minutes or weeks, at t₂The residual activity was measured.

The term "protease" is understood herein as an enzyme having protease activity. The term "protease activity" means the ability of a protein to be degraded by proteases into amino acids, which is the catabolism of proteins by hydrolysis of peptide bonds that link amino acids together in polypeptide chains to form proteins.

The term "wild-type protease" means a protease expressed by a naturally occurring wild-type organism such as a bacterium, archaea, yeast, fungus, plant or animal occurring in nature. An example of a wild-type protease is subtilisin 309, i.e., SEQ ID NO:2, amino acids 1 to 269.

The term "parent" or "parent protease" means a protease that has been altered to produce a variant as defined herein, i.e., a starting protease that is used to describe a protease that has been mutated to obtain a protease variant as defined herein.

The term "mature polypeptide" is defined herein as a polypeptide having the activity of a protease variant, which is the final form of the polypeptide formed after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, autocatalytic activation, etc.

By "protease variant" is herein understood a polypeptide comprising an alteration or modification, such as a substitution, insertion and/or deletion of one or more (several) amino acid residues at one or more (several) specific positions compared to its parent. Protease variants can be obtained by culturing a microorganism comprising a gene encoding the protease variant under conditions suitable for expression of the gene by the microorganism. Genes encoding protease variants can be obtained by human intervention by modifying the gene sequence encoding the wild-type protease.

And (3) replacement: for amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, substitution of glutamine at position 12 with lysine is designated "Q123K" or "Asn 123 Lys". Multiple mutations may be separated by a plus sign ("+"), e.g., "Q123K + L234M" or "Asn 123Lys + Leu234 Met", representing the substitution of glutamine (Q) with lysine (K) and leucine (L) with methionine (M) at positions 123 and 234, respectively.

Deletion (c): for amino acid deletions, the following nomenclature is used: original amino acids, positions. Thus, the deletion of asparagine at position 155 is designated "Asn 155" or "N155". Multiple deletions may be separated by ("+") as described above for substitutions.

Inserting: for amino acid insertions, the following nomenclature is used: initial amino acid, position, initial amino acid, newly inserted amino acid. For example, insertion of a lysine after glycine at position 195 is designated "G195 GK". When more than one amino acid residue is inserted, for example Lys and Ala are inserted after G195, this is expressed as: gly195GlyLysAla or G195 GKA.

In the case where the substitution and insertion occur at the same position, it can be represented as S99SD + S99A or abbreviated as S99 AD.

Different changes: when a different alteration can be introduced at a position, the different alterations are separated by a comma, e.g., "Asn 184Asp, Gly, Ser" indicates that the asparagine at position 184 is replaced by aspartic acid or glycine or serine. Alternatively, different alterations or optional permutations may be indicated in parentheses, for example, Asn184[ Asp, Gly, Ser ] or Asn184{ Asp, Gly, Ser } or abbreviated N184[ D, G, S ] or N184{ D, G, S }.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For The purposes of The present invention, The sequence identity between two amino acid sequences is determined using The Needman-WeChat algorithm (Needman and Wensh (Wunsch), 1970, J.Mol.biol.) (48: 443-. The parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of EBLOSUM 62) substitution matrix. The niedel (Needle) output labeled "longest agreement" (obtained using the-non-simplified (nobrief) option) is used as the percent agreement and calculated as follows:

(consensus residue 100)/(alignment length-total number of vacancies in alignment)

The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell for mutations that occur during replication.

The present invention relates to protease variants comprising a polypeptide having an amino acid sequence identical to SEQ ID NO:2, wherein the protease is a subtilisin variant comprising the substitutions G97D + a98R + S99N, having an amino acid sequence of at least 95%, 96%, 97%, 98%, or 99% sequence identity.

Preferably, the protease has at least 96.6%, 97.0%, 97.4%, 97.8%, 98.1%, 98.5%, 98.9%, 99.3%, 99.6% or 100% sequence identity to SEQ ID NO 3. Thus, the protease has an amino acid sequence identical to SEQ ID NO:3 by less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid change. The amino acid change is a conservative amino acid substitution or deletion that does not significantly affect the folding and/or activity of the protein, typically a substitution or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids;

examples of conservative substitutions are in the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not normally alter a particular activity are described by h.neurath and r.l.hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, 35Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly. Alternatively, the amino acid change has the property of causing a change in the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermostability, change substrate specificity, change the pH optimum, etc. of a polypeptide.

