Oral care compositions containing enzymes

Abstract

The present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, and a glucoamylase, a pharmaceutical use of said composition, a use of said composition in the treatment of an oral disease, a method of treatment comprising administering said composition to a human subject, a method of preventing or removing an oral biofilm comprising contacting said oral biofilm with said composition, a method for reducing the risk of forming an oral biofilm, and a kit-of-parts comprising said composition.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, and a glucoamylase, a pharmaceutical use of said composition, a use of said composition in the treatment of an oral disease, a method of treatment comprising administering said composition to a human subject, a method of preventing or removing an oral biofilm comprising contacting said oral biofilm with said composition, a method for reducing the risk of forming an oral biofilm, and a kit-of-parts comprising said composition. [Background technology]

[0003] Biofilms are bacterial communities found on solid surfaces in many different environments, including surfaces in the oral cavity. Oral biofilms, or dental plaque, contain many bacteria that are associated with oral health problems such as bad breath, demineralization, caries, tooth decay, potential tooth loss, and periodontal disease (gingivitis and periodontitis).

[0004] Oral biofilm formation occurs in three stages, known as the lag phase, growth phase, and stationary state. During the lag phase, glycoproteins derived from saliva bind to oral surfaces, such as teeth, forming structures called pellicles that serve as binding sites for bacteria. During the growth phase, coaggregation occurs, i.e., secondary colonizers attach to primary colonizers, resulting in the multi-species biofilm growth and maturation. During the stationary state, biofilm growth slows and eventually stops. This stage-based formation cycle results in biofilms existing in multiple successive layers, making them more difficult to physically detach.

[0005] In biofilms, the bacterial cells present are distributed in an extracellular polymeric matrix consisting mainly of water, proteins, exopolysaccharides, lipopolysaccharides, lipids, surfactants, and extracellular DNA, with exopolysaccharides making up a large proportion of the dry weight of the biofilm (HC Flemming, and J. Wingender (2010), Nat. Rev. Microbiol. 8, 623-633). Exopolysaccharides are primarily homopolymers of glucose and fructose, including (1-3)-α-D-glucan, (1-4)-α-D-glucan, (1-6)-α-D-glucan, and (2-6)-β-D-fructan. These polysaccharides are synthesized from incorporated sucrose by glucosyltransferases and fructosyltransferases secreted by oral bacteria such as Streptococcus spp., Lactobacillus spp., and Actinomyces spp. Streptococcus mutans and dextran are glucans of particular importance in the formation of dental plaque. Streptococcus mutans has a highly branched structure that contains a backbone made of glucose molecules linked by (1-3)-α bonds and (1-6)-α-glycosidic bonds in its side chains. Dextran is a high molecular weight polymer of glucose that contains multiple consecutive (1-6)-α-linkages in the backbone and side chains, starting with a (1-3)-α-linkage (M. Pleszczynska et al. (2016), Biotechnol. Appl. Biochem. 64(3), 337-346). Fructans are essentially linear polysaccharides, consisting mainly of β-(2,6)-linked fructosyl residues and some β-(2,1)-linked branches.

[0006] Due to the increasing resistance to antimicrobial agents and the mechanical properties of biofilms, many current oral care products are rather ineffective in combating biofilm formation and alleviating the associated oral health problems.The focus of biofilm removal has been on mechanical peeling.However, this approach is made difficult by the multi-layered nature of biofilms, and is further exacerbated by the fact that mechanically removing biofilms, for example by brushing teeth, expands and intensifies the areas in the oral cavity where biofilms are attached and spread, and thus may increase rather than alleviate the severity of the problem.

[0007] Considering the important role of biofilms in oral diseases, there is a need in the art for oral care compositions that can effectively target oral biofilms. WO 1997 / 38669 (Novozymes) describes oral care compositions that include mutanase and dextranase, WO 1998 / 57653 (Novozymes) provides oral care compositions that include dextranase and pullulanase, WO 2000 / 17331 discloses oral care compositions that include Paenibacillus fructanase, and WO 2020 / 099490 (Novozymes) describes oral care compositions that include mutanase and DNase. However, there is still a need for oral care compositions that target oral biofilms. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides oral care compositions comprising invertase, beta-glucosidase, and glucoamylase that are useful for preventing the formation of oral biofilms. [Means for solving the problem]

[0009] In a first aspect, the present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. In a preferred embodiment of the first aspect, the oral care composition further comprises an alpha-amylase.

[0010] In a second aspect, the present invention relates to a composition according to the first aspect for use as a medicament.

[0011] In a third aspect, the present invention relates to a composition according to the first aspect for use in the treatment of an oral disease.

[0012] In a fourth aspect, the present invention relates to a method for preventing or removing an oral biofilm comprising contacting said oral biofilm with an oral care composition according to the first aspect.

[0013] In a fifth aspect, the present invention relates to a method for reducing the risk of oral biofilm formation comprising contacting an oral biofilm with an oral care composition according to the first aspect.

[0014] In a sixth aspect, the present invention provides a method for producing a composition comprising the steps of: a) an oral composition according to a first aspect; b) Concerning a kit-of-parts including instructions for use.

[0015] definition In accordance with this Detailed Description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0016] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0017] Alpha-amylase: The term "alpha-amylase" refers to a polypeptide having alpha-amylase activity that catalyzes the endohydrolysis of (1→4)-α-D-glucosidic linkages in polysaccharides containing three or more (1→4)-α-linked D-glucose units. The systematic name of alpha-amylase is 4-α-D-glucan glucanohydrolase (EC 3.2.1.1). The terms "alpha-amylase" and "α-amylase", as well as the expression "polypeptide having alpha-amylase activity", are used interchangeably throughout this application. For the purposes of the present invention, alpha-amylase activity may be measured according to alpha-amylase activity assay I or alpha-amylase activity assay II described in Example 2 below.

[0018] Beta-glucosidase: The term "beta-glucosidase" refers to a polypeptide having beta-glucosidase activity that catalyzes the hydrolysis of terminal non-reducing β-D-glucosyl residues with the release of β-D-glucose. The systematic name of beta-glucosidase is β-D-glucoside glucohydrolase (EC 3.2.1.21). The terms "beta-glucosidase" and "β-glucosidase", as well as the expression "polypeptide having beta-glucosidase activity" are used interchangeably throughout this application. For the purposes of the present invention, beta-glucosidase activity can be measured according to the beta-glucosidase activity assay described in Example 2 below.

[0019] Biofilm prevention: The term "biofilm prevention" refers to the ability of a polypeptide to reduce the amount of biofilm growing under defined conditions. For purposes of the present invention, biofilm prevention may be measured according to Example 4 or Example 5 herein.

[0020] Dentures: The term "dentures" is meant to include false teeth such as braces, aligners, and retainers as well.

[0021] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide, which fragment has the enzymatic activity of the mature polypeptide. A fragment of invertase has invertase activity, a fragment of beta-glucosidase has beta-glucosidase activity, a fragment of glucoamylase has glucoamylase activity, and a fragment of alpha-amylase has alpha-amylase activity.

[0022] Glucoamylase: The term "glucoamylase" refers to a polypeptide having glucoamylase activity that catalyzes the hydrolysis of terminal (1→4) linked α-D-glucose residues successively from the non-reducing end of the chain with the release of β-D-glucose. Glucoamylase is also known as glucan 1,4-α-glucosidase, and the systematic name of glucoamylase is 4-α-D-glucan glucohydrolase (EC 3.2.1.3). The terms "glucoamylase" and the expressions "polypeptide having glucoamylase activity" are used interchangeably throughout this application. For the purposes of the present invention, glucoamylase activity can be measured according to the glucoamylase activity assay described in Example 2 below.

[0023] Invertase: The term "invertase" refers to a polypeptide having invertase activity that catalyzes the hydrolysis of terminal non-reducing β-D-fructofuranoside residues of β-D-fructofuranosides. Invertase is also known as β-fructofuranosidase, and the systematic name of invertase is β-D-fructofuranoside fructohydrolase (EC 3.2.1.26). The terms "invertase" and the expressions "polypeptide having invertase activity" are used interchangeably throughout this application. For the purposes of the present invention, invertase activity can be measured according to the invertase activity assay described in Example 2 below.

[0024] Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its mature form following N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0025] Parent: The term "parent" refers to an enzyme that is modified to produce an enzyme variant. In one aspect, the parent is a parent invertase that is modified to produce an invertase variant. In one aspect, the parent is a parent beta-glucosidase that is modified to produce a beta-glucosidase variant. In one aspect, the parent is a parent glucoamylase that is modified to produce a glucoamylase variant. In one aspect, the parent is a parent alpha-amylase that is modified to produce an alpha-amylase variant.

[0026] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is expressed by the parameter "sequence identity."

[0027] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 6.6.0 or later, as the "longest identity" output. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The output of Needle, labeled "longest identity", is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0028] For the purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later, as the "longest identity" output. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. For the Needle program to report the longest identity, the nobrief option must be specified on the command line. The output of Needle, displayed as "longest identity", is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0029] Variant: The term "variant" refers to an invertase, beta-glucosidase, glucoamylase, or alpha-amylase that contains an artificial mutation, i.e., a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation) at one or more positions. A substitution refers to replacing an amino acid occupying a position with a different amino acid, a deletion refers to removing an amino acid occupying a position, and an insertion refers to adding 1-5 amino acids (e.g., 1-3 amino acids, 1 amino acid) adjacent to and immediately following the amino acid occupying a position. [Brief description of the drawings]

[0030] [Figure 1]Examples of thermal stability data generated using the nanoDSF instrument are shown. Panel A is an example of triplicate acquired data for SEQ ID NO:3 (ratio of fluorescence emission at 350 nm to 330 nm) as a function of temperature. Panel B shows the first derivative of the raw data in Panel A. The peak maximum in the plot of the first derivative corresponds to the midpoint of the thermal unfolding transition, referred to as the Tm. In this example, the Tm corresponds to 61.9° C. at pH 6.0 for SEQ ID NO:3, and is highly reproducible within three replicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Overview of Arrays SEQ ID NO:1 is the gDNA sequence of an invertase obtained from Aspergillus niger.

[0032] SEQ ID NO:2 is the translation product (including the signal peptide) obtained from SEQ ID NO:2.

[0033] SEQ ID NO:3 is an invertase (mature polypeptide) obtained from Aspergillus niger.

[0034] SEQ ID NO:4 is the gDNA sequence of a beta-glucosidase obtained from Aspergillus niger.

[0035] SEQ ID NO:5 is the translation product (including the signal peptide) obtained from SEQ ID NO:4.

[0036] SEQ ID NO:6 is a beta-glucosidase (mature polypeptide) obtained from Aspergillus niger.

[0037] SEQ ID NO:7 is the gDNA sequence of the glucoamylase obtained from Aspergillus niger.

[0038] SEQ ID NO:8 is the translation product (including the signal peptide) obtained from SEQ ID NO:7.

[0039] SEQ ID NO:9 is the glucoamylase (mature polypeptide) obtained from Aspergillus niger.

[0040] SEQ ID NO:10 is the gDNA sequence of the invertase obtained from Bipolaris sorokiniana.

[0041] SEQ ID NO:11 is the translation product (including the signal peptide) obtained from SEQ ID NO:10.

[0042] SEQ ID NO:12 is an invertase (mature polypeptide) obtained from Bipolaris sorokiniana.

[0043] SEQ ID NO:13 is the gDNA sequence of an invertase obtained from Aspergillus aculeatus.

[0044] SEQ ID NO:14 is the translation product (including the signal peptide) obtained from SEQ ID NO:13.

[0045] SEQ ID NO:15 is an invertase (mature polypeptide) obtained from Aspergillus aculeatus.

[0046] SEQ ID NO:16 is the gDNA sequence of the invertase obtained from Pestalotiopsis vismiae.

[0047] SEQ ID NO:17 is the translation product (including the signal peptide) obtained from SEQ ID NO:16.

[0048] SEQ ID NO:18 is an invertase (mature polypeptide) obtained from Pestalotiopsis vismiae.

[0049] SEQ ID NO:19 is the gDNA sequence of the invertase obtained from Aspergillus avenaceus.

[0050] SEQ ID NO:20 is the translation product (including the signal peptide) obtained from SEQ ID NO:19.

[0051] SEQ ID NO:21 is the invertase (mature polypeptide) obtained from Aspergillus avenaceus.

[0052] SEQ ID NO:22 is the gDNA sequence of an invertase obtained from Aspergillus sclerotiorum.

[0053] SEQ ID NO:23 is the translation product (including the signal peptide) obtained from SEQ ID NO:22.

[0054] SEQ ID NO:24 is an invertase (mature polypeptide) obtained from Aspergillus sclerotiorum.

[0055] SEQ ID NO:25 is the gDNA sequence of an invertase obtained from Fusarium avenaceum.

[0056] SEQ ID NO:26 is the translation product (including the signal peptide) obtained from SEQ ID NO:25.

[0057] SEQ ID NO:27 is an invertase (mature polypeptide) obtained from Fusarium avenaceum.

[0058] SEQ ID NO:28 is the gDNA sequence of an invertase obtained from Penicillium coprophilum.

[0059] SEQ ID NO:29 is the translation product (including the signal peptide) obtained from SEQ ID NO:28.

[0060] SEQ ID NO:30 is the invertase (mature polypeptide) obtained from Penicillium coprophilum.

[0061] SEQ ID NO:31 is the gDNA sequence of an invertase obtained from Penicillium murcianum.

[0062] SEQ ID NO:32 is the translation product (including the signal peptide) obtained from SEQ ID NO:31.

[0063] SEQ ID NO:33 is the invertase (mature polypeptide) obtained from Penicillium murcianum.

[0064] SEQ ID NO:34 is the gDNA sequence of an invertase obtained from Penicillium venetum.

[0065] SEQ ID NO:35 is the translation product (including the signal peptide) obtained from SEQ ID NO:34.

[0066] SEQ ID NO:36 is an invertase (mature polypeptide) obtained from Penicillium venetum.

[0067] SEQ ID NO:37 is the gDNA sequence of the invertase obtained from Curvularia spicifera.

[0068] SEQ ID NO:38 is the translation product (including the signal peptide) obtained from SEQ ID NO:37.

[0069] SEQ ID NO:39 is the invertase (mature polypeptide) obtained from Curvularia spicifera.

[0070] SEQ ID NO: 40 is the gDNA sequence of an invertase obtained from Alternaria sp.

[0071] SEQ ID NO:41 is the translation product (including the signal peptide) obtained from SEQ ID NO:40.

[0072] SEQ ID NO:42 is the invertase (mature polypeptide) obtained from Alternaria sp.

[0073] SEQ ID NO:43 is the gDNA sequence of an invertase obtained from Fusarium temperatum.

[0074] SEQ ID NO:44 is the translation product (including the signal peptide) obtained from SEQ ID NO:43.

[0075] SEQ ID NO:45 is the invertase (mature polypeptide) obtained from Fusarium temperatum.

[0076] SEQ ID NO:46 is the gDNA sequence of invertase obtained from Aspergillus japonicus.

[0077] SEQ ID NO:47 is the translation product (including the signal peptide) obtained from SEQ ID NO:46.

[0078] SEQ ID NO:48 is an invertase (mature polypeptide) obtained from Aspergillus japonicus.

[0079] SEQ ID NO:49 is the coding sequence for an alpha-amylase obtained from Bacillus amyloliquefaciens.

[0080] SEQ ID NO:50 is the alpha-amylase (mature polypeptide) obtained from Bacillus amyloliquefaciens.

[0081] SEQ ID NO:51 is the secretion signal from Bacillus licheniformis.

[0082] SEQ ID NO:52 is a His affinity tag.

[0083] Detailed Description of the Invention The present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. As illustrated in the examples of the present application, the inventors have determined that invertase, beta-glucosidase, and glucoamylase, especially those of microbial origin, are highly effective in preventing the formation of oral biofilms and / or reducing the risk of oral biofilm formation. Furthermore, these enzymes are highly stable in the presence of a wide range of oral care ingredients, making them highly suitable for oral care formulations. Without wishing to be bound by theory, the combination of invertase, beta-glucosidase, and glucoamylase is particularly effective in degrading dietary carbohydrates that serve as substrates for oral bacteria, as well as exopolysaccharides found in oral biofilms, thereby providing a preventative effect on the formation of oral biofilms.

[0084] The present inventors have further determined that a combination of invertase, beta-glucosidase, glucoamylase, and alpha-amylase can potently remove biofilms already present in the oral cavity.

[0085] Thus, in a first aspect, the present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0086] Invertase The invertase may be derived from any organism. Preferably, the invertase is of microbial origin, most preferably, the invertase is of bacterial or fungal origin. In a preferred embodiment, the invertase is derived from Aspergillus niger. In a preferred embodiment, the invertase is derived from Bipolaris sorokiniana. In a preferred embodiment, the invertase is derived from Aspergillus aculeatus. In a preferred embodiment, the invertase is derived from Pestalotiopsis vismiae. In a preferred embodiment, the invertase is derived from Aspergillus avenaceus. In a preferred embodiment, the invertase is derived from Aspergillus sclerotiorum. In a preferred embodiment, the invertase is derived from Fusarium avenaceum. The invertase may be derived from any organism. In a preferred embodiment, the invertase is derived from Penicillium coprophilum. In a preferred embodiment, the invertase is derived from Penicillium murcianum. In a preferred embodiment, the invertase is derived from Penicillium venetum. In a preferred embodiment, the invertase is derived from Curvularia spicifera. In a preferred embodiment, the invertase is derived from Alternaria sp. In a preferred embodiment, the invertase is derived from Fusarium temperatum. In a preferred embodiment, the invertase is derived from Aspergillus japonicus.

[0087] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:2; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:1 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:2, the mature polypeptide of SEQ ID NO:2, or SEQ ID NO:3 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0088] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2 or a mature polypeptide of SEQ ID NO:2. A preferred mature polypeptide of SEQ ID NO:2 corresponds to amino acid residues 16 to 628 of SEQ ID NO:2.

[0089] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3.

[0090] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:2, or the mature polypeptide of SEQ ID NO:2. A preferred mature polypeptide of SEQ ID NO:2 corresponds to amino acid residues 16 to 628 of SEQ ID NO:2.

[0091] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:3, or a fragment thereof.

[0092] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0093] In another embodiment, the invertase is derived from SEQ ID NO: 2 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 2 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 3 by substitution, deletion or addition of one or more amino acids.

