Oral care compositions comprising invertase

Abstract

The present invention relates to an oral care composition comprising an invertase, the use of said composition as a medicament, the use of said composition in the treatment of oral diseases, methods of treatment comprising administration of said composition to a human subject, methods of prevention or removal of oral biofilms comprising contacting the oral biofilms with said composition, and methods of use of said composition in the treatment of oral diseases. A method for reducing the risk of oral biofilm formation, and a kit comprising the composition.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a Sequence Listing in computer-readable form. The computer-readable form is hereby incorporated by reference herein. Field of the Invention

[0003] The present invention relates to an oral care composition comprising an invertase, the use of said composition as a medicament, the use of said composition in the treatment of oral diseases, a treatment method comprising administering said composition to a human subject, a method for preventing or removing oral biofilm comprising contacting oral biofilm with said composition, a method for reducing the risk of oral biofilm formation, and a kit comprising said composition. Background Art

[0004] Biofilms are bacterial communities found on solid surfaces (including oral surfaces) in many different environments. Oral biofilms or dental plaque contain many bacteria associated with oral health problems such as oral malodor, demineralization, dental caries, tooth decay, potential tooth loss, and gum diseases (gingivitis and periodontitis).

[0005] The formation of oral biofilm occurs in three stages, respectively called the lag phase, the growth phase, and the steady state. In the lag phase, glycoproteins from saliva bind to oral surfaces (such as teeth) and form a structure called a pellicle, which serves as an attachment site for bacteria. In the growth phase, co-aggregation occurs, i.e., secondary bacterial colonizers attach to primary bacterial colonizers, causing an increase in biofilm diversity and biofilm growth and maturation. In the steady state, biofilm growth slows down and eventually stops. This stage-based formation cycle results in the biofilm existing in several consecutive layers, which makes the physical abrasion of the biofilm more difficult.

[0006] Within the biofilm, the resident bacterial cells are distributed in an extracellular polymeric matrix that consists mainly of water, proteins, exopolysaccharides, lipopolysaccharides, lipids, surfactants, and extracellular DNA, with exopolysaccharides making up the major portion of the biofilm dry weight (H.C. Flemming and J. Wingender (2010), Nat. Rev. Microbiol. 8, 623 - 633). Exopolysaccharides are mainly homopolymers of glucose and fructose, including (1 - 3)-α-D-glucans, (1 - 4)-α-D-glucans, (1 - 6)-α-D-glucans, and (2 - 6)-β-D-fructans. These polysaccharides are synthesized from ingested sucrose by glucosyltransferases and fructosyltransferases secreted by oral bacteria such as species of Streptococcus, Lactobacillus, and Actinomyces. Mutans and dextran are glucans that are particularly important in the formation of dental plaque. Mutans has a highly branched structure, with a backbone composed of glucose molecules linked by (1 - 3)-α bonds and (1 - 6)-α-glycosidic bonds in the side chains. Dextran is also a high-molecular-weight polymer of glucose, containing multiple consecutive (1 - 6)-α-bonds in the backbone and side chains, starting with a (1 - 3)-α-bond (M. Pleszczynska et al. (2016), Biotechnol. Appl. Biochem. 64(3), 337 - 346). Fructans are mainly linear polysaccharides and consist mainly of fructosyl residues linked by β-(2,6) and some branches linked by β-(2,1).

[0007] Due to increased resistance to antimicrobial agents and the mechanical properties of the biofilm, many current oral care products are highly inefficient in addressing biofilm formation and alleviating related oral health problems. The focus of biofilm removal lies in mechanical abrasion. However, the multi-layered nature of the biofilm poses difficulties for mechanical abrasion, and the mechanical removal of the biofilm (e.g., by brushing) expands and deepens the area of biofilm attachment and spread in the oral cavity, potentially increasing rather than reducing the severity of the problem, thus further affecting mechanical abrasion.

[0008] In view of 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 an oral care composition comprising mutanase and dextranase, WO 1998 / 57653 (Novozymes) provides an oral care composition comprising dextranase and pullulanase, WO 2000 / 17331 discloses an oral care composition comprising a Paenibacillus fructanase, and WO 2020 / 099490 (Novozymes) describes an oral care composition comprising mutanase and DNase. However, there is still a need for oral care compositions that target oral biofilms. Summary of the Invention

[0009] The present invention provides polypeptides having β-fructofuranosidase activity (i.e., invertases) and oral care compositions comprising said polypeptides, which are stable in the presence of oral care ingredients and provide biofilm prevention.

[0010] In a first aspect, the present invention relates to an oral care composition comprising an invertase selected from the group consisting of:

[0011] a) a polypeptide having at least 70% sequence identity with SEQ ID NO:3;

[0012] b) a polypeptide having at least 70% sequence identity with SEQ ID NO:6;

[0013] c) a polypeptide having at least 70% sequence identity with SEQ ID NO:9;

[0014] d) a polypeptide having at least 70% sequence identity with SEQ ID NO:12;

[0015] e) a polypeptide having at least 70% sequence identity with SEQ ID NO:15;

[0016] f) a polypeptide having at least 70% sequence identity with SEQ ID NO:18;

[0017] g) a polypeptide having at least 70% sequence identity with SEQ ID NO:21;

[0018] h) a polypeptide having at least 70% sequence identity with SEQ ID NO:24;

[0019] i) a polypeptide having at least 70% sequence identity with SEQ ID NO:27;

[0020] j) a polypeptide having at least 70% sequence identity with SEQ ID NO:30;

[0021] k) a polypeptide having at least 70% sequence identity with SEQ ID NO:33;

[0022] l) a polypeptide having at least 70% sequence identity with SEQ ID NO:36; and

[0023] m) a polypeptide having at least 70% sequence identity with SEQ ID NO:39;

[0024] wherein the polypeptide has β-fructofuranosidase activity and wherein the oral care composition further comprises at least one oral care ingredient.

[0025] The present invention also relates to the use of the oral care composition of the present invention as a medicament, a method of using the oral care composition of the present invention, and a kit comprising the oral composition of the present invention.

[0026] In a second aspect, the present invention relates to a polypeptide having β-fructofuranosidase activity, the polypeptide being selected from the group consisting of:

[0027] a) a polypeptide having at least 70% sequence identity with SEQ ID NO:3;

[0028] b) a polypeptide having at least 70% sequence identity with SEQ ID NO:6;

[0029] c) a polypeptide having at least 70% sequence identity with SEQ ID NO:9;

[0030] d) a polypeptide having at least 70% sequence identity with SEQ ID NO:12;

[0031] e) a polypeptide having at least 70% sequence identity with SEQ ID NO:15;

[0032] f) a polypeptide having at least 70% sequence identity with SEQ ID NO:18;

[0033] g) a polypeptide having at least 70% sequence identity with SEQ ID NO:21;

[0034] h) a polypeptide having at least 70% sequence identity with SEQ ID NO:24;

[0035] i) a polypeptide having at least 70% sequence identity with SEQ ID NO:27;

[0036] j) a polypeptide having at least 70% sequence identity with SEQ ID NO:30;

[0037] (k) a polypeptide having at least 70% sequence identity with SEQ ID NO:33;

[0038] (l) a polypeptide having at least 70% sequence identity with SEQ ID NO:36; and

[0039] (m) a polypeptide having at least 70% sequence identity with SEQ ID NO:39.

[0040] The present invention also relates to polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs, expression vectors, host cells comprising these polynucleotides; and methods for producing these polypeptides.

[0041] Definitions

[0042] For the purposes of this detailed description, the following definitions apply. Note that the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0043] Unless otherwise defined or clearly indicated by the 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 pertains.

[0044] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcribing a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA, which is processed through a series of steps (including splicing) and then presented as mature, spliced mRNA.

[0045] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically determined by an open reading frame that begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA). A coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0046] Control sequence: The term "control sequence" means a nucleic acid sequence involved in regulating the expression of a polynucleotide, either in vivo or in vitro, in a particular organism. Each control sequence may be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, prepeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiation and termination sequences. At a minimum, the control sequences include a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites that facilitate the ligation of the control sequences to the coding region of the polynucleotide encoding the polypeptide, these control sequences may be provided with multiple linkers.

[0047] Denture: The term "denture" is intended to cover the denture itself as well as orthodontic appliances, invisible braces, retainers, etc.

[0048] Expression: The term "expression" means any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0049] Expression vector: An "expression vector" is a linear or circular DNA construct that contains a DNA sequence encoding a polypeptide, the coding sequence being operably linked to appropriate control sequences capable of effecting the expression of the DNA in a suitable host. Such control sequences may include a promoter that affects transcription, optional operator sequences that control transcription, sequences encoding a suitable ribosome binding site on the mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0050] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, where the "extended" polypeptide has β-fructofuranosidase activity.

[0051] Fragment: The term "fragment" means a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminus of the mature polypeptide, where the fragment has β-fructofuranosidase activity.

[0052] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of the polypeptide of the present invention. The fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide with the polynucleotide of the present invention or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Inteins can also be used to construct fusion polypeptides, where the fusion polypeptide is generated after translation (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). The fusion polypeptide may further comprise a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0053] Heterologous: For a host cell, the term "heterologous" means that a polypeptide or nucleic acid is not naturally present in the host cell. For a polypeptide or nucleic acid, the term "heterologous" means that a control sequence (e.g., the promoter of a polypeptide or nucleic acid) is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0054] Host strain or host cell: "Host strain" or "host cell" refers to an organism into which an expression vector, phage, virus, or other DNA construct (including a polynucleotide encoding a polypeptide of interest (e.g., amylase)) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from the cells.

[0055] Introduce: In the case of inserting a nucleic acid sequence into a cell, the term "introduce" means "transfect", "transform", or "transduce", as known in the art.

[0056] Invertase: The term "invertase" means a polypeptide having β-fructofuranosidase activity, which catalyzes the hydrolysis of the terminal non-reducing β-D-fructofuranoside residue in β-D-fructofuranoside. Invertase is also known as β-fructofuranosidase, and the scientific name of invertase is β-D-fructofuranoside fructohydrolase (EC 3.2.1.26). The terms "invertase" and "polypeptide having β-fructofuranosidase activity" are used interchangeably throughout the application. For the purposes of the present invention, β-fructofuranosidase (invertase) activity can be determined according to the β-fructofuranosidase (invertase) activity assay described in Example 2 below.

[0057] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific material or component that has been separated from at least one other material or component (including but not limited to, other proteins, nucleic acids, cells, etc.). Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form that does not exist in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted polypeptides expressed in host cells.

[0058] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form after N-terminal and / or C-terminal processing (e.g., removal of the signal peptide).

[0059] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having β-fructofuranosidase activity.

[0060] Native: The term "native" means a nucleic acid or polypeptide that naturally occurs in a host cell.

[0061] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid can be single-stranded or double-stranded and can include chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' orientation.

[0062] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a way that does not exist in nature to contain a segment of nucleic acid or is synthetic and contains one or more control sequences operably linked to a nucleic acid sequence.

[0063] Operably linked: The term "operably linked" means that the designated components are in a relationship (including but not limited to juxtaposition) that allows them to function in the intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0064] Parent: The term "parent" or means the enzyme that is altered to produce an enzyme variant. In one aspect, the parent is the parent invertase that is altered to produce an invertase variant. In one aspect, the parent is the parent β-glucosidase that is altered to produce a β-glucosidase variant. In one aspect, the parent is the parent glucoamylase that is altered to produce a glucoamylase variant. In one aspect, the parent is the parent α-amylase that is altered to produce an α-amylase variant.

[0065] Purified: The term "purified" means a nucleic acid, polypeptide, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation, a purified polypeptide or nucleic acid can form discrete bands). A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., weight percentage or molar percentage). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is significantly increased after applying purification or enrichment techniques. The term "enriched" means that a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component is present in a composition at a relative or absolute concentration higher than that in the starting composition.

[0066] In one aspect, as used herein, the term "purified" refers to a polypeptide or cell that is substantially free of components from the production organism (especially insoluble components). In other aspects, the term "purified" refers to a polypeptide that is substantially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some soluble components of the organism and the culture medium from which it is recovered. The polypeptide can be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0067] Accordingly, the polypeptide can be purified such that only small amounts of other proteins, especially other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components present in the cells from which the polypeptide is derived, especially other proteins and most especially other enzymes. The polypeptide can be "substantially pure", i.e., free of other components from the organism that produces it (e.g., the host organism used for recombinant production of the polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other polypeptide material that is naturally or recombinantly associated with the polypeptide.

[0068] Thus, preferably, by weight of the total polypeptide material present in the preparation, a substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is substantially free of other polypeptide material that is naturally or recombinantly associated with it). For example, this can be achieved by preparing the polypeptide using well-known recombinant methods or using classical purification methods.

[0069] Recombinant: The term "recombinant" is used in its conventional meaning and refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group that is different from the group of sequences found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses a gene that is not found within a cell in its natural (non-recombinant) form, or expresses a native gene at a different level or under different conditions compared to that found in nature. The terms "recombinant" and "genetically modified" and "transgenic" are synonymous.

[0070] Recovery: The term "recover" or "recovery" means the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials. For example, the polypeptide is recovered from the whole fermentation broth or from the cell-free fermentation broth by the following methods: by polypeptide crystal harvesting, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in a cassette filter, drum filtration, rotary drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter or the like, sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, ultrafiltration in cross-flow, dynamic cross-flow or dead-end operation)), or by centrifugation (using a horizontal centrifuge, disc stack centrifuge, hydro cyclone or the like) or by precipitating the polypeptide and using the associated solid-liquid separation method to harvest the polypeptide from the broth medium by using size fractionation. Recovery encompasses the separation and / or purification of the polypeptide.