The key amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at each residue in the molecule, and the resulting mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also Hilton et al, 1996, J.biol.chem.271: 4699-4708. The active site of an enzyme or other biological interaction may also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations in putative contact site amino acids. See, e.g., de Vos et al,1992, Science 255: 306-); smith et al,1992, J.mol.biol.224: 899-904; wlodaver et al,1992, FEBS Lett.309: 59-64. For BPN' (SEQ ID NO:1), the catalytic triad comprising amino acids S221, H64 and D32 is essential for the protease activity of the enzyme. Preferably, a protease variant as defined herein comprises these essential amino acids and is free of substitutions of these amino acids.

Preferably, the protease of the invention has an amino acid sequence with at least 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 3, comprising the substitutions G97D + a98R + S99N (positions corresponding to the amino acid positions of the polypeptide of SEQ ID No. 1), and preferably further comprising one or more alterations selected from: S3T, R10F, V11I, Q12K, a13T, P14Q, a15Q, L21I, T22F, S24N, V26A, K27R, S36A, T38S, N43R, R45R, V51R, P52R, G53R, T58R, Q59R, G61R, E89R, S101R, V104R, S105R, I107R, L111R, G115R, G118R, V121R, a 122R, L124R, P129R, P131R, a R, Q R, S137R, T143R, S143 36144, R145, V36147, V36149, V151, N153, N75, N185, N72, N185, N85, N185, N72, N235, N185, N235, N85, N72, N185, N85, N72, N85, N72, N85, N72, N143R, N240, N85, N72, N240, N85, N72, N240, N85, N72, N240, N72, N240, N85, N240, N42, N240, N72, N240, N R, N240, N R, N240, N42R, N240, N R, N42, N240, N42, N72, N42, N240, N R, N240, N42, N R, N42, N240, N R, N42, N R, N42, N240, N36, N42, N240, N R, N42, N R, N42, N240, N R, N: Q12K, S101R, L124M, A151G, N155, A161R, N184G, A194P, N218S, wherein the positions correspond to the amino acid positions of the polypeptide of SEQ ID NO. 1. More preferably, the protease enzyme suitable for use in a toothpaste or liquid oral care composition of the present invention has an amino acid sequence represented by SEQ ID NO. 3.

Preferably, the toothpaste and liquid oral care protease of the invention is a variant of subtilase derived from B.lentus, more preferably a variant of subtilisin 309 having at least 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO. 2 and comprising the substitutions G97D + A98R + S99N as the amino acid sequence of SEQ ID NO. 2. It may also be stated above that preferably the protease variant suitable for use in a toothpaste or liquid oral care composition according to the invention is a subtilisin 309 variant comprising at least 95% sequence identity to SEQ ID No. 2, said variant comprising at least the following amino acid substitutions: G97D + A98R + S99N, wherein the positions correspond to the amino acid positions of the polypeptide of SEQ ID NO: 1.

Most preferably, the protease comprises the substitution G97D + a98R + S99N and further comprises at least 1, 2, 3, 4, 5, 6, 7 or all amino acid substitutions selected from: S3T, R10F, V11I, Q12K, a13T, P14Q, a15Q, L21Q, T22Q, S24Q, V26Q, K27Q, S36Q, T38Q, N43Q, R45Q, V51Q, P52Q, G53Q, T58Q, Q59Q, G61Q, E89Q, S101Q, V104Q, S105Q, I107Q, L111Q, G115Q, G118Q, V121Q, a 122Q, L124, P129Q, P131Q, a Q, Q Q, S137Q, S143Q, S36144, R145, V36147, V36149, V149, N151, N161, N185, N72, N75, N185, N72, N85, N185, N166, N72, N185, N72, N235, N85, N72, N85, N72, N85, N143Q, N85, N72, N85, N143, N72, N143Q, N143, N85, N143, N72, N85, N143, N72, N85, N72, N85, N143, N85, N72, N85, N72, N143, N85, N143, N72, N85, N143, N85, N143, N85, N143, N72, N85, N72, N85, N143, N72, N85, N72, N85, N72, N85, N240, N85, N72, N85, N240, N143, N85, N240, N145, N85, N145, N240, N145, N72, N85, N240, N145, N85, N145, N85, N240, N145, N85, N145, N85, N145, N72, N240, N145, N240, N145, N72, N145, N72, N145, N72, N145.

The protease enzyme for inclusion in the toothpaste or liquid oral care composition of the present invention may be prepared using suitable methods known to those skilled in the art, preferably using common targeted gene mutation techniques and expression systems.

Preferably, the oral care composition comprising the protease variant of the invention may be a toothpaste, a mouthwash or an oral spray. Preferably as a toothpaste. The toothpaste and liquid oral care composition may be the toothpaste and oral care composition of the examples, or a commercial toothpaste and liquid oral care composition such as black, yunnan white drug, Crest jiajieshi, Colgate gaoze, china, kodao, beautiful tooth health, black sister, suke Saky, clorsuling or any other brand extension and oral care composition form.