[0094] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 3, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 3, and the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 3 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0095] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:11; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:11; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:10 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:11, the mature polypeptide of SEQ ID NO:11, or SEQ ID NO:12 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0096] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11 or a mature polypeptide of SEQ ID NO: 11. A preferred mature polypeptide of SEQ ID NO: 11 corresponds to amino acid residues 22 to 637 of SEQ ID NO: 11.

[0097] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:12.

[0098] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:11, or the mature polypeptide of SEQ ID NO:11. A preferred mature polypeptide of SEQ ID NO:11 corresponds to amino acid residues 22 to 637 of SEQ ID NO:11.

[0099] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:12, or a fragment thereof.

[0100] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0101] In another embodiment, the invertase is derived from SEQ ID NO: 11 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 11 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 12 by substitution, deletion or addition of one or more amino acids.

[0102] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 12, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 12, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 12 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0103] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:15; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 14; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:13 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:14, the mature polypeptide of SEQ ID NO:14, or SEQ ID NO:15 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0104] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 14 or a mature polypeptide of SEQ ID NO: 14. A preferred mature polypeptide of SEQ ID NO: 14 corresponds to amino acid residues 17 to 651 of SEQ ID NO: 14.

[0105] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:15.

[0106] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 14, or the mature polypeptide of SEQ ID NO: 14. The preferred mature polypeptide of SEQ ID NO: 14 corresponds to amino acid residues 17 to 651 of SEQ ID NO: 14.

[0107] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:15, or a fragment thereof.

[0108] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0109] In another embodiment, the invertase is derived from SEQ ID NO: 14 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 14 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 15 by substitution, deletion or addition of one or more amino acids.

[0110] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 15, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 15, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 15 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0111] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:17; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 17; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 16 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:17, the mature polypeptide of SEQ ID NO:17, or SEQ ID NO:18 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0112] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or a mature polypeptide of SEQ ID NO: 17. A preferred mature polypeptide of SEQ ID NO: 17 corresponds to amino acid residues 21 to 621 of SEQ ID NO: 17.

[0113] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18.

[0114] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 17, or the mature polypeptide of SEQ ID NO: 17. The preferred mature polypeptide of SEQ ID NO: 17 corresponds to amino acid residues 21 to 621 of SEQ ID NO: 17.

[0115] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:18, or a fragment thereof.

[0116] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0117] In another embodiment, the invertase is derived from SEQ ID NO: 17 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 17 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 18 by substitution, deletion or addition of one or more amino acids.

[0118] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 18, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 18, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 18 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0119] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:20; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:19 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:20, the mature polypeptide of SEQ ID NO:20, or SEQ ID NO:21 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0120] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20, or a mature polypeptide of SEQ ID NO:20. A preferred mature polypeptide of SEQ ID NO:20 corresponds to amino acid residues 19 to 624 of SEQ ID NO:20.

[0121] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21.

[0122] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:20, or the mature polypeptide of SEQ ID NO:20. A preferred mature polypeptide of SEQ ID NO:20 corresponds to amino acid residues 19 to 624 of SEQ ID NO:20.

[0123] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:21, or a fragment thereof.

[0124] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0125] In another embodiment, the invertase is derived from SEQ ID NO: 20 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 20 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 21 by substitution, deletion or addition of one or more amino acids.

[0126] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 21, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 21, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 21 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0127] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:23; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:22 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or SEQ ID NO:24 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0128] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23 or a mature polypeptide of SEQ ID NO:23. A preferred mature polypeptide of SEQ ID NO:23 corresponds to amino acid residues 19 to 621 of SEQ ID NO:23.

[0129] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24.

[0130] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 23, or the mature polypeptide of SEQ ID NO: 23. A preferred mature polypeptide of SEQ ID NO: 23 corresponds to amino acid residues 19 to 621 of SEQ ID NO: 23.

[0131] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:24, or a fragment thereof.

[0132] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0133] In another embodiment, the invertase is derived from SEQ ID NO: 23 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 23 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 24 by substitution, deletion or addition of one or more amino acids.

[0134] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 24, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 24, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 24 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0135] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:26; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:25 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:26, the mature polypeptide of SEQ ID NO:26, or SEQ ID NO:27 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0136] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26 or a mature polypeptide of SEQ ID NO:26. A preferred mature polypeptide of SEQ ID NO:26 corresponds to amino acid residues 16 to 619 of SEQ ID NO:26.

[0137] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27.

[0138] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 26, or the mature polypeptide of SEQ ID NO: 26. A preferred mature polypeptide of SEQ ID NO: 26 corresponds to amino acid residues 16 to 619 of SEQ ID NO: 26.

[0139] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:27, or a fragment thereof.

[0140] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0141] In another embodiment, the invertase is derived from SEQ ID NO: 26 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 26 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 27 by substitution, deletion or addition of one or more amino acids.

[0142] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 27, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 27, and the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 27 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0143] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:29; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:28 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:29, the mature polypeptide of SEQ ID NO:29, or SEQ ID NO:30 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0144] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29 or a mature polypeptide of SEQ ID NO: 29. A preferred mature polypeptide of SEQ ID NO: 29 corresponds to amino acid residues 18 to 620 of SEQ ID NO: 29.

[0145] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30.

[0146] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:29, or the mature polypeptide of SEQ ID NO:29. A preferred mature polypeptide of SEQ ID NO:29 corresponds to amino acid residues 18 to 620 of SEQ ID NO:29.

[0147] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:30, or a fragment thereof.

[0148] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0149] In another embodiment, the invertase is derived from SEQ ID NO: 29 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 29 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 30 by substitution, deletion or addition of one or more amino acids.

[0150] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 30, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 30, and the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 30 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0151] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:32; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:33; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:32; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:31 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:32, the mature polypeptide of SEQ ID NO:32, or SEQ ID NO:33 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0152] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, or a mature polypeptide of SEQ ID NO: 32. A preferred mature polypeptide of SEQ ID NO: 32 corresponds to amino acid residues 18 to 623 of SEQ ID NO: 32.

[0153] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:33.

[0154] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 32, or the mature polypeptide of SEQ ID NO: 32. A preferred mature polypeptide of SEQ ID NO: 32 corresponds to amino acid residues 18 to 623 of SEQ ID NO: 32.

[0155] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:33, or a fragment thereof.

[0156] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0157] In another embodiment, the invertase is derived from SEQ ID NO: 32 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 32 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 33 by substitution, deletion or addition of one or more amino acids.

[0158] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 33, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 33, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 33 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0159] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:35; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 35; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 34 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:35, the mature polypeptide of SEQ ID NO:35, or SEQ ID NO:36 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0160] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, or a mature polypeptide of SEQ ID NO: 35. A preferred mature polypeptide of SEQ ID NO: 35 corresponds to amino acid residues 18 to 621 of SEQ ID NO: 35.

[0161] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36.

[0162] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 35, or the mature polypeptide of SEQ ID NO: 35. A preferred mature polypeptide of SEQ ID NO: 35 corresponds to amino acid residues 18 to 621 of SEQ ID NO: 35.

[0163] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:36, or a fragment thereof.

[0164] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0165] In another embodiment, the invertase is derived from SEQ ID NO: 35 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 35 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 36 by substitution, deletion or addition of one or more amino acids.

[0166] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 36, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 36, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 36 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0167] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:38; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:39; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 38; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 37 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:38, the mature polypeptide of SEQ ID NO:38, or SEQ ID NO:39 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0168] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38, or a mature polypeptide of SEQ ID NO: 38. A preferred mature polypeptide of SEQ ID NO: 38 corresponds to amino acid residues 24 to 637 of SEQ ID NO: 38.

[0169] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:39.

[0170] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 38, or the mature polypeptide of SEQ ID NO: 38. A preferred mature polypeptide of SEQ ID NO: 38 corresponds to amino acid residues 24 to 637 of SEQ ID NO: 38.

[0171] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:39, or a fragment thereof.

[0172] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0173] In another embodiment, the invertase is derived from SEQ ID NO: 38 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 38 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 39 by substitution, deletion or addition of one or more amino acids.

[0174] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 39, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 39, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 39 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0175] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:41; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:42; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:41; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 40 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:41, the mature polypeptide of SEQ ID NO:41, or SEQ ID NO:42 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0176] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41, or a mature polypeptide of SEQ ID NO: 41. A preferred mature polypeptide of SEQ ID NO: 41 corresponds to amino acid residues 23 to 626 of SEQ ID NO: 41.

[0177] In a preferred embodiment, the invertase has at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:42.

[0178] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 41, or the mature polypeptide of SEQ ID NO: 41. A preferred mature polypeptide of SEQ ID NO: 41 corresponds to amino acid residues 23 to 626 of SEQ ID NO: 41.

[0179] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:42, or a fragment thereof.

[0180] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0181] In another embodiment, the invertase is derived from SEQ ID NO: 41 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 41 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 42 by substitution, deletion or addition of one or more amino acids.

[0182] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 42, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 42, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 42 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0183] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:44; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:45; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:44; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:43 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO: 44, the mature polypeptide of SEQ ID NO: 44, or SEQ ID NO: 45 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0184] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44 or a mature polypeptide of SEQ ID NO: 44. A preferred mature polypeptide of SEQ ID NO: 44 corresponds to amino acid residues 16 to 618 of SEQ ID NO: 44.

[0185] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:45.

[0186] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 44, or the mature polypeptide of SEQ ID NO: 44. A preferred mature polypeptide of SEQ ID NO: 44 corresponds to amino acid residues 16 to 618 of SEQ ID NO: 44.

[0187] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:45, or a fragment thereof.

[0188] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0189] In another embodiment, the invertase is derived from SEQ ID NO: 44 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 44 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 45 by substitution, deletion or addition of one or more amino acids.

[0190] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 45, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 45, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 45 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0191] In one aspect, the invertase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:47; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:48; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:47; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:46 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:47, the mature polypeptide of SEQ ID NO:47, or SEQ ID NO:48 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has invertase activity.

[0192] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47 or a mature polypeptide of SEQ ID NO: 47. A preferred mature polypeptide of SEQ ID NO: 47 corresponds to amino acid residues 20 to 653 of SEQ ID NO: 47.

[0193] In a preferred embodiment, the invertase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:48.

[0194] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO: 47, or the mature polypeptide of SEQ ID NO: 47. A preferred mature polypeptide of SEQ ID NO: 47 corresponds to amino acid residues 20 to 653 of SEQ ID NO: 47.

[0195] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:48, or a fragment thereof.

[0196] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0197] In another embodiment, the invertase is derived from SEQ ID NO: 47 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 47 by substitution, deletion or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 48 by substitution, deletion or addition of one or more amino acids.

[0198] In some embodiments, the invertase is a variant of a parent invertase, preferably SEQ ID NO: 48, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 48, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 48 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0199] Important 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 every residue in a molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for invertase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224;899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of important amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms within a polypeptide or protein family that includes related polypeptides or polypeptides / proteins that are typically derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used in protein structure modeling to identify important amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0200] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0201] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0202] The oral care compositions of the present invention may contain invertase in any effective amount or concentration. In a preferred embodiment, the oral care composition contains from about 1 ppm to about 500 ppm of invertase, preferably from about 1 ppm to about 100 ppm, more preferably from about 5 ppm to about 75 ppm, even more preferably from about 10 ppm to about 60 ppm, and most preferably from 10 ppm to 60 ppm of invertase.

[0203] In a preferred embodiment, the oral care composition comprises an invertase in an amount of at least 1 ppm, e.g., at least 5 ppm, at least 10 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, at least 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, at least 85 ppm, at least 90 ppm, at least 95 ppm, at least 100 ppm, or more.

[0204] In a particularly preferred embodiment, the oral care composition comprises at least 10 ppm of invertase. In another particularly preferred embodiment, the oral care composition comprises at least 60 ppm of invertase.

[0205] Invertase prevents oral biofilm formation. Preferably, invertase improves the effect of preventing oral biofilm. In one embodiment, invertase prevents oral biofilm formation by at least 5%, for example, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. For the purposes of the present invention, prevention of oral biofilm can be measured, for example, according to Example 4 below.

[0206] The invertase reduces the risk of oral biofilm formation. Preferably, the invertase reduces the risk of oral biofilm formation by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0207] Invertase can also remove oral biofilm. Preferably, invertase improves the effect of removing oral biofilm. In one embodiment, invertase removes at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of oral biofilm.

[0208] Invertase is highly stable in dosage forms and / or formats suitable for oral care, especially in dosage forms or formats such as toothpaste, mouthwash, lozenges, mints, gum, candy, etc. High stability, e.g., comparable or improved stability, can be comparable or improved physical stability and / or chemical stability. Comparable or improved chemical stability, i.e. comparable or improved stability in the presence of another agent (e.g., another enzyme, active ingredient, excipient, or solvent), can occur when the invertase and the other agent are coformulated and / or coadministered, preferably coformulated.

[0209] In a preferred embodiment, the invertase has a comparable or improved thermostability. In the context of the present invention, the term "comparable thermostability" means that the thermostability of the invertase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is within ±5% of the thermostability of the same invertase alone (i.e., in the absence of said oral care ingredient). In the context of the present invention, the term "improved thermostability" means that the thermostability of the invertase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is improved by at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or even more, compared to the thermostability of the same invertase alone (i.e., in the absence of said oral care ingredient). For purposes of the present invention, thermal stability may be measured according to Example 3 below and is defined as the midpoint of the thermal unfolding transition (Tm).

[0210] In one embodiment, the invertase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0211] In a preferred embodiment, the invertase has similar or improved thermostability at pH 4-8, e.g., pH 4, 5, 6, 7, or 8. Preferably, the invertase has similar or improved thermostability at pH 5-7, more preferably pH 5-6, most preferably pH 5 and / or pH 6.

[0212] In one embodiment, the oral care composition comprises a benzoate, e.g., sodium benzoate, and an invertase having similar or improved thermal stability in the presence of a benzoate, e.g., sodium benzoate. Preferably, the invertase has similar or improved thermal stability in the presence of 0.01-5% benzoate (e.g., sodium benzoate), more preferably 0.05-2.5% benzoate, even more preferably 0.1-1% benzoate, and most preferably 0.1-0.5% benzoate. Preferably, the invertase has similar or improved thermal stability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0213] In one embodiment, the oral care composition comprises EDTA and an invertase that has similar or improved thermostability in the presence of EDTA. Preferably, the invertase has similar or improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0214] In one embodiment, the oral care composition comprises ethanol and an invertase having similar or improved thermostability in the presence of ethanol. Preferably, the invertase has similar or improved thermostability in the presence of 0.1-20% ethanol, more preferably 1-10% ethanol, even more preferably 2.5-7.5% ethanol, and most preferably 5% ethanol. Preferably, the invertase has similar or improved thermostability in the presence of 1-100000 mM ethanol, more preferably 100-10000 mM ethanol, and most preferably 1000 mM ethanol.

[0215] In one embodiment, the oral care composition comprises a fluoride, such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride, and an invertase having the same or improved thermal stability in the presence of a fluoride, such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride. Preferably, the invertase has the same or improved thermal stability in the presence of 1-5000 ppm of fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm of fluoride, and most preferably 1,000-1500 ppm of fluoride. Preferably, the invertase has the same or improved thermal stability in the presence of 1-100 mM of fluoride (e.g., sodium fluoride), more preferably 5-75 mM of fluoride, even more preferably 10-50 mM of fluoride, and most preferably 20-40 mM of fluoride.

[0216] In one embodiment, the oral care composition comprises glycerol and an invertase having similar or improved thermostability in the presence of glycerol. Preferably, the invertase has similar or improved thermostability in the presence of 1-50% glycerol, more preferably 5-40% glycerol, and most preferably 10-30% glycerol. Preferably, the invertase has similar or improved thermostability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0217] In one embodiment, the oral care composition comprises a peroxide, e.g., hydrogen peroxide, and an invertase that has similar or improved thermal stability in the presence of a peroxide, e.g., hydrogen peroxide. Preferably, the invertase has similar or improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, and most preferably 100-500 mM peroxide.

[0218] In one embodiment, the oral care composition comprises mannitol and an invertase that has similar or improved thermostability in the presence of mannitol. Preferably, the invertase has similar or improved thermostability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0219] In one embodiment, the oral care composition comprises a phosphate, such as sodium or potassium phosphate, and an invertase that has similar or improved thermostability in the presence of a phosphate, such as sodium or potassium phosphate. Preferably, the invertase has similar or improved thermostability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0220] In one embodiment, the oral care composition comprises sodium dodecyl sulfate (SDS) and an invertase that has similar or improved thermostability in the presence of SDS. Preferably, the invertase has similar or improved thermostability in the presence of 10-50 mM SDS, more preferably 15-25 mM SDS, and most preferably 17 mM SDS.

[0221] In one embodiment, the oral care composition comprises a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate, and an invertase that has similar or improved thermal stability in the presence of a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate. Preferably, the invertase has similar or improved thermal stability in the presence of 0.01-5% sorbate (e.g., potassium sorbate), more preferably 0.05-2.5% sorbate, even more preferably 0.1-1% sorbate, and most preferably 0.1-0.5% sorbate. Preferably, the invertase has similar or improved thermal stability in the presence of 1-100 mM sorbate (e.g., potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0222] In one embodiment, the oral care composition comprises sorbitol and an invertase having similar or improved thermal stability in the presence of sorbitol. Preferably, the invertase has similar or improved thermal stability in the presence of 0.1-70% sorbitol, more preferably 1-60% sorbitol, even more preferably 5-50% sorbitol, and most preferably 10-40% sorbitol. Preferably, the invertase has similar or improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0223] Beta-glucosidase The beta-glucosidase may be derived from any organism. Preferably, the beta-glucosidase is of microbial origin, and most preferably, the beta-glucosidase is of bacterial or fungal origin. In a preferred embodiment, the beta-glucosidase is derived from Aspergillus niger.

[0224] In one aspect, the beta-glucosidase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:5; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:4 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or SEQ ID NO:6 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has beta-glucosidase activity.

[0225] In preferred embodiments, the beta-glucosidase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5, or a mature polypeptide of SEQ ID NO:5. A preferred mature polypeptide of SEQ ID NO:5 corresponds to amino acid residues 20 to 860 of SEQ ID NO:5.

[0226] In preferred embodiments, the beta-glucosidase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0227] In a preferred embodiment, the beta-glucosidase comprises, consists essentially of, or consists of SEQ ID NO:5, or the mature polypeptide of SEQ ID NO:5. The preferred mature polypeptide of SEQ ID NO:5 corresponds to amino acid residues 20 to 860 of SEQ ID NO:5.

