A dental care composition for animals

Abstract

The present invention relates to a composition comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases, use of the composition as medicaments, use of the composition in treatment of oral disease, methods of treatment comprising administering the composition to animals, methods of preventing or removing oral biofilm comprising contacting an oral biofilm with the composition, methods for reducing the risk of oral biofilm formation, methods for removing the oral biofilm formed, and kits of parts comprising the composition.

Description

[0001] A DENTAL CARE COMPOSITION FOR ANIMALS

[0002] Reference to sequence listing

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a composition comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases; use of the composition as medicaments of animals; use of the composition in treatment of dental care of animals; methods of treatment comprising administering the composition to animals; methods of preventing or removing oral biofilm comprising contacting an oral biofilm with the composition; methods for reducing the risk of oral biofilm formation; and kits of parts comprising the composition.

[0006] BACKGROUND OF THE INVENTION

[0007] A lot of animal owner focus on dental care of their animals specially companion animals like dogs and cats to avoid dental disease, which undesirably causes decayed teeth, gum bleeding, and even teeth losing. If bacteria living on the teeth enter the animal’s body, further, the animals may have bad influences on their heart, lung, kidney and so on, which undesirably causes other diseases. It is estimated that over 80 percent of all dogs have severe oral problems. When the veterinarian meets the dog for vaccination it also often ends up in an investigation of the general health of the dog. The dental health is the most frequent cause for immediate action. So as to solve such problems, recently, dental care products only for pet animals, especially, pet dogs are used to prevent dental disease in animals. However, along with prevention, there is a need of dental care products which also removes any bacteria biofilms on the teeth of animals to avoid dental diseases, or avoid severity in dental diseases.

[0008] Thus, there is a need of an improved dental care product for animals to avoid and reduce dental disease in animals.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides compositions comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases useful for prevention of dental biofilm formation and / or removal of dental biofilm formation. In a first aspect, the invention relates to a composition for dental care of an animal comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases.

[0011] In a second aspect, the invention relates to a dental care composition comprising combination of enzymes and at least one dental care ingredient. The combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases.

[0012] In a third aspect, the invention relates to a composition according to the first aspect or second aspect for use as a medicament.

[0013] In a fourth aspect, the invention relates to a method for preventing or removing oral biofilm in an animal, the method comprising contacting the oral biofilm with a composition according to the first aspect or second aspect.

[0014] In a fifth aspect, the invention relates to a method for reducing the risk of oral biofilm formation in an animal, the method comprising contacting the oral biofilm with a composition according to the first aspect or second aspect.

[0015] In a sixth aspect, the invention relates to a method of preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal, the method comprising contacting a composition according to the first or second aspect with the teeth of the animal. A related aspect is directed to the use of a composition or a dental care composition of the invention for preventing or removing of dental plaque or reducing the formation of dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal.

[0016] A further aspect of the invention is directed to a dental care composition comprising a combination of enzymes and at least one dental care ingredient, the combination of enzymes comprising: a. one or more polypeptides having alpha amylase activity, wherein the polypeptide has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2, b. one or more polypeptides having glucanase activity selected from consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6; iii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8; iv. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10; and v. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12, c. one or more polypeptides having xylanase activity, wherein the polypeptide has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16; and d. one or more polypeptides having lipase activity, wherein the lipase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ I D NO: 18.

[0017] A further aspect of the invention is directed to a composition of the invention for use in the prevention of biofilm formation on the dentition of an animal or the removal of biofilm on the dentition of an animal. A related aspect of the invention is directed to a composition of the invention for use in the prevention of dental plaque formation in an animal or for use in the removal of dental plaque in an of animal. An aspect of the invention is directed to a method of removing oral biofilm formed in an animal, the method comprising contacting the oral biofilm with a composition or a dental care composition of the invention. An aspect of the invention is directed to a method of removing oral biofilm formed in an animal, the method comprising contacting the oral biofilm with a composition or a dental care composition of the invention.

[0018] SEQUENCE OVERVIEW

[0019] SEQ ID NO:1 is an amino acid sequence of a GH13_5 alpha amylase obtained from Bacillus amyloliquefaciens (including the signal peptide).

[0020] SEQ ID NO:2 is an amino acid sequence of a GH13_5 alpha amylase obtained from Bacillus amyloliquefaciens (mature polypeptide).

[0021] SEQ ID NO:3 is an amino acid sequence of a GH5 beta-glucanase obtained from Trichoderma reesei (including the signal peptide).

[0022] SEQ ID NO:4 is an amino acid sequence of a GH5 beta-glucanase obtained from Trichoderma reesei (mature polypeptide).

[0023] SEQ ID NO:5 is an amino acid sequence of a GH6 glucanase obtained from Trichoderma reesei (including the signal peptide).

[0024] SEQ ID NO:6 is an amino acid sequence of a GH6 glucanase obtained from Trichoderma reesei obtained from (mature polypeptide).

[0025] SEQ ID NO:7 is an amino acid sequence of a GH7 endo-glucanase obtained from Trichoderma reesei (including the signal peptide).

[0026] SEQ ID NO:8 is an amino acid sequence of a GH7 endo-glucanase obtained from Trichoderma reesei (mature polypeptide).

[0027] SEQ ID NO:9 is an amino acid sequence of a GH7 exo-glucanase obtained from Trichoderma reesei (including the signal peptide).