[0071] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0072] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity", which is implemented as in the needle program of the EMBOSS software 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. In order for the needle program to report the longest identity, the non-simplified (-nobrief) option must be specified on the command line. The output of the "longest identity" marked by needle is calculated as follows:

[0073] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0074] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity", which is implemented as in the needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (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 NCBINUC4.4) substitution matrix. In order for the needle program to report the longest identity, the non-simplified option must be specified on the command line. The output of "longest identity" marked by needle is calculated as follows:

[0075] (Number of identical deoxyribonucleotides × 100) / (Alignment length – total number of gaps in the alignment)

[0076] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates the secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is excised during the secretion process.

[0077] Subsequence: The term "subsequence" means a polynucleotide in which one or more nucleotides are deleted from the 5'-end and / or 3'-end of the mature polypeptide coding sequence; wherein the subsequence encodes a fragment having β-fructofuranosidase activity.

[0078] Variant: The term "variant" means an invertase that contains artificial mutations (i.e., substitutions, insertions (including extensions) and / or deletions (e.g., truncations)) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0079] Wild type: When referring to an amino acid sequence or a nucleic acid sequence, the term "wild type" means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., protein, amino acid or nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences). Description of the Drawings

[0080] Figure 1Shows an example of thermal stability data generated using a nanoDSF instrument. Panel A is an example of data (ratio of fluorescence emission at 350 nm to 330 nm) of the invertase of SEQ ID NO:40 obtained in triplicate as a function of temperature. Panel B shows the first derivative of the raw data in Panel A. The peak maximum in the first derivative plot corresponds to the midpoint of the thermal unfolding transition, called Tm. In this example, for SEQ ID NO:40, the Tm at pH 6.0 corresponds to 61.9 °C and is highly reproducible within three replicates.

[0081] Sequence summary

[0082] SEQ ID NO:1 is the gDNA sequence of the invertase obtained from Bipolaris sorokiniana.

[0083] SEQ ID NO:2 is the translation product (including the signal peptide) obtained from SEQ ID NO:10.

[0084] SEQ ID NO:3 is the invertase (mature polypeptide) obtained from Bipolaris sorokiniana.

[0085] SEQ ID NO:4 is the gDNA sequence of the invertase obtained from Aspergillus aculeatus.

[0086] SEQ ID NO:5 is the translation product (including the signal peptide) obtained from SEQ ID NO:13.

[0087] SEQ ID NO:6 is the invertase (mature polypeptide) obtained from Aspergillus aculeatus.

[0088] SEQ ID NO:7 is the gDNA sequence of the invertase obtained from Pestalotiopsis vismiae.

[0089] SEQ ID NO:8 is the translation product (including the signal peptide) obtained from SEQ ID NO:16.

[0090] SEQ ID NO:8 is the invertase (mature polypeptide) obtained from Pestalotiopsis vismiae.

[0091] SEQ ID NO:10 is the gDNA sequence of the invertase obtained from Aspergillus avenaceus.

[0092] SEQ ID NO:11 is the translation product (including the signal peptide) obtained from SEQ ID NO:19.

[0093] SEQ ID NO:12 is an invertase (mature polypeptide) obtained from Aspergillus avenae.

[0094] SEQ ID NO:13 is the gDNA sequence of an invertase obtained from Aspergillus sclerotiorum.

[0095] SEQ ID NO:14 is the translation product (including the signal peptide) obtained from SEQ ID NO:22.

[0096] SEQ ID NO:15 is an invertase (mature polypeptide) obtained from Aspergillus sclerotiorum.

[0097] SEQ ID NO:16 is the gDNA sequence of an invertase obtained from Fusarium avenaceum.

[0098] SEQ ID NO:17 is the translation product (including the signal peptide) obtained from SEQ ID NO:25.

[0099] SEQ ID NO:18 is an invertase (mature polypeptide) obtained from Fusarium avenaceum.

[0100] SEQ ID NO:19 is the gDNA sequence of an invertase obtained from Penicillium coprophilum.

[0101] SEQ ID NO:20 is the translation product (including the signal peptide) obtained from SEQ ID NO:28.

[0102] SEQ ID NO:21 is an invertase (mature polypeptide) obtained from Penicillium coprophilum.

[0103] SEQ ID NO:22 is the gDNA sequence of an invertase obtained from Penicillium murcianum.

[0104] SEQ ID NO:23 is the translation product (including the signal peptide) obtained from SEQ ID NO:31.

[0105] SEQ ID NO:24 is an invertase (mature polypeptide) obtained from Penicillium murcianum.

[0106] SEQ ID NO:25 is the gDNA sequence of an invertase obtained from Penicillium venetum.

[0107] SEQ ID NO:26 is the translation product (including the signal peptide) obtained from SEQ ID NO:34.

[0108] SEQ ID NO:27 is the invertase (mature polypeptide) obtained from Penicillium waksmanii.

[0109] SEQ ID NO:28 is the gDNA sequence of the invertase obtained from Curvularia spicifera.

[0110] SEQ ID NO:29 is the translation product (including the signal peptide) obtained from SEQ ID NO:37.

[0111] SEQ ID NO:30 is the invertase (mature polypeptide) obtained from Curvularia spicifera.

[0112] SEQ ID NO:31 is the gDNA sequence of the invertase obtained from Alternaria sp.

[0113] SEQ ID NO:32 is the translation product (including the signal peptide) obtained from SEQ ID NO:40.

[0114] SEQ ID NO:33 is the invertase (mature polypeptide) obtained from Alternaria sp.

[0115] SEQ ID NO:34 is the gDNA sequence of the invertase obtained from Fusarium temperatum.

[0116] SEQ ID NO:35 is the translation product (including the signal peptide) obtained from SEQ ID NO:43.

[0117] SEQ ID NO:36 is the invertase (mature polypeptide) obtained from Fusarium temperatum.

[0118] SEQ ID NO:37 is the gDNA sequence of the invertase obtained from Aspergillus japonicus.

[0119] SEQ ID NO:38 is the translation product (including the signal peptide) obtained from SEQ ID NO:46.

[0120] SEQ ID NO:39 is the invertase (mature polypeptide) obtained from Aspergillus japonicus.

[0121] SEQ ID NO:40 is the invertase (mature polypeptide) obtained from Aspergillus niger. Detailed Description of the Invention

[0122] The present invention relates to an oral care composition comprising an invertase and at least one oral care ingredient. As shown in the examples of the present application, the inventors of the present invention have identified certain invertases of microbial origin that are highly effective in preventing the formation of oral biofilms and / or reducing the risk of oral biofilm formation. In addition, these enzymes are highly stable in the presence of a wide range of oral care ingredients, making them highly suitable for oral care formulations.

[0123] Invertase

[0124] In one aspect, the invertase is selected from the group consisting of:

[0125] (a) a polypeptide having 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 with SEQ ID NO:2;

[0126] (b) a polypeptide having 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 with SEQ ID NO:3;

[0127] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:2;

[0128] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:1 or its cDNA sequence;

[0129] (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;

[0130] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids; and

[0131] (g) A fragment of the polypeptide of (a), (b), (c), (d) or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0132] 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 with SEQ ID NO:2 or the mature polypeptide of SEQ ID NO:2. The preferred mature polypeptide of SEQ ID NO:2 corresponds to amino acid residues 22 to 637 of SEQ ID NO:2. <s>0000297< / s>

[0133] 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 with SEQ ID NO:3.

[0134] 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. The preferred mature polypeptide of SEQ ID NO:2 corresponds to amino acid residues 22 to 637 of SEQ ID NO:2.

[0135] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:3 or a fragment thereof.

[0136] The invertase may have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0137] On the other hand, the invertase is derived from SEQ ID NO:2 by substitution, deletion or addition of one or several amino acids. On the other hand, the polypeptide is derived from the mature polypeptide of SEQ ID NO:2 by substitution, deletion or addition of one or several amino acids. On the other hand, the polypeptide is derived from SEQ ID NO:3 by substitution, deletion or addition of one or more amino acids.

[0138] Note: There seems to be a formatting issue with the " " in the original text which might be a special tag or something not properly formatted. I've kept it as is but it might need further clarification depending on its actual meaning.In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:3) that contains 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, such as 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; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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, such as a polyhistidine segment, an epitope, or a binding module.

[0139] In one aspect, the invertase is selected from the group consisting of:

[0140] (a) a polypeptide having 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 with SEQ ID NO:5;

[0141] (b) a polypeptide having 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 with SEQ ID NO:6;

[0142] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:5;

[0143] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:4 or its cDNA sequence;

[0144] (e) Derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or the polypeptide of SEQ ID NO:6 by substitution, deletion, or addition of one or several amino acids;

[0145] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0146] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0147] 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 with 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 17 to 651 of SEQ ID NO:5.

[0148] 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 with SEQ ID NO:6.

[0149] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:5 or SEQ ID NO:5. The preferred mature polypeptide of SEQ ID NO:5 corresponds to amino acid residues 17 to 651 of SEQ ID NO:5.

[0150] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:6 or a fragment thereof.

[0151] The invertase may have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0152] In another aspect, the invertase is derived from SEQ ID NO:5 by substitution, deletion or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:6 by substitution, deletion or addition of one or more amino acids.

[0153] In some embodiments, the invertase is a variant of the parental invertase (preferably SEQ ID NO:6) that contains substitutions, deletions and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO:6, and the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:6 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a conservative nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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, such as a polyhistidine segment, an epitope, or a binding module.

[0154] In one aspect, the invertase is selected from the group consisting of:

[0155] (a) a polypeptide having 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 with SEQ ID NO:8;

[0156] (b) a polypeptide having 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 with SEQ ID NO:9;

[0157] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:8;

[0158] (d) a polypeptide encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:7 or its cDNA sequence;

[0159] (e) a polypeptide derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or the polypeptide of SEQ ID NO:9 by substitution, deletion, or addition of one or several amino acids;

[0160] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0161] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0162] 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:8 or the mature polypeptide of SEQ ID NO:8. The preferred mature polypeptide of SEQ ID NO:8 corresponds to amino acid residues 21 to 621 of SEQ ID NO:8.

[0163] 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:9.

[0164] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:8 or SEQ ID NO:8. The preferred mature polypeptide of SEQ ID NO:8 corresponds to amino acid residues 21 to 621 of SEQ ID NO:8.

[0165] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:9 or a fragment thereof.

[0166] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0167] In another aspect, the invertase is derived from SEQ ID NO:8 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:9 by substitution, deletion, or addition of one or more amino acids.

[0168] In some embodiments, the invertase is a variant of the parental invertase (preferably SEQ ID NO:9) that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO:9, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:9 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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 (such as a polyhistidine segment, an epitope, or a binding module).

[0169] In one aspect, the invertase is selected from the group consisting of:

[0170] (a) a polypeptide having 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 with SEQ ID NO:11;

[0171] (b) a polypeptide having 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 with SEQ ID NO:12;

[0172] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:11;

[0173] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:10 or its cDNA sequence;

[0174] (e) a polypeptide derived from SEQ ID NO:11, the mature polypeptide of SEQ ID NO:11, or the polypeptide of SEQ ID NO:12 by substitution, deletion, or addition of one or several amino acids;

[0175] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0176] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[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 with the mature polypeptide of SEQ ID NO:1 or SEQ ID NO:11. The preferred mature polypeptide of SEQ ID NO:11 corresponds to amino acid residues 19 to 624 of SEQ ID NO:11.

[0178] 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 with SEQ ID NO:12.

[0179] In a preferred embodiment, the invertase comprises the mature polypeptide of SEQ ID NO:11 or SEQ ID NO:11, consists essentially of it, or consists of it. The preferred mature polypeptide of SEQ ID NO:11 corresponds to amino acid residues 19 to 624 of SEQ ID NO:11.

[0180] In a preferred embodiment, the invertase comprises SEQ ID NO:12 or a fragment thereof, consists essentially of it, or consists of it.

[0181] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0182] In another aspect, the invertase is derived from SEQ ID NO:11 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:21 by substitution, deletion, or addition of one or more amino acids.

[0183] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:12) that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO:12, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:12 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module).

[0184] In one aspect, the invertase is selected from the group consisting of:

[0185] (a) a polypeptide having 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 with SEQ ID NO:14;

[0186] (b) a polypeptide having 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 with SEQ ID NO:15;

[0187] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:14;

[0188] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:13 or its cDNA sequence;

[0189] (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;

[0190] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0191] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0192] 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 with 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 19 to 621 of SEQ ID NO:14.

[0193] 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 with SEQ ID NO:15.

[0194] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:14 or SEQ ID NO:14. The mature polypeptide of preferred SEQ ID NO:14 corresponds to amino acid residues 19 to 621 of SEQ ID NO:14.

[0195] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:15 or a fragment thereof.

[0196] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0197] In another aspect, the invertase is derived from SEQ ID NO:14 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:15 by substitution, deletion, or addition of one or more amino acids.

[0198] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:15) that contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO:15, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:15 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module).

[0199] In one aspect, the invertase is selected from the group consisting of:

[0200] A polypeptide having 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 with SEQ ID NO:17;

[0201] A polypeptide having 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 with SEQ ID NO:18;

[0202] A polypeptide having 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 with the mature polypeptide of SEQ ID NO:17;

[0203] A polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:16 or its cDNA sequence;

[0204] 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;

[0205] A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0206] A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0207] 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 with 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 16 to 619 of SEQ ID NO:17.

[0208] 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 with SEQ ID NO:18.

[0209] 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 16 to 619 of SEQ ID NO:17.

[0210] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:18 or a fragment thereof.

[0211] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0212] In another aspect, the invertase is derived from SEQ ID NO:17 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:18 by substitution, deletion, or addition of one or more amino acids.