If the commercial oral care composition used contains enzymes, the enzymes are inactivated by incubation in a boiling water bath for 10min prior to use.

The present inventors have found that the protease stability during aging in the context of toothpaste and liquid oral care compositions is unexpectedly improved in comparison to the protease stability of toothpaste and oral care formulations that differ only in that the subtilase variant has been replaced by an equal weight/weight% of its parent, under a variety of oral care composition contexts.

Under the same conditions (as described in example 5), preferably using the toothpaste formulation shown in table 2, a residual protease activity of about 95% after 4 weeks of aging at 37 ℃ was found, a residual enzyme activity of about 85% after 4 weeks of aging at 45 ℃ was found compared to the protease stability of a toothpaste composition comprising the protease variant as described herein, except that the protease variant had been replaced by an equal amount of its parent, compared to a residual activity of about 72% after 4 weeks of aging at 37 ℃ and a residual activity of less than 50% after 4 weeks of aging at 45 ℃ for a comparative formulation comprising the parent protease but not the variant. The improved stability of the protease variants compared to their parents was evident after 1 week of storage at 37 ℃ and 45 ℃.

When tested under the same conditions, preferably using the toothpaste and liquid oral care formulations shown in tables 2 and 7 and aged at 37 ℃ or 45 ℃ for 1 week, toothpaste and liquid oral care compositions comprising a protease that is at least 96% identical to, but different from, SEQ ID No. 3 exhibit a protease stability of at least 73%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% during aging as compared to the same toothpaste and liquid oral care formulation except that the protease has been replaced by a protease having the amino acid sequence of SEQ ID No. 3. And also shows an improved or equivalent tooth whitening performance during ageing compared to toothpaste and liquid oral care compositions differing only in that the protease has been replaced by a protease having the amino acid sequence of SEQ ID No. 3, when tested under the same conditions, preferably using the toothpaste and liquid oral care formulations shown in tables 2 and 7 and aged at 37 ℃ and 45 ℃ for 1 week.

As detailed in the examples herein (example 7), the liquid oral care compositions comprising the protease variants showed significantly improved efficacy in whitening teeth by aging at 37 ℃ and 45 ℃ compared to the liquid oral compositions shown in table 7 comprising the parent protease.

Examples

Example 1: construction and expression of protease variants

Variants of the invention may be constructed and expressed by methods known to those skilled in the art. The following is a possible example of how to make variants of the invention:

directional mutation:

preparation of protease variants of the invention all DNA manipulations are performed by PCR, using conventional Cloning of PCR-generated DNA fragments (e.g., Sambrook et al; Molecular Cloning; Cold Spring Harbor Laboratory Press) containing oligos of the desired mutation. Recombinant subtillis encoding protease variants are used to express the protease variants. Cultured by inoculating with an appropriate medium (e.g., 20g/L glucose, 30g/L soybean peptone, 5g/L yeast powder). The culture was usually carried out at 30 ℃ for 3 days with shaking at 250 rpm.

Example 2: purification of protease variants

After completion of the fermentation, the culture broth was centrifuged at 14000g at 4 ℃ for 20 minutes, and the centrifuged supernatant was recovered. The recovered supernatant was filtered through a 0.22 μm filtration device to remove the remaining bacillus host cells. The pH of the 0.22 μ M filtrate was adjusted to pH8.0 with 3M Tris base solution for column purification. 20mM Tris/HCl, 1mM CaCl were prepared₂The buffer solution was equilibrated at pH8.0, and the MEPhopercel column (Pall Corporation) was equilibrated with the solution. After washing the column with equilibration buffer, the pH-adjusted 0.22 μm filtrate was injected and 20mM CH was added₃COOH/NaOH、1mM CaCl₂The column was eluted stepwise with a solution of pH 4.5. Fractions from the column were assayed for protease activity using the Suc-AAPF-pNA assay at pH9.0 and peak fractions were pooled. With 20% (v/v) CH₃The pH of the pooled peak fractions from the MEP Hypercel column were adjusted to pH6.0 with COOH or 3M Tris alkaline solution and the pH adjusted pool was diluted with deionized water to 20mM MES/NaOH, 2mM CaCl₂And pH6.0 solution. The diluted pool was applied to a sample of 20mM MES/NaOH, 2mM CaCl₂pH6.0 in a Fast Flow column (GE Healthcare) equilibrated with equilibration buffer. After washing the column with equilibration buffer, the protease variant was eluted with a linear NaCl gradient (0 → 0.5M) of the same buffer, eluting at least five column volumes. Fractions from the column were analyzed for protease activity using the Suc-AAPF-pNA assay at pH9.0 and the active fractions were analyzed by SDS-PAGE. Fractions in which only one band was observed on coomassie stained SDS-PAGE gels were pooled, and this was the purified subtilisin preparation and used for further experiments.