[0228] In a preferred embodiment, the beta-glucosidase comprises, consists essentially of, or consists of SEQ ID NO:6, or a fragment thereof.

[0229] The beta-glucosidase can have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0230] In another embodiment, the beta-glucosidase is derived from SEQ ID NO: 5 by substitution, deletion, or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 5 by substitution, deletion, or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 6 by substitution, deletion, or addition of one or more amino acids.

[0231] In some embodiments, the beta-glucosidase is a variant of a parent beta-glucosidase, preferably SEQ ID NO: 6, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the beta-glucosidase is a variant of SEQ ID NO: 6, wherein the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 6 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., polyhistidine tracts, antigenic epitopes, or binding modules.

[0232] Important 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 every residue in a molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for beta-glucosidase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224;899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of important amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms within a polypeptide or protein family that includes related polypeptides or polypeptides / proteins that are typically derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used in protein structure modeling to identify important amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0233] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0234] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0235] The oral care compositions of the present invention may contain any effective amount or concentration of beta-glucosidase. In a preferred embodiment, the oral care compositions contain from about 1 ppm to about 500 ppm of beta-glucosidase, preferably from about 1 ppm to about 100 ppm, more preferably from about 5 ppm to about 75 ppm, even more preferably from about 10 ppm to about 60 ppm, and most preferably from 10 ppm to 60 ppm of beta-glucosidase.

[0236] In a preferred embodiment, the oral care composition comprises beta-glucosidase in an amount of at least 1 ppm, e.g., at least 5 ppm, at least 10 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, at least 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, at least 85 ppm, at least 90 ppm, at least 95 ppm, at least 100 ppm, or more.

[0237] In a particularly preferred embodiment, the oral care composition comprises at least 10 ppm of beta-glucosidase. In another particularly preferred embodiment, the oral care composition comprises at least 60 ppm of beta-glucosidase.

[0238] Beta-glucosidase prevents the formation of oral biofilm. Preferably, beta-glucosidase improves the effect of preventing oral biofilm. In one embodiment, beta-glucosidase prevents the formation of oral biofilm by at least 5%, for example, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. For purposes of the present invention, prevention of oral biofilm can be measured, for example, according to Example 4 below.

[0239] The beta-glucosidase reduces the risk of oral biofilm formation. Preferably, the beta-glucosidase reduces the risk of oral biofilm formation by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0240] Beta-glucosidase can also remove oral biofilm. Preferably, the beta-glucosidase improves the effectiveness of removing oral biofilm. In one embodiment, the beta-glucosidase removes at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the oral biofilm.

[0241] Beta-glucosidase is highly stable in dosage forms and / or formats suitable for oral care, particularly in dosage forms or formats such as toothpaste, mouthwash, lozenges, mints, gum, candy, etc. High stability, e.g., comparable or improved stability, can be comparable or improved physical stability and / or chemical stability. Comparable or improved chemical stability, i.e., comparable or improved stability in the presence of another agent (e.g., another enzyme, active ingredient, excipient, or solvent), can occur when beta-glucosidase and another agent are coformulated and / or coadministered, preferably coformulated.

[0242] In a preferred embodiment, the beta-glucosidase has comparable or improved thermostability. In the context of the present invention, the term "comparable thermostability" means that the thermostability of a beta-glucosidase in the presence (or, alternatively, co-formulated with) a particular oral care ingredient or component is within ±5% of the thermostability of the same beta-glucosidase alone (i.e., in the absence of said oral care ingredient). In the context of the present invention, the term "improved thermostability" means that the thermostability of a beta-glucosidase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is improved by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or even more, compared to the thermostability of the same beta-glucosidase alone (i.e., in the absence of said oral care ingredient). For purposes of the present invention, thermostability can be measured according to Example 3 below and is defined as the midpoint (Tm) of the thermal unfolding transition.

[0243] In one embodiment, the beta-glucosidase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0244] In a preferred embodiment, the beta-glucosidase has similar or improved thermostability at pH 4-8, e.g., pH 4, 5, 6, 7, or 8. Preferably, the beta-glucosidase has similar or improved thermostability at pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0245] In one embodiment, the oral care composition comprises a benzoate, e.g., sodium benzoate, and a beta-glucosidase that has similar or improved thermal stability in the presence of a benzoate, e.g., sodium benzoate. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 0.01-5% benzoate (e.g., sodium benzoate), more preferably 0.05-2.5% benzoate, even more preferably 0.1-1% benzoate, and most preferably 0.1-0.5% benzoate. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0246] In one embodiment, the oral care composition comprises EDTA and a beta-glucosidase that has similar or improved thermal stability in the presence of EDTA. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0247] In one embodiment, the oral care composition comprises ethanol and a beta-glucosidase that has similar or improved thermostability in the presence of ethanol. Preferably, the beta-glucosidase has similar or improved thermostability in the presence of 0.1-20% ethanol, more preferably 1-10% ethanol, even more preferably 2.5-7.5% ethanol, and most preferably 5% ethanol. Preferably, the beta-glucosidase has similar or improved thermostability in the presence of 1-100000 mM ethanol, more preferably 100-10000 mM ethanol, and most preferably 1000 mM ethanol.

[0248] In one embodiment, the oral care composition comprises a fluoride, e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride, and a beta-glucosidase that has similar or improved thermal stability in the presence of a fluoride, e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-5000 ppm fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm fluoride, and most preferably 1,000-1500 ppm fluoride. Preferably, the beta-glucosidase has similar or improved thermostability in the presence of 1-100 mM fluoride (e.g., sodium fluoride), more preferably 5-75 mM fluoride, even more preferably 10-50 mM fluoride, and most preferably 20-40 mM fluoride.

[0249] In one embodiment, the oral care composition comprises glycerol and a beta-glucosidase that has similar or improved thermostability in the presence of glycerol. Preferably, the beta-glucosidase has similar or improved thermostability in the presence of 1-50% glycerol, more preferably 5-40% glycerol, and most preferably 10-30% glycerol. Preferably, the beta-glucosidase has similar or improved thermostability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0250] In one embodiment, the oral care composition comprises a peroxide, e.g., hydrogen peroxide, and a beta-glucosidase that has similar or improved thermal stability in the presence of a peroxide, e.g., hydrogen peroxide. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, and most preferably 100-500 mM peroxide.

[0251] In one embodiment, the oral care composition comprises mannitol and a beta-glucosidase that has similar or improved thermal stability in the presence of mannitol. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0252] In one embodiment, the oral care composition comprises a phosphate, such as sodium or potassium phosphate, and a beta-glucosidase that has similar or improved thermal stability in the presence of a phosphate, such as sodium or potassium phosphate. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0253] In one embodiment, the oral care composition comprises sodium dodecyl sulfate (SDS) and a beta-glucosidase that has similar or improved thermal stability in the presence of SDS. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-1000 mM SDS, more preferably 10-500 mM SDS, more preferably 15-150 mM SDS, and most preferably 17-170 mM SDS.

[0254] In one embodiment, the oral care composition comprises a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate, and a beta-glucosidase that has similar or improved thermal stability in the presence of a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 0.01-5% sorbate (e.g., potassium sorbate), more preferably 0.05-2.5% sorbate, even more preferably 0.1-1% sorbate, and most preferably 0.1-0.5% sorbate. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 1-100 mM sorbate (e.g., potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0255] In one embodiment, the oral care composition comprises sorbitol and a beta-glucosidase that has similar or improved thermal stability in the presence of sorbitol. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 0.1-70% sorbitol, more preferably 1-60% sorbitol, even more preferably 5-50% sorbitol, and most preferably 10-40% sorbitol. Preferably, the beta-glucosidase has similar or improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0256] Glucoamylase The glucoamylase may be derived from any organism. Preferably, the glucoamylase is of microbial origin, and most preferably, the glucoamylase is of bacterial or fungal origin. In a preferred embodiment, the glucoamylase is derived from Aspergillus niger.

[0257] In one aspect, the glucoamylase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8; (b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:8; (d) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:7 or its cDNA sequence; (e) a polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or SEQ ID NO:9 by substitution, deletion, or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), (c), (d) or (e), wherein the polypeptide has glucoamylase activity.

[0258] In a preferred embodiment, the glucoamylase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8, or a mature polypeptide of SEQ ID NO:8. A preferred mature polypeptide of SEQ ID NO:8 corresponds to amino acid residues 19 to 640 of SEQ ID NO:8.

[0259] In a preferred embodiment, the glucoamylase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9.

[0260] In a preferred embodiment, the glucoamylase comprises, consists essentially of, or consists of SEQ ID NO:8, or the mature polypeptide of SEQ ID NO:8. A preferred mature polypeptide of SEQ ID NO:8 corresponds to amino acid residues 19 to 640 of SEQ ID NO:8.

[0261] In a preferred embodiment, the glucoamylase comprises, consists essentially of, or consists of SEQ ID NO:9, or a fragment thereof.

[0262] The glucoamylase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0263] In another embodiment, the glucoamylase is derived from SEQ ID NO: 8 by substitution, deletion, or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 8 by substitution, deletion, or addition of one or several amino acids. In another embodiment, the polypeptide is derived from SEQ ID NO: 9 by substitution, deletion, or addition of one or more amino acids.

[0264] In some embodiments, the glucoamylase is a variant of a parent glucoamylase, preferably SEQ ID NO: 9, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the glucoamylase is a variant of SEQ ID NO: 9, wherein the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 9 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., methionine residues at the amino terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0265] Important 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 every residue in a molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for glucoamylase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224;899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of important amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms within a polypeptide or protein family that includes related polypeptides or polypeptides / proteins that are typically derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used in protein structure modeling to identify important amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0266] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0267] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0268] The oral care compositions of the present invention may contain glucoamylase in any effective amount or concentration. In a preferred embodiment, the oral care composition contains from about 1 ppm glucoamylase to about 500 ppm glucoamylase, preferably from about 1 ppm to about 100 ppm, more preferably from about 5 ppm to about 75 ppm, even more preferably from about 10 ppm to about 60 ppm, and most preferably from 10 ppm to 60 ppm glucoamylase.

[0269] In a preferred embodiment, the oral care composition comprises glucoamylase in an amount of at least 1 ppm, e.g., at least 5 ppm, at least 10 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, at least 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, at least 85 ppm, at least 90 ppm, at least 95 ppm, at least 100 ppm, or more.

[0270] In a particularly preferred embodiment, the oral care composition comprises at least 10 ppm of glucoamylase. In another particularly preferred embodiment, the oral care composition comprises at least 60 ppm of glucoamylase.

[0271] Glucoamylase prevents the formation of oral biofilm. Preferably, glucoamylase improves the effect of preventing oral biofilm. In one embodiment, glucoamylase prevents the formation of oral biofilm by at least 5%, for example, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. For purposes of the present invention, prevention of oral biofilm can be measured, for example, according to Example 4 below.

[0272] The glucoamylase reduces the risk of oral biofilm formation. Preferably, the glucoamylase reduces the risk of oral biofilm formation by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0273] Glucoamylase can also remove oral biofilm. Preferably, the glucoamylase improves the effectiveness of removing oral biofilm. In one embodiment, the glucoamylase removes at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the oral biofilm.

[0274] The glucoamylase is highly stable in dosage forms and / or formats suitable for oral care, particularly in dosage forms or formats such as toothpaste, mouthwash, lozenges, mints, gum, candy, etc. High stability, e.g., comparable or improved stability, can be comparable or improved physical stability and / or chemical stability. Comparable or improved chemical stability, i.e., comparable or improved stability in the presence of another agent (e.g., another enzyme, active ingredient, excipient, or solvent), can occur when the glucoamylase and the other agent are coformulated and / or coadministered, preferably coformulated.

[0275] In a preferred embodiment, the glucoamylase has comparable or improved thermostability. In the context of the present invention, the term "compare thermostability" means that the thermostability of the glucoamylase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is within ±5% of the thermostability of the same glucoamylase alone (i.e., in the absence of said oral care ingredient). In the context of the present invention, the term "improved thermostability" means that the thermostability of the glucoamylase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is improved by at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or even more, compared to the thermostability of the same glucoamylase alone (i.e., in the absence of said oral care ingredient). For purposes of the present invention, thermal stability may be measured according to Example 3 below and is defined as the midpoint of the thermal unfolding transition (Tm).

[0276] In one embodiment, the glucoamylase has similar or improved thermostability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0277] In a preferred embodiment, the glucoamylase has similar or improved thermostability at pH 4-8, e.g., pH 4, 5, 6, 7, or 8. Preferably, the glucoamylase has similar or improved thermostability at pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0278] In one embodiment, the oral care composition comprises a benzoate, e.g., sodium benzoate, and a glucoamylase having similar or improved thermostability in the presence of a benzoate, e.g., sodium benzoate. Preferably, the glucoamylase has similar or improved thermostability in the presence of 0.01-5% benzoate (e.g., sodium benzoate), more preferably 0.05-2.5% benzoate, even more preferably 0.1-1% benzoate, and most preferably 0.1-0.5% benzoate. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0279] In one embodiment, the oral care composition comprises EDTA and a glucoamylase that has similar or improved thermostability in the presence of EDTA. Preferably, the glucoamylase has similar or improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0280] In one embodiment, the oral care composition comprises ethanol and a glucoamylase having similar or improved thermostability in the presence of ethanol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 0.1-20% ethanol, more preferably 1-10% ethanol, even more preferably 2.5-7.5% ethanol, and most preferably 5% ethanol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-100000 mM ethanol, more preferably 100-10000 mM ethanol, and most preferably 1000 mM ethanol.

[0281] In one embodiment, the oral care composition comprises a fluoride, such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride, and a glucoamylase having similar or improved thermostability in the presence of a fluoride, such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-5000 ppm of fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm of fluoride, and most preferably 1,000-1500 ppm of fluoride. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-100 mM of fluoride (e.g., sodium fluoride), more preferably 5-75 mM of fluoride, even more preferably 10-50 mM of fluoride, and most preferably 20-40 mM of fluoride.

[0282] In one embodiment, the oral care composition comprises glycerol and a glucoamylase that has similar or improved thermostability in the presence of glycerol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-50% glycerol, more preferably 5-40% glycerol, and most preferably 10-30% glycerol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0283] In one embodiment, the oral care composition comprises a peroxide, e.g., hydrogen peroxide, and a glucoamylase that has similar or improved thermostability in the presence of a peroxide, e.g., hydrogen peroxide. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, and most preferably 100-500 mM peroxide.

[0284] In one embodiment, the oral care composition comprises mannitol and a glucoamylase that has similar or improved thermostability in the presence of mannitol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0285] In one embodiment, the oral care composition comprises a phosphate, such as sodium or potassium phosphate, and a glucoamylase that has similar or improved thermostability in the presence of a phosphate, such as sodium or potassium phosphate. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0286] In one embodiment, the oral care composition comprises sodium dodecyl sulfate (SDS) and a glucoamylase that has similar or improved thermostability in the presence of SDS. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-1000 mM SDS, more preferably 10-500 mM SDS, more preferably 15-150 mM SDS, and most preferably 17-170 mM SDS.

[0287] In one embodiment, the oral care composition comprises a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate, and a glucoamylase having similar or improved thermostability in the presence of a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate. Preferably, the glucoamylase has similar or improved thermostability in the presence of 0.01-5% sorbate (e.g., potassium sorbate), more preferably 0.05-2.5% sorbate, even more preferably 0.1-1% sorbate, and most preferably 0.1-0.5% sorbate. Preferably, the glucoamylase has similar or improved thermostability in the presence of 1-100 mM sorbate (e.g., potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0288] In one embodiment, the oral care composition comprises sorbitol and a glucoamylase having similar or improved thermostability in the presence of sorbitol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 0.1-70% sorbitol, more preferably 1-60% sorbitol, even more preferably 5-50% sorbitol, and most preferably 10-40% sorbitol. Preferably, the glucoamylase has similar or improved thermostability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0289] Alpha-amylase The alpha-amylase may be derived from any organism. Preferably, the alpha-amylase is of microbial origin, and most preferably, the alpha-amylase is of bacterial or fungal origin. In a preferred embodiment, the alpha-amylase is derived from Bacillus amyloliquefaciens.

[0290] In one aspect, the alpha-amylase is (a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:50; (b) a polypeptide encoded by a polynucleotide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO:49 or its cDNA sequence; (c) a polypeptide derived from SEQ ID NO: 50 by substitution, deletion or addition of one or several amino acids; (f) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N-terminus and / or C-terminus are extended by the addition of one or more amino acids; and (g) a fragment of a polypeptide of (a), (b), or (c), wherein the polypeptide has alpha-amylase activity.

[0291] In a preferred embodiment, the alpha-amylase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:50.

[0292] In a preferred embodiment, the alpha-amylase comprises, consists essentially of, or consists of SEQ ID NO:50, or a fragment thereof.

[0293] The alpha-amylase may have an N-terminal and / or C-terminal extension of one or more amino acids, for example, from 1 to 5 amino acids.

[0294] In another embodiment, the alpha-amylase is derived from SEQ ID NO: 50 by substitution, deletion, or addition of one or several amino acids. In another embodiment, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 50 by substitution, deletion, or addition of one or several amino acids.

[0295] In some embodiments, the alpha-amylase is a variant of a parent alpha-amylase, preferably SEQ ID NO: 50, comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the alpha-amylase is a variant of SEQ ID NO: 50, wherein the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 50 is up to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes may be of a minor nature, i.e. conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine tract, an antigenic epitope, or a binding module.

[0296] Important 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 every residue in a molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for alpha-amylase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224;899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of important amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms within a polypeptide or protein family that includes related polypeptides or polypeptides / proteins that are typically derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used in protein structure modeling to identify important amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0297] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0298] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0299] The oral care compositions of the present invention may contain any effective amount or concentration of alpha-amylase. In a preferred embodiment, the oral care compositions contain from about 1 ppm alpha-amylase to about 500 ppm alpha-amylase, preferably from about 1 ppm to about 100 ppm, more preferably from about 5 ppm to about 75 ppm, even more preferably from about 10 ppm to about 60 ppm, and most preferably from 10 ppm to 60 ppm alpha-amylase.

[0300] In a preferred embodiment, the oral care composition comprises an alpha-amylase in an amount of at least 1 ppm, e.g., at least 5 ppm, at least 10 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, at least 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, at least 85 ppm, at least 90 ppm, at least 95 ppm, at least 100 ppm, or more.