[0213] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO: 18), which variant contains substitutions, deletions, and / or insertions at one or more positions. In one aspect, the invertase is a variant of SEQ ID NO: 18, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 18 is up to 15, such as 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 the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module.

[0214] In one aspect, the invertase is selected from the group consisting of:

[0215] (a) a polypeptide having 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 with SEQ ID NO: 20;

[0216] (b) a polypeptide having 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 with SEQ ID NO: 21;

[0217] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO: 20;

[0218] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO: 19 or its cDNA sequence;

[0219] (e) Derived from SEQ ID NO:20, the mature polypeptide of SEQ ID NO:20, or the polypeptide of SEQ ID NO:21 by substitution, deletion, or addition of one or several amino acids;

[0220] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0221] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0222] 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 with SEQ ID NO:20 or the mature polypeptide of SEQ ID NO:20. The preferred mature polypeptide of SEQ ID NO:20 corresponds to amino acid residues 18 to 620 of SEQ ID NO:20.

[0223] 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 with SEQ ID NO:21.

[0224] 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. The preferred mature polypeptide of SEQ ID NO:20 corresponds to amino acid residues 18 to 620 of SEQ ID NO:20.

[0225] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:21 or a fragment thereof.

[0226] The invertase may have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0227] In another aspect, the invertase is derived from SEQ ID NO:20 by substitution, deletion or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO210 by substitution, deletion or addition of one or more amino acids.

[0228] In some embodiments, the invertase is a variant of the parental invertase (preferably SEQ ID NO:21) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a conservative nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as the methionine residue 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 (such as a polyhistidine segment, an epitope, or a binding module).

[0229] In one aspect, the invertase is selected from the group consisting of:

[0230] (a) a polypeptide having 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 with SEQ ID NO:23;

[0231] (b) a polypeptide having 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 with SEQ ID NO:24;

[0232] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:23;

[0233] (d) a polypeptide encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:22 or its cDNA sequence;

[0234] (e) a polypeptide derived from SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or the polypeptide of SEQ ID NO:24 by substitution, deletion, or addition of one or more amino acids;

[0235] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0236] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0237] 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:23 or the mature polypeptide of SEQ ID NO:23. The preferred mature polypeptide of SEQ ID NO:23 corresponds to amino acid residues 18 to 623 of SEQ ID NO:23.

[0238] 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.

[0239] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:23 or SEQ ID NO:23. The preferred mature polypeptide of SEQ ID NO:23 corresponds to amino acid residues 18 to 623 of SEQ ID NO:23.

[0240] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:24 or a fragment thereof.

[0241] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0242] In another aspect, the invertase is derived from SEQ ID NO:23 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:24 by substitution, deletion, or addition of one or more amino acids.

[0243] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:24) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module).

[0244] In one aspect, the invertase is selected from the group consisting of:

[0245] (a) a polypeptide having 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:26;

[0246] (b) a polypeptide having 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:27;

[0247] (c) a polypeptide having 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 the mature polypeptide of SEQ ID NO:26;

[0248] (d) a polypeptide encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:25 or its cDNA sequence;

[0249] (e) a polypeptide derived from the mature polypeptide of SEQ ID NO:36, SEQ ID NO:26, or the polypeptide of SEQ ID NO:27 by substitution, deletion, or addition of one or several amino acids;

[0250] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0251] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0252] 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 the mature polypeptide of SEQ ID NO:26 or SEQ ID NO:36. The preferred mature polypeptide of SEQ ID NO:26 corresponds to amino acid residues 18 to 621 of SEQ ID NO:26.

[0253] 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:27.

[0254] In a preferred embodiment, the invertase comprises the mature polypeptide of SEQ ID NO:26 or SEQ ID NO:26, consists essentially of the mature polypeptide of SEQ ID NO:26, or consists of the mature polypeptide of SEQ ID NO:26. The preferred mature polypeptide of SEQ ID NO:26 corresponds to amino acid residues 18 to 621 of SEQ ID NO:26.

[0255] In a preferred embodiment, the invertase comprises SEQ ID NO:27 or a fragment thereof, consists essentially of SEQ ID NO:27 or a fragment thereof, or consists of SEQ ID NO:27 or a fragment thereof.

[0256] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1-5 amino acids).

[0257] In another aspect, the invertase is derived from SEQ ID NO:26 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:27 by substitution, deletion, or addition of one or more amino acids.

[0258] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:27) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a conservative nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module).

[0259] In one aspect, the invertase is selected from the group consisting of:

[0260] (a) a polypeptide having 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:29;

[0261] (b) a polypeptide having 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 with SEQ ID NO:30;

[0262] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:29;

[0263] (d) a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:28 or its cDNA sequence;

[0264] (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;

[0265] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0266] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0267] 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 with SEQ ID NO:29 or the mature polypeptide of SEQ ID NO:29. The preferred mature polypeptide of SEQ ID NO:29 corresponds to amino acid residues 24 to 637 of SEQ ID NO:29.

[0268] 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 with SEQ ID NO:30.

[0269] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:29 or SEQ ID NO:29. The preferred mature polypeptide of SEQ ID NO:29 corresponds to amino acid residues 24 to 637 of SEQ ID NO:29.

[0270] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:30 or a fragment thereof.

[0271] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0272] In another aspect, the invertase is derived from SEQ ID NO:29 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:30 by substitution, deletion, or addition of one or more amino acids.

[0273] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:30) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid alterations can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal 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 segment, an epitope, or a binding module).

[0274] In one aspect, the invertase is selected from the group consisting of:

[0275] a polypeptide having 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 with SEQ ID NO:32;

[0276] a polypeptide having 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 with SEQ ID NO:33;

[0277] a polypeptide having 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 with the mature polypeptide of SEQ ID NO:32;

[0278] a polypeptide encoded by a polynucleotide having 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 with the mature polypeptide coding sequence of SEQ ID NO:31 or its cDNA sequence;

[0279] 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;

[0280] a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0281] a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0282] 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 with SEQ ID NO:32 or the mature polypeptide of SEQ ID NO:32. The preferred mature polypeptide of SEQ ID NO:32 corresponds to amino acid residues 23 to 626 of SEQ ID NO:32.

[0283] 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 with SEQ ID NO:33.

[0284] 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. The preferred mature polypeptide of SEQ ID NO:32 corresponds to amino acid residues 23 to 626 of SEQ ID NO:32.

[0285] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:42 or a fragment thereof.

[0286] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0287] In another aspect, the invertase is derived from SEQ ID NO:32 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:33 by substitution, deletion, or addition of one or more amino acids.

[0288] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:33) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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, such as a polyhistidine segment, an epitope, or a binding module.

[0289] In one aspect, the invertase is selected from the group consisting of:

[0290] (a) a polypeptide having 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:35;

[0291] (b) a polypeptide having 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;

[0292] (c) a polypeptide having 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 the mature polypeptide of SEQ ID NO:35;

[0293] (d) a polypeptide encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:34 or its cDNA sequence;

[0294] (e) A polypeptide derived from SEQ ID NO:35, the mature polypeptide of SEQ ID NO:35, or the polypeptide of SEQ ID NO:36 by substitution, deletion, or addition of one or several amino acids;

[0295] (f) A polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0296] (g) A fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0297] 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 with SEQ ID NO:35 or the mature polypeptide of SEQ ID NO:35. The preferred mature polypeptide of SEQ ID NO:35 corresponds to amino acid residues 16 to 618 of SEQ ID NO:35.

[0298] 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 with SEQ ID NO:36.

[0299] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:35 or SEQ ID NO:35. The preferred mature polypeptide of SEQ ID NO:35 corresponds to amino acid residues 16 to 618 of SEQ ID NO:35.

[0300] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:36 or a fragment thereof.

[0301] The invertase may have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0302] In another aspect, the invertase is derived from SEQ ID NO:35 by substitution, deletion or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:36 by substitution, deletion or addition of one or more amino acids.

[0303] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:36) that contains 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, such as 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 the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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, such as a polyhistidine segment, an epitope, or a binding module.

[0304] In one aspect, the invertase is selected from the group consisting of:

[0305] (a) a polypeptide having 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 with SEQ ID NO:38;

[0306] (b) a polypeptide having 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 with SEQ ID NO:39;

[0307] (c) a polypeptide having 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 with the mature polypeptide of SEQ ID NO:38;

[0308] (d) a polypeptide encoded by a polynucleotide having 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 the mature polypeptide coding sequence of SEQ ID NO:37 or its cDNA sequence;

[0309] (e) a polypeptide derived from SEQ ID NO:38, the mature polypeptide of SEQ ID NO:38, or the polypeptide of SEQ ID NO:39 by substitution, deletion, or addition of one or several amino acids;

[0310] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d), or (e), wherein the N-terminus and / or C-terminus has been extended by addition of one or more amino acids; and

[0311] (g) a fragment of the polypeptide of (a), (b), (c), (d), or (e); wherein the polypeptide has β-fructofuranosidase activity.

[0312] 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:38 or the mature polypeptide of SEQ ID NO:38. The preferred mature polypeptide of SEQ ID NO:38 corresponds to amino acid residues 20 to 653 of SEQ ID NO:38.

[0313] 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.

[0314] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:38 or SEQ ID NO:38. The preferred mature polypeptide of SEQ ID NO:38 corresponds to amino acid residues 20 to 653 of SEQ ID NO:38.

[0315] In a preferred embodiment, the invertase comprises, consists essentially of, or consists of SEQ ID NO:39 or a fragment thereof.

[0316] The invertase may have an N-terminal and / or C-terminal extension of one or more amino acids (e.g., 1 - 5 amino acids).

[0317] In another aspect, the invertase is derived from SEQ ID NO:38 by substitution, deletion, or addition of one or several amino acids. In another aspect, 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 aspect, the polypeptide is derived from SEQ ID NO:39 by substitution, deletion, or addition of one or more amino acids.

[0318] In some embodiments, the invertase is a variant of a parental invertase (preferably SEQ ID NO:39) that contains 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can have a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1 - 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as a methionine residue 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 (such as a polyhistidine segment, an epitope, or a binding module).

[0319] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at each residue in the molecule, and the β-fructofuranosidase activity of the resulting molecule is tested to identify the amino acid residues critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutagenesis of putative contact-site amino acids. See, for example, 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. The identity of essential amino acids can also be inferred from alignment with related polypeptides, and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a polypeptide or protein family that share a common ancestor (typically having similar three-dimensional structures, functions, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0320] Using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by 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; US 5,223,409; WO 92 / 06204), and site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0321] The mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of the mutagenized polypeptides cloned and expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenized DNA molecules encoding the active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the polypeptide.

[0322] The oral care composition of the present invention can contain any effective amount or concentration of invertase. 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.

[0323] In a preferred embodiment, the oral care composition contains at least 1 ppm, such as 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 of invertase.

[0324] 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.

[0325] Invertase prevents the formation of oral biofilm. Preferably, invertase has an improved effect on oral biofilm prevention. In an embodiment, invertase prevents the formation of oral biofilm by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%. For the purposes of the present invention, oral biofilm prevention can be determined, for example, according to Example 4 below.

[0326] Invertase reduces the risk of oral biofilm formation. Preferably, 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%.

[0327] Invertase can also remove oral biofilm. Preferably, invertase has an improved effect on oral biofilm removal. In an embodiment, invertase removes at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the oral biofilm.

[0328] Invertase is highly stable in formulations and / or forms suitable for oral care (especially in formulations or forms such as toothpaste, mouthwash, lozenges, mints, chewing gum, confectionery, etc.). High stability, for example, comparable or improved stability, can be comparable or improved physical and / or chemical stability. When invertase is co-formulated and / or co-administered with another agent, preferably during co-formulation, comparable or improved chemical stability can occur, i.e., comparable or improved stability in the presence of another agent (such as another enzyme, active ingredient, excipient, or solvent).

[0329] In a preferred embodiment, the invertase has comparable or improved thermal stability. In the context of the present invention, the term "comparable thermal stability" means that in the presence of a specific oral care ingredient or component (or alternatively stated as formulated with a specific oral care ingredient or component), the thermal stability of the invertase is within + / - 5% of the thermal stability of the same invertase alone (i.e., in the absence of the oral care ingredient). In the context of the present invention, the term "improved thermal stability" means that the thermal stability of the invertase 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 higher, compared to the thermal stability of the same invertase alone (i.e., in the absence of the oral care ingredient), in the presence of a specific oral care ingredient or component (or alternatively stated as formulated with a specific oral care ingredient or component). For the purposes of the present invention, thermal stability can be determined according to Example 3 below and is defined by the midpoint of the thermal unfolding transition (Tm).

[0330] In one embodiment, the invertase has comparable or improved thermal stability in the presence of at least one, such as 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: benzoates (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphates (preferably sodium phosphate), SDS, sorbates (preferably potassium sorbate), and sorbitol.

[0331] In a preferred embodiment, the invertase has comparable or improved thermal stability at pH 4 - 8, such as at pH 4, 5, 6, 7, or 8. Preferably, the invertase has comparable or improved thermal stability at pH 5 - 7, more preferably at pH 5 - 6, and most preferably at pH 5 and / or pH 6.

[0332] In one embodiment, the oral care composition comprises a benzoate (such as sodium benzoate), and in the presence of the benzoate (such as sodium benzoate), the invertase has comparable or improved thermal stability. Preferably, the invertase has comparable or improved thermal stability in the presence of 0.01% - 5% benzoate (such as 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 comparable or improved thermal stability in the presence of 1 - 100 mM benzoate (such as sodium benzoate), more preferably 5 - 50 mM benzoate, and most preferably 10 - 35 mM benzoate.