[0028] SEQ ID NO: 10 is an amino acid sequence of a GH7 exo-glucanase obtained from Trichoderma reesei (mature polypeptide).

[0029] SEQ ID NO:11 is an amino acid sequence of a GH7 beta-glucanase obtained from Humicola insolens (including the signal peptide).

[0030] SEQ ID NO: 12 is an amino acid sequence of a GH7 beta-glucanase obtained from Humicola insolens (mature polypeptide). SEQ ID N0:13 is an amino acid sequence of a GH16 beta-glucanase obtained from Bacillus amyloliquefaciens (including the signal peptide).

[0031] SEQ ID NO:14 is an amino acid sequence of a GH16 beta-glucanase obtained from Bacillus amyloliquefaciens (mature polypeptide).

[0032] SEQ ID NO: 15 is an amino acid sequence of a GH10 xylanase obtained from Humicola insolens (including the signal peptide).

[0033] SEQ ID NO:16 is an amino acid sequence of a GH10 xylanase obtained from Humicola insolens (mature polypeptide).

[0034] SEQ ID NO: 17 is an amino acid sequence of a GH10 xylanase obtained from Aspergillus aculeatus (including the signal peptide).

[0035] SEQ ID NO: 18 is an amino acid sequence of a GH10 xylanase obtained from Aspergillus aculeatus (mature polypeptide).

[0036] SEQ ID NO: 19 is an amino acid sequence of a lipase obtained from Thermomyces lanuginosus (including the signal peptide).

[0037] SEQ ID NQ:20 is an amino acid sequence of a lipase obtained from Thermomyces lanuginosus (mature polypeptide).

[0038] SEQ ID NO:21 is an amino acid sequence of a phosphodiesterase obtained from Metabacillus indicus (including the signal peptide).

[0039] SEQ ID NO:22 is an amino acid sequence of a phosphodiesterase obtained from Metabacillus indicus (mature polypeptide).

[0040] SEQ ID NO:23 is a nucleic acid sequence of a GH13_5 alpha amylase isolated from Bacillus amyloliquefaciens.

[0041] SEQ ID NO:24 is a nucleic acid sequence of a GH5 beta-glucanase obtained from Trichoderma reesei.

[0042] SEQ ID NO:25 is a nucleic acid sequence of a GH6 glucanase obtained from Trichoderma reesei.

[0043] SEQ ID NO:26 is a nucleic acid sequence of a GH7 endo-glucanase obtained from Trichoderma reesei.

[0044] SEQ ID NO:27 is a nucleic acid sequence of a GH7 exo-glucanase obtained from Trichoderma reesei. SEQ ID NO:28 is a nucleic acid sequence of a GH16 beta-glucanase obtained from Bacillus amyloliquefaciens.

[0045] SEQ ID NO:29 is a nucleic acid sequence of a GH10 xylanase obtained from Humicola insolens.

[0046] SEQ ID NQ:30 is a nucleic acid sequence of a GH10 xylanase obtained from Aspergillus aculeatus.

[0047] SEQ ID NO:31 is a nucleic acid sequence of a lipase obtained from Thermomyces lanuginosus.

[0048] SEQ ID NO:32 is a nucleic acid sequence of a phosphodiesterase obtained from Metabacillus indicus.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention relates to compositions for dental care of animals comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases. As illustrated in the Examples of the present application, the present inventors have identified, alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases are effective in preventing formation of oral biofilm and / or reducing the risk of oral biofilm formation in animals. The present inventors have further identified that the combination of alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases is able to potently remove biofilm already present in the oral cavity of the animals.

[0051] Thus, in a first aspect, the present invention relates to dental care compositions comprising a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases; and at least one dental care ingredient.

[0052] DEFINITIONS

[0053] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0054] Unless defined otherwise 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. Alpha-amylase: The term “alpha-amylase” means a polypeptide alpha-amylase activity that catalyzes the endohydrolysis of (1^4)-a-D-glucosidic linkages in polysaccharides containing three or more (1^4)-a-linked D-glucose units. The systematic name for alpha-amylase is 4-a-D- glucan glucanohydrolase (EC 3.2.1.1). The terms “alpha-amylase” and “a-amylase” and the expression “a polypeptide having alpha-amylase activity” are used interchangeably throughout this application. For purposes of the present invention, alpha-amylase activity may be determined according to Alpha-amylase activity assay I or Alpha-amylase activity assay II described in Example 2 below.

[0055] Glucanase: The term "glucanase" refers to any enzyme that is capable of hydrolyzing a glycoside bond.

[0056] Xylanase: The term "xylanase" refers a xylanase enzyme defined according to that known in the art as endo-1 ,4-0-xylanase, or 4-0-D-xylan xylanohydrolase, known to catalyze the endohydrolysis of (1 - >4)-0-D-xylosidic linkages in xylene.

[0057] Lipase: The term “lipase" refers to subset of Carboxylic Ester Hydrolases which catalyze the hydrolysis of ester bounds in lipids including triglycerides, phospholipids and galactolipids.

[0058] Phosphodiesterase: The term "phosphodiesterase (PDE)" refers to a type of phosphatase which breaks down bonds of phosphodiesters such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and the like, and is an enzyme that catalyzes a reaction for generating a phosphomonoester and an alcohol through hydrolysis and is classified into a total of 12 subtypes. The term "Phosphodiesterase," used interchangeably with the term "PDE," refers to a member of the cyclic nucleotide phosphodiesterases families.