[0301] In a particularly preferred embodiment, the oral care composition comprises at least 10 ppm of alpha-amylase. In another particularly preferred embodiment, the oral care composition comprises at least 60 ppm of alpha-amylase.

[0302] Alpha-amylase prevents the formation of oral biofilm. Preferably, the alpha-amylase improves the effectiveness of preventing oral biofilm. In one embodiment, the alpha-amylase prevents oral biofilm formation by at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. For purposes of the present invention, prevention of oral biofilm may be measured, for example, according to Example 4 below.

[0303] The alpha-amylase reduces the risk of oral biofilm formation. Preferably, the alpha-amylase reduces the risk of oral biofilm formation by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0304] Alpha-amylases can also remove oral biofilms. Preferably, the alpha-amylase improves the effectiveness of removing oral biofilms. In one embodiment, the alpha-amylase removes at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the oral biofilm.

[0305] The alpha-amylase is highly stable in dosage forms and / or formats suitable for oral care, particularly in dosage forms or formats such as toothpaste, mouthwash, lozenges, mints, gum, candy, etc. High stability, e.g., comparable or improved stability, can be comparable or improved physical stability and / or chemical stability. Comparable or improved chemical stability, i.e., comparable or improved stability in the presence of another agent (e.g., another enzyme, active ingredient, excipient, or solvent), can occur when the alpha-amylase and the other agent are co-formulated and / or co-administered, preferably co-formulated.

[0306] In a preferred embodiment, the alpha-amylase has comparable or improved thermostability. In the context of the present invention, the term "compare thermostability" means that the thermostability of the alpha-amylase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is within ±5% of the thermostability of the same alpha-amylase alone (i.e., in the absence of said oral care ingredient). In the context of the present invention, the term "improved thermostability" means that the thermostability of the alpha-amylase in the presence (or, alternatively, co-formulated with) of a particular oral care ingredient or component is improved by at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or even more, compared to the thermostability of the same alpha-amylase alone (i.e., in the absence of said oral care ingredient). For purposes of the present invention, thermal stability may be measured according to Example 3 below and is defined as the midpoint of the thermal unfolding transition (Tm).

[0307] In one embodiment, the alpha-amylase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0308] In a preferred embodiment, the alpha-amylase has similar or improved thermostability at pH 4-8, such as pH 4, 5, 6, 7, or 8. Preferably, the alpha-amylase has similar or improved thermostability at pH 5-7, more preferably pH 5-6, most preferably pH 5 and / or pH 6.

[0309] In one embodiment, the oral care composition comprises a benzoate, e.g., sodium benzoate, and an alpha-amylase that has similar or improved thermostability in the presence of a benzoate, e.g., sodium benzoate. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 0.01-5% benzoate (e.g., sodium benzoate), more preferably 0.05-2.5% benzoate, even more preferably 0.1-1% benzoate, and most preferably 0.1-0.5% benzoate. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0310] In one embodiment, the oral care composition comprises EDTA and an alpha-amylase that has similar or improved thermostability in the presence of EDTA. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0311] In one embodiment, the oral care composition comprises ethanol and an alpha-amylase that has similar or improved thermostability in the presence of ethanol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 0.1-20% ethanol, more preferably 1-10% ethanol, even more preferably 2.5-7.5% ethanol, and most preferably 5% ethanol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-100,000 mM ethanol, more preferably 100-10,000 mM ethanol, and most preferably 1,000 mM ethanol.

[0312] In one embodiment, the oral care composition comprises a fluoride, e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride, and an alpha-amylase that has similar or improved thermal stability in the presence of a fluoride, e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride. Preferably, the alpha-amylase has similar or improved thermal stability in the presence of 1-5000 ppm fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm fluoride, and most preferably 1,000-1500 ppm fluoride. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-100 mM fluoride (e.g., sodium fluoride), more preferably 5-75 mM fluoride, even more preferably 10-50 mM fluoride, and most preferably 20-40 mM fluoride.

[0313] In one embodiment, the oral care composition comprises glycerol and an alpha-amylase that has similar or improved thermostability in the presence of glycerol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-50% glycerol, more preferably 5-40% glycerol, and most preferably 10-30% glycerol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0314] In one embodiment, the oral care composition comprises a peroxide, e.g., hydrogen peroxide, and an alpha-amylase that has similar or improved thermal stability in the presence of a peroxide, e.g., hydrogen peroxide. Preferably, the alpha-amylase has similar or improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, and most preferably 100-500 mM peroxide.

[0315] In one embodiment, the oral care composition comprises mannitol and an alpha-amylase that has similar or improved thermostability in the presence of mannitol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0316] In one embodiment, the oral care composition comprises a phosphate, such as sodium or potassium phosphate, and an alpha-amylase that has similar or improved thermostability in the presence of a phosphate, such as sodium or potassium phosphate. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0317] In one embodiment, the oral care composition comprises sodium dodecyl sulfate (SDS) and an alpha-amylase that has similar or improved thermostability in the presence of SDS. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 10-50 mM SDS, more preferably 15-25 mM SDS, and most preferably 17 mM SDS.

[0318] In one embodiment, the oral care composition comprises a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate, and an alpha-amylase that has similar or improved thermal stability in the presence of a sorbate, such as sodium sorbate, potassium sorbate, or calcium sorbate. Preferably, the alpha-amylase has similar or improved thermal stability in the presence of 0.01-5% sorbate (e.g. potassium sorbate), more preferably 0.05-2.5% sorbate, even more preferably 0.1-1% sorbate, and most preferably 0.1-0.5% sorbate. Preferably, the alpha-amylase has similar or improved thermal stability in the presence of 1-100 mM sorbate (e.g. potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0319] In one embodiment, the oral care composition comprises sorbitol and an alpha-amylase that has similar or improved thermostability in the presence of sorbitol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 0.1-70% sorbitol, more preferably 1-60% sorbitol, even more preferably 5-50% sorbitol, and most preferably 10-40% sorbitol. Preferably, the alpha-amylase has similar or improved thermostability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0320] Oral care ingredients and formats The oral care compositions of the present invention comprise an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0321] In one embodiment, the oral care composition comprises: a) an invertase, a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3; b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15; d) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18; e) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21; f) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24; g) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; h) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30; i) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; j) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36; k) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; l) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; m) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45; n) an invertase selected from the group consisting of polypeptides having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48; b) a beta-glucosidase having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6; and c) a glucoamylase having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9; and, optionally, d) includes an alpha-amylase having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:50.

[0322] In one embodiment, the oral care composition comprises: a) an invertase comprising, consisting essentially of, or consisting of SEQ ID NO:3; b) a beta-glucosidase comprising, consisting essentially of, or consisting of SEQ ID NO:6; c) a glucoamylase comprising, consisting essentially of, or consisting of SEQ ID NO:9; and, optionally, d) comprises an alpha-amylase comprising, consisting essentially of, or consisting of SEQ ID NO:50.

[0323] The oral care ingredients may vary according to different types of oral care compositions and the desired properties and / or activities of the oral care composition. For purposes of the present invention, the terms "ingredients" and "components" are used interchangeably with respect to oral care compositions.

[0324] The oral care compositions of the present invention may be oral care compositions such as toothpaste or toothpaste tablets, dental creams, mouthwash or mouthwash tablets, mouthrinses, lozenges, troches, chewing gums, confectioneries, candies, and the like, designed to remove biofilms in the oral cavity, for example biofilms present on teeth, soft tissues of the oral cavity, and dentures present in the oral cavity.

[0325] The oral care compositions of the present invention may also be extraoral care compositions such as denture cleaning solutions, denture cleaning tablets, denture cleaning powders, and the like, designed to remove biofilm from dentures that have been removed from the oral cavity for cleaning.

[0326] In a preferred embodiment, the oral care composition is an intraoral care composition and the at least one oral care component is selected from the group consisting of abrasives, humectants, solvents, thickeners, binders, buffering agents, foaming agents, foam control agents, sweeteners, softeners, plasticizers, flavoring agents, colorants, therapeutic agents, antimicrobial agents, anti-tartar agents, fluoride sources, preservatives, detergents, surfactants, colorants, buffering agents, softeners, plasticizers, whitening agents, bleaching agents, gum base components, and fillers.

[0327] The oral care ingredients referred to herein are categorized under general headings according to functionality, however, this should not be construed as limiting as the ingredients may include additional functionality as will be appreciated by those of skill in the art.

[0328] Toothpaste, dental cream, mouthwash, and mouth rinse Oral care compositions of the present invention in the form of toothpastes, dental creams, mouthwashes, and mouthrinses may contain ingredients and / or materials selected from the following categories:

[0329] [Table 1]

[0330] [Table 2]

[0331] Toothpaste Toothpastes and dental creams / gels typically include oral care ingredients such as abrasives, solvents, humectants, detergents / surfactants, thickeners and binders, buffers, flavors, sweeteners, fluoride sources, therapeutic agents, colorants, and preservatives.

[0332] In a preferred embodiment, the present invention relates to an oral care composition in the form of a toothpaste or dental cream comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient, the at least one oral care ingredient being selected from the following ingredients:

[0333] [Table 3]

[0334] [Table 4]

[0335] In a preferred embodiment, the present invention relates to an oral care composition in the form of a toothpaste or dental cream comprising an invertase, a beta-glucosidase, a glucoamylase, an alpha-amylase, and at least one oral care ingredient.

[0336] The oral care composition of the present invention may be a toothpaste comprising the following ingredients (in % by weight of the final toothpaste composition): Abrasive: 10~70% Wetting agent: 0~80% Thickener: 0.1-20% Binder: 0.01-10% Sweetener: 0.1-5% Foaming agent: 0~15% Enzymes (invertase, beta-glucosidase, glucoamylase, optionally alpha-amylase): 0.01-20%

[0337] Mouthwash Mouthwashes and mouthrinses of the present invention, including tartar removing solutions, typically include as oral care ingredients a carrier liquid, detergents / surfactants, buffers, flavoring agents, humectants, sweetening agents, therapeutic agents, fluoride sources, coloring agents, and preservatives.

[0338] In a preferred embodiment, the present invention relates to an oral care composition in the form of a mouthwash or mouthrinse comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0339] [Table 5]

[0340] In a preferred embodiment, the present invention relates to an oral care composition in the form of a mouthwash or mouthrinse comprising an invertase, a beta-glucosidase, a glucoamylase, an alpha-amylase, and at least one oral care ingredient.

[0341] The oral care composition of the present invention may be a mouthwash comprising the following ingredients (in % by weight of the final mouthwash composition): Water: 0~70% Ethanol: 0-20% Wetting agent: 0~20% Surfactant: 0-2% Enzymes (invertase, beta-glucosidase, glucoamylase, optionally alpha-amylase): 0.01-20% Other Ingredients: 0-2% (e.g., flavors, sweeteners, fluoride sources).

[0342] The mouthwash composition may be buffered with a suitable buffer, for example sodium citrate or sodium phosphate, in the pH range of 6 to 7.5.

[0343] The relevant oral care components suitable for toothpaste, dental cream, mouthwash and mouth rinse are described in more detail below.Those skilled in the art can modify the oral care composition according to the type of oral care composition and the desired properties and / or activity of a particular oral care composition.The oral care composition does not necessarily have to include all the components mentioned.

[0344] Abrasives Abrasives may be incorporated into the oral care compositions of the present invention, which include, according to the present invention, alumina and its hydrates, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, bentonite, silicon dioxide, sodium bicarbonate, and also powdered plastics, such as polyvinyl chloride, polyamide, polymethyl methacrylate, polystyrene, phenol-formaldehyde resins, melamine-formaldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels.

[0345] Also suitable as abrasives are calcium pyrophosphate, water-insoluble alkaline metaphosphates, polymetaphosphates, dicalcium phosphate and / or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxyapatite, etc. Mixtures of these substances can also be used.

[0346] Silica dental abrasives of various kinds are preferred due to their unique advantages of excellent tooth cleaning and polishing performance without excessively abrading tooth enamel or dentin, and their good compatibility with other potential ingredients such as metal ions and fluoride.

[0347] Depending on the oral care composition, the abrasive product may be present at 0-70% by weight, preferably 1%-70% by weight.

[0348] In toothpaste, the abrasive content is typically present in the range of 10% to 70% by weight of the final toothpaste product.

[0349] Wetting Agent Humectants are used, for example, to prevent the loss of moisture from toothpaste and to prevent toothpaste from hardening when exposed to air. Some humectants also impart a desirable sweetness of flavor to toothpaste and mouthwash compositions. Suitable humectants for use in the oral care compositions herein include the following compounds and mixtures thereof: glycerol, polyols, sorbitol, xylitol, maltitol, lactitol, polyoxyethylene, polyethylene glycol (PEG), polypropylene glycol, propylene glycol, 1,3-propanediol, 1,4-butanediol, hydrogenated partially hydrolyzed polysaccharides, etc., coconut oil fatty acids, amides of N-methyl-taurine, and Pluronic®.

[0350] The humectant is generally present at from 0% to 80% by weight, preferably from 5 to 70% by weight.

[0351] Thickeners / Binding Agents Suitable thickening and / or binding agents include silica, starch, tragacanth gum, xanthan gum, karaya gum, carrageenan (extract of Irish moss), gum arabic, alginates, pectin, cellulose derivatives such as hydroxyethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose and hydroxyethylpropylcellulose, polyacrylic acid and its salts, polyvinylpyrrolidone and carboxyvinyl polymers, and inorganic thickening agents such as amorphous silica compounds. These agents stabilize the oral care compositions of the present invention.

[0352] Thickeners may be present in toothpastes, dental creams and gels, and mouthwashes in amounts of 0.1% to 20% by weight, and binders may be present in the range of 0.01 to 10% by weight of the final product.

[0353] Foaming agents and foam regulators As foaming agents, soap, anionic, cationic, nonionic, amphoteric and / or zwitterionic surfactants can be used alone or in combination. These may be present at levels of 0% to 15%, preferably 0.1% to 13%, more preferably 0.25% to 10% by weight of the final product. The surfactant is preferably one that does not simply have the effect of inactivating enzymes and other components contained in the oral care composition. Useful surfactants include anionic, nonionic and amphoteric compounds, with anionic compounds being preferred.

[0354] Examples of suitable surfactants include salts of higher alkyl sulfates such as sodium lauryl sulfate or other suitable alkyl sulfates having 8 to 18 carbon atoms in the alkyl group; salts of sulfonated monoglycerides of higher fatty acids such as sodium lauryl sulfoacetate, sodium coconut oil monoglyceride sulfonate or other suitable sulfonated monoglycerides of fatty acids of 10 to 18 carbon atoms; salts of amides of higher fatty acids with lower aliphatic amino acids, for example, acids of 12 to 16 carbon atoms, for example, sodium-N-methyl-N-palmitoyl tauride, N-lauro ... salts of esters of such fatty acids with isotopic acids or glycerol monosulfate; for example, the sodium salt of monosulfated monoglyceride of hydrogenated coconut fatty acid; olefin sulfonates, for example, salts of alkenesulfonates or alkenesulfonates having 12 to 16 carbon atoms in the carbon chain of the molecule or mixtures thereof; and soaps of higher fatty acids of 12 to 18 carbon atoms, such as coconut fatty acid.

[0355] The cation of the salt may be sodium, potassium, or mono-, di-, or triethanolamine.Nonionic surfactants include sucrose / fatty acid ester, maltose / fatty acid ester, maltitol / fatty acid ester, maltotriitol / fatty acid ester, maltotetraitol / fatty acid ester, maltopentaitol / fatty acid ester, maltohexaitol / fatty acid ester, maltoheptaitol / fatty acid ester, sorbitan / fatty acid ester, lactose / fatty acid ester, lactinose / fatty acid ester, polyoxyethylene / polyoxypropylene copolymer, polyoxyethylene alkyl ether, polyoxyethylene / fatty acid ester, fatty acid alkanolamide, polyoxyethylene sorbitan / fatty acid ester, polyoxyethylene / hydrogenated castor oil, and polyglycerin / fatty acid ester.

[0356] Most preferred are sodium lauryl sulfate, sodium dodecylbenzenesulfonate and sodium lauryl sarcosinate.

[0357] Preferred suds control agents include polyethylene glycol.

[0358] The blowing agents and foam control agents may be present in an amount of from 0% to 15% by weight, preferably from 0.01% to 10% by weight.

[0359] Sweetener Suitable sweeteners include, but are not limited to, saccharin and its water soluble salts, dextrose, sucrose, lactose, maltose, levulose, aspartame, cyclamate, D-tryptophan, dihydrochalcones, acesulfame, stevioside, rebaudioside, glycyrrhizin, perartine, thaumatin, p-methoxycinnamaldehyde, hydrogenated starch hydrolysates, xylitol, sorbitol, erythritol, mannitol, and mixtures thereof.

[0360] The sweetener may be present in an amount of from 0.001% to 60% by weight, preferably from 0.01% to 50% by weight.

[0361] Flavoring Agent Flavoring agents are typically present in small amounts such as from 0.01% to about 5% by weight, especially from 0.1% to 5% by weight. Flavors that may be used in the present invention include, but are not limited to, oil of wintergreen, peppermint oil, spearmint oil, clove bud oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, 1-menthyl acetate, sage, eugenol, parsley oil, oxanone, alpha-irisone, marjoram, lemon, orange, cranberry, propenyl guethol, cinnamon, vanillin, ethyl vanillin, heliotropin, 4-cis-heptenal, diacetyl, para-tert-butylphenyl methyl acetate, carvone, cineole, menthone, cinnamic aldehyde, limonene, ocimene, n-decyl alcohol, citronellol, alpha-terpineol, methyl acetate, citronellyl acetate, methyl eugenol, linalool, thymol, rosemary oil, pimento oil, diatomaceous oil, eucalyptus oil, and mixtures thereof.

[0362] Cooling agents may also be part of the flavor system or may be added separately to the composition.Preferred cooling agents in the present composition are paramenthan carboxamide agents such as N-ethyl-p-menthane-3-carboxamide (commercially known as "WS-3"), menthol, 3-1-menthoxypropane-1,2-diol ("TK-10"), menthone glycerol acetal ("MGA"), menthyl lactate, and mixtures thereof.

[0363] Whitening Agent / Bleach Whitening agents / bleaching agents include H2O2 and may be added in amounts of less than 5%, preferably 0.05-4%, calculated on the weight of the final composition.