[0333] In one embodiment, the oral care composition comprises EDTA, and in the presence of EDTA, the invertase has comparable or improved thermal stability. Preferably, in the presence of 0.1 - 10 mM EDTA, more preferably 0.5 - 5 mM EDTA, and most preferably 1 mM EDTA, the invertase has comparable or improved thermal stability.

[0334] In one embodiment, the oral care composition comprises ethanol, and in the presence of ethanol, the invertase has comparable or improved thermal stability. Preferably, 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, the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 100000 mM ethanol, more preferably 100 - 10000 mM ethanol, and most preferably 1000 mM ethanol, the invertase has comparable or improved thermal stability.

[0335] In one embodiment, the oral care composition comprises fluoride (such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), and in the presence of fluoride (such as sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 5000 ppm fluoride (such as sodium fluoride), more preferably 500 - 2500 ppm fluoride, and most preferably 1000 - 1500 ppm fluoride, the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 100 mM fluoride (such as sodium fluoride), more preferably 5 - 75 mM fluoride, even more preferably 10 - 50 mM fluoride, and most preferably 20 - 40 mM fluoride, the invertase has comparable or improved thermal stability.

[0336] In one embodiment, the oral care composition comprises glycerol, and in the presence of glycerol, the invertase has comparable or improved thermal stability. Preferably, in the presence of 1% - 50% glycerol, more preferably 5% - 40% glycerol, and most preferably 10% - 30% glycerol, the invertase has comparable or improved thermal stability. Preferably, 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, the invertase has comparable or improved thermal stability.

[0337] In one embodiment, the oral care composition comprises a peroxide (such as hydrogen peroxide), and in the presence of the peroxide (such as hydrogen peroxide), the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 1000 mM peroxide, more preferably 50 - 750 mM peroxide, and most preferably 100 - 500 mM peroxide, the invertase has comparable or improved thermal stability.

[0338] In one embodiment, the oral care composition comprises mannitol, and in the presence of mannitol, the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 1000 mM mannitol, more preferably 150 - 750 mM mannitol, and most preferably 250 - 550 mM mannitol, the invertase has comparable or improved thermal stability.

[0339] In one embodiment, the oral care composition comprises a phosphate (such as sodium phosphate or potassium phosphate), and in the presence of the phosphate (such as sodium phosphate or potassium phosphate), the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 50 mM phosphate (such as sodium phosphate), more preferably 2.5 - 25 mM phosphate, and even more preferably 5 - 10 mM phosphate, the invertase has comparable or improved thermal stability.

[0340] In one embodiment, the oral care composition comprises sodium dodecyl sulfate (SDS), and in the presence of SDS, the invertase has comparable or improved thermal stability. Preferably, in the presence of 10 - 50 mM SDS, more preferably 15 - 25 mM SDS, and most preferably 17 mM SDS, the invertase has comparable or improved thermal stability.

[0341] In one embodiment, the oral care composition comprises a sorbate (such as sodium sorbate, potassium sorbate, or calcium sorbate), and in the presence of the sorbate (such as sodium sorbate, potassium sorbate, or calcium sorbate), the invertase has comparable or improved thermal stability. Preferably, in the presence of 0.01% - 5% sorbate (such as potassium sorbate), more preferably 0.05% - 2.5% sorbate, even more preferably 0.1% - 1% sorbate, and most preferably 0.1% - 0.5% sorbate, the invertase has comparable or improved thermal stability. Preferably, in the presence of 1 - 100 mM sorbate (such as potassium sorbate), more preferably 5 - 75 mM sorbate, even more preferably 7.5 - 50 mM sorbate, and most preferably 10 - 35 mM sorbate, the invertase has comparable or improved thermal stability.

[0342] In one embodiment, the oral care composition comprises sorbitol and, in the presence of sorbitol, the invertase has comparable or improved thermal stability. Preferably, 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, the invertase has comparable or improved thermal stability. Preferably, 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, the invertase has comparable or improved thermal stability.

[0343] Source of invertase

[0344] The polypeptide having β-fructofuranosidase activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain into which the polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0345] In one aspect, the polypeptide is a polypeptide obtained from Bipolaris sorokiniana.

[0346] In one aspect, the polypeptide is a polypeptide obtained from Aspergillus aculeatus.

[0347] In one aspect, the polypeptide is a polypeptide obtained from Pestalotiopsis versicolor.

[0348] In one aspect, the polypeptide is a polypeptide obtained from Aspergillus avenae.

[0349] In one aspect, the polypeptide is a polypeptide obtained from Aspergillus sclerotiorum.

[0350] In one aspect, the polypeptide is a polypeptide obtained from Fusarium avenaceum.

[0351] In one aspect, the polypeptide is a polypeptide obtained from Penicillium coprophilum.

[0352] In one aspect, the polypeptide is a polypeptide obtained from Penicillium murcianum.

[0353] In one aspect, the polypeptide is a polypeptide obtained from Curvularia spicifera.

[0354] In one aspect, the polypeptide is a polypeptide obtained from Alternaria species.

[0355] In one aspect, the polypeptide is a polypeptide obtained from Fusarium temperatum.

[0356] In one aspect, the polypeptide is a polypeptide obtained from Aspergillus japonicus.

[0357] It should be understood that for the foregoing species, the present invention encompasses the perfect and imperfect stages as well as other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0358] The above-mentioned probes can be used to identify and obtain polypeptides from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples directly obtained from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Then, polynucleotides encoding the polypeptide can be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide has been detected with the probe, the polynucleotide can be isolated or cloned by using techniques known to those of ordinary skill in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0359] Polynucleotide

[0360] The present invention also relates to polynucleotides encoding the polypeptides of the present invention, as described herein.

[0361] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof. The polynucleotide can be cloned from strains of the genus Bipolaris, Aspergillus, Pestalotiopsis, Fusarium, Penicillium, Curvularia, Alternaria or related organisms, and thus, for example, can be a polynucleotide sequence encoding a variant of the polypeptide of the present invention.

[0362] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from Bipolaris cells, preferably Bipolaris sorokiniana cells.

[0363] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from Aspergillus cells, preferably Aspergillus aculeatus cells, Aspergillus avenaceus cells, Aspergillus sclerotiorum cells or Aspergillus japonicus cells.

[0364] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from Pestalotiopsis cells, preferably Pestalotiopsis versicolor cells.

[0365] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Fusarium cell, preferably a Fusarium avenaceum cell or a Fusarium temperatum cell.

[0366] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Penicillium cell, preferably a Penicillium coprophilum cell or a Penicillium murcianum cell.

[0367] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Curvularia cell, preferably a Curvularia lunata cell.

[0368] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from an Alternaria cell, preferably an Alternaria species cell.

[0369] These polynucleotides can also be constructed by introducing nucleotide substitutions that do not result in changes in the amino acid sequence of the polypeptide but correspond to the codon usage of the host organism intended for production of the enzyme, or can be mutated by introducing nucleotide substitutions that may result in different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0370] In one aspect, the polynucleotide is isolated.

[0371] In another aspect, the polynucleotide is purified.

[0372] Nucleic acid construct

[0373] The present invention also relates to a nucleic acid construct comprising the polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0374] The polynucleotide can be manipulated in a variety of ways to provide for the expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0375] Promoter

[0376] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell for expression of the polynucleotide encoding the polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that shows transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0377] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York; Davis et al., 2012, ibid.; and Song et al., 2016, PLOS One 11(7):e0158447.

[0378] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in filamentous fungal host cells are promoters obtained from cells of the genera Aspergillus, Fusarium, Rhizomucor, and Trichoderma, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0379] Examples of useful promoters for expression in yeast hosts are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae) and Schmoll and 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0380] Terminator

[0381] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0382] Preferred terminators for bacterial host cells can be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0383] Preferred terminators for filamentous fungal host cells are obtainable from Aspergillus or Trichoderma species, such as from the genes for Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase 1, and Trichoderma reesei endoglucanase 1, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications,” and Schmoll et al., 2014. 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0384] Preferred terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0385] mRNA stabilizer

[0386] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0387] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0388] Examples of mRNA stabilizer regions of fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0389] Leader sequence

[0390] The control sequence can also be a leader sequence, i.e., an mRNA untranslated region that is important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

[0391] Suitable leader sequences for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12):3051-3059 and Kaberdin and 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0392] Preferred leader sequences for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0393] Suitable leader sequences for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0394] Polyadenylation sequence

[0395] The control sequence can also be a polyadenylation sequence, i.e., a sequence operably linked to the 3'-end of the polynucleotide that is recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0396] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from genes of the following: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0397] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0398] Signal peptide

[0399] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the polypeptide and directs the polypeptide into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may itself contain a signal peptide coding sequence that is naturally linked to the coding sequence segment of the polypeptide to be encoded in the translation reading frame. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, the heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence in order to enhance the secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the host cell may be used.

[0400] Effective signal peptide coding sequences for bacterial host cells are signal peptide coding sequences obtained from genes of the following: Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0401] Effective signal peptide coding sequences for filamentous fungal host cells are signal peptide coding sequences obtained from genes of the following: Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic protease, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0402] Useful signal peptides of yeast host cells are obtained from the genes of the following: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, ibid.

[0403] Propeptide

[0404] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or pro-polypeptide (or in some cases is called a zymogen). Pro-polypeptides are usually inactive and can be converted into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the pro-polypeptide. Propeptide coding sequences can be obtained from the genes of the following: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease, and Saccharomyces cerevisiae α-factor.

[0405] In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both a signal peptide sequence and a propeptide sequence are present, the polypeptide can contain only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide can contain a mixture of the mature polypeptide and a polypeptide containing a partial or full-length propeptide sequence and / or signal peptide sequence.

[0406] Regulatory sequence

[0407] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides related to host cell growth. Examples of regulatory sequences are those that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 system or GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein gene amplified with heavy metals.

[0408] Transcription factor

[0409] The control sequence may also be a transcription factor, i.e., a polynucleotide encoding a polypeptide that specifically binds DNA, which controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can act alone and / or in combination with one or more other polypeptides or transcription factors in a complex to act by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can directly (i.e., by binding to its promoter to activate the transcription of a gene encoding a protein of interest) or indirectly (i.e., such as by binding to the promoter of another transcription factor that regulates the transcription of a gene encoding a protein of interest to activate the transcription of the other transcription factor) regulate the expression of a protein of interest. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0410] Expression vector

[0411] The present invention also relates to a recombinant expression vector comprising the polynucleotide, promoter, and transcription and translation termination signals of the present invention. The polynucleotides and control sequences can be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into an appropriate vector for expression. In producing the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

[0412] The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and can cause the expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or a closed circular plasmid.

[0413] The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that integrates into the genome when introduced into a host cell and replicates with the chromosome into which it has been integrated. Moreover, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell can be used, or a transposon can be used.

[0414] The vector preferably contains one or more selectable markers that allow for the convenient selection of cells such as transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide resistance or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, etc.

[0415] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0416] For integration into the genome of the host cell, the vector can rely on a polynucleotide sequence encoding a polypeptide or any other element of the vector for integration into the genome by homologous recombination (such as homologous directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0417] For autonomous replication, the vector can further contain an origin of replication that enables the vector to replicate autonomously in the host cell under discussion. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The term "origin of replication" or "plasmid replicon" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0418] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of the polypeptide. For example, 2 or 3 or 4 or 5 or more copies can be inserted into the host cell. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene together with the polynucleotide, where cells containing the amplified copy of the selectable marker gene and thus the additional copy of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective reagent.

[0419] Host cell

[0420] The present invention also relates to recombinant host cells that contain the polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the polypeptide of the present invention.

[0421] A construct or vector comprising a polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as a self - replicating extra - chromosomal vector, as described earlier. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. In addition, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell can contain a single copy or at least two copies of the polynucleotide of the present invention, such as three, four, five or more copies.

[0422] The host cell can be any microbial cell useful for the recombinant production of the polypeptide of the present invention, such as a prokaryotic cell or a fungal cell.

[0423] Prokaryotic host cells can be any Gram - positive or Gram - negative bacterium. Gram - positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram - negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0424] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In the examples, the Bacillus cells are Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cells.

[0425] For the purposes of the present invention, Bacillus species / genus / species should be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 70: 406-438.

[0426] The bacterial host cell can also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

[0427] The bacterial host cell can also be any Streptomyces cell, including but not limited to: Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0428] Methods for introducing DNA into prokaryotic host cells are well known in the art and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. A person skilled in the art will be able to readily determine a suitable method for introducing DNA into a given prokaryotic cell depending, for example, on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294, Choi et al., 2006, J. Microbiol. Methods 64:391-397, and Donald et al., 2013, J. Bacteriol. 195(11):2612-2620.

[0429] The host cell can be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the Oomycota and all mitosporic fungi (such as defined by Hawksworth et al. in Ainsworth and Bisby’s Dictionary of The Fungi, 8th Edition, 1995, CAB International, University Press, Cambridge, UK).

[0430] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (as reviewed in Li et al., 2017, Microbial Cell Factories 16:168) and the procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474; Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as a linearized or circular polynucleotide.

[0431] The fungal host cell can be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). For the purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0432] The yeast host cell can be a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as Pichia pastoris (Komagataella phaffii) cell.

[0433] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeast (such as Saccharomyces cerevisiae) is by budding of single cell thalli and carbon catabolism can be fermentative.

[0434] The filamentous fungal host cell may be a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is a cell of Aspergillus, Trichoderma or Fusarium. In another preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei or Fusarium venenatum cell.

[0435] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, FusariumSarcochroum), Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Rhizomucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cells.

[0436] In one aspect, the host cell is isolated.