[0059] Animal: The term "animal" means organisms that form the biological kingdom Animalia, for example, domesticated animals such as dogs, cats, cattle, horses, pigs, etc. and undomesticated animals such as deer, elk, alpacas, lamas, and moose, carnivores, such as bears, wolves, lions, and tigers, rodents, such as rats, mice, and beaver, bats, marsupials, such as kangaroos and opossums and cetaceans, such as whales, dolphins, and porpoises.

[0060] Biofilm: Biofilm formation in the present context is a process whereby microorganisms attach to and grow on a surface within the oral cavity and produce extracellular polymers that facilitate attachment and matrix formation. Biofilm formation leads to plaque formation on teeth. Biofilms within the oral cavity differ in their microbial members, with variations depending on the species of the animal.

[0061] Biofilm prevention: The term “biofilm prevention” means the ability of a polypeptide to decrease the amount of a biofilm grown under defined conditions. For purposes of the present invention, biofilm prevention may be determined according to Example 1 herein. Fragment: The term “fragment” means a polypeptide having one or more amino acids absent from the amino and / or carboxyl terminus of the mature polypeptide, wherein the fragment has the enzyme activity of mature polypeptide. A fragment of an glucanase has glucanase activity, a fragment of a xylanase has xylanase activity, a fragment of a lipase has lipase activity, a fragment of a phosphodiesterase has phosphodiesterase activity and a fragment of an alphaamylase has alpha-amylase activity.

[0062] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide).

[0063] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0064] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) 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), preferably version 6.6.0 or later. 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 -no brief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0065] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0066] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” 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), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. For the Needle program to report the longest identity, the no brief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0067] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0068] Variant: The term “variant” means an alpha amylase, a glucanase, a xylanase, a lipase, or a phosphodiesterase comprising a man-made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means 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.

[0069] Alpha amylase

[0070] The alpha amylase may be derived from any organism. Preferably, the alpha amylase is of microbial origin; most preferably, the alpha amylase is of bacterial or fungal origin. In a preferred embodiment, the alpha amylase is derived from Bacillus amyloliquefaciens. The alpha amylase is GH 13 alpha amylase. In a preferred embodiment, the alpha amylase is GH13_5 alpha amylase.

[0071] In one aspect, the alpha amylase is selected from the group consisting of:

[0072] (a) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2;

[0073] (b) a polypeptide encoded by a polynucleotide having at least 80%, e.g., 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 the cDNA sequence thereof;

[0074] (c) a polypeptide derived from SEQ ID NO:1 , a mature polypeptide of SEQ ID NO:1 by substitution, deletion, or addition of one or several amino acids;

[0075] (d) a polypeptide derived from the polypeptide of (a), (b), and (c); wherein the N- and / or C- terminal end has been extended by the addition of one or more amino acids; and

[0076] (e) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has alpha amylase activity.

[0077] In a preferred embodiment, the alpha amylase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2.

[0078] Glucanase

[0079] The glucanase may be derived from any organism. Preferably, the glucanase is of microbial origin; most preferably, the glucanase is of bacterial or fungal origin. In a preferred embodiment, the glucanase is derived from Trichoderma reesei. The glucanase is selected from a group consisting of GH5 beta-glucanase, GH6 glucanase, GH7 endo-glucanase, GH7 exo-glucanase and GH16 beta-glucanase. In one aspect, the glucanase is selected from the group consisting of:

[0080] (a) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4;

[0081] (b) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6;

[0082] (c) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8;

[0083] (d) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10;

[0084] (e) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11, or to the polypeptide of SEQ ID NO: 12;

[0085] (f) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ I D NO: 14;

[0086] (g) a polypeptide encoded by a polynucleotide having at least 80%, e.g., 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:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 the cDNA sequence thereof;

[0087] (h) a polypeptide derived from SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 , a mature polypeptide of SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 SEQ ID NO: 11 or SEQ ID NO: 13 by substitution, deletion, or addition of one or several amino acids;

[0088] (i) a polypeptide derived from the polypeptide of (a), (b), (c), (d) (e), (f), (g) or (h) wherein the N- and / or C-terminal end has been extended by the addition of one or more amino acids; and

[0089] (g) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g) or (h); wherein the polypeptide has glucanase activity.

[0090] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4.

[0091] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6.

[0092] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8.

[0093] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10.

[0094] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12.

[0095] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ ID NO: 14.

[0096] Xylanase

[0097] The xylanase may be derived from any organism. Preferably, the xylanase is of microbial origin; most preferably, the glucanase is of bacterial or fungal origin. In a preferred embodiment, the xylanase is derived from Humicola insolens. The xylanase is GH10 xylanase.

[0098] In one aspect, the xylanase is selected from the group consisting of:

[0099] (a) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ I D NO: 16;

[0100] (b) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ I D NO: 18.

[0101] (c) a polypeptide encoded by a polynucleotide having at least 80%, e.g., 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:15, or SEQ ID NO: 17 or the cDNA sequence thereof;

[0102] (d) a polypeptide derived from SEQ ID NO: 15, or SEQ ID NO: 17, a mature polypeptide of SEQ ID NO:13, or SEQ ID NO: 15 by substitution, deletion, or addition of one or several amino acids;

[0103] (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d); wherein the N- and / or C- terminal end has been extended by the addition of one or more amino acids; and

[0104] (f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has xylanase activity.