[0364] Other bleaching components that may be comprised according to the present invention include peroxydiphosphates, urea, peroxides, metal peroxides such as calcium peroxide, sodium peroxide, strontium peroxide, magnesium peroxide, hypochlorites such as sodium hypochlorite, and salts of perboric acid, persilicate, perphosphate and percarbonate such as sodium perborate, potassium persilicate and sodium percarbonate. The peroxide compounds may be stabilized by adding triphenylmethane dyes, chelating agents or antioxidants such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT).

[0365] solvent A solvent is typically added to a composition of the invention in an amount sufficient to render the composition flowable, e.g., if the composition is a toothpaste, dental cream, or gel, or in an amount sufficient to dissolve the other components of the composition, e.g., if the composition is a mouthwash or mouthrinse.

[0366] Suitable solvents include water, ethanol, and water / ethanol mixtures, which may be present in amounts from 0.1% to 70%.

[0367] Antibacterial agents The present invention may also include water soluble sources of certain metal ions such as zinc, copper, silver and tin (e.g., zinc chloride, copper chloride and stannous chloride, and silver nitrate), as well as water soluble antimicrobial agents such as chlorhexidine, triclosan, digluconate, hexetidine, alexidine, quaternary ammonium antimicrobial compounds.

[0368] Sparingly soluble zinc salts such as zinc citrate, zinc C14 alkyl maleate, zinc benzoate, zinc caproate, zinc carbonate, etc. may also be used and included in the compositions of the present invention to prolong the antimicrobial effect of the zinc ions due to the slow dissolution of these zinc salts in saliva.

[0369] The antimicrobial agent may be present in an amount of from 0% to 50% by weight, preferably from 0.01% to 40% by weight, and most preferably from 0.1% to 30% by weight.

[0370] Anti-tartar agent The compositions of the present invention may include an anti-tartar agent, such as an inorganic anti-tartar agent including any of the pyrophosphate salts, such as disodium pyrophosphate, dipotassium pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, and mixtures thereof.

[0371] Organophosphite compounds which may function as anti-tartar agents include polyphosphonates such as disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), methane diphosphonic acid, and 2-phosphonobutane-1,2,4-tricarboxylic acid.

[0372] The anti-tartar agent may be present in an amount of from 0% to 10% by weight, preferably from 0.1% to 5% by weight.

[0373] Preservatives Suitable preservatives include sodium benzoate, potassium sorbate, p-hydroxybenzoic acid esters, methylparaben, ethylparaben, propylparaben, citric acid, calcium citrate, and mixtures thereof.

[0374] Preservatives may be present in an amount of from 0% to 40% by weight, preferably from 0.01% to 30% by weight.

[0375] Fluoride Source The compositions of the present invention may also contain ingredients that can be used as fluoride sources. Preferred soluble fluoride sources include sodium fluoride, potassium fluoride, stannous fluoride, indium fluoride, sodium monofluorophosphate, sodium hexafluorosilicate, zinc fluoride, lithium fluoride, aluminum fluoride, acid fluoride phosphate, ammonium difluoride, titanium tetrafluoride, and amine fluorides.

[0376] Particularly preferred are sodium fluoride and sodium monofluorophosphate.

[0377] The fluoride source may be present in an amount of 0% to 20% by weight, preferably 0.01% to 15% by weight, and most preferably 0.1% to 10% by weight.

[0378] In a preferred embodiment, at least one oral care ingredient is a fluoride source, preferably the fluoride source is selected from the group consisting of sodium fluoride, calcium fluoride, stannous fluoride, or sodium monofluorophosphate.

[0379] Coloring agent Suitable colorants or pigments for the oral care compositions of the present invention include non-toxic, water-insoluble inorganic pigments such as titanium dioxide and chromium oxide greens, ultramarine blue and pink, and ferric oxide, as well as water-insoluble dye lakes prepared by spreading the calcium or aluminum salts of FD&C dyes onto alumina, such as FD&C Green No. 1 Lake, FD&C Blue No. 2 Lake, FD&C Red No. 30 Lake, FD&C Yellow No. 16 Lake, and FD&C Yellow No. 10.

[0380] A preferred opacifying agent is titanium dioxide.

[0381] The colorant may be present in an amount of from 0% to 20% by weight, preferably from 0.01% to 15% by weight, and most preferably from 0.1% to 10% by weight.

[0382] Buffer The oral care compositions of the present invention may also include buffers, i.e., pH adjusting agents, such as alkali metal hydroxides, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazoles, and mixtures thereof.

[0383] Specific buffering agents include monosodium phosphate, trisodium phosphate, sodium hydroxide, potassium hydroxide, alkali metal carbonates, sodium carbonate, imidazole, pyrophosphate, sodium citrate, hydrochloric acid, sodium hydroxide, triethanolamine, triethylamine, lactic acid, malic acid, fumaric acid, tartaric acid, phosphoric acid, and mixtures thereof.

[0384] The buffer may be present in an amount of from 0% to 10% by weight, preferably from 0.01% to 5% by weight.

[0385] Chewing gum If the oral composition according to the invention is a chewing gum, it may be any known type of chewing gum, such as optionally coated chewing gum pieces and sticks, or a chewing gum with any desired shape depending on the intended use. The chewing gum preparation may be of any quality, including bubble gum properties.

[0386] In a preferred embodiment, the present invention relates to an oral care composition in the form of a chewing gum comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient selected from elastomers, softeners, plasticizers, emulsifiers, waxes, colorants, sweeteners, flavoring agents, fillers, and thickeners. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0387] Gum Base Ingredients Chewing gum is conventionally considered to be composed of a water-insoluble or base part and a water-soluble part that includes flavors, sweeteners, and colorants. The gum base part of the gum is the chewing substance that imparts the chewing properties to the final product. The gum base part defines the flavor and sweetness release profile and plays an important role in gum products. Flavor, sweetness, and color can be considered to provide the sensory appealing aspects of chewing gum. There is no limitation regarding the chewing gum base used in the chewing gum preparation according to the present invention. Conventional chewing gum bases available, for example, from Dansk Tyggegummi Fabrik A / S, LADreyfus, or Cafasa Gum SIA are usually suitable, although specially prepared formulations can also be used. The formulation depends on the desired type of chewing gum or the desired type of structure. Ingredients suitable for gum base include those materials complying with the U.S. Chewing Gum Base Regulations (21 Code of Federal Regulations, Part 172,615) as well as other national and international lists (or positive lists), including elastomers, resins, waxes, polyvinyl acetates, oils, fats, emulsifiers, fillers, and antioxidants.

[0388] The gum base is usually contained in the final product in an amount of 15 to 90% by weight, preferably 30 to 40% by weight, and more preferably 5 to 25% by weight.

[0389] Elastomers provide chewiness, elasticity or resilience to the base and control the release of foam and flavor in the final chewing gum. They can be any water insoluble polymer known in the art. Elastomers include both natural and synthetic styrene butadiene copolymers (SBR) and non-SBR types. Examples of natural elastomers include, but are not limited to, rubbers such as rubber latex (natural rubber) and guayule, and gums such as chicle, jelutong, balata, gutta percha, reticapsi, sorba, crown gum, nispero, rosidingha, perillo, nigargutta, tunu, gutta kei, pendare, leche de baka, chiquibul, crown gum, and mixtures thereof. Examples of synthetic elastomers include, but are not limited to, polyisobutylene, isobutylene-isoprene copolymers (butyl rubber), polyethylene, polybutadiene, styrene butadiene copolymers, polyisoprene, and mixtures thereof.

[0390] The amount of elastomer (rubber) used in a gum base composition can vary widely depending on a variety of factors, such as the type of gum base used (sticky, or conventional, foamy, or standard), the consistency of the gum base composition desired, and the other ingredients used in the composition to produce the final chewing gum product. Generally, the elastomer is present in the gum base composition in an amount of from about 15% to about 60% by weight, preferably from about 25% to about 30% by weight, based on the total weight of the gum base composition.

[0391] Elastomer solvents help soften or plasticize the elastomer components, thereby providing bulk for chewing.

[0392] Elastomer solvents include, but are not limited to, natural rosin esters and synthetic derivatives, such as terpenes. Examples of elastomeric solvents suitable for use herein include tall oil rosin esters; partially hydrogenated wood and gum rosins; glycerol esters of wood and gum rosins, partially hydrogenated wood / gum rosins, partially dimerized wood and gum rosins, polymerized wood and gum rosins, and tall oil rosins; deodorized glycerol esters of wood rosins; pentaerythritol esters of wood and gum rosins; partially hydrogenated wood and gum rosins; methyl esters of partially hydrogenated wood rosins; rosins and modified rosins, such as methyl, glycerol, and pentaerythritol esters of hydrogenated, dimerized, and polymerized rosins; terpene resins, such as polymers of α-pinene or β-pinene, terpene hydrocarbon resins; polyterpenes, and the like, and mixtures thereof. The elastomer solvent may be used in the gum base composition in an amount of from about 2% to about 40% by weight, and preferably from about 7% to about 15% by weight of the gum base composition.

[0393] Polyvinyl acetate provides stretch or elasticity to the gum base. It also influences chewing bulk, softness, and foaming properties, hydrophilic properties, and flavor release.

[0394] The amount of polyvinyl acetate of different molecular weights present in the gum base composition should be an amount effective to impart the desired chewing properties to the finished chewing gum, such as integrity, softness, chewing bulk, film forming properties, hydrophilic properties, and flavor release. The total amount of polyvinyl acetate used in the gum base composition is typically about 45% to about 92% by weight based on the total gum base composition. The vinyl polymer may have a molecular weight ranging from about 2000 Da up to about 95000 Da.

[0395] Typically, low molecular weight polyvinyl acetates have a weight average molecular weight of about 2,000 Da to about 14,000 Da. Medium molecular weight polyvinyl acetates typically have a weight average molecular weight of about 15,000 Da to about 55,000 Da. High molecular weight polyvinyl acetates typically have a weight average molecular weight of about 55,000 Da to about 95,000 Da, but can range as high as 500,000 Da.

[0396] The waxes, fats and oils plasticize the elastomer mixture and improve the elasticity of the gum base. The waxes provide a soft or hard chew, affect flavor release, and can provide bulk and smoothness to the gum base. The fats and oils provide a soft chew. The fats, oils and waxes may be used individually or in combination, and the gum base may be a wax-free gum base.

[0397] Waxes, if used, may be of mineral, animal, vegetable or synthetic origin. Non-limiting examples of mineral waxes include petroleum waxes such as paraffin and microcrystalline wax, animal waxes include beeswax, vegetable waxes include carnauba, candelilla, rice bran, esparto, flax and sugar cane, synthetic waxes include those produced by Fischer-Tropsch synthesis, and mixtures thereof.

[0398] Suitable oils and fats which can be used in the gum composition include hydrogenated or partially hydrogenated vegetable or animal fats such as cottonseed oil, soybean oil, coconut oil, palm kernel oil, beef tallow, hydrogenated tallow, lard, cocoa butter, lanolin, etc.; fatty acids or esters of palmitic acid, oleic acid, stearic acid, linoleic acid, lauric acid, myristic acid, caproic acid, caprylic acid, decanoic acid, etc. and salts such as sodium stearate and potassium stearate. These ingredients, if used, are generally present in an amount up to about 7% by weight of the gum composition, preferably up to about 3.5% by weight of the gum composition.

[0399] Hydrogenated vegetable oils are preferred as softeners, including soybean oil and cottonseed oil, which may be used alone or in combination. These softeners provide good texture and soft chewing characteristics to the gum base composition. These softeners are generally used in amounts of about 5% to about 14% by weight of the gum base composition.

[0400] Emulsifiers help disperse the immiscible components of the gum base composition into a single stable system. Emulsifiers impart hydrophilic properties to the gum base and help plasticize the resins and polyvinyl acetate. They also affect the softness and foam properties of the base. Typical emulsifiers include acetylated monoglycerides, glyceryl monostearate, lecithin, fatty acid monoglycerides, diglycerides, propylene glycol monostearate, lecithin, triacetin, glyceryl triacetate, and the like, and mixtures thereof.

[0401] Preferred emulsifiers are glyceryl monostearate and acetylated monoglycerides, which function as plasticizers. The emulsifiers may be used in an amount of about 2% to about 15% by weight of the gum base composition, preferably about 7% to about 11% by weight of the gum base composition.

[0402] Fats, oils, waxes, emulsifiers, and certain sugar fillers are often grouped together and referred to as softeners. Softeners are able to penetrate the gum base's substructure due to the low molecular weight of these ingredients, making the gum base plastic and less viscous. Useful plasticizers and softeners include lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate, propylene glycol nonastearate, acetylated monoglycerides, glycerin, fully unsaturated vegetable oils such as non-hydrogenated cottonseed oil, hydrogenated vegetable oils, petroleum waxes, sorbitan monostearate, tallow, and the like, and mixtures thereof, as well as high fructose corn syrup, corn syrup, sorbitol solution, hydrogenated starch hydrolysates, and the like, and mixtures thereof.

[0403] The amount of softener should be present in an amount effective to provide a finished chewing gum with the desired chewing bulk and softness. When used as softeners, these materials are generally used in the gum base composition in amounts up to about 25%, preferably from about 1% to about 17%, by weight of the gum base composition.

[0404] The gum base may further comprise a surfactant. Examples of suitable surfactants include polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, polyethylene (4) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, polyoxyethylene (5) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, sorbitan monolaurate, and the like. The amount of surfactant should be present in an amount effective to provide the finished chewing gum with the desired softness. Typically, the surfactant is used in the base in an amount of about 0.5% to about 3.0% by weight, based on the total weight of the gum base.

[0405] The gum base compositions of the present invention may also contain effective amounts of fillers, sometimes referred to as bulking agents. These materials impart hardness and bulk and affect the texture and flavor release of the chewing gum. Useful fillers include organic and inorganic compounds (mineral auxiliaries) such as calcium carbonate, magnesium carbonate, ground limestone, magnesium silicate, calcium phosphate, cellulose polymers, clays, alumina, aluminum hydroxide, aluminum silicate, tale, tricalcium phosphate, dicalcium phosphate, and the like, and mixtures thereof. These fillers or auxiliaries may be used in the gum base compositions in various amounts. The amount of filler should be present in an amount effective to provide a finished chewing gum with the desired flavor release and integrity. Typically, fillers are used in the gum base compositions in an amount of about 1% to about 40%, preferably about 5% to about 20%, by weight of the gum base composition.

[0406] The gum base may also contain antioxidants to improve stability, reduce any oily taste, and provide a longer shelf life.Typical, non-limiting examples of antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate.Mixtures thereof may also be used.

[0407] Other gum ingredients The remaining components of the chewing gum composition are conventional and typically comprise from 10 to 85% by weight of the final product.

[0408] Examples of these are sweeteners, softeners, colorants, fillers, thickeners, and flavoring agents of the types and amounts conventionally used in chewing gum.

[0409] Suitable flavoring agents are those known to those skilled in the art, such as natural and artificial flavors. These flavors can be selected from synthetic flavor oils and flavoring aromatics and / or oils derived from plants, leaves, flowers, fruits, etc., oleoresins and extracts, and combinations thereof. Non-limiting representative flavor oils include spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, nutmeg oil, allspice, sage oil, nutmeg, bitter almond oil, and cassia oil. Other useful flavors are artificial, natural, and synthetic fruit flavors, such as vanilla, and citrus oils, including lemon, orange, lime, grapefruit, and fruit essences, including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, etc. These flavoring agents may be used in liquid or solid form and may be used individually or in admixture. Commonly used flavors include mints such as peppermint, menthol, artificial vanilla, cinnamon derivatives, and various fruit flavors, whether used individually or in admixture.

[0410] Other useful flavoring agents may be used, including aldehydes and esters such as cinnamyl acetate, cinnamic aldehyde, diethyl acetal citrate, dihydrocarbyl acetate, eugenyl formate, p-methylanisole, etc. In general, any flavor additive or food additive may be used.

[0411] Further examples of aldehyde flavourings include acetaldehyde (apple), benzaldehyde (cherry, almond), anisaldehyde (licorice, anise), cinnamic aldehyde (cinnamon), citral, i.e. α-citral (lemon, lime), neral, i.e. β-citral (lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, cream), heliotrope, i.e. piperonal (vanilla, cream), vanillin (vanilla, cream), α-amyl cinnamic aldehyde (spicy fruity flavour), butyraldehyde (butter, cheese), valeraldehyde (butter, cheese), Examples of aldehydes include, but are not limited to, aldehydes C-8 (citrus), aldehydes C-9 (citrus), aldehydes C-12 (citrus), 2-ethylbutyraldehyde (berries), hexenal, i.e., trans-2-hexenal (berries), tolylaldehyde (cherries, almonds), veratraldehyde (vanilla), 2,6-dimethyl-5-heptenal, i.e., melonal (melon), 2,6-dimethyloctanal (green fruits), and 2-dodecenal (citrus, mandarin), cherry, grape, strawberry shortcake, and mixtures thereof.

[0412] The amount of flavoring agent used herein is generally a matter of preference depending on factors such as the type of final chewing gum composition, the particular flavor, the gum used, and the intensity of flavor desired. Thus, the amount of flavoring agent may be varied to obtain the desired result in the final product, and such variations are within the ability of one of ordinary skill in the art without undue experimentation. In gum compositions, flavoring agents are generally present in an amount of about 0.02% to about 5% by weight of the chewing gum composition.

[0413] Chewing gum compositions generally contain fillers. These fillers (bulking agents) may be water-soluble and include, but are not limited to, monosaccharides, disaccharides, polysaccharides, sugar alcohols, and mixtures thereof; sorbitol, xylitol, maltitol, mannitol, isomalt (Palatinit according to Suddeutsche Zucker); (商標) and a racemic mixture of α-D-glucopyranosyl-1,6-mannitol and α-D-glucopyranosyl-1,6-sorbitol manufactured under the trade name of Lycasin®, glycerol, aspartame, Lycasin® glycerol, galactitol, acesulfame K, saccharin and its salts, cyclamic acid and its salts, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin and sucralose and mixtures thereof or mixtures thereof with other suitable sweeteners, maltodextrins; hydrogenated starch hydrolysates; hydrogenated hexoses; hydrogenated disaccharides; minerals such as calcium carbonate, talc, titanium dioxide, dicalcium phosphate, cellulose, and the like, and mixtures thereof. The fillers may be used in an amount of up to about 60% by weight of the chewing gum composition, preferably about 25% to 60% by weight.