[0437] In another aspect, the host cell is purified.

[0438] Production method

[0439] The present invention also relates to methods for producing the polypeptides of the present invention, which methods comprise (a) culturing a cell that produces the polypeptide in its wild-type form under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0440] In one aspect, the cell is a Bipolaris cell, preferably a Bipolaris sorokiniana cell.

[0441] In one aspect, the cell is an Aspergillus cell, preferably an Aspergillus aculeatus cell, an Aspergillus avenaceus cell, an Aspergillus sclerotiorum cell or an Aspergillus japonicus cell.

[0442] In one aspect, the cell is a Pestalotiopsis cell, preferably a Pestalotiopsis weirii cell.

[0443] In one aspect, the cell is a Fusarium cell, preferably a Fusarium avenaceum cell or a Fusarium temperatum cell.

[0444] In one aspect, the cell is a Penicillium cell, preferably a Penicillium coprophilum cell or a Penicillium murcianum cell.

[0445] In one aspect, the cell is a Curvularia cell, preferably a Curvularia spicifera cell.

[0446] In one aspect, the cell is an Alternaria cell, preferably an Alternaria species cell.

[0447] The present invention also relates to methods for producing the polypeptides of the present invention, which methods comprise (a) culturing the recombinant host cells of the present invention under conditions conducive to the production of the polypeptide; and optionally (b) recovering the polypeptide.

[0448] In one aspect, the recombinant host cell is a Bacillus subtilis cell.

[0449] In one aspect, the recombinant host cell is a Bacillus licheniformis cell.

[0450] In one aspect, the recombinant host cell is an Aspergillus niger cell.

[0451] In one aspect, the recombinant host cell is an Aspergillus oryzae cell.

[0452] In one aspect, the recombinant host cell is a Trichoderma reesei cell.

[0453] In one aspect, the recombinant host cell is a Pichia pastoris (Komagataella phaffii) cell.

[0454] The host cells are cultured in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid-state and / or microcarrier-based fermentations) in a suitable medium and under conditions that permit the expression and / or isolation of the polypeptide. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0455] Polypeptides can be detected using methods specific for the polypeptide known in the art, including but not limited to the use of specific antibodies, enzyme product formation, enzyme substrate disappearance, or assays that measure the relative or specific activity of the polypeptide.

[0456] Polypeptides can be recovered from the medium using methods known in the art, which methods include but are not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0457] Polypeptides can be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, for example, Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0458] In an alternative aspect, the polypeptide is not recovered.

[0459] Invertase particles

[0460] The present invention also relates to enzyme particles comprising the polypeptides of the present invention. In embodiments, the particles comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0461] The diameter of the core (measured as the equivalent spherical diameter (volume-based average particle size)) can be 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm. The core diameter measured as the equivalent spherical diameter can be determined using laser diffraction such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0462] In embodiments, the core comprises a polypeptide having the β-fructofuranosidase activity of the present invention.

[0463] The core can include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity regulators, light spheres, plasticizers, salts, lubricants and fragrances.

[0464] The core can include binders, such as synthetic polymers, waxes, fats or carbohydrates.

[0465] The core typically can include, as a homogeneous blend, salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components.

[0466] The core can contain inert particles onto which the polypeptide is adsorbed within the inert particles or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0467] The diameter of the core can be 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.

[0468] The core can be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during processing or for coloring the granules. Optional coatings can include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC) and polyvinyl alcohol (PVA).

[0469] The coating can be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10% or at least 15%) by weight of the core. The amount can be at most 100%, 70%, 50%, 40% or 30%.

[0470] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm or at least 5 μm thick. In some embodiments, the thickness of the coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0471] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating having very few or no holes, such that the core unit has very few or no uncoated areas. The layer or coating should be uniform in thickness.

[0472] The coating can further contain other materials known in the art, such as fillers, anti-adhesives, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0473] The salt coating can contain at least 60% by weight of salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

[0474] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm or at least 8 μm. In particular embodiments, the thickness of the salt coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0475] The salt can be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the fine particles are less than 50 μm, e.g., less than 10 μm or less than 5 μm.

[0476] The salt coating can contain a single salt or a mixture of two or more salts. The salt can be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20 °C, preferably at least 0.5 g / 100 g of water, e.g., at least 1 g / 100 g of water, e.g., at least 5 g / 100 g of water.

[0477] The salt can be an inorganic salt such as a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or a salt of a simple organic acid (less than 10 carbon atoms, such as 6 or fewer carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions or metal ions of the first transition series such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0478] The salt in the coating may have a constant humidity of more than 60%, especially more than 70%, more than 80% or more than 85% at 20 °C, or it may be another hydrate form (e.g., anhydrous) of such a salt. The salt coating can be as described in WO00 / 01793 or WO 2006 / 034710.

[0479] Specific examples of suitable salts are NaCl (CH 20 °C = 76%), Na2CO3 (CH 20 °C = 92%), NaNO3 (CH 20 °C = 73%), Na2HPO4 (CH 20 °C = 95%), Na3PO4 (CH 25 °C = 92%), NH4Cl (CH 20 °C = 79.5%), (NH4)2HPO4 (CH 20 °C = 93.0%), NH4H2PO4 (CH 20 °C = 93.1%), (NH4)2SO4 (CH 20 °C = 81.1%), KCl (CH 20 °C = 85%), K2HPO4 (CH 20 °C = 92%), KH2PO4 (CH 20 °C = 96.5%), KNO3 (CH 20 °C = 93.5%), Na2SO4 (CH 20 °C = 93%), K2SO4 (CH 20 °C = 98%), KHSO4 (CH 20°C = 86%), MgSO4(CH 20 °C = 90%), ZnSO4(CH 20 °C = 90%) and sodium citrate (CH 25 °C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2 and magnesium acetate.

[0480] The salt can be in anhydrous form, or it can be a hydrated salt, i.e., a crystalline salt hydrate having one or more bound waters of crystallization, for example, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4 . 7H2O), zinc sulfate heptahydrate (ZnSO4 . 7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4 . 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0481] Preferably, the salt is used as a salt solution, for example, using a fluidized bed.

[0482] The coating material can be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, where the alcohol contains 12 to 20 carbon atoms and where there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and glycerol monoesters, and glycerol di-esters, and glycerol tri-esters of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1483591.

[0483] The particles can optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0484] The core can be prepared by blending granulation components, for example, by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction methods, drum granulation and / or high shear granulation.

[0485] Methods for preparing cores can be found in the Handbook of Powder Technology; Particle size enlargement by C.E. Capes; Volume 1; 1980; Elsevier. The preparation methods include known feed and granulation techniques, such as:

[0486] (a) Spray-dried products, where a liquid polypeptide solution is atomized in a spray-drying tower to form small droplets, which are dried as they descend in the drying tower to form polypeptide-containing particulate material. In this way, very small particles can be produced (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Volume 71; pages 140-142; Marcel Dekker).

[0487] (b) Layered products, where the polypeptide is coated in layers around preformed inert core particles. Usually, a polypeptide-containing solution is atomized in a fluidized-bed apparatus, where the preformed core particles are fluidized and the polypeptide-containing solution adheres to the core particles and is dried until a dry polypeptide layer remains on the surface of the core particles. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this way. Products of this type are described, for example, in WO 97 / 23606.

[0488] (c) Absorbed core particles, where instead of coating the polypeptide in layers around the core, the polypeptide is absorbed onto and / or into the surface of the core. Such methods are described in WO 97 / 39116.

[0489] (d) Extruded or pelletized products, where a polypeptide-containing paste is pressed into pellets or extruded through small openings under pressure and cut into particles, which are then dried. Such particles usually have a relatively large size, since the material with the extrusion openings (usually a flat plate with drilled holes) limits the pressure drop allowable through the extrusion openings. In addition, when using small openings, very high extrusion pressures increase the heat generation in the polypeptide paste, which is harmful to the polypeptide (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Volume 71; pages 140-142; Marcel Dekker).

[0490] (e) Spray granulation products, where the polypeptide-containing powder is suspended in molten wax and the suspension is sprayed (e.g., through a rotary atomizer) into a cooling chamber where the droplets solidify rapidly (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140 - 142; Marcel Dekker). The resulting product is one where the polypeptide is uniformly distributed throughout the inert material rather than concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

[0491] (f) Mixer granulation products, where the polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and the powder are mixed in a suitable ratio, and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate, and the particles will accumulate to form granules containing the polypeptide. Such methods are described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. The granules composed of polypeptide, filler, binder, etc. are mixed with cellulose fibers to strengthen the particles, thus producing so-called T-granules. The strengthened particles are more robust and release less enzyme dust.

[0492] (g) Size reduction, where cores are produced by milling or crushing larger particles, pellets, tablets, briquettes, etc. containing the polypeptide. The desired core particle fraction is obtained by sieving the milled or crushed product. Oversize and undersize particles can be recycled. Size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0493] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wet and sticky. The sticky particles collide with other particles and attach to them to form granules.

[0494] (i) These cores can be subjected to drying, for example in a fluidized bed dryer. Those skilled in the art can use other known methods for drying granules in the feed or enzyme industry. The drying is preferably carried out at a product temperature of 25 °C to 90 °C. For some polypeptides, it is important that the core containing the polypeptide contains a small amount of water before coating with salt. If a water-sensitive polypeptide is coated with salt before removing the excess water, the excess water will be trapped in the core and may have a negative impact on the activity of the polypeptide. After drying, these cores preferably contain 0.1 - 10% w / w water.

[0495] Dust-free particles can be produced as disclosed, for example, in US 4,106,991 and US 4,661,452, and can optionally be coated by methods known in the art.

[0496] The particles can further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. Then, each enzyme will be present in more particles, ensuring a more uniform distribution of the enzymes and reducing the physical separation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-particles are disclosed in the ip.com disclosure IPCOM000200739D.

[0497] Another example of formulating a polypeptide using co-particles is disclosed in WO 2013 / 188331.

[0498] The invention also relates to a protected polypeptide prepared by the method disclosed in EP 238216.

[0499] Liquid formulations

[0500] The invention also relates to a liquid composition comprising a polypeptide of the invention. The composition can comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borates, or phenylboric acid derivatives such as 4-formylphenylboric acid).

[0501] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, these compositions contain from about 5% to about 90% of such materials.

[0502] In one aspect, the liquid formulation comprises 20% - 80% w / w polyol. In one embodiment, the liquid formulation comprises 0.001% - 2% w / w preservative.

[0503] In another embodiment, the present invention relates to a liquid formulation comprising:

[0504] (A) 0.001% - 25% w / w of a polypeptide having the β - fructofuranosidase activity of the present invention;

[0505] (B) 20% - 80% w / w polyol;

[0506] (C) Optionally 0.001% - 2% w / w preservative; and

[0507] (D) Water.

[0508] In another embodiment, the present invention relates to a liquid formulation comprising:

[0509] (A) 0.001% - 25% w / w of a polypeptide having the β - fructofuranosidase activity of the present invention;

[0510] (B) 0.001% - 2% w / w preservative;

[0511] (C) Optionally 20% - 80% w / w polyol; and

[0512] (D) Water.

[0513] In another embodiment, the liquid formulation comprises one or more formulations, such as formulations selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2 - propylene glycol or 1,3 - propylene glycol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600 and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0514] In another embodiment, the liquid formulation comprises 20% - 80% polyol (i.e., the total amount of polyol), such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol. In one embodiment, the liquid formulation comprises 20% - 80% polyol, such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol, wherein the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2 - propylene glycol or 1,3 - propylene glycol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600 and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% - 80% polyol (i.e., the total amount of polyol), such as 25% - 75% polyol, 30% - 70% polyol, 35% - 65% polyol or 40% - 60% polyol, wherein the polyol is selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG).

[0515] In another embodiment, the preservative is selected from the group consisting of: sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02% - 1.5% w / w preservative, such as 0.05% - 1% w / w preservative or 0.1% - 0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001% - 2% w / w preservative (i.e., the total amount of preservative), such as 0.02% - 1.5% w / w preservative, 0.05% - 1% w / w preservative or 0.1% - 0.5% w / w preservative, wherein the preservative is selected from the group consisting of: sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0516] In another embodiment, the liquid formulation further comprises one or more additional enzymes, for example, hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0517] Oral care ingredients and forms

[0518] The oral care composition of the present invention comprises invertase and at least one oral care ingredient.

[0519] Thus, in one aspect, the present invention relates to an oral care composition comprising invertase selected from the group consisting of:

[0520] a) a polypeptide having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 SEQ ID NO: 3;

[0521] b) a polypeptide having at least 90%, for example, 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 SEQ ID NO: 6;

[0522] c) a polypeptide having at least 65%, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 SEQ ID NO: 9;

[0523] d) a polypeptide having at least 65%, such as at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0524] e) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0525] f) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0526] g) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0527] h) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0528] i) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0529] j) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0530] k) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0531] l) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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; and

[0532] m) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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;

[0533] wherein the polypeptide has β - fructofuranosidase activity and wherein the oral care composition further comprises at least one oral care ingredient.