[0105] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16.

[0106] In a preferred embodiment, the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO:18.

[0107] Lipase

[0108] The lipase may be derived from any organism. Preferably, the lipase is of microbial origin; most preferably, the lipase is of bacterial or fungal origin. In a preferred embodiment, the lipase is derived from Thermomyces lanuginosus.

[0109] In one aspect, the lipase is selected from the group consisting of:

[0110] (f) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20;

[0111] (g) a polypeptide encoded by a polynucleotide having at least 80%, e.g., 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 the cDNA sequence thereof;

[0112] (h) a polypeptide derived from SEQ ID NO: 19, a mature polypeptide of SEQ ID NO: 19 by substitution, deletion, or addition of one or several amino acids;

[0113] (i) a polypeptide derived from the polypeptide of (a), (b), and (c); wherein the N- and / or C- terminal end has been extended by the addition of one or more amino acids; and

[0114] (j) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has lipase activity.

[0115] In a preferred embodiment, the alpha amylase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20. Phosphodiesterase

[0116] The phosphodiesterase may be derived from any organism. Preferably, the phosphodiesterase is of microbial origin; most preferably, the phosphodiesterase is of bacterial or fungal origin. In a preferred embodiment, the phosphodiesterase is derived from Metabacillus indicus.

[0117] In one aspect, the phosphodiesterase is selected from the group consisting of:

[0118] (a) a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 21 , or to the polypeptide of SEQ ID NO: 22;

[0119] (b) a polypeptide encoded by a polynucleotide having at least 80%, e.g., 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:21 or the cDNA sequence thereof;

[0120] (c) a polypeptide derived from SEQ ID NO:21 a mature polypeptide of SEQ ID NO:21 by substitution, deletion, or addition of one or several amino acids;

[0121] (d) a polypeptide derived from the polypeptide of (a), (b), and (c); wherein the N- and / or C- terminal end has been extended by the addition of one or more amino acids; and

[0122] (e) a fragment of the polypeptide of (a), (b), or (c); wherein the polypeptide has lipase activity.

[0123] In a preferred embodiment, the alpha amylase has at least 80%, e.g., 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: 21 , or to the polypeptide of SEQ ID NO: 22.

[0124] Dental care ingredients

[0125] The dental care compositions of the invention comprises a combination of enzymes comprising alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases; and at least one dental care ingredient.

[0126] The dental care ingredients for animals may be varied according to the different types of dental care composition as well as the desired characteristics and / or activities of the dental care compositions. For the purpose of the present invention, the terms “ingredient” and “component” are used interchangeably in relation to dental care compositions.

[0127] An dental care composition of the invention may be compositions such as toothpaste, a tooth gel, a tooth cream, a tooth powder, dentifrice, oral spray, a chewable product, a mouth- freshener, a mouthwash, a mouth rinse, and a dental pack, and the like, which is designed to remove biofilm inside the oral cavity, e.g., biofilm residing on teeth, on soft tissues of the oral cavity, and on dentures residing in the oral cavity.

[0128] In a preferred embodiment, the dental care composition is at least one dental care component is selected from the group consisting of abrasives, humectants, solvents, thickening agents, binding agents, buffering agents, foaming agents, foaming modulators, sweetening agents, softening agents, plasticizing agents, flavoring agents, coloring agents, therapeutic agents, anti-microbial agents, tartar-controlling agents, fluoride sources, preservatives, detergents, surfactants, coloring agents, buffering agents, softeners, plasticizers, whitening agents, bleaching agents, gum-base ingredients, and bulking agents.

[0129] The dental care composition may further comprise various color and flavoring additives, binders, emulsifiers, preservatives, and antioxidants, and may be formulated as an aqueous solution, a non-aqueous solution, a suspension, a gel, a foam, a paste, a powder, a dust, a solid, or an emulsion.

[0130] The dental care composition may also be applied to a variety of animal food types and treats for consumption.

[0131] The dental care composition may further comprise additives including, but not limited to, sorbitol, dextrin, maltodextrin, collagen, glycerine, hexoses, glucosamine, and mixtures thereof.

[0132] The dental care composition may further comprise natural polymers, including but not limited to, starches, for example potato starch, corn starch, wheat starch, tapioca starch, and pea starch, pectins, gum Arabic, acacia gum, guar gum, Xanthan gum, proteins, for example, egg whites, pea protein, and glutein, and oils, for example, coconut oil, canola oil, or corn oil.

[0133] The dental care composition may further comprise a sugar, an edible toothpaste, a nutraceutical, a water-soluble thickener, or a combination thereof.

[0134] Sugars may include, but are not limited to, agave nectar, barley malt syrup, barley sugar birch syrup, brown sugar, caramel, coconut sugar, corn syrup, date sugar, dextrose, sucrose, lactose, maltose, fructose, galactose, glucose, golden syrup, high fructose corn syrup, high maltose corn syrup, honey, confectioners' sugar, inositol, inverted sugar syrup, jiggery, mannose, maple sugar, maple syrup, molasses (from sugar beets), molasses (from sugar cane), palm sugar, ribose, rhamnose, toffee, treacle, trehalose, xylose, and mixtures thereof.