[0414] The chewing gum composition may also include a high intensity sweetener (sweetener). High intensity sweeteners have a sweetness intensity substantially greater than that of sucrose. Examples of suitable high intensity sweeteners include: a) water-soluble naturally occurring intense sweeteners such as dihydrochalcones, monellin, stevioside, glycyrrhizin, dihydroflavonols, and L-aminodicarboxylic acid aminoalkonoic acid ester amides as disclosed in U.S. Pat. No. 4,619,834, and mixtures thereof; b) Water-soluble artificial sweeteners, including soluble saccharin salts such as sodium or calcium saccharin salts, cyclamates, the sodium, ammonium, or calcium salts of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, the potassium salt of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide (acesulfame-K), the free acid form of saccharin, and the like, and mixtures thereof; c) dipeptide sweeteners, including sweeteners derived from L-aspartic acid, such as 1-aspartyl-L-phenylalanine methyl ester (aspartame) and the substances described in U.S. Pat. No. 3,492,131, L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate (alitame), the methyl esters of L-aspartyl-L-phenylglycerin and L-aspartyl-L-2,5-dihydrophenyl-glycine, L-aspartyl-2,5-dihydro-L-phenylalanine, L-aspartyl-L-(1-cyclohexene)-alanine, and the like, and mixtures thereof; d) Water-soluble intense sweeteners derived from naturally occurring water-soluble sweeteners, such as chlorinated derivatives of normal sugars (sucrose), such as derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose, known under the trade name Sucralose®; examples of chlorodeoxysucrose derivatives and chlorodeoxygalactosucrose derivatives include, but are not limited to, 1-chloro-1'-deoxysucrose; 4-chloro-4-deoxy-α-D-galactopyranosyl-α-D-fructofuranoside, or 4-chloro-4-deoxygalactosucrose; 4-chloro-4-deoxy-α-D-galactopyranosyl-1-chloro-l-deoxy-β-D-fructo-furanoside, or 4,1'-dichloro-4,1'-dideoxygalactosucrose; 1',6'-dichloro-1',6'-dideoxy 4-chloro-4-deoxy-α-D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructofuranoside, or 4,1',6'-trichloro-4,1',6'-trideoxygalactosucrose; 4,6-dichloro-4,6-dideoxy-α-D-galactopyranosyl-6-chloro-6-deoxy-β-D-fructofuranoside, or 4,6,6'-trichloro-4,6,6'-trideoxygalactosucrose Lactosucrose; 6,1',6'-trichloro-6,1',6'-trideoxysucrose; 4,6-dichloro-4,6-dideoxy-α-D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructofuranoside, or 4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxygalacto-sucrose; and 4,6,1',6'-tetradeoxy-sucrose, and mixtures thereof; and e) Proteinaceous intense sweeteners such as Thaumaoccous daniclii (thaumatin I and II). The amount of sweetener used in the chewing gum composition will vary depending on the sweetener selected for the particular chewing gum. Thus, for any given sweetener, a sufficient amount of sweetener is used to provide the desired sweetness level. The saccharide sweeteners and sugar alcohols described above are typically used in an amount of about 1% to about 70% by weight, preferably about 40% to about 50% by weight, based on the total weight of the chewing gum composition. The intense sweeteners described above are typically used in an amount of up to about 1% by weight, preferably about 0.05% to about 0.4% by weight, based on the total weight of the chewing gum composition.

[0415] Colorants useful in the present invention are used in amounts effective to produce the desired color. These colorants include pigments and may be incorporated in amounts up to about 6% by weight of the gum composition. Titanium dioxide, a preferred pigment, may be incorporated in amounts up to 2% by weight of the gum composition, and preferably less than about 1% by weight. Colorants may also include natural food colors and dyes suitable for food, drug, and cosmetic applications. These colorants are known as FD&C dyes and lakes. Materials acceptable for the foregoing uses are preferably water soluble. Illustrative non-limiting examples include the indigoid dye known as FD&C Blue No. 2, which is the disodium salt of 5,5-indigostin sulfonic acid. Similarly, the dye known as FD&C Green No. 1 comprises a triphenylmethane dye and is the monosodium salt of 4-[4-(N-ethyl-Np-sulfonium benzylamino)diphenylmethylene]-[1-(N-ethyl-Np-sulfonium benzyl)-delta-2,5-cyclo-hexadienimine].

[0416] Examples of thickening agents include methylcellulose, alginates, carrageenan, xanthan gum, gelatin, carob, tragacanth, and locust bean, emulsifiers such as lecithin and glyceryl monostearate, acidulants such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid, and mixtures thereof.

[0417] The plasticizers, softeners, emulsifiers, waxes, and antioxidants described above as suitable for use in gum bases may also be used in the chewing gum compositions.

[0418] Active gum ingredients The oral care compositions of the present invention in the form of chewing gum may also contain a variety of active ingredients such as antimicrobial agents, Zn salts, fluoride, and urea.

[0419] In addition, the oral compositions according to the present invention may contain any other active ingredients, if desired, such as anti-caries agents, anti-tartar agents, anti-plaque agents, anti-periodontal disease agents, anti-fungal agents, smoking cessation agents, anti-cold agents, agents to prevent gingivitis, and the like.

[0420] The antibacterial agent used in the composition may be any of a wide range of cationic antibacterial agents such as quaternary ammonium compounds (e.g., cetylpyridinium chloride) and substituted guanidines such as chlorhexidine and the corresponding compound alexidine. Mixtures of cationic antibacterial agents may also be used in the present invention.

[0421] Antibacterial quaternary ammonium compounds include those in which one or two of the substituents on the quaternary nitrogen have a carbon chain length of 8 to 20, typically 10 to 18 carbon atoms (typically an alkyl group), while the remaining substituents (typically an alkyl or benzyl group) have fewer carbon atoms, for example 1 to 7 carbon atoms, typically a methyl or ethyl group. Dodecyltrimethylammonium bromide, tetradecylpyridinium chloride, tetradecylethylpyridinium chloride, dodecyldimethyl(2-phenoxyethyl)ammonium bromide, benzyldimethylstearylammonium chloride, cetylpyridinium chloride, quaternized 5-amino-1,3-bis(2-ethyl-hexyl)-5-methylhexahydropyrimidine, and benzethonium chloride are representative examples of typical quaternary ammonium antibacterial agents. Other compounds are the bis[4-(R-amino)-1-pyridinium]alkanes as disclosed in U.S. Patent No. 4,206,215 (Bailey, June 3, 1980), which is incorporated herein by reference. Preferred quaternary ammonium compounds are pyridinium compounds.

[0422] Cationic antimicrobial agents are generally employed in the present compositions at levels of from about 0.02% to about 1%, preferably from about 0.3% to about 0.7%, and most preferably from about 0.3% to about 0.5%.

[0423] As the readily soluble zinc salt, it is in principle possible to use any physiologically acceptable readily soluble zinc salt of an inorganic or organic acid, which is capable of releasing zinc ions and is approved for use in foodstuffs, cosmetics or medicines, etc. Non-limiting examples are, for example, zinc citrate, zinc sulfate, zinc lactate, zinc chloride, zinc acetate and mixtures thereof. Among these, the salt of zinc acetate is preferred.

[0424] The zinc salt used must be readily soluble to ensure that an effective amount of zinc ions is released in the oral cavity for the intended purpose within a reasonable period of time.

[0425] Advantageously, the zinc salt is present in the oral composition in an amount of 0.001 to 1.25% by weight. The amount used will depend on the dosage form and intended use and will be adapted to release an effective amount of zinc ions for the intended use.

[0426] As the taste-masking salt, at least one salt selected from sodium chloride, ammonium chloride, and physiologically acceptable alkali metals, alkaline earth metals and / or ammonium carbonate is used.

[0427] The alkali metals are in particular sodium or potassium, while the alkaline earth metals are advantageously calcium or magnesium. Particularly preferred taste masking salts are sodium, potassium and magnesium carbonates, sodium chloride, ammonium chloride and mixtures thereof.

[0428] The taste masking salt is advantageously used in the oral composition in an amount of 0.05 to 6.25% by weight, more preferably 0.25 to 3.50% by weight, for example 0.50 to 2.50% by weight.

[0429] The amount of taste-masking salt used to mask the taste of zinc in any given case can be determined by one skilled in the art and will depend on the particular zinc salt in question and the form of administration selected.

[0430] Urea is used as an anti-caries product to neutralize the acids produced in plaque after eating and drinking. In addition to urea, the composition can also contain pharmacologically acceptable substances capable of releasing urea under the conditions prevailing in the mouth. Examples thereof are salts and addition compounds of urea with inorganic compounds such as magnesium sulfate, calcium phosphate, sodium chloride, etc.

[0431] The urea content of the composition according to the invention varies between 0.05% and 80% by weight, preferably between 0.2% and 25% by weight.

[0432] The chewing gum compositions can be prepared using standard techniques and equipment known to those skilled in the art. Equipment useful according to the present invention also includes mixing and stirring equipment.

[0433] Lozenges and Troches Lozenges are flavored medicinal dosage forms intended to be sucked and retained in the mouth or throat. They may contain vitamins, antibiotics, antiseptics, local anesthetics, antihistamines, decongestants, corticosteroids, astringents, analgesics, flavorings, demulcents, or combinations of these ingredients. Lozenges may take a variety of shapes, the most common being flat, round, octagonal, and biconvex shapes. Another type is called a rod, which is in the form of a short rod or cylinder. The soft variety of lozenges is called a troche, and consists of a medicine in a gelatin or glycerogelatin base, or in a base of acacia, sucrose, and water (HALieberman, Pharmaceutical Dosage Forms; Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, NY).

[0434] In a preferred embodiment, the present invention relates to an oral care composition in the form of a lozenge or troche comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient, the at least one oral care ingredient being selected from lubricants, fillers, sweeteners, and flavoring agents. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0435] Lubricants The use of lubricants in the manufacture of compressed lozenges is to facilitate the release of the lozenges from the mold in which they are formed. The lubricants used in the present invention are solid materials that are uncharged and do not interfere (e.g., form complexes) with the cationic antimicrobial agents. The material should preferably be water insoluble. One class of suitable materials that meet these requirements are non-toxic hydrocarbon fats or derivatives. Examples include hydrogenated tallow and hydrogenated vegetable oils. Polyethylene glycols may also be used as lubricants, so long as they are solid materials (meaning that they have a molecular weight generally in the range of 4000 Da to 6000 Da). These materials may also be used as fillers, as described below.

[0436] Mixtures of lubricants may also be used in the present invention. Lubricants are used at levels of from about 0.1% to about 4.0%, preferably from about 0.5% to about 2%.

[0437] Lozenge Vehicle The term "lozenge vehicle" is used herein to refer to the materials that hold the active ingredients, i.e., enzymes, and lubricants. These materials are also known as bulking agents or fillers. The vehicle is non-cariogenic, which requires that the vehicle be free of sucrose and similar materials.

[0438] Acceptable filler materials include mannitol, sorbitol, xylitol, polyethylene glycol and non-cariogenic dextran. Fillers may be used alone or in combination.

[0439] Mannitol is a natural sugar alcohol and is available as a fine powder. Mannitol is only about 50% as sweet as sucrose. However, the negative calorific value of a solution of mannitol can provide a pleasant cooling sensation in the mouth as the lozenge dissolves.

[0440] Sorbitol is a chemical isomer of mannitol and has a similar sweetness. The negative calorific value of its solution also produces a pleasant cooling sensation in the mouth. Sorbitol is available as a free-flowing granule or a crystalline powder. Polyethylene glycols (PEGs) can also be used in the present compositions. These materials have the general formula HOCH2(CH2OCH2) n It is a polymer of ethylene oxide with CH2OH. The use of PEG alone is not preferred, but its use in combination with other fillers is acceptable. The molecular weight found to be most desirable is between 4000 Da and 6000 Da.

[0441] Fillers are generally used in the present invention at levels of from about 85% to about 99.8%, preferably from about 90% to about 98%, and most preferably from about 94% to about 97%.

[0442] Other lozenge components Acceptable lozenges may be manufactured simply using the active ingredient, lubricant and filler materials as outlined above. However, materials such as spray-dried or encapsulated flavors, or liquid flavors adsorbed on a suitable diluent, are generally included to make the lozenge more aesthetically acceptable. Spray-dried or encapsulated flavors are preferred. Suitable flavors include oil of peppermint, oil of wintergreen, oil of sassafras, oil of spearmint, and oil of clove. Sweeteners are also acceptable for use in the present compositions. Suitable agents include aspartame, acesulfame, saccharin, dextrose and levulose. Sweeteners and flavors are generally used in the compositions of the present invention at levels of about 0.1% to about 2%, preferably about 0.25% to about 1.5%.

[0443] It is also acceptable to have a water-soluble fluoride compound in solid form present in the lozenge in an amount sufficient to obtain a fluoride concentration of about 0.0025% to about 5.0% by weight, preferably about 0.005% to about 2.0% by weight, to provide additional caries prevention benefits. Preferred fluorides are sodium fluoride, stannous fluoride, indium fluoride and sodium monofluorophosphate. The lozenges may also contain various active ingredients such as antimicrobial agents, Zn salts, fluoride, and urea (see above).

[0444] Confectionery and candy In a preferred embodiment, the present invention relates to an oral care composition in the form of a confectionery or candy comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient, the at least one oral care ingredient being selected from colorants, sweeteners, flavoring agents, and oil modifiers. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0445] The preparation of confectionery formulations is historically well known and has changed little over the years. Confectionery articles are classified as either "hard" or "soft" confectionery. The volatile oil modifier of the present invention can be incorporated into conventional hard and soft confectionery by blending the modifier.

[0446] The hard confectionery may be processed and formulated by conventional means. Generally, hard confectionery has a base consisting of a mixture of sugar and other carbohydrate fillers held in an amorphous or glassy state. This form is considered to be a syrup solid of sugar, generally having about 0.5% to about 1.5% water. Such materials usually contain up to about 92% corn syrup, up to about 55% sugar, and about 0.1% to about 5% water by weight of the final composition. The syrup component is generally prepared from high fructose corn syrup, but may contain other ingredients. Additionally, ingredients such as flavors, sweeteners, acidulants, colorants, and the like may also be added.

[0447] Such confections may be conveniently prepared by conventional methods such as those including open-air cookers, vacuum cookers, and scraped-surface cookers, also known as high-speed atmospheric cookers.

[0448] Fire cookers involve the traditional method of making candy bases. In this method, the desired amount of carbohydrate filler is dissolved in water by heating the agent in a kettle until the filler dissolves. Additional filler may then be added and cooking continues until a final temperature of 145-156°C is reached. The batch is then cooled and processed into a resinous mass for the incorporation of flavors, colors, and other additives.

[0449] High speed atmospheric cookers use a heat exchanger surface, which involves spreading a film of candy over a heat exchange surface, and heat the candy to 165-170°C in a few minutes. The candy is then quickly cooled to 100-120°C and processed into a resinous mass that can incorporate flavors, colors, and other additives.

[0450] The carbohydrate filler is boiled in a sous vide cooker to 125-132°C and a vacuum is applied to boil off additional water without additional heat. When cooking is complete, the mass is semi-solid and has a resinous consistency. At this point, flavors, colors, and other additives are mixed into the mass by conventional mechanical mixing operations.

[0451] During the manufacture of conventional hard confectioneries, the optimum mixing required to uniformly incorporate flavors, colors, and other additives is determined by the time required to achieve uniform distribution of the ingredients. Typically, mixing times of 4 to 10 minutes have been found to be acceptable.

[0452] The candy mass may be properly sized and then cut into workable portions or molded into the desired shape. Depending on the shape and size of the final product desired, various molding techniques may be used. A general description of the composition and preparation of hard confections may be found in HALieberman, Pharmaceutical Dosage Forms; Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, NY.

[0453] Equipment useful in accordance with the present invention includes cooking and mixing equipment well known in the confectionery arts, and the selection of particular equipment will be apparent to one skilled in the art. In contrast, compressed tablet confections contain particular ingredients and are formed into a structure under pressure.

[0454] These confections generally contain sugar in an amount of up to about 95% by weight of the composition, as well as typical tablet excipients such as binders and lubricants, as well as flavorings, colorants, etc. As with hard confections, soft confections may also be used in the present invention. The preparation of soft confections such as nougat involves traditional methods such as the combination of two main ingredients, namely (1) high boiling syrups such as corn syrup, hydrogenated starch hydrolysates, and (2) sugarless dairy-derived compounds such as egg albumin, gelatin, soybean-derived compounds, milk proteins, and mixtures thereof, into relatively light texture frappes that are generally prepared. The frappes are generally relatively light, and may range in density from about 0.5 to about 0.7 grams / cc, for example.

[0455] Confectionery flavor ingredients are flavors that have an associated bitter or other unpleasant aftertaste. These flavor ingredients can be selected from natural and synthetic flavor liquids, such as volatile oils derived from plants, leaves, flowers, fruits, stems, and combinations thereof, synthetic flavor oils, flavoring fragrances, and oils, liquids, oleoresins, or extracts. Non-limiting representative examples of volatile oils include spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, menthol, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, nutmeg oil, allspice oil, sage oil, nutmeg extract, bitter almond oil, and cassia oil. In addition, the confectionery may also contain artificial, natural, or synthetic flavors, including fruit flavors such as vanilla, and citrus oils, including lemon, orange, grape, lime, and grapefruit, and fruit essences, including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, and the like, either individually or in admixture.

[0456] Other useful flavoring agents include aldehydes and esters such as benzaldehyde (cherry, almond), citral, i.e., alpha-citral (lemon, lime), neral, i.e., beta-citral (lemon, lime), decanal (orange, lemon), aldehyde C-8 (citrus), aldehyde C-9 (citrus), aldehyde C-12 (citrus), tolylaldehyde (cherry, almond), 2,6-dimethyl-octanal (green fruits), and 2-dodecenal (citrus, mandarin), mixtures thereof, and the like.

[0457] When sweeteners are used, the present invention contemplates including those sweeteners well known in the art, including both natural and artificial sweeteners. The sweeteners may be selected from the following non-limiting list: sugars such as sucrose, glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof, saccharin and its various salts such as sodium or calcium salts; cyclamic acid and its various salts such as sodium salts; dipeptide sweeteners such as aspartame, dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (stevioside); chloro derivatives of sucrose; dihydroflavonols; hydroxyguaiacol esters; L-aminodicarboxylic acid gem-diamines; L-aminodicarboxylic acid aminoalkenoic acid ester amides; and sugar alcohols such as sorbitol, sorbitol syrup, mannitol, xylitol. Also contemplated is the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly its potassium (acesulfame-K), sodium and calcium salts.