Example 3: detection of protease Activity

Proteolytic activity was determined by a method using Suc-AAPF-pNA as a substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-proline-phenylalanine-p-nitroanilide and is a blocking peptide that can be cleaved by endoproteases. After cleavage, free PNA molecules are released, which appear yellow and can be detected by visible spectrophotometry at a wavelength of 405 nm. Suc-AAPF-PNA substrate was manufactured by Bachem (Cat. No. L1400, dissolved in DMSO). The protease sample to be analyzed was in active buffer (100mM Tris,10mM CaCl)₂0.005% TritonX-100, pH 8.6). The assay was performed by transferring 180. mu.l of the diluted enzyme sample to a 96-well microtiter plate and adding 20. mu.l of the substrate working solution (1mg/ml in 100mM Tris pH8.6). The solutions were mixed at room temperature and the absorbance was measured at OD 405nm, every 10 seconds, 40 times. The slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the activity of the protease in question under a given set of conditions. The protease samples were diluted to a level where the slope was linear.

Example 4: determination of tooth whitening efficacy

The tooth whitening efficacy was measured by the following method

1. Dyeing dense hydroxyapatite:

a compact hydroxyapatite sheet to be used in a tooth brushing machine is cut and ground into a standard sheet with the length of 30mm, the width of 20mm and the thickness of 4 mm. After the cutting and grinding are finished, the standard sheet is washed by water and soaked in softened water for 3 hours. And fixing the naturally dried standard piece on a dyeing machine, dyeing the standard piece by adopting a mixture of artificial saliva, tea extract and coffee extract on the dyeing machine, carrying out dyeing on the standard piece in a cycle of dyeing liquid soaking, heating and aging and dyeing liquid soaking, finishing dyeing after 12 hours, and drying for later use.

2. Decontamination experiment of a tooth brushing machine:

fixing the required amount of the dyed hydroxyapatite sheets, immersing the dyed hydroxyapatite sheets in the aqueous solution of the composition to be detected, brushing the dyed hydroxyapatite sheets for 20min by using a tooth brushing machine, immediately taking off the dyed hydroxyapatite sheets, washing the dyed hydroxyapatite sheets with clear water, and drying the dyed hydroxyapatite sheets for later use.

3. Measurement of tooth whitening effect:

and (3) adopting a whiteness meter to measure the whiteness without a tooth brushing machine decontamination experiment, and then measuring the whiteness after the tooth brushing decontamination experiment, wherein the difference value is the effect of the whitening substance.

Example 5: comparison of enzyme activity stability of protease variants and parent protease in toothpaste

1% of the subtilase variant represented by SEQ ID NO. 3 and 1% of the parent protease, respectively, were added to the composition in the base toothpaste.

The base toothpaste formulations were prepared as shown in table 2 below.

TABLE 2 basic toothpaste compositions

The test method comprises the following steps: after storing the different toothpastes to which the parent protease and the protease variant represented by SEQ ID NO. 3 were added at 37 ℃ and 45 ℃ for 1, 2, 4 and 8 weeks, respectively, 180. mu.l of the reaction solution was taken to measure OD at 405nm, respectively, according to the enzyme activity test method described in example 3₄₀₅The relative residual enzyme activity was calculated as a measure of the stability of the different proteases in the toothpaste, taking the initial value of the respective protease as 100%. The results of the tests at 37 ℃ and 45 ℃ are shown in tables 3-4 below:

TABLE 337 ℃ comparison of protease Activities stored for 1-8 weeks

TABLE 445 ℃ comparison of protease enzyme activities stored for 1-8 weeks

The experimental results shown in tables 3-4 indicate that the protease variants are more stable in toothpaste compositions than the parent protease.

Example 6: comparison of stability of tooth whitening function of protease variants and parent protease in toothpaste

1% of the subtilase variant represented by SEQ ID NO. 3 and 1% of the parent protease are added to the base toothpaste composition, respectively. The base toothpaste composition ingredients were as described in example 5.