[0135] An edible toothpaste, may include, but is not limited to, dental grade silica abrasive, dicalcium phosphate dihydrate, pumice, polyphosphate, microcrystalline cellulose, magnesium stearate, and mixtures thereof. Nutraceuticals may include, but are not limited to, brewer's yeast, Echinacea purpurea, Ascophyllum nodosum, flax seed, polyunsaturated fatty acids, calcium, vitamins and minerals, and combinations thereof.

[0136] Water soluble thickeners, may include, but are not limited to, gelatin, carboxymethylcellulose, agar, starch, carrageen, and mixtures thereof.

[0137] The dental care composition may further comprise a color additive, a flavoring additive (palatant enhancer), a binder, an emulsifier, a preservative, an antioxidant, or a combination thereof.

[0138] The dental care composition may be formulated as an aqueous solution, a non-aqueous solution, a suspension, a gel, a foam, a paste, a powder, a dust, a solid, or an emulsion.

[0139] Although the dental care ingredients mentioned herein are categorized by a general header according to a functionality, this is not to be construed as a limitation, as an ingredient may comprise additional functionalities as will be appreciated by the skilled person.

[0140] Application of dental care compositions

[0141] The dental care compositions of the invention are suitable for use in the treatment of oral disease in animals, wherein prevention or removal of oral biofilm is desired. The compositions of the invention are particularly suitable for preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal.

[0142] The biofilm is typically the biofilm in the oral cavity of a companion animal, such as a such as a dog or a cat or a horse or a pig, more typically a dog or cat or hose, even more typically a dog or cat. In a typical embodiment, the animal is a companion animal, such as a dog or a cat or a horse or a pig, more typically a dog or cat or hose, even more typically a dog or cat.

[0143] In a preferred embodiment, the dental care composition is administered to the oral cavity of the animal. In a more typical embodiment, the dental care composition is applied to the teeth or dentition of the animal. The composition may be applied by any variety of means, such as by use of a toothbrush or brushed onto the teeth using another device or a finger, applied as a formulation which adheres to the animal’s teeth (sticky formulation), such as a fluid formulation such as fluid formulation applied to the animal’s food and which adheres temporarily to the dentition, or integrated within a chew toy.

[0144] A dental care composition of the invention may be added to and contained in a chew toy that releases the dental care composition while the dog chews on it. In addition, the chew toy has a mechanical effect on the established dental biofilm. The chew toy can then be refilled with gel before given to the dog. There are several different types of chew toys, including rubber, rawhide, wood, paper, and mineral. Chew toys are commonly associated with puppies and dogs, though they may also be used for cats, horses, pigs, birds, rodents, and rabbits. In one aspect, the present invention relates to a dental care composition a combination of enzymes comprising an alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases, and at least one dental care ingredient for use as a medicament.

[0145] In one aspect, the present invention relates to a dental care composition a combination of enzymes comprising an alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases, and at least one dental care ingredient for use in the treatment of oral disease.

[0146] In a preferred embodiment, the present invention relates to a dental care composition a combination of enzymes comprising an alpha amylases, glucanases and / or xylanases, and lipases and / or phosphodiesterases, and at least one dental care ingredient for use in preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal.

[0147] As known to the skilled person, the concentration of each enzyme will depend on its enzymatic activity. The composition of the invention comprises the enzymes in a concentration suitable to reduce the rate of biofilm formation in the animal, or to remove the biofilm or plaque resulting therefrom after repeated application. Furthermore, the concentration of the enzyme will depend on the manner of the application of the composition to the animal in that a different concentration may be needed if the application method is tooth-brushing compared to the concentration if the application method is administration via the means of a chew toy, especially since the frequency of the application of the composition may differ between application methods. In a typical embodiment, each enzyme class has a concentration of 1-100 ppm, such as 5-80 ppm, more typically 10-60 ppm.

[0148] PREFERRED EMBODIMENTS

[0149] PARAGRAPHS:

[0150] 1 . A composition for dental care of an animal, the composition comprising a combination of enzymes, the combination of enzymes comprising: i. one or more alpha amylases ii. one or more glucanases and / or one or more xylanases; and iii. one or more lipases and / or one or more phosphodiesterases. 2. The composition according to paragraph 1 , wherein the composition further comprises at least one dental care ingredient.

[0151] 3. The composition according to paragraph 1 , wherein the alpha amylase is GH13 alpha amylase.

[0152] 4. The composition according to paragraphs 1 or 3, wherein the alpha amylase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2.

[0153] 5. The composition according to paragraph 4, wherein the alpha amylase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2.

[0154] 6. The composition according to paragraph 1 , wherein the glucanase is selected from a group consisting of GH5 beta-glucanase, GH6 glucanase, GH7 endo-glucanase, GH7 exo-glucanase, GH7 beta glucanase and GH16 beta-glucanase.

[0155] 7. The composition according to paragraphs 1 or 6, wherein the glucanase is selected from a group consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6; iii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8; iv. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10; and v. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12, vi. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ ID NO: 14.

[0156] 8. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4.

[0157] 9. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6.

[0158] 10. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8.

[0159] 11. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10.

[0160] 12. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12.