[0458] The confectionery may also include coloring agents. Coloring agents are suitable for food, drug, and cosmetic applications and may be selected from any of the numerous dyes known as FD&C dyes and the like. Materials acceptable for the aforementioned range of uses are preferably water-soluble. An illustrative example includes the indigoid dye known as FD&C Blue No. 2, which is the disodium salt of 5,5'-indigo tin disulfonic acid. Similarly, the dye known as FD&C Green No. 1 includes triphenylmethane dyes and is the monosodium salt of 4-[4-N-ethyl-p-sulfobenzylamino)diphenylmethylene]-[1-(N-ethyl-Np-sulfonium benzyl)-2-5-cyclohexadienimine]. A complete description of the FD&C and D&C dyes and their corresponding chemical structures may be found in Volume 5 of the Kirk-Othmer Encyclopedia of Chemical Technology.

[0459] The confectionery may also contain a volatile oil modifier, such as capsicum oleoresin. The oil modifier is present in an amount that is not detectable as an individual component in the oral cavity, yet is capable of altering the sensory perception of the volatile oil. The oil modifier is present in an amount of about 1 to about 150 ppm of the confectionery. Capsicum is available from Capsicum minimum, Capsicum frutescens, Capsicum annuum, and similar varieties. Commercially, capsicum fruits are referred to as chilies or peppers. These fruits are known to be extremely hot, pungent, and have a distinctive odor.

[0460] With regard to confectionery compressed tablet formulations, such formulations include a tablet granulation base and various additives, such as sweeteners and flavorings. The tablet granulation base used will vary depending on factors such as the type of base used, the desired friability, and other components used to make the final product. These confectioneries generally contain sugar in amounts up to 95% by weight of the composition.

[0461] The confectionery compressed tablets may further include tablet excipients such as binders or lubricants, as well as flavors, colorants, and volatile oils and volatile oil modifiers.

[0462] The variations that may be practiced with respect to these confectioneries are extensive and within the ability of one skilled in the art, particularly with regard to additional composition fillers, the use of flavoring agents, the use of coloring agents, and the like.

[0463] Extraoral care compositions Extraoral care formulations, such as denture cleaning solutions, denture cleaning tablets, denture cleaning powders, etc., may contain ingredients and / or materials selected from the following categories:

[0464] [Table 6]

[0465] In preferred embodiments, the at least one oral care ingredient is selected from the group consisting of carrier liquids, disinfectants and bleaches, cleaning agents, detergents and surfactants, foaming agents, preservatives, and flavoring agents.

[0466] In one aspect, the oral care composition of the present invention may also be included in a filament suitable for use in dental cleaning, such as a filament useful as dental floss.Preferably, the oral care composition is coated on the outside of the filament.Thus, in a preferred embodiment, the present invention relates to a filament suitable for dental cleaning, comprising an oral care composition comprising an invertase, a beta-glucosidase, and a glucoamylase.

[0467] Application of oral care compositions The oral care compositions of the present invention are suitable for use in the treatment of oral diseases where prevention or removal of oral biofilm is desired. The compositions of the present invention are particularly suitable for treating periodontal disease and dental caries.

[0468] Periodontal disease, also known as gum disease, is a series of inflammatory conditions caused by bacterial infection and subsequent buildup of biofilm on the tissues surrounding the teeth. Periodontal disease can be divided into the following classifications in terms of severity: gingivitis (including plaque-induced gingivitis), chronic periodontitis, aggressive periodontitis, periodontitis as a symptom of systemic disease, necrotizing ulcerative gingivitis / periodontitis, periodontal abscess, and combined periodontal-endodontic lesions. Periodontal disease can further be considered as either localized or systemic, depending on the extent of the affected area.

[0469] Dental caries, also known as tooth decay or cavities, is caused by organic acids such as lactic acid released by specific biofilm-forming bacteria that are resident in the oral cavity, including Streptococcus mutans and some Lactobacillus species. Dental caries can be accompanied by further complications such as inflammation of the tissues surrounding the tooth, tooth loss, and the formation of infections or abscesses. Dental caries can be classified according to the site, etiology, rate of progression, and the hard tissues affected, for example according to the GV Black classification (classes I, II, III, IV, V, and VI).

[0470] In one aspect, the present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient for use as a pharmaceutical. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0471] In one aspect, the present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient for use in treating an oral disease. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0472] In a preferred embodiment, the present invention relates to an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient for use in the treatment of periodontal disease and / or dental caries. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0473] In one aspect, the present invention relates to the use of an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient for the therapeutic or prophylactic treatment of a human subject. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0474] In one aspect, the present invention relates to a method of treating a human subject comprising administering to the human subject an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. In a preferred embodiment, the oral care composition further comprises an alpha-amylase. In a preferred embodiment, the oral care composition is administered to the oral cavity of the human subject.

[0475] In one aspect, the present invention relates to a method for preventing or removing oral biofilms, comprising contacting the biofilm with an oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient. In a preferred embodiment, the oral care composition further comprises an alpha-amylase. In one embodiment, the oral care composition is an extraoral care composition, and the biofilm is present on an object, preferably the object is a denture. In one embodiment, the object is present inside or outside the oral cavity.

[0476] Preferred Embodiments 1) An oral care composition comprising an invertase, a beta-glucosidase, a glucoamylase, and at least one oral care ingredient.

[0477] 2) The oral care composition of embodiment 1, wherein the invertase, beta-glucosidase, and glucoamylase are of microbial origin, preferably the invertase, beta-glucosidase, and glucoamylase are independently of bacterial or fungal origin.

[0478] 3) The oral care composition of any one of embodiments 1-2, wherein invertase, beta-glucosidase, and glucoamylase are each present in an effective amount, preferably from about 1 ppm to about 500 ppm.

[0479] 4) Invertase is a) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:2 or the polypeptide of SEQ ID NO:3; b) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:11 or SEQ ID NO:12; c) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:14 or SEQ ID NO:15; d) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:17 or SEQ ID NO:18; e) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:20 or SEQ ID NO:21; f) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:23 or SEQ ID NO:24; g) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:26 or SEQ ID NO:27; h) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:29 or SEQ ID NO:30; i) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:32 or SEQ ID NO:33; j) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:35 or SEQ ID NO:36; k) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 38 or SEQ ID NO: 39; l) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:41 or SEQ ID NO:42; m) a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 44 or SEQ ID NO: 45; and n) a polypeptide having at least 70%, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 47 or SEQ ID NO: 48. An oral care composition according to any one of embodiments 1 to 3.

[0480] 5) Invertase is a) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:3; b) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:11 or SEQ ID NO:12; c) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:14 or SEQ ID NO:15; d) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:17 or SEQ ID NO:18; e) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:20 or SEQ ID NO:21; f) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:23 or SEQ ID NO:24; g) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:26 or SEQ ID NO:27; h) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:29 or SEQ ID NO:30; i) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:32 or SEQ ID NO:33; j) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:35 or SEQ ID NO:36; k) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO: 38 or SEQ ID NO: 39; l) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO:41 or SEQ ID NO:42; m) a polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO: 44 or SEQ ID NO: 45; and n) A polypeptide comprising, consisting essentially of, or consisting of the mature polypeptide of SEQ ID NO: 47 or SEQ ID NO: 48. An oral care composition according to any one of the preceding embodiments.

[0481] 6) An oral care composition according to any one of embodiments 1 to 5, wherein the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:2 or SEQ ID NO:3.

[0482] 7) The oral care composition of any one of embodiments 1 to 3, wherein the beta-glucosidase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:5 or the polypeptide of SEQ ID NO:6, and preferably the beta-glucosidase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:5 or the polypeptide of SEQ ID NO:6.

[0483] 8) The oral care composition according to any one of the preceding embodiments, wherein the glucoamylase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 8 or the polypeptide of SEQ ID NO: 9, and preferably the glucoamylase comprises, essentially consists of, or consists of the mature polypeptide of SEQ ID NO: 8 or the polypeptide of SEQ ID NO: 9.

[0484] 9) The oral care composition of any one of embodiments 1-6, wherein the invertase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0485] 10) The oral care composition according to embodiment 9, wherein the invertase has similar or improved thermal stability at pH 4-8, preferably pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0486] 11) The oral care composition of any one of embodiments 9 to 10, wherein the thermal stability is measured as described in Example 3.

[0487] 12) The oral care composition of any of embodiments 1-3 or 7, wherein the beta-glucosidase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0488] 13) The oral care composition of embodiment 12, wherein the beta-glucosidase has similar or improved thermal stability at pH 4-8, preferably pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0489] 14) The oral care composition of any one of embodiments 12-13, wherein the thermal stability is measured as described in Example 3.

[0490] 15) The oral care composition of any of embodiments 1-3 or 8, wherein the glucoamylase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0491] 16) The oral care composition of embodiment 15, wherein the beta-glucosidase has similar or improved thermal stability at pH 4-8, preferably pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0492] 17) The oral care composition of any one of embodiments 15-16, wherein the thermal stability is measured as described in Example 3.

[0493] 18) The oral care composition of any one of embodiments 1-6 or 9-11, wherein the invertase prevents oral biofilm formation by at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0494] 19) The oral care composition of any one of embodiments 1-3, 7, or 12-14, wherein the beta-glucosidase prevents the formation of oral biofilms by at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0495] 20) The oral care composition of any one of embodiments 1-3, 8, or 15-17, wherein the glucoamylase prevents oral biofilm formation by at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0496] 21) An oral care composition according to any one of embodiments 18 to 20, wherein prevention of oral biofilm is measured as described in Example 4 or Example 5.

[0497] 22) The oral care composition of any one of embodiments 1 to 21, further comprising alpha-amylase.

[0498] 23) The oral care composition of embodiment 20, wherein the alpha-amylase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:50, and most preferably the alpha-amylase comprises, consists essentially of, or consists of the polypeptide of SEQ ID NO:50.

[0499] 24) The oral care composition of any of embodiments 22-23, wherein the alpha-amylase has similar or improved thermal stability in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients selected from the group consisting of benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0500] 25) The oral care composition of embodiment 24, wherein the alpha-amylase has similar or improved thermal stability at pH 4-8, preferably pH 5-7, more preferably pH 5-6, and most preferably pH 5 and / or pH 6.

[0501] 26) The oral care composition of any one of embodiments 24-25, wherein the thermal stability is measured as described in Example 3.

[0502] 27) The oral care composition of any one of embodiments 22-26, wherein the alpha-amylase removes at least 5%, e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the oral biofilm.

[0503] 28) The oral care composition of embodiment 27, wherein removal of oral biofilm is measured as described in Example 6.

[0504] 29) The oral care composition according to any one of the preceding embodiments, in the form of an oral care composition, preferably in the form of a toothpaste or toothpaste tablet, dental cream, mouthwash or mouthwash tablet, mouthrinse, lozenge, troche, chewing gum, confectionery or candy.

[0505] 30) An oral care composition according to any one of the preceding embodiments in the form of an extraoral care composition, preferably in the form of a denture cleaning solution, a denture cleaning tablet or a denture cleaning powder.

[0506] 31) An oral care composition according to any one of embodiments 1 to 30 for use as a medicament.

[0507] 32) An oral care composition according to any of embodiments 1 to 30 for use in the treatment of oral diseases, preferably for use in the treatment of periodontal disease and / or dental caries.

[0508] 33) Use of an oral care composition according to any one of embodiments 1 to 30 for the therapeutic or prophylactic treatment of a human subject.

[0509] 34) A method of treating a human subject, comprising administering to the human subject an oral care composition according to any one of embodiments 1-30, preferably administering the oral care composition to the oral cavity of the human subject.

[0510] 35) A method for preventing or removing an oral biofilm, comprising contacting the oral biofilm with an oral care composition according to any one of embodiments 1 to 30.

[0511] 36) The method of embodiment 35, wherein the oral biofilm is present on an object, preferably a denture.

[0512] 37) The method of embodiment 34, wherein the denture is present inside or outside the oral cavity.

[0513] 38) A method for reducing the risk of formation of an oral biofilm, comprising contacting the oral biofilm with an oral care composition according to any one of embodiments 1 to 30.

[0514] 39) a) an oral care composition according to any one of embodiments 1 to 30; b) A kit of parts, including instructions for use. EXAMPLES

[0515] Materials and Methods Media and Solutions YP+2% maltose medium consisted of 1% yeast extract, 2% bacto peptone and 2% maltose.

[0516] YP+2% glucose medium consisted of 1% yeast extract, 2% bacto peptone and 2% dextrose.

[0517] DAP4C-1 medium consisted of 1.1% MgSO4 / 7H2O, 0.1% KH2PO4, 0.2% C6H8O7 / H2O (citric acid), 2% dextrose, 1% maltose, 0.52% K3PO4 / H2O, 0.05% yeast extract, 0.05% trace metals, 0.1% DowFax 63N10, and 0.05% CaCO3.

[0518] PDA plates were composed of 39 g potato dextrose agar, 50 mL glycerol, 20 g agar, and deionized water up to 1 L. The medium was sterilized by autoclaving at 15 psi for 15 min (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0519] LB agar plates were composed of 37 g LB agar (Sigma Aldrich L3027), 5 g soluble starch 0.5% (Merck 101252), 10 mL K2PO4 1M, 20 mL 20% glucose solution and deionized water up to 1 L.

[0520] LB medium was composed of 25 g LB broth (Fluka L3152) and deionized water up to 1 L. The medium was sterilized by autoclaving at 15 psi for 15 min (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0521] COVE sucrose plates for selection of Aspergillus transformants consisted of 342 g sucrose, 20 g agar powder, 20 mL COVE salts solution, and deionized water up to 1 L. The medium was sterilized by autoclaving at 15 psi for 15 min (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The medium was cooled to 60°C and 10 mM NaNO3 was added.

[0522] The COVE salt solution was composed of 26 g MgSO4 / 7H2O, 26 g KCL, 76 g KH2PO4, 50 ml COVE trace metals solution, and deionized water up to 1 L.

[0523] The COVE trace metals solution was composed of 0.04 g Na2B4O7 / 10H2O, 0.4 g CuSO4 / 5H2O, 1.2 g FeSO4 / 7H2O, 0.7 g MnSO4 / H2O, 0.8 g Na2MoO4 / 2H2O, 10 g ZnSO4 / 7H2O, and deionized water up to 1 L.

[0524] Example 1a: Cloning and expression of invertase, beta-glucosidase, and glucoamylase SEQ ID NOs:1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, and 46 were identified from their corresponding donor organisms and cloned according to the scheme described in US Patent Application Publication No. 2019 / 0225988 using three overlapping fragments for integration into the niiA / niiD locus of strain ColS1300 using the DSMS system (described in US Patent Application Publication No. 2019 / 0225988).

[0525] ColS1300 protoplasts were prepared and transformed according to the method described in WO 2012 / 003379 using approximately 200-500 ng of each overlapping DNA fragment. Transformants were plated on COVE sucrose 10 mM NaNO3 plates and those capable of producing each mature polypeptide (SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48, respectively) were selected by SDS-PAGE electrophoresis.

[0526] Fermentation was carried out in 250 mL baffled shake flasks filled with 100 mL of fermentation medium and incubated with spores of the selected strains expressing each mature polypeptide. Fermentation was carried out at 30° C. for 4 days under agitation at 150 rpm. Invertases (SEQ ID NO: 3, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48) were expressed using DAP4C-1 medium, beta-glucosidase (SEQ ID NO: 6) was expressed using YP2% glucose medium, and glucoamylase (SEQ ID NO: 9) was expressed using YP2% maltose medium. Culture broth was harvested by filtration using a 0.2 μm filter device.

[0527] Invertase and beta-glucosidase were purified by hydrophobic interaction chromatography. The integrity of the mature polypeptides was confirmed by SDS-PAGE electrophoresis, and the concentration of the purified enzymes was measured by absorbance at 280 nm. Glucoamylase was not purified, but was used as a negative control in the ColS1300 host background fermented under similar conditions to exclude unwanted host background activity.

[0528] Example 1b: Cloning and expression of alpha-amylase DNA encoding the mature polypeptide of the alpha-amylase gene from Bacillus amyloliquefaciens (SEQ ID NO: 49) was ordered from Twist Bioscience. This synthetic DNA fragment was directionally assembled into the Bacillus expression vector described in WO 12 / 025577 by standard Golden Gate cloning methods using BsaI and T4 DNA ligase enzymes. Briefly, the DNA encoding the mature peptide of the alpha-amylase gene was cloned in frame into the Bacillus licheniformis secretion signal containing the sequence MKQQKRLYARLLTLLFALIFLLPHSAAAA (SEQ ID NO: 51), replacing the native secretion signal of the alpha-amylase gene. An affinity tag containing the sequence HHHHHH (SEQ ID NO: 52) was added downstream of this gene to allow for purification procedures.

[0529] The alpha-amylase fusion gene was integrated into the Bacillus subtilis host cell genome by homologous recombination during transformation. Transformants were selected on LB medium agar supplemented with 6 micrograms of chloramphenicol per ml. One recombinant Bacillus subtilis clone containing the alpha-amylase expression construct was selected and cultured in a 500 ml baffled Erlenmeyer flask containing 100 ml of yeast extract-based medium on a rotating shaker platform. After 3 days of incubation at 30°C, the enzyme-containing supernatant was harvested by centrifugation and the enzyme was purified by His-tag purification.

[0530] The mature alpha-amylase (SEQ ID NO: 50) containing a C-terminal His tag (SEQ ID NO: 52) was incubated with Ni as the metal ion. 2+The enzyme was purified on a 5 mL HisTrap Excel column (GE Healthcare Life Sciences) by immobilized metal chromatography (IMAC) using 100 mM NaCl at pH 7. Purification was performed at pH 7 and bound protein was eluted with imidazole. After buffer exchange into 50 mM HEPES, 100 mM NaCl, pH 7.0, the purity of the purified enzyme was confirmed by SDS-PAGE and the enzyme concentration was determined by measuring the absorbance at 280 nm.

[0531] Example 2: Activity Assay Invertase activity assay Invertase activity can be measured by incubating 50 ppm invertase (0.05 mg / mL in 25 mM universal buffer containing acetate, MES, HEPES, and glycine, pH 6) with sucrose substrate (1% weight / volume) at 37° C. for 60 min at 1400 rpm, with a sample without added enzyme used as control. After incubation, samples are centrifuged at 16100×G for 5 min at room temperature and the supernatant is centrifuged on a Dionex PD10 column (ThermoFisher Scientific) with the following gradient, as well as fructose and glucose (Sigma) as standard references: (商標) - Analyzed by liquid chromatography using an IC300 system.