And comparing the tooth whitening performance of the toothpaste containing the protease variant and the parent protease variant, and performing a tooth whitening efficacy test. The basic toothpaste, to which 1% of the parent protease and 1% of the protease variant represented by SEQ ID NO. 3 were added, was stored at 37 ℃ and 45 ℃ for 1, 2, 4, and 8 weeks, respectively, and then subjected to the tooth whitening efficacy test as described in example 4. The results of the testing of the samples stored at 37 ℃ and 45 ℃ are shown in tables 5-6.

TABLE 537 ℃ comparison of tooth whitening efficacy of toothpaste stored for 1-8 weeks

The results shown in table 5 indicate that the toothpaste containing SEQ ID NO:3 has remarkable improvement on the maintenance of tooth whitening efficacy under the condition of 37 ℃ heat storage.

TABLE 645 deg.C storage for 1-8 weeks comparison of tooth whitening efficacy of toothpaste

The experimental results shown in table 6 indicate that the toothpaste containing SEQ ID NO:3, the maintenance of the tooth whitening effect of the protease variant toothpaste under the condition of 45 ℃ heat storage is obviously improved.

Example 7: comparison of stability of tooth whitening function of protease variants and parent protease in mouthwash

1% of the subtilase variant represented by SEQ ID NO. 3 and 1% of the parent protease, respectively, were added to the base mouthwash composition. The base mouthwash composition ingredients are shown in table 7.

TABLE 7 basic mouthwash composition

Tooth whitening performance of the mouthwash containing the protease variant and the parent protease variant was compared and a tooth whitening efficacy test was performed. The basic mouth rinse composition with 1% of the parent protease and 1% of the protease variant represented by SEQ ID No. 3 added was stored at 37 ℃ and 45 ℃ for 1, 2, 4 and 8 weeks, respectively, before being subjected to the tooth whitening efficacy test as described in example 4. The results of the testing of the samples stored at 37 ℃ and 45 ℃ are shown in tables 8-9.

TABLE 837 ℃ comparison of teeth whitening efficacy of mouthwash stored for 1-8 weeks

The results shown in table 8 indicate that the toothpaste containing SEQ ID NO:3, the maintenance of tooth whitening efficacy of the mouthwash of the protease variant shown in the specification under the condition of 37 ℃ heat storage is remarkably improved.

Table 945 ℃ comparison of tooth whitening efficacy of mouthwash stored for 1-8 weeks

The experimental results shown in table 9 indicate that the toothpaste containing SEQ ID NO:3, the maintenance of the tooth whitening effect of the protease variant mouthwash under the condition of 45 ℃ heat storage is obviously improved.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which are made without departing from the spirit and principle of the present invention and are equivalent to the replacement of the above embodiments are included in the scope of the present invention.

Sequence listing

<110> Guangzhou Libai Enterprise group Co Ltd

<120> oral care compositions and uses thereof

<130> JY181-2803932

<141> 2018-12-19

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 275

<212> PRT

<213> Artificial

<400> 1

Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu

1 5 10 15

His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30

Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45

Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His

50 55 60

Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly

65 70 75 80

Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu

85 90 95

Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110

Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125

Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala

130 135 140

Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly

145 150 155 160

Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala

165 170 175

Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Glu Glu Val

180 185 190

Gly Pro Glu Leu Asp Val Met Ala Pro Gln Val Ser Ile Gln Ser Thr

195 200 205

Leu Pro Gln Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Xaa Met Ala Ser

210 215 220

Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Pro His Asn

225 230 235 240

Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys

245 250 255

Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270

Ala Ala Gln

275

<210> 2

<211> 269

<212> PRT

<213> Artificial

<400> 2

Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile

180 185 190

Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile

225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu

245 250 255

Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

<210> 3

<211> 268

<212> PRT

<213> Artificial

<400> 3

Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Lys Ala Pro Ala Ala

1 5 10 15

His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

20 25 30

Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

35 40 45

Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

50 55 60

His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Asp Arg

85 90 95

Asn Gly Arg Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Met Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Gly Ala Ser Gly Ser Gly Arg Gly Ser Ile Ser Tyr

145 150 155 160

Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn

165 170 175

Gly Asn Arg Ala Ser Phe Ser Gln Tyr Gly Pro Gly Leu Asp Ile Val

180 185 190

Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala

195 200 205

Ser Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala

210 215 220

Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg

225 230 235 240

Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr

245 250 255

Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg

260 265

Claims (3)

1. An oral care composition characterized by: comprising a protease variant which is a variant of a parent protease which is a subtilisin, said protease variant having the amino acid sequence represented by SEQ ID NO. 3.

2. The oral care composition of claim 1, wherein: also comprises one or more of catalase, lipase, protease, laccase, dextranase and lysozyme.

3. Use of an oral care composition characterized by: use of the oral care composition of any one of claims 1-2 in a toothpaste or a liquid oral care composition.

Images