[0161] 13. The composition according to paragraph 7, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ ID NO: 14. The composition according to paragraph 1 , wherein the xylanase is GH10 xylanase. The composition according to paragraphs 1 or 14, wherein the xylanase is selected from a group consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO: 18. The composition according to paragraph 14, wherein the xylanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16. The composition according to paragraph 14, wherein the xylanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO: 18. The composition according to paragraph 1 , wherein the lipase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20. The composition according to paragraphs 1 or 18, wherein the lipase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20. The composition according to paragraph 1 , wherein the phosphodiesterase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 21 , or to the polypeptide of SEQ ID NO: 22. The composition according to paragraph 1 or 20, wherein the phosphodiesterase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 21 , or to the polypeptide of SEQ ID NO: 22. The composition according to paragraph 2, wherein the dental care ingredient is 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. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more phosphodiesterases. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more lipases. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more xylanases and one or more lipases. The composition according to paragraphs 1 to 22, wherein the combination of enzymes comprises one or more alpha amylases, one or more xylanases and one or more phosphodiesterases. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, and one or more lipases. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, and one or more phosphodiesterases. The composition according to paragraphs 1-22, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, one or more lipases, and one or more phosphodiesterases. 30. The composition according to any of paragraphs 1-29, wherein the animal is a companion animal selected from the group consisting of a dog, a cat, a horse, or a lama.

[0162] 31. The composition according to any of paragraphs 1-30, wherein the companion animal is a dog.

[0163] 32. A dental care composition comprising a combination of enzymes and at least one dental care ingredient, the combination of enzymes comprising: i. one or more alpha amylases ii. one or more glucanase and / or one or more xylanases; and iii. one or more lipases and / or one or more phosphodiesterases.

[0164] 33. The dental care composition according to paragraph 32, wherein the alpha amylase is GH13 alpha amylase.

[0165] 34. The dental care composition according to paragraph 32 or 33, wherein the alpha amylase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2.

[0166] 35. The dental care composition according to paragraphs 34, wherein the alpha amylase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2.

[0167] 36. The dental care composition according to paragraph 32, wherein the glucanase is selected from a group consisting of GH5 beta-glucanase, GH6 glucanase, GH7 endo-glucanase, GH7 exo-glucanase, GH7 beta glucanase and GH16 beta-glucanase.

[0168] 37. The dental care composition according to paragraphs 32 or 36, wherein the glucanase is selected from a group consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6; iii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8; iv. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10; v. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12; and vi. v. a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least

[0169] 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ ID NO: 14.

[0170] 38. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4.

[0171] 39. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6.

[0172] 40. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8. 41. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10.

[0173] 42. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ I D NO: 12.

[0174] 43. The dental care composition according to paragraph 37, wherein the glucanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 13, or to the polypeptide of SEQ ID NO: 14.

[0175] 44. The dental care composition according to paragraph 32, wherein the xylanase is GH10 xylanase.

[0176] 45. The dental care composition according to paragraphs 32 or 44, wherein the xylanase is selected from a group consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO: 18.

[0177] 46. The dental care composition according to paragraph 45, wherein the xylanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16.

[0178] 47. The dental care composition according to paragraph 45, wherein the xylanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO: 18.

[0179] 48. The dental care composition according to paragraph 32, wherein the lipase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20.

[0180] 49. The dental care composition according to paragraphs 48, wherein the lipase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 19, or to the polypeptide of SEQ ID NO: 20.

[0181] 50. The dental care composition according to paragraph 32, wherein the phosphodiesterase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 21 , or to the polypeptide of SEQ ID NO: 22.

[0182] 51. The dental care composition according to paragraphs 50, wherein the phosphodiesterase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 21 , or to the polypeptide of SEQ ID NO: 22.

[0183] 52. The dental care composition according to paragraph 32, wherein the dental care ingredient is 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.

[0184] 53. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more phosphodiesterases.

[0185] 54. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more lipases.

[0186] 55. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more xylanases and one or more lipases. 56. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more xylanases and one or more phosphodiesterases.

[0187] 57. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, and one or more lipases.

[0188] 58. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, and one or more phosphodiesterases.

[0189] 59. The dental care composition according to paragraphs 32-52, wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases, one or more xylanases, one or more lipases, and one or more phosphodiesterases.

[0190] 60. A dental care composition comprising a combination of enzymes and at least one dental care ingredient, the combination of enzymes comprising: b. alpha amylase, wherein the alpha amylase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2, c. a group of glucanases consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6; iii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8; iv. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10; and v. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12, d. xylanase, wherein the xylanase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16; and e. lipase, wherein the lipase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ ID NO: 18. The dental care composition according to paragraph 60, wherein the composition comprises: a. an alpha amylase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 1 , or the polypeptide of SEQ ID NO: 2 b. glucanases comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 , or the polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; c. a xylanase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 15, or the polypeptide of SEQ ID NO: 16 d. a lipase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 17, or the polypeptide of SEQ ID NO: 18 62. The dental care composition according to paragraph 60, wherein the dental care ingredient is 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.

[0191] 63. The dental care composition according to any of paragraphs 32-62 for use as a medicament for dental health of animals.

[0192] 64. The dental care composition according to paragraph 63, wherein the medicament is toothpaste, a tooth gel, a tooth cream, a tooth powder, dentifrice, oral spray, a chewable product, a mouth-freshener, a mouthwash, a mouth rinse, and a dental pack.

[0193] 65. The dental care composition according to any of paragraph 63, wherein the animal is a companion animal.