[0532] [Table 7]

[0533] For SEQ ID NO:3, the table below shows the release of fructose after incubation measured by liquid chromatography as area under the curve in nanocoulombs per minute (nC*min), where the area under the curve is proportional to the release of fructose. Liquid chromatography analysis revealed that invertase can break down sucrose into its component monosaccharides (fructose and sucrose), indicating invertase activity.

[0534] [Table 8]

[0535] Beta-glucosidase activity assay Beta-glucosidase activity was measured by mixing enzyme solution (20 μL of 0.1–10 μg / mL enzyme in 0.01% Triton® X-100), assay buffer (50 μL, 100 mM acetic acid, 1 mM CaCl2, 150 mM KCl, 0.01% Triton® X-100, adjusted to pH 5 with NaOH), and substrate solution (50 μL, 2 mM 4-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich) in 0.01% Triton® X-100). N-7006) (prepared from a substrate stock solution of 100 mM 4-nitrophenyl-β-D-glucopyranoside dissolved in MilliQ water) can be measured by a colorimetric assay by incubating at 37°C for 20 minutes, adding 50 μL of stop solution (1 M Na2CO3) and measuring the absorbance at 405 nm. A sample without enzyme solution is used as a control.

[0536] Glucoamylase activity assay Glucoamylase activity can be measured by colorimetric assay by incubating enzyme solution (20 μL of 3 μg / mL in 50 mM NaOAc, 0.02% Triton® X-100, pH 4.3) and substrate solution (100 μL of 30 mg / mL maltodextrin (Sigma, De16.5-19.5) in 50 mM NaOAc, pH 4.3) at 37° C. and 800 rpm for 60 min in a 96-well plate (the 96-well plate is pre-incubated with the substrate solution at 37° C. for 3 min before adding the enzyme solution). The reaction is stopped by adding stop solution (100 μL of 1 M Tris-HCl, pH 8.2). After transferring 10 μL of the reacted sample to another 96-well plate, 200 μL of Glucose CII test kit solution (Wako, 432-90913) is added to each well. The mixture is left at room temperature for 15 min and the absorbance is measured at 505 nm. 10 μL of glucose standards (0, 0.25, 0.50, 0.75, 1.0, 1.5, and 2.0 mg / mL glucose dissolved in HO) are used as controls.

[0537] Alpha-amylase activity assay I Alpha-amylase activity can be measured, for example, using a reducing sugar assay with corn starch substrate. The number of reducing ends formed by hydrolysis of the α-1,4-glycosidic bonds found in starch by alpha-amylase is measured by reaction with p-hydroxybenzoic acid hydrazide (PHBAH). After reaction with PHBAH, the number of reducing ends can be measured by absorbance at 405 nm, and the concentration of reducing ends is proportional to the alpha-amylase activity in the sample.

[0538] Corn starch substrate (3 mg / ml) is solubilized by cooking in MilliQ water for 5 min and cooled before assay. 50 μl of activity buffer is mixed with 50 μl of corn starch substrate and 50 μl of enzyme, and incubated for 5 min after mixing. The reaction is stopped by adding 75 μl of stop solution (Ka-Na-tartrate 50 g / L; NaOH 20 g / L; PHBAH 15 mg / mL) and then incubated at 95 °C for 10 min and absorbance at 405 nm is measured. Alpha-amylase samples to be analyzed should be diluted to give an absorbance at 405 nm of 0-2.2, which is within the linear range of the activity assay.

[0539] Alpha-amylase activity assay II Alpha-amylase activity can be measured by a method using Phadevas substrate (e.g., Magle Life Sciences, Lund, Sweden). Phadevas tablets contain linked starch polymers in the form of water-insoluble spherical microspheres. A blue dye is covalently attached to these microspheres. The linked starch polymers in the microspheres are degraded at a rate proportional to the alpha-amylase activity. When alpha-amylase degrades the starch polymers, the released blue dye dissolves in water and the concentration of the dye can be measured by measuring the absorbance at 620 nm. The concentration of the dye is proportional to the alpha-amylase activity.

[0540] The alpha-amylase sample to be analyzed is diluted in activity buffer of the desired pH value. One substrate tablet is suspended in 5 mL of activity buffer under magnetic stirring. 30 μl of diluted alpha-amylase sample is added to 150 μl of substrate in the microtiter plate, followed by mixing and incubation at 37 °C for 15 min. The reaction is stopped by adding 30 μl of 1 M NaOH, followed by mixing. The microtiter plate is centrifuged at 4000 × g for 5 min and 100 μl of the supernatant is transferred to a new microtiter plate. The absorbance is measured at 620 nm. The alpha-amylase sample to be analyzed should be diluted to obtain an absorbance at 620 nm between 0 and 2.2, which is within the linear range of the activity assay.

[0541] Example 3: Measurement of thermal stability Preparation of oral care formulations for measuring thermal stability The thermal stability, i.e., the midpoint of thermal unfolding transition (Tm), of selected invertases (SEQ ID NO:3, 12, 15, and 36), beta-glucosidase (SEQ ID NO:6), glucoamylase (SEQ ID NO:9), and alpha-amylase (SEQ ID NO:50) were measured in the presence of oral care components in the concentration ranges commonly used in oral care product formulations and selected oral care commercial products. The Tm parameter was used to evaluate thermal stability since it is the temperature at which the populations of folded and unfolded protein molecules are equal and is a widely accepted parameter to use when evaluating thermal stability. High purity and biotechnology grade reagents were obtained from various sources and stock solutions were freshly prepared using MilliQ water. These formulation chemicals and their stocks as well as the final concentrations used for Tm measurements are listed in Table 1.

[0542] [Table 9]

[0543] Purified preparations of enzyme samples were diluted to a stock concentration of 2 mg / ml and then further diluted 10-fold (corresponding to a final protein concentration of 0.2 mg / ml) in an oral care formulation consisting of the individual compounding chemicals, phosphate citrate buffer (McIlvaine buffer, see below) and MilliQ water. All dilutions were made using a robotic arm in a 384-well low volume deep well plate (Greiner Bio-One International, item no. 784201) in a final volume of 70 μl, which was used for the thermostability measurements. Tm measurements for each enzyme were performed using McIlvaine buffers at pH 5.0 and pH 6.0, approximately in the physiological pH range of the oral cavity. 100 ml of McIlvaine buffer at pH 5.0 was prepared by mixing 51.50 ml of 0.2 M Na2HPO4 and 48.50 ml of 0.1 M citric acid, whereas 100 ml of McIlvaine buffer at pH 6 was prepared by mixing 63.15 ml of 0.2 M Na2HPO4 and 36.85 ml of 0.1 M citric acid.

[0544] Measurement of Tm Thermal stability measurements were performed using a capillary-based nano-differential scanning fluorescence instrument (nanoDSF); Prometheus NT.Plex (NanoTemper Technologies GmbH, Munchen, Germany). Standard nanoDSF grade capillary tips were used (NanoTemper Technologies, catalog number: PR-AC002). Enzyme samples were loaded into the capillary by capillary action (triplicates for each sample). Emission intensities at 330 nm and 350 nm were optimized by changing the LED power of the instrument to ensure sufficient signal. Fluorescence signals at 330 nm and 350 nm were continuously monitored as a function of temperature (the heating rate used for thermal unfolding was 20 °C to 95 °C at 3.3 °C per minute). Data were analyzed using PR.StabilityAnalysis 1.1.0.11077 software provided by the manufacturer. The analysis was model-independent and simply took the peak maximum of the first derivative corresponding to the midpoint of the approximate thermal unfolding transition and defined as the Tm (see FIG. 1).

[0545] Reproducibility of thermal stability data FIG. 1 shows an example of thermal stability data generated using the nanoDSF instrument. Panel A is an example of triplicate acquired data for SEQ ID NO:3 (ratio of fluorescence emission at 350 nm to 330 nm) as a function of temperature. Panel B shows the first derivative of the raw data in Panel A. The peak maximum in the plot of the first derivative corresponds to the midpoint of the thermal unfolding transition, referred to as the Tm. In this example, the Tm corresponds to 61.9° C. at pH 6.0 and is highly reproducible within three replicates.

[0546] The data shown in Figure 1 is an example of the type of data generated for various enzymes in different formulations using nanoDSF. In all cases, the data showed clear unfolding transitions and clearly defined peaks in the first derivative, and were highly reproducible.

[0547] Thermostability of invertase, beta-glucosidase, glucoamylase, and alpha-amylase in the presence of oral care ingredients Tables 2 and 3 show the average thermal stability of invertase (SEQ ID NO:3), beta-glucosidase (SEQ ID NO:6), and glucoamylase (SEQ ID NO:9) derived from triplicate measurements at pH 5.0 and pH 6.0, respectively, in the presence of a range of commonly used oral care ingredients.

[0548] Tables 4 and 5 show the average thermal stability of three invertases (SEQ ID NOs: 36, 12 and 15) in the presence of a range of commonly used oral care ingredients derived from triplicate measurements at pH 5.0 and pH 6.0, respectively.

[0549] Table 6 shows the average thermal stability of alpha-amylase (SEQ ID NO:50) in the presence of a range of commonly used oral care ingredients derived from triplicate measurements at pH 5.0 and pH 6.0.

[0550] These data demonstrate that these ingredients individually do not adversely affect the thermostability of invertase, beta-glucosidase, glucoamylase, and alpha-amylase, and that these enzymes have similar or even improved stability in the presence of these ingredients under simulated oral conditions, making the enzymes suitable for oral care formulations and applications in the oral cavity.

[0551] [Table 10]

[0552] [Table 11]

[0553] [Table 12]

[0554] [Table 13]

[0555] [Table 14]

[0556] Example 4: Human saliva biofilm prevention assay with purified enzymes Human saliva biofilm prevention assays were performed using the method described in WO 2020 / 099490 with minor modifications. Briefly, biofilms were grown in 96-well plates in the presence of either 50 mM HEPES buffer containing 100 mM NaCl, pH 7 as a control, or enzyme solutions containing invertase (SEQ ID NO: 3, 10 ppm and 60 ppm), beta-glucosidase (SEQ ID NO: 6, 10 ppm and 60 ppm), glucoamylase (SEQ ID NO: 9, 10 ppm and 60 ppm), alpha-amylase (SEQ ID NO: 50, 60 ppm), or invertase, beta-glucosidase, glucoamylase, and alpha-amylase (SEQ ID NO: 3, 6, 9, and 50, 60 ppm of each enzyme) in 50 mM HEPES buffer containing 100 mM NaCl, pH 7.

[0557] Plates were incubated in a Thermo Scientific (商標) Rectangular AnaeroBox (商標) The vessels (ThermoScientific AnaeroGen 2.5L, #AN0025A) were incubated without agitation for 24 hours at 37° C. Enzyme and control samples were evaluated in eight replicates.

[0558] After incubation, planktonic bacteria were removed by gentle washing twice with 100 μl of 0.9% NaCl, and biofilms were stained with 0.095% crystal violet solution for 15 min at room temperature. Plates were rinsed twice with 100 μL of 0.9% NaCl, and the attached dye was dissolved in 96% ethanol and 0.1% acetic acid solution. Absorbance was measured at 600 nm using a microplate reader (SpectraMax M3, Molecular Devices).

[0559] For data processing, the absorbance was considered proportional to the extent of biofilm remaining after treatment with enzyme or control. Results were expressed as a percentage of biofilm prevention and calculated as follows: 100-((A600nm enzyme treated sample) / (A600nm buffer control treated sample) x 100) where A600nm represents the average of eight absorbance measurements at 600nm for either enzyme or control treated samples. The results, listed in Table 7, show that all three enzymes exhibited anti-biofilm effects at 10 ppm and 60 ppm, with invertase having the highest activity at both concentrations.

[0560] [Table 15]

[0561] Example 5: Human saliva biofilm prevention assay with supernatants Human saliva biofilm prevention assays were performed using the method described in WO 2020 / 099490 with minor modifications. Briefly, biofilms were grown in 96-well plates in the presence of either 50 mM HEPES buffer containing 100 mM NaCl, pH 7 (as a control) or enzyme solutions containing invertases (SEQ ID NOs: 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48). Enzymes were evaluated using 10 μl of supernatant samples from recombinant Aspergillus oryzeae strains expressing the respective enzymes.

[0562] Plates were incubated in a Thermo Scientific (商標) Rectangular AnaeroBox (商標) The vessels (ThermoScientific AnaeroGen 2.5L, #AN0025A) were incubated without agitation for 24 hours at 37° C. Enzyme and control samples were evaluated in eight replicates.

[0563] After incubation, planktonic bacteria were removed by gentle washing twice with 100 μl of 0.9% NaCl, and biofilms were stained with 0.095% crystal violet solution for 15 min at room temperature. Plates were rinsed twice with 100 μL of 0.9% NaCl, and the attached dye was dissolved in 96% ethanol and 0.1% acetic acid solution. Absorbance was measured at 600 nm using a microplate reader (SpectraMax M3, Molecular Devices).

[0564] For data processing, the absorbance was considered proportional to the extent of biofilm remaining after treatment with enzyme or control. Results were expressed as a percentage of biofilm prevention and calculated as follows: 100-((A600nm enzyme treated sample) / (A600nm buffer control treated sample) x 100) where A600nm represents the average of eight absorbance measurements at 600nm for either enzyme or control treated samples. The results, listed in Table 8, show that all evaluated invertases exhibit strong anti-biofilm effects.

[0565] [Table 16]

[0566] Example 6: Human saliva biofilm removal assay Biofilm removal assays were performed using biofilms grown from human saliva in 96-well microtiter plates according to the method described in WO 2020 / 099490 with minor modifications. Briefly, biofilm samples grown for 24 hours were treated with either 200 μL of citrate phosphate buffer (McIlvaine buffer, pH 6, prepared by mixing 12.63 ml of 0.2 M Na2HPO4 and 7.37 ml of 0.1 M citric acid) as a control, an enzyme solution containing invertase, beta-glucosidase, and glucoamylase (SEQ ID NOs: 3, 6, and 9, 60 ppm of each enzyme) in McIlvaine buffer, pH 6, or an enzyme solution containing invertase, beta-glucosidase, glucoamylase, and alpha-amylase (SEQ ID NOs: 3, 6, 9, and 50, 60 ppm of each enzyme) in McIlvaine buffer, pH 6, for 30 minutes at 37° C. with agitation at 50 rpm. Enzyme samples and control samples were run in eight replicates.

[0567] After enzyme treatment, the samples were rinsed twice with 200 μL of 0.9% sodium chloride and stained with 0.095% crystal violet solution for 15 min at room temperature. The samples were rinsed three times with 200 μL of 0.9% sodium chloride, the dye was extracted with 33% acetic acid solution, and the absorbance was measured at 600 nm.

[0568] For data processing, the absorbance was considered proportional to the extent of biofilm remaining after treatment with enzyme or control. Results were expressed as the percentage of biofilm removal and calculated as follows: 100-((A600nm enzyme treated sample) / (A600nm buffer control treated sample) x 100) where A600nm represents the average of eight absorbance measurements at 600nm using a SpectraMax M3, Molecular Devices, of samples treated with enzyme or control. The results are listed in Table 9 and show that the combination of invertase, beta-glucosidase, glucoamylase, and alpha-amylase provides a significant improvement in biofilm removal compared to the combination of invertase, beta-glucosidase, and glucoamylase.

[0569] [Table 17]

Claims

1. An oral care composition comprising invertase, beta-glucosidase, glucoamylase, and at least one oral care component.

2. The oral care composition according to claim 1, wherein the invertase, beta-glucosidase, and glucoamylase are of microbial origin, and preferably, the invertase, beta-glucosidase, and glucoamylase are independently of bacterial or fungal origin.

3. The oral care composition according to claim 1 or 2, wherein the invertase, beta-glucosidase, and glucoamylase are each present in an effective amount, preferably in an amount of about 1 ppm to about 500 ppm.

4. The oral care composition according to claim 1, wherein the invertase has at least 70%, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3, and preferably the invertase contains, is essentially made from, or consists of the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO:

3.

5. The oral care composition according to claim 1, wherein the beta-glucosidase has at least 70%, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 5 or the polypeptide of SEQ ID NO: 6, and preferably the beta-glucosidase comprises, is essentially derived from, or consists of the mature polypeptide of SEQ ID NO: 5 or the polypeptide of SEQ ID NO:

6.

6. The oral care composition according to claim 1, wherein the glucoamylase has at least 70%, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 8 or the polypeptide of SEQ ID NO: 9, and preferably the glucoamylase contains, is essentially derived from, or consists of the mature polypeptide of SEQ ID NO: 8 or the polypeptide of SEQ ID NO:

9.

7. The oral care composition according to claim 1 or 4, wherein the invertase has the same or improved thermal stability in the presence of at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care components selected from the group consisting of benzoates (preferably sodium benzoate), EDTA, ethanol, fluorides (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphates (preferably sodium phosphate), SDS, sorbates (preferably potassium sorbate), and sorbitol.

8. The oral care composition according to claim 1 or 5, wherein the beta-glucosidase has the same or improved thermal stability in the presence of at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care components selected from the group consisting of benzoates (preferably sodium benzoate), EDTA, ethanol, fluorides (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphates (preferably sodium phosphate), SDS, sorbates (preferably potassium sorbate), and sorbitol.

9. The oral care composition according to claim 1 or 6, wherein the glucoamylase has the same or improved thermal stability in the presence of at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care components selected from the group consisting of benzoates (preferably sodium benzoate), EDTA, ethanol, fluorides (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphates (preferably sodium phosphate), SDS, sorbates (preferably potassium sorbate), and sorbitol.

10. An oral care composition according to claim 1, further comprising alpha-amylase, wherein the alpha-amylase has at least 70%, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO: 50, and most preferably the alpha-amylase comprises, essentially comprises, or consists of the polypeptide of SEQ ID NO:

50.

11. An oral care composition according to claim 1 or 10, for use as a pharmaceutical.

12. An oral care composition according to any one of claims 1 or 10, for use in the treatment of oral diseases, preferably for use in the treatment of periodontal disease and / or dental caries.

13. A method for preventing or removing an oral biofilm, comprising contacting the oral biofilm with the oral care composition described in claim 1 or 10.

14. A method for reducing the risk of oral biofilm formation, comprising contacting the oral biofilm with the oral care composition described in claim 1 or 10.

15. It is a kit of parts, a) The oral care composition according to any one of claims 1 or 10, b) Kit of parts, including instructions for use.

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