[0534] Preferably, the invertase is selected from the group consisting of:

[0535] a) a polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3;

[0536] b) a polypeptide comprising SEQ ID NO:6, consisting essentially of SEQ ID NO:6, or consisting of SEQ ID NO:6;

[0537] c) a polypeptide comprising SEQ ID NO:9, consisting essentially of SEQ ID NO:9, or consisting of SEQ ID NO:9;

[0538] d) a polypeptide comprising SEQ ID NO:12, consisting essentially of SEQ ID NO:12, or consisting of SEQ ID NO:12;

[0539] e) a polypeptide comprising SEQ ID NO:15, consisting essentially of SEQ ID NO:15, or consisting of SEQ ID NO:15;

[0540] f) a polypeptide comprising SEQ ID NO:18, consisting essentially of SEQ ID NO:18, or consisting of SEQ ID NO:18;

[0541] g) a polypeptide comprising SEQ ID NO:21, consisting essentially of SEQ ID NO:21, or consisting of SEQ ID NO:21;

[0542] h) a polypeptide comprising SEQ ID NO:24, consisting essentially of SEQ ID NO:24, or consisting of SEQ ID NO:24;

[0543] i) a polypeptide comprising SEQ ID NO:27, consisting essentially of SEQ ID NO:27, or consisting of SEQ ID NO:27;

[0544] j) a polypeptide comprising SEQ ID NO:30, consisting essentially of SEQ ID NO:30, or consisting of SEQ ID NO:30;

[0545] k) a polypeptide comprising SEQ ID NO:33, consisting essentially of SEQ ID NO:33, or consisting of SEQ ID NO:33;

[0546] l) a polypeptide comprising SEQ ID NO:36, consisting essentially of SEQ ID NO:36, or consisting of SEQ ID NO:36; and

[0547] m) a polypeptide comprising SEQ ID NO:39, consisting essentially of SEQ ID NO:39, or consisting of SEQ ID NO:39.

[0548] The oral care ingredients can vary depending on the different types of oral care compositions and the desired characteristics and / or activities of the oral care compositions. For the purposes of the present invention, the terms "ingredient" and "component" can be used interchangeably when referring to oral care compositions.

[0549] The oral care compositions of the present invention can be internal oral care compositions, such as toothpaste or toothpaste tablets, dental cream, mouthwash or mouthwash tablets, oral rinses, lozenges, soft lozenges, chewing gums, confections, candies, etc., which are designed to remove biofilms in the oral cavity, such as biofilms residing on teeth, oral soft tissues, and dentures residing in the oral cavity.

[0550] The oral care compositions of the present invention can also be external oral care compositions, such as denture cleaning solutions, denture cleaning tablets, denture cleaning powders, etc., which are designed to remove biofilms from dentures that have been removed from the oral cavity for cleaning.

[0551] 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, buffers, foaming agents, foam modifiers, sweetening agents, softeners, plasticizers, flavoring agents, colorants, therapeutic agents, anti-microbial agents, tartar control agents, fluoride ion sources, preservatives, detergents, surfactants, colorants, buffers, softeners, plasticizers, whitening agents, bleaching agents, gum base components, and swelling agents.

[0552] Although the oral care ingredients mentioned herein are classified by general headings according to functionality, this is not to be construed as limiting, since as will be understood by those skilled in the art, an ingredient may comprise additional functionality.

[0553] Toothpastes, tooth creams, mouthwashes and oral rinses

[0554] The intraoral care compositions of the present invention are in the form of toothpastes, tooth creams, mouthwashes, and oral rinses, and these intraoral care compositions may include ingredients and / or substances selected from the following categories:

[0555]

[0556]

[0557] Toothpaste

[0558] Toothpastes and tooth creams / teeth gels typically include abrasives, solvents, humectants, detergents / surfactants, thickeners and binders, buffers, flavoring agents, sweetening agents, fluoride ion sources, therapeutic agents, colorants, and preservatives as oral care ingredients.

[0559] In a preferred embodiment, the present invention relates to an oral care composition in the form of a toothpaste or a tooth cream, the oral care compositions comprising invertase and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0560]

[0561]

[0562] The oral care composition of the present invention may be a toothpaste comprising the following ingredients (by weight % of the final toothpaste composition):

[0563] Abrasives: 10% to 70%

[0564] Humectants: 0% to 80%

[0565] Thickeners: 0.1% to 20%

[0566] Binder: 0.01% to 10%

[0567] Sweetener: 0.1% to 5%

[0568] Foaming agent: 0% to 15%

[0569] Enzyme (invertase): 0.01% to 20%

[0570] Mouthwash

[0571] The mouthwash and oral cleansing agent (including plaque removal solution) of the present invention typically include a carrier liquid, a detergent / surfactant, a buffer, a flavoring agent, a humectant, a sweetener, a therapeutic agent, a fluoride ion source, a coloring agent, and a preservative as oral care ingredients.

[0572] In a preferred embodiment, the present invention relates to an oral care composition in the form of a mouthwash or an oral cleansing agent, which oral care compositions contain invertase and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0573]

[0574]

[0575] The oral care composition of the present invention can be a mouthwash containing the following ingredients (by weight% of the final mouthwash composition):

[0576] Water: 0% to 70%

[0577] Ethanol: 0% to 20%

[0578] Humectant: 0% to 20%

[0579] Surfactant: 0% to 2%

[0580] Enzyme (invertase): 0.01% to 20%

[0581] Other ingredients: 0% to 2% (e.g., fragrance, sweetener, fluoride ion source).

[0582] The mouthwash composition can be buffered with a suitable buffer (e.g., sodium citrate or sodium phosphate) in the pH range of 6 - 7.5.

[0583] The relevant oral care components applicable to toothpaste, tooth cream, mouthwash, and oral cleansing agent are further detailed below. Those skilled in the art can change the oral care components according to the type of the oral care composition and the desired characteristics and / or activities of the specific oral care composition. The oral care composition does not necessarily contain all the mentioned ingredients.

[0584] Abrasive

[0585] A polishing abrasive material can be incorporated into the oral care compositions of the present invention. According to the present invention, the polishing abrasive material includes alumina and its hydrates (such as α-aluminum trihydrate), magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates (such as calcined aluminosilicate and aluminosilicate), calcium carbonate, zirconium silicate, bentonite, silica, sodium bicarbonate, and powdered plastics (such as polyvinyl chloride), polyamide, polymethyl methacrylate, polystyrene, phenolic resin, melamine-formaldehyde resin, urea-formaldehyde resin, epoxy resin, powdered polyethylene, silica xerogel, hydrogel, and aerogel, etc.

[0586] Also suitable as abrasives are calcium pyrophosphate, water-insoluble alkali metaphosphates, polyphosphates, dicalcium phosphate and / or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxyapatite, etc. Mixtures of these substances may also be used.

[0587] Various types of silica tooth abrasives are preferred because of their unique benefits of excellent tooth cleaning and polishing performance without excessive enamel or dentin wear, and their good compatibility with other possible ingredients such as metal ions and fluoride.

[0588] Depending on the oral care composition, the abrasive product can be present in an amount of 0% to 70% by weight, preferably 1% to 70%.

[0589] For toothpaste, the abrasive material content is typically in the range of 10% to 70% by weight of the final toothpaste product.

[0590] Humectant

[0591] Humectants are used to prevent water loss from, for example, toothpaste and to avoid hardening of the toothpaste when exposed to air. Some humectants also impart the desired sweetness to toothpaste and mouthwash compositions. Humectants suitable for the oral care compositions according to the present invention include the following compounds and their mixtures: 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

[0592] Humectants are typically present in an amount of 0% to 80% by weight, preferably 5% to 70%.

[0593] Thickener / binder

[0594] Suitable thickeners and / or binders include silica, starch, tragacanth, xanthan gum, karaya gum, carrageenan (extract of Irish moss), gum arabic, alginates, pectin, cellulose derivatives (such as hydroxyethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose and hydroxyethylpropyl cellulose), polyacrylic acid and its salts, polyvinylpyrrolidone and carboxyvinyl polymers, and inorganic thickeners (such as amorphous silica compounds). These agents stabilize the oral care compositions of the present invention.

[0595] The thickener can be present in toothpaste, tooth cream and gels, and mouthwashes in an amount of 0.1% to 20% by weight of the final product, and the binder can be present in a range of 0.01% to 10% by weight of the final product.

[0596] Foaming agent and foam regulator

[0597] As foaming agents, soaps, anionic, cationic, nonionic, amphoteric and / or zwitterionic surfactants can be used alone or in combination. These can 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 only suitable to the extent that it does not inactivate the enzymes and other components contained in the oral care composition. Useful surfactants include anionic, nonionic, and amphoteric compounds, preferably anionic compounds.

[0598] 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; sodium lauryl sulfoacetate, salts of sulfonated monoglycerides of higher fatty acids, such as sodium coconut monoglyceride sulfonate or other suitable sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms; amide salts of higher fatty acids (such as acids having 12 to 16 carbon atoms) with lower aliphatic amino acids, such as sodium N-methyl-N-palmitoyl taurate, sodium N-lauroyl sarcosinate, sodium N-myristoyl sarcosinate, and sodium N-palmitoyl sarcosinate; salts of esters of such fatty acids with isothionic acids or with glycerol monosulfate; for example, the sodium salt of the monosulfate monoglyceride of hydrogenated coconut oil fatty acid; olefin sulfonates, such as olefin sulfonates or mixtures thereof having 12 to 16 carbon atoms in the carbon chain of the molecule; and soaps of higher fatty acids, such as those having 12 to 18 carbon atoms, such as coconut oil fatty acid.

[0599] The cation of the salt can be sodium, potassium, or monoethanolamine, diethanolamine, or triethanolamine. Nonionic surfactants include sucrose / fatty acid esters, maltose / fatty acid esters, maltitol / fatty acid esters, maltotriitol / fatty acid esters, maltotetraitol / fatty acid esters, maltopentaitol / fatty acid esters, maltohexaitol / fatty acid esters, mahoheptaitol / fatty acid esters, sorbitan / fatty acid esters, lactose / fatty acid esters, lactinose / fatty acid esters, polyoxyethylene / polyoxypropylene copolymers, polyoxyethylene alkyl ethers, polyoxyethylene / fatty acid esters, fatty acid alkanolamides, polyoxyethylene sorbitan / fatty acid esters, polyoxyethylene / hydrogenated castor oil, and polyglycerol / fatty acid esters.

[0600] Most preferably, they are sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium lauroyl sarcosinate.

[0601] Preferred foam regulators include polyethylene glycol.

[0602] The foaming agent and the foam regulator can be present in an amount of 0% to 15% by weight, preferably 0.01% to 10% by weight.

[0603] Sweetener

[0604] Suitable sweeteners include, but are not limited to, saccharin and its water-soluble salts, dextrose, sucrose, lactose, maltose, levulose, aspartame, cyclamate, D-tryptophan, dihydrochalcone, acesulphame, stevioside, levaudioside, glycyrrhizin, pellartine, thaumatin, paramethoxycinnamaldehyde, hydrogenated starch hydrolysates, xylitol, sorbitol, erythritol, mannitol, and mixtures thereof.

[0605] The sweetener can be present in an amount of 0.001% to 60% by weight, preferably 0.01% to 50% by weight.

[0606] Flavoring agent

[0607] Flavoring agents are typically present in small amounts, for example, from 0.01% to about 5%, especially 0.1% to 5% by weight. Flavors useful in the present invention include, but are not limited to, wintergreen oil, peppermint oil, spearmint oil, clove flower oil, menthol, anethole, methyl salicylate, cineole, cinnamon, 1-menthyl acetate, sage, eugenol, celery oil, oxanone, α-ionone, marjoram, lemon, orange, cranberry, allyl ethyl guaiacol, cinnamon, vanillin, ethyl vanillin, piperonal, 4-cis-heptenal, diacetyl, methyl p-tert-butylphenylacetate, carvone, eucalyptol, menthone, cinnamaldehyde, limonene, ocimene, n-decanol, citronellol, α-terpineol, methyl acetate, citronellyl acetate, methyl eugenol, linalool, thymol, rosemary oil, pimento oil, diatomaceous oil, eucalyptus oil, and mixtures thereof.

[0608] Cooling agents can also be part of the flavoring system or added separately to the composition. Preferred cooling agents in the compositions of the present invention are p-menthane carboxamido reagents such as N-ethyl-p-menthane-3-carboxamide (commercially known as "WS-3"), menthol, 3-1-menthoxy-1,2-propanediol ("TK-10"), menthone glycerol acetal ("MGA"), menthyl lactate, and mixtures thereof.

[0609] Whitening agent / bleaching agent

[0610] Whitening agents / bleaching agents include H2O2 and can be added in an amount less than 5%, preferably 0.05% to 4% based on the weight of the final composition.

[0611] Other bleaching components that can be included in 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 perborates, persilicates, perphosphates, and percarbonates (such as sodium perborate, potassium persilicate, and sodium percarbonate). Peroxide compounds can be stabilized by adding triphenylmethane dyes, chelating agents, or antioxidants (such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)).

[0612] Solvent

[0613] Solvents are typically added to the compositions of the present invention in an amount sufficient to render the composition in a flowable form in the case of compositions such as toothpaste, tooth cream, or gel, or to dissolve other components of the composition in the case of, for example, mouthwash or oral rinse.

[0614] Suitable solvents include water, ethanol, and water / ethanol mixtures, which can be present in an amount of 0.1% to 70%.

[0615] Antimicrobial agent

[0616] The present invention also includes water-soluble antimicrobial agents such as chlorhexidine, triclosan, digluconates, hexetidine, alexidine, quaternary ammonium antibacterial compounds; and may also include water-soluble sources of certain metal ions such as zinc, copper, silver, and stannous (e.g., zinc chloride, copper chloride, stannous chloride, and silver nitrate).

[0617] Due to the slow dissolution of these zinc salts in saliva, slightly soluble zinc salts such as zinc citrate, zinc C14-alkyl maleate, zinc benzoate, zinc hexanoate, and zinc carbonate may also be included in the compositions of the present invention to prolong the antimicrobial efficacy of zinc ions.

[0618] The antimicrobial agent can 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.

[0619] Dental calculus control agent

[0620] The compositions of the present invention may contain tartar control agents such as inorganic phosphorus tartar control agents, which include any pyrophosphates such as disodium pyrophosphate, dipotassium pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, and mixtures thereof.

[0621] Organic phosphorus compounds that can be used as tartar control agents include polyphosphonates such as disodium ethane-1-hydroxy-1,1-diphosphate (EHDP), methanediphosphonic acid, and 2-phosphonobutane-1,2,4-tricarboxylic acid.

[0622] The tartar control agent can be present in an amount of from 0% to 10% by weight, preferably from 0.1% to 5% by weight.

[0623] Preservative

[0624] Suitable preservatives include sodium benzoate, potassium sorbate, parabens, methyl paraben, ethyl paraben, propyl paraben, citric acid, calcium citrate, and mixtures thereof.

[0625] The preservative can be present in an amount of from 0% to 40% by weight, preferably from 0.01% to 30% by weight.

[0626] Fluoride ion source

[0627] The compositions of the present invention may also contain ingredients that can be used as a source of fluoride ions. Preferred soluble fluoride ion sources include sodium fluoride, potassium fluoride, stannous fluoride, indium fluoride, sodium monofluorophosphate, sodium hexafluorosilicate, zinc fluoride, lithium fluoride, aluminum fluoride, acid fluorophosphates, ammonium bifluoride, titanium tetrafluoride, and amine fluorides.

[0628] Particularly preferred are sodium fluoride and sodium monofluorophosphate.

[0629] The fluoride ion source can 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.

[0630] In a preferred embodiment, at least one oral care ingredient is a fluoride ion source; preferably, the fluoride ion source is selected from the group consisting of sodium fluoride, calcium fluoride, stannous fluoride, or sodium monofluorophosphate.

[0631] Colorant

[0632] Colorants or pigments suitable for the oral care compositions of the present invention include non-toxic water-insoluble inorganic pigments such as titanium dioxide and chromium oxide green, ultramarine blue and pink, and iron oxides, as well as water-insoluble dye lakes prepared by extending calcium or aluminum salts of FD&C dyes on 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 Lake.

[0633] A preferred opacifier is titanium dioxide.

[0634] The colorant can 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.

[0635] Buffer

[0636] The oral care compositions of the present invention may further include buffering agents, i.e., pH regulators, such as alkali metal hydroxides, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazoles, and mixtures thereof.

[0637] Specific buffering agents include monosodium phosphate, trisodium phosphate, sodium hydroxide, potassium hydroxide, alkali metal carbonates, sodium carbonate, imidazole, pyrophosphates, sodium citrate, hydrochloric acid, sodium hydroxide, triethanolamine, triethylamine, lactic acid, malic acid, fumaric acid, tartaric acid, phosphoric acid, and mixtures of these.

[0638] The buffering agent can be present in an amount of from 0% to 10% by weight, preferably from 0.01% to 5% by weight.

[0639] Chewing gum

[0640] When the oral composition according to the present invention is chewing gum, it can be any known type of chewing gum, such as optionally coated chewing gum tablets, and bars or chewing gums provided in any desired shape in response to the intended use. The chewing gum product can have any quality, including bubble gum quality.

[0641] In a preferred embodiment, the present invention relates to an oral care composition in the form of a chewing gum, the oral care compositions comprising invertase and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from elastomers, softeners, plasticizers, emulsifiers, waxes, colorants, sweeteners, flavorants, bulking agents and thickeners.

[0642] Gum base component

[0643] Chewing gums are traditionally considered to consist of a water-insoluble or matrix part and a water-soluble part containing flavorants, sweeteners and colorants. The gum base part of the chewing gum is the masticatory substance that imparts the chewing characteristics to the final product. It defines the release profile of the flavor and sweetener and plays a significant role in chewing gum products. Flavorants, sweeteners and colorants can be considered to provide the sensory appeal of the chewing gum. There is no restriction on the chewing gum matrix used in the chewing gum products according to the present invention. Conventional chewing gum matrices can be obtained from, for example, Dansk Tyggegummi Fabrik A / S, L.A. Dreyfus or Cafasa Gum SIA are generally suitable, but specially manufactured formulations can also be used. The formulation depends on the type of chewing gum desired or the type of structure desired. Suitable raw materials for the gum base include substances according to the U.S. Chewing Gum Base Regulations - Title 21 of the Code of Federal Regulations, Section 172,615 and according to other national and international lists (or positive lists), and include elastomers, resins, waxes, polyvinyl acetate, oils, fats, emulsifiers, fillers, and antioxidants.

[0644] The gum base generally comprises from 15% to 90% by weight, preferably from 30% to 40% by weight, more preferably from 5% to 25% by weight of the final product.

[0645] Elastomers provide chewiness, elasticity or resilience to the matrix and control the release of bubbles and flavors in the final chewing gum. They can be any water-insoluble polymers known in the art. They include styrene-butadiene copolymers (SBR) and non-SBR types, including both natural and synthetic. 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, lechi capsi, sorva, crown gum, nispero, rosidinha, perillo, nigergutta, tunu, gutta kay, pendare, leche de vaca, chiquibul, crown gum, etc.), and mixtures thereof. Examples of synthetic elastomers include, but are not limited to, polyisobutylene, isobutene-isoprene copolymer (butyl rubber), polyethylene, polybutadiene, styrene-butadiene copolymer, polyisoprene, etc., and mixtures thereof.

[0646] The amount of elastomer (rubber) used in the gum base composition will vary widely depending on various factors such as the type of gum base used (tacky or conventional, bubbly or standard), the desired consistency of the gum base composition, and the other components used in the composition to prepare the final chewing gum product. Generally, based on the total weight of the gum base composition, the elastomer is present in the gum base composition in an amount of about 15% to about 60% by weight, preferably about 25% to about 30% by weight.

[0647] Elastomer solvents help to soften or plasticize the elastomer component. In this way, they provide expansibility to the chew.

[0648] Elastomeric 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 rosin and gum rosin; glycerol esters of wood rosin and gum rosin, glycerol esters of partially hydrogenated wood rosin / gum rosin, glycerol esters of partially dimerized wood rosin and gum rosin, glycerol esters of polymerized wood rosin and gum rosin, and glycerol esters of tall oil rosin; deodorized glycerol esters of wood rosin; pentaerythritol esters of wood rosin and gum rosin; partially hydrogenated wood rosin and gum rosin; methyl esters of partially hydrogenated wood rosin; methyl esters, glycerol esters, and pentaerythritol esters of rosin and modified rosins (such as hydrogenated, dimerized, and polymerized rosins); terpene resins (such as polymers of α-pinene or β-pinene), terpene olefin resins; polyterpenes; etc. and mixtures thereof. The elastomeric solvent can be used in the gum base composition in an amount of about 2% to about 40%, preferably about 7% to about 15% by weight of the gum base composition.

[0649] Polyvinyl acetate provides extensibility or elasticity to the gum base. They also affect chew expansion, softness, and bubble, hydrophilic characteristics, and flavor release.

[0650] The amounts of polyvinyl acetate of different molecular weights present in the gum base composition should be effective to provide the desired chewing characteristics of the finished chewing gum, such as integrity, softness, chew expansion, film-forming characteristics, hydrophilic characteristics, 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 can have a molecular weight of about 2000 Da to about 95,000 Da.

[0651] Typically, low molecular weight polyvinyl acetate has a weight average molecular weight of about 2,000 Da to about 14,000 Da. Medium molecular weight polyvinyl acetate typically has a weight average molecular weight of about 15,000 Da to 55,000 Da. High molecular weight polyvinyl acetate typically has a weight average molecular weight of 55,000 Da to about 95,000 Da, but can be as high as 500,000 Da.

[0652] Waxes, fats, and oils plasticize the elastomeric mixture and improve the elasticity of the gum base. Waxes can provide a soft or hard chew, affect flavor release, and provide expansion and smoothness to the gum base. Fats and oils provide a soft chew. Fats, oils, and waxes can be used alone or in combination, or the gum base can be a wax-free gum base.

[0653] When wax is used, it can be of mineral, animal, plant, or synthetic origin. Non-limiting examples of mineral waxes include petroleum waxes (such as paraffin wax and microcrystalline wax), animal waxes (including beeswax), plant waxes (including carnauba wax, candelilla wax, rice bran wax, esparto wax, linseed wax, and sugarcane wax), synthetic waxes (including those produced by the Fischer-Tropsch process), and mixtures thereof.

[0654] Suitable oils and fats that can be used in the chewing gum composition include hydrogenated or partially hydrogenated vegetable or animal fats, such as cottonseed oil, soybean oil, coconut oil, palm kernel oil, tallow, hydrogenated tallow, lard, cocoa butter, lanolin, and the like; fatty acids such as palmitic acid, oleic acid, stearic acid, linoleic acid, lauric acid, myristic acid, caproic acid, capric acid, or esters and salts (such as sodium stearate and potassium stearate). When used, these ingredients are typically present in amounts up to about 7% by weight of the chewing gum composition, and preferably up to about 3.5% by weight of the chewing gum composition.

[0655] Preferred as softening agent is hydrogenated vegetable oil, and comprises soybean oil and cottonseed oil, and they can be used alone or in combination.These softening agents provide good texture and soft chewing characteristics for gum base composition.These softening agents are usually used with the amount of about 5% to about 14% by gum base composition weight.

[0656] Emulsifiers help to disperse the immiscible components of the gum base composition into a single stable system. They provide hydrophilic characteristics for the gum base and help plasticizing resins and polyvinyl acetate. They also affect the softness of the matrix and the bubble characteristics of the matrix. Typical emulsifiers include acetylated monoglycerides, glyceryl monostearate, lecithin, fatty acid monoglycerides, diglycerides, propylene glycol monostearate, lecithin, triacetin, glyceryl triacetate, etc. and mixtures thereof.

[0657] Preferred emulsifiers are glyceryl monostearate and acetylated monoglycerides. These serve as plasticizers. The emulsifier can be used in an amount of about 2% to about 15% by weight of the gum base composition, and preferably in an amount of about 7% to about 11% by weight of the gum base composition.

[0658] Fat, oil, wax, emulsifier and some sugar swelling agent are combined together and are referred to as softening agent usually.Due to the low molecular weight of these compositions, softening agent can penetrate the basic structure of gum base, makes it have plasticity and less viscosity.Above-mentioned useful plasticizer and softening agent comprise lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, triacetin, glyceryl lecithin, glyceryl monostearate, propylene glycol monostearate, acetylated monoglyceride, glycerine, completely unsaturated vegetable oil (for example non-hydrogenated cottonseed oil, hydrogenated vegetable oil, petroleum wax, sorbitan monostearate, tallow etc.) and composition thereof, and also comprise high fructose corn syrup, corn syrup, sorbitol solution, hydrogenated starch hydrolysate etc. and composition thereof.

[0659] The softener should be present in an amount effective to provide the desired chewing swellability and softness to the finished chewing gum. When used as softeners, these materials are typically used in gum base compositions in an amount up to about 25%, preferably about 1% to about 17%, by weight of the gum base composition.

[0660] The gum base may further contain a surfactant. Examples of suitable surfactants include polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (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 present should effectively provide the desired softness to the finished chewing gum. Typically, based on the total weight of the gum base, the surfactant is used in the matrix in an amount of about 0.5% to about 3.0% by weight.

[0661] The gum base composition of the present invention may also include an effective amount of a filler, sometimes referred to as a bulking agent. These materials increase hardness and bulk and affect the texture and flavor release of the chewing gum. Useful fillers include organic and inorganic compounds (mineral adjuvants) such as calcium carbonate, magnesium carbonate, heavy calcium carbonate, magnesium silicate, calcium phosphate, cellulose polymers, clays, alumina, aluminum hydroxide, aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate, and the like, and mixtures thereof. These fillers or adjuvants can be used in the gum base composition in various amounts. The amount of filler present should effectively provide the desired flavor release and integrity to the finished chewing gum. Typically, the filler can be used in the gum base composition in an amount of about 1% to about 40%, and preferably about 5% to about 20% by weight of the gum base composition.

[0662] The gum base may also contain an antioxidant to provide improved 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 can also be used.

[0663] Other gum components

[0664] The remaining components in the chewing gum composition are conventional and typically comprise from 10% to 85% by weight of the final product.

[0665] Examples thereof are sweeteners, softeners, colorants, bulking agents, thickeners, and flavoring agents of the types and amounts conventionally used in chewing gums.

[0666] Suitable flavoring agents are those spices known to the person skilled in the art, such as natural and artificial spices. These flavoring agents can be selected from synthetic flavor oils and flavoring fragrances and / or oils, oleoresins, and extracts derived from plants, leaves, flowers, fruits, etc., 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, sweet pepper oil, sage oil, mace oil, bitter almond oil, and cassia oil. Other useful flavoring agents 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 can be used in liquid or solid form and can be used alone or in combination. Commonly used spices include mint, such as peppermint, menthol, artificial vanilla, cinnamon derivatives, and various fruit flavors, which can be used alone or in combination.

[0667] Other useful flavoring agents include aldehydes and esters, for example, cinnamyl acetate, cinnamaldehyde, diethyl acetal citrate, dihydrocarvyl acetate, eugenyl formate, p-methyl anisole, etc. can be used. Generally, any flavoring agent or food additive can be used.

[0668] Additional examples of aldehyde flavoring agents include, but are not limited to, acetaldehyde (apple), benzaldehyde (cherry, almond), anisaldehyde (licorice, anise), cinnamaldehyde (cinnamon), citral (α-citral) (lemon, lime), neral (β-citral) (lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, cream), heliotropin (vanilla, cream), vanillin (vanilla, cream), α-amyl cinnamaldehyde (spicy fruity fragrance), butyraldehyde (butter, cheese), valeraldehyde (butter, cheese), citronellal (various types), decanal (citrus fruits), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrus fruits), aldehyde C-12 (citrus fruits), 2-ethyl butyraldehyde (berry fruits), hexenal (trans-2-hexenal) (berry fruits), tolyl aldehyde (cherry, almond), veratraldehyde (vanilla), 2,6-dimethyl-5-heptenal (melon aldehyde) (melon), 2,6-dimethyloctanal (green fruits), and 2-dodecanal (citrus, tangerine), cherry, grape, strawberry shortcake, and mixtures thereof, etc.

[0669] The amount of flavoring agent used herein is generally influenced by factors such as the type of the final chewing gum composition, the individual flavor, the gum used, and the desired flavor intensity. Thus, the amount of flavoring agent can be varied to obtain the desired result in the final product, and such variations are within the capabilities of those skilled in the art without undue experimentation. In the chewing gum composition, the flavoring agent is generally present in an amount of about 0.02% to about 5% by weight of the chewing gum composition.

[0670] Chewing gum compositions generally include bulking agents. These bulking agents (carriers, fillers) can be water-soluble and include bulking agents selected from the group consisting of (but not limited to): monosaccharides, disaccharides, polysaccharides, sugar alcohols, and mixtures thereof; sorbitol, xylitol, maltitol, mannitol, isomalt (racemic mixture of α-D-glucopyranosyl-1,6-mannitol and α-D-glucopyranosyl-1,6-sorbitol, manufactured by Suddeutsche Zucker under the trade name Palatinit TM manufactured), glycerin, aspartame, glycerol, galactitol, acesulfame potassium, saccharin and its salts, cyclohexylsulfamate and its salts, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, and sucralose and mixtures thereof or mixtures thereof with other suitable sweeteners, maltodextrin; hydrogenated starch hydrolysates; hydrogenated hexoses; hydrogenated disaccharides; minerals such as calcium carbonate, talc, titanium dioxide, dicalcium phosphate, cellulose, etc., and mixtures thereof. The bulking agent can be used in an amount of up to about 60% by weight of the chewing gum composition, preferably about 25% to about 60% by weight.

[0671] Chewing gum compositions may also include high-intensity sweeteners (sweetening agents). The sweetening intensity of high-intensity sweeteners is substantially greater than that of sucrose. Examples of suitable intense sweeteners include:

[0672] a) water-soluble naturally occurring intense sweeteners such as dihydrochalcones, monellin, steviosin, glycyrrhizin, flavanonols, and L-amino dicarboxylic acids, amino alkanoate amides such as those disclosed in U.S. Patent No. 4,619,834, and mixtures thereof;

[0673] b) water-soluble artificial sweeteners, including soluble saccharin salts (such as sodium saccharin or calcium saccharin salts), cyclohexylsulfamates, sodium, ammonium, or calcium salts of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, potassium salt of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide (acesulfame potassium), free acid form of saccharin, etc., and mixtures thereof;

[0674] c) Dipeptide-based sweeteners, including sweeteners derived from L-aspartic acid (e.g., L-aspartyl-L-phenylalanine methyl ester (aspartame) and the materials described in U.S. Patent No. 3,492,131), L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate (alitame), L-aspartyl-L-phenylglycerol and methyl esters of L-aspartyl-L-2,5-dihydrophenyl-glycine, L-aspartyl-2,5-dihydro-L-phenylalanine, L-aspartyl-L-(1-cyclohexenyl)-alanine, etc. and mixtures thereof;

[0675] d) Water-soluble intense sweeteners derived from naturally occurring water-soluble sweeteners, such as chlorinated derivatives of common sugar (sucrose), such as chlorodeoxysugar derivatives (e.g., derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose), known, for example, under the product name; Examples of chlorodeoxysucrose 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-deoxy-β-D-fructofuranoside, or 4,1'-dichloro-4,1'-dideoxygalactosucrose; 1',6'-dichloro-1',6'-dideoxysucrose; 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; 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'-tetradeoxygalactosucrose; and 4,6,1',6'-tetradeoxy-sucrose, and mixtures thereof; and

[0676] e) Protein-based intense sweeteners such as Thaumaoccous daniclii (thaumatin I and II). The amount of sweetener used in the chewing gum composition will vary with the sweetener selected for a particular chewing gum. Thus, for any given sweetener, an amount sufficient to provide the desired level of sweetness is used. Based on the total weight of the chewing gum composition, the above-mentioned saccharide sweeteners and sugar alcohols are generally 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 above-mentioned intense sweeteners are generally used in an amount of up to about 1% by weight, preferably about 0.05% to about 0.4% by weight.

[0677] The colorants useful in the present invention are used in an amount effective to produce the desired color. These colorants include pigments, which can be incorporated in amounts up to about 6% by weight of the chewing gum composition. A preferred pigment, titanium dioxide, can be incorporated in amounts up to about 2% by weight of the chewing gum composition, preferably less than about 1% by weight. Colorants can also include natural food pigments and dyes suitable for food, pharmaceutical, and cosmetic applications. These colorants are referred to as F.D.&C. dyes and lakes. Acceptable materials for the foregoing uses are preferably water-soluble. Illustrative non-limiting examples include indigo carmine, known as F.D.&C. Blue No. 2, which is the disodium salt of 5,5-indigodisulfonic acid. Similarly, the dye known as F.D.&C. Green No. 1 contains a triphenylmethane dye and is the monosodium salt of 4-[4-(N-ethyl-N-p-toluenesulfonamido)diphenylmethylene]-[1-(N-ethyl-N-p-toluenesulfonamido)-δ-2,5-cyclohexadienimine].

[0678] Examples of thickeners include methylcellulose, alginates, carrageenans, xanthan gum, gelatin, locust bean gum, gum tragacanth, and guar gum, emulsifiers (such as lecithin and glycerol monostearate), acidulants (such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid), and mixtures thereof.

[0679] The plasticizers, softeners, emulsifiers, waxes, and antioxidants discussed above, which are suitable for the gum base, can also be used in the chewing gum composition.

[0680] Active chewing gum components

[0681] The oral care composition in the form of a chewing gum according to the present invention may also contain various active ingredients such as antimicrobial agents, Zn salts, fluorides, and ureas.

[0682] In addition, if desired, the oral composition according to the present invention may include any other active ingredients such as anticaries agents, anticalculus agents, antigingivitis agents, antiperiodontal agents, antifungal agents, antismoking agents, antihypothermia agents, anti-gingivitis agents, etc.

[0683] The antimicrobial agent used in the composition can be any of a variety of cationic antimicrobial agents, such as quaternary ammonium compounds (e.g., cetylpyridinium chloride) and substituted guanidines (e.g., chlorhexidine and the corresponding compound alexidine). Mixtures of cationic antimicrobial agents can also be used in the present invention.

[0684] Antimicrobial quaternary ammonium compounds include those in which one or two substituents on the quaternary nitrogen have a carbon chain length of about 8 to 20, typically 10 to 18, carbon atoms (typically alkyl groups), while the remaining substituents (typically alkyl or benzyl groups) have a smaller number of carbon atoms (e.g., 1 to 7 carbon atoms), typically methyl or ethyl groups. Examples of typical quaternary ammonium antibacterial agents are dodecyltrimethylammonium bromide, cetylpyridinium chloride, cetylpyridinium ethyl chloride, dodecyldimethyl(2-phenoxyethyl)ammonium bromide, benzyldimethylstearylammonium chloride, cetylpyridinium chloride, quaternized 5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidine, and benzethonium chloride. Other compounds are bis[4-(R-amino)-1-pyridine]alkanes as disclosed in U.S. Patent 4,206,215 to Bailey, issued June 3, 1980, which is incorporated herein by reference. Pyridine compounds are preferred quaternary ammonium compounds.

[0685] The cationic antimicrobial agent is generally used in the compositions of the present invention at a level of about 0.02% to about 1%, preferably about 0.3% to about 0.7%, and most preferably about 0.3% to about 0.5%.

[0686] As the soluble zinc salt, in principle, any physiologically acceptable, soluble zinc salt of an inorganic or organic acid can be used, which salt can release zinc ions and is approved for the intended use, such as in food, cosmetic, or pharmaceutical products. Non-limiting examples are, for example, zinc citrate, zinc sulfate, zinc lactate, zinc chloride, zinc acetate, and mixtures thereof. Among these salts, zinc acetate is preferred.

[0687] The zinc salt used must be soluble to ensure that the amount of zinc ions released in the oral cavity over a suitable period of time is effective for the targeted purpose.

[0688] Advantageously, the zinc salt is present in the oral composition in an amount of 0.001% to 1.25% by weight. The amount used depends on the form of administration and the intended use and is adjusted such that the amount of zinc ions released is effective for the intended use.

[0689] 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.

[0690] Alkali metals, especially sodium or potassium, and alkaline earth metals are advantageously calcium or magnesium. Particularly preferred taste-masking salts are sodium carbonate, potassium carbonate, and magnesium carbonate, sodium chloride, ammonium chloride, and mixtures thereof.

[0691] The taste-masking salt is advantageously used in the oral composition in an amount of from 0.05% to 6.25% by weight, more preferably from 0.25% to 3.50% by weight, for example from 0.50% to 2.50% by weight.

[0692] In each case, the amount of the taste-masking salt used to mask the taste of zinc can be determined by a person skilled in the art and depends on the particular zinc salt in question and the chosen form of administration.

[0693] Urea is used as an anti-caries product for neutralizing acids produced in dental plaque after eating or drinking. In addition to urea, the composition may also contain a pharmaceutically acceptable substance capable of releasing urea under the prevailing conditions in the mouth. Examples thereof are salts and addition compounds between urea and inorganic compounds (such as magnesium sulfate, calcium phosphate, sodium chloride, etc.).

[0694] 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.

[0695] Standard techniques and equipment known to those skilled in the art can be used to prepare the chewing gum composition. Useful devices according to the invention also include mixing and kneading devices.

[0696] Troches and lozenges

[0697] Troches are flavored pharmaceutical dosage forms intended to be inhaled and held in the mouth or pharynx. They may contain vitamins, antibiotics, antiseptics, local anesthetics, antihistamines, decongestants, corticosteroids, astringents, analgesics, flavoring agents, emollients, or combinations of these ingredients. Troches can be in various shapes, most commonly flat, round, octagonal, and biconvex forms. Another type called bacilli is in the form of short rods or cylinders. The soft variety of troches is called lozenges and consists of a drug in a gelatin or glycerogelatin matrix or in an acacia, sucrose, and water matrix (H.A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, N.Y.).

[0698] In a preferred embodiment, the present invention relates to oral care compositions in the form of lozenges or troches, which oral care compositions comprise invertase and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from lubricants, swelling agents, sweeteners and flavoring agents.

[0699] Lubricant

[0700] Lubricants are used in the manufacture of compressed lozenges to facilitate the release of the lozenge from the mold in which it is formed. The lubricants used in the present invention are solid materials that are uncharged and do not interfere with (e.g., complex) cationic antimicrobial agents. The material should preferably be insoluble in water. One type of suitable material that meets these requirements is non-toxic hydrocarbon fats or derivatives. Examples include hydrogenated tallow and hydrogenated vegetable oils. Polyethylene glycols can also be used as lubricants provided they are solid materials, which generally means that the polyethylene glycols have a molecular weight in the range of 4000 Da to 6000 Da. As described below, these materials can also be used as fillers.

[0701] Mixtures of lubricants can also be used in the present invention. The lubricants are used at levels of from about 0.1% to about 4.0%, preferably from about 0.5% to about 2%.

[0702] Troche carrier

[0703] As used herein, the term "lozenge carrier" is used to denote one or more materials that carry the active ingredient (i.e., the enzyme) as well as the lubricant. These materials are also referred to as swelling agents or fillers. Since the carrier is non-cariogenic, the carrier should be free of sucrose and similar materials.

[0704] Acceptable filler materials include mannitol, sorbitol, xylitol, polyethylene glycol and non-cariogenic dextran. The fillers can be used alone or in combination.

[0705] Mannitol is a naturally occurring sugar alcohol and can be obtained as a fine powder. Its sweetness is only about 50% of the sweetness of sucrose. However, the negative heat of solution of mannitol enables it to impart a pleasant, cooling sensation in the mouth when the lozenge dissolves.

[0706] Sorbitol is a chemical isomer of mannitol and has a similar degree of sweetness. Its heat of solution (which is negative) also provides a pleasant, cooling sensation in the mouth. Sorbitol can be obtained as free-flowing granules or as a crystalline powder. Polyethylene glycol (PEG) can also be used in the compositions of the present invention. These materials are polymers of ethylene oxide having the general formula HOCH2(CH2OCH2) n CH2OH. It is not advantageous to use PEG alone, but they are acceptable when combined with other fillers. The most desirable molecular weight has been found to be between 4000 Da and 6000 Da.

[0707] The filler is generally used in the compositions of the present invention at a level of from about 85% to about 99.8%, preferably from about 90% to about 98%, and most preferably from about 94% to about 97%.

[0708] Other troche components

[0709] Acceptable lozenges can be made using only the active ingredient, lubricant, and filler materials outlined above. However, to make the lozenges more acceptable from an aesthetic point of view, they generally include materials such as spray-dried or encapsulated flavorings or liquid flavorings adsorbed onto a suitable diluent. Spray-dried or encapsulated flavorings are preferred. Suitable flavorings include peppermint oil, wintergreen oil, sassafras oil, spearmint oil, and clove oil. Sweetening agents are also acceptable for use in the compositions of the present invention. Suitable agents include aspartame, acesulfame, saccharin, dextrose, and levulose. Sweetening agents and flavoring agents are generally used in the compositions of the present invention at a