[0194] 66. The dental care composition according to paragraph 65, wherein the companion animal is selected from a group consisting of a dog, a cat, a horse, and a lama.

[0195] 67. The dental care composition according to any of paragraphs 65-66, wherein the companion animal is a dog.

[0196] 68. A method for preventing or removing oral biofilm in an animal, the method comprising contacting the oral biofilm with a composition according to any of paragraphs 1-32, or a dental care composition according to any of paragraphs 32-67.

[0197] 69. A method for reducing the risk of oral biofilm formation in an animal, the method comprising contacting the oral biofilm with a composition according to any of paragraphs 1-32, or a dental care composition according to any of paragraphs 32-67.

[0198] 70. A method of removing oral biofilm formed in an animal, the method comprising contacting the oral biofilm with a composition according to any of paragraphs 1-31 , or a dental care composition according to any of paragraphs 32-67.

[0199] 71. A method of preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal, said method contacting the composition of paragraphs 1-67 with the teeth of said animal.

[0200] 72. Use of a composition as defined in any of paragraphs 1-31 or a dental care composition according to any of paragraphs 33-67 for preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal.

[0201] 73. A composition as defined in any of paragraphs 1 to 67 for use in the prevention of biofilm formation on the dentition of an animal or the removal of biofilm on the dentition of an animal. 74. A composition as defined in any of paragraphs 1 to 67 for use in the prevention of dental plaque formation in an animal or the removal of dental plaque in an of animal.

[0202] 75. A kit of parts comprising: i. a dental care composition according to any of paragraphs 32-67; and ii. instructions for use.

[0203] EXAMPLES

[0204] Samples:

[0205] Example 1 : Canine multispecies biofilm prevention assay

[0206] The biofilm prevention effect of different enzyme blends listed in the samples above were tested on a multispecies biofilm grown on 96-well microtiter plates consisting of five canine dental pathogens: Neisseria zoodegmatis DSM21643, Pasteurella canis DSM22968, Pseudomonas fragi DSM3456, Capnocytophaga canis DSM101831 and Enterococcus faecalis DSM20478 (see References below). 96-well microtiter plates (Nunclon Delta surface ThermoScientific #167008) were filled with 75 l of Tripticase Soy Broth (TSB) +2% glucose containing 1 x 107 CFU / ml bacterial inoculum of each of the five strains mentioned above and 25 pl of enzyme solution in Citrate-phosphate buffer pH 6 (Mcllvaine buffer) to yield a final concentration of 60ppm. For control samples the enzyme solution was replaced with Mcllvaine buffer pH 6. Plates were incubated at 30°C for 72 hours without shaking in a Thermo ScientificTM Rectangular AnaeroBoxTM Container under microaerophilic conditions (ThermoScientific AnaeroGen 2.5L #AN0025A). Enzyme and control samples were carried out with 8 replicates. Citrate-phosphate buffer pH 6 (Mcllvaine buffer) was prepared by mixing 12.63 ml 0.2M

[0207] Na2HPC>4 + 7.37 ml 0.1 M citric acid. After incubation, planktonic bacteria were removed by two gentle washes with 100 pl 0.9% sodium chloride and biofilms were stained with 0.095% crystal violet solution for 15 min at room temperature. Plates were rinsed twice with 100 pL 0.9% sodium chloride and adhered dye was dissolved with a solution of 96% ethanol and 0.1 % acetic acid in water. Absorbance was measured at 600 nm with a microplate reader SpectraMax M3, Molecular Devices.

[0208] For the data processing, the absorbance was taken to be proportional to the extent of remaining biofilm after enzyme or buffer treatment. The results were expressed as percentage of biofilm prevention and was calculated as follows:

[0209] 100-((A600nm enzyme treated sample) / (A600nm buffer control treated sample) x100), where A600nm refers to the average of 8 absorbances measured at 600 nm of either enzyme or buffer treated samples.

[0210] Results are listed in Table 1.

[0211] Table 1. Canine Biofilm prevention assay

[0212] Results showed that the enzyme blends listed above had a prevention effect on formation of canine multispecies biofilm.

[0213] The experiments were repeated and again showed high degree of prevention with the combinations within the scope of the invention.

[0214] Table 1.2 Example 2: Canine multispecies biofilm removal assay

[0215] The biofilm removal effect of different enzymes blends as listed in the samples above was tested on a multispecies biofilm grown on 96-well microtiter plates consisting of five canine dental pathogens: Neisseria zoodegmatis DSM21643, Pasteurella canis DSM22968, Pseudomonas fragi DSM3456, Capnocytophaga canis DSM101831 and Enterococcus faecalis DSM20478 (see References below). 96-well microtiter plates (Nunclon Delta surface ThermoScientific #163320) were filled with 200 l of Tripticase Soy Broth (TSB) + 2% glucose containing 1 x 107CFU / ml bacterial inoculum of each of the five strains mentioned above. Plates were incubated at 30°C for 72 hours without shaking in a Thermo ScientificTM Rectangular AnaeroBoxTM Container under microaerophilic conditions (ThermoScientific AnaeroGen 2.5L #AN0025A).

[0216] After incubation, the supernatant containing the planktonic bacteria was removed from each well and samples were treated either with 200 pL Citrate-phosphate buffer pH 8 (Mcllvaine buffer) as a control or 60ppm enzyme blends as listed in the samples above in Mcllvaine buffer pH 8 for 30 minutes at 37°C, 50rpm shaking. Enzyme and control samples were carried out with 8 replicates.

[0217] Citrate-phosphate buffer pH 8 (Mcllvaine buffer) was prepared by mixing 19.45 ml 0.2M Na2HPC>4 + 0.55 ml 0.1 M citric acid.

[0218] After enzyme treatment, samples were rinsed twice with 200 pL 0.9% sodium chloride and stained with 0.095% crystal violet solution for 15 minutes at room temperature. Samples were rinsed three times with 200 pL 0.9% sodium chloride and the dye was extracted with 33% acetic acid solution and absorbance at 600nm was measured.

[0219] For the data processing, the absorbance was taken to be proportional to the extent of remaining biofilm after enzyme or buffer treatment. The percentage of biofilm removal was calculated as follows:

[0220] 100-((A600nm enzyme treated sample) / (A600nm buffer control treated sample)x100), where A600nm refers to the average of 8 absorbances measured at 600 nm using SpectraMax M3, Molecular Devices, of either enzyme or buffer treated samples. Results are listed in Table 2.

[0221] Table 2. Canine biofilm removal assay

[0222] Results showed that the enzyme blends listed above had a removal effect on formation of canine multispecies biofilm.

[0223] References: 1.Cunha et al. BMC Veterinary Research (2020) 16:469. A polymicrobial biofilm model for testing the antimicrobial potential of a nisin-biogel for canine periodontal disease control

[0224] 2. Holcombe et al., 2014, PLOS ONE | DOI:10.1371 / journal.pone.0113744. Early Canine Plaque Biofilms: Characterization of Key Bacterial Interactions Involved in Initial Colonization of Enamel

[0225] 3. A. Manuel, J.V. Rao, K. John & J.M. Aranjani. 2014. Biofilm Production and Antibiotic Susceptibility of Planktonic and Biofilm Bacteria of Canine Dental Tartar Isolates. Acta Scientiae Veterinariae. 42: 1202.

Claims

CLAIMS1 . A composition for dental care of an animal, the composition comprising a combination of enzymes, the combination of enzymes comprising: i. one or more alpha amylases ii. one or more glucanases and / or one or more xylanases; and iii. one or more lipases and / or one or more phosphodiesterases.

2. The composition according to claim 1 , wherein the alpha amylase is GH13 alpha amylase.

3. The composition according to claim 1 , wherein the glucanase is selected from a group consisting of GH5 beta-glucanase, GH6 glucanase, GH7 endo-glucanase, GH7 exo- glucanase, GH7 beta-glucanase and GH16 beta-glucanase.

4. The composition according to claim 1 , wherein the xylanase is GH10 xylanase.

5. The composition according to claim 1 , wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more phosphodiesterases.

6. The composition according to claim 1 , wherein the combination of enzymes comprises one or more alpha amylases, one or more glucanases and one or more lipases.

7. The composition according to claim 1 , wherein the combination of enzymes comprises one or more alpha amylases, one or more xylanases and one or more lipases.

8. A dental care composition comprising a combination of enzymes and at least one dental care ingredient, the combination of enzymes comprising: i. one or more alpha amylases ii. one or more glucanase and / or one or more xylanases; and iii. one or more lipases and / or one or more phosphodiesterases.

9. A dental care composition comprising a combination of enzymes and at least one dental care ingredient, the combination of enzymes comprising: a. one or more polypeptides having alpha amylase activity, wherein the polypeptide has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , or to the polypeptide of SEQ ID NO: 2, b. one or more polypeptides having glucanase activity selected from consisting of: i. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, or to the polypeptide of SEQ ID NO: 4; ii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 5, or to the polypeptide of SEQ ID NO: 6; iii. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 7, or to the polypeptide of SEQ ID NO: 8; iv. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 9, or to the polypeptide of SEQ ID NO: 10; and v. a polypeptide having at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 , or to the polypeptide of SEQ ID NO: 12, c. one or more polypeptides having xylanase activity, wherein the polypeptide has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 15, or to the polypeptide of SEQ ID NO: 16; and d. one or more polypeptides having lipase activity, wherein the lipase has at least 80%, e.g., 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%, at least 100% sequence identity to the mature polypeptide of SEQ ID NO: 17, or to the polypeptide of SEQ I D NO: 18.

10. A method for preventing or removing oral biofilm in an animal, the method comprising contacting the oral biofilm with a composition according to any of claims 1-7, or a dental care composition according to any of claims 8-9.

11. A method for reducing the risk of oral biofilm formation in an animal, the method comprising contacting the oral biofilm with a composition according to any of claims 1-7, or a dental care composition according to any of claims 8-9.

12. A method of removing oral biofilm formed in an animal, the method comprising contacting the oral biofilm with a composition according to any of claims 1-7, or a dental care composition according to any of claims 8-9.

13. A method of preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal, said method contacting the composition of claims 1-9 with the teeth of said animal.

14. Use of a composition as defined in any of claims 1-7 or a dental care composition according to any of claims 8-9 for preventing or removing dental plaque, reducing the likelihood of dental cavities, or reducing gingivitis in an animal.

15. A composition as defined in any of claims 1-7 or a dental care composition according to any of claims 8-9 for use in the prevention of biofilm formation on the dentition of an animal or for use in the removal of biofilm on the dentition of an animal.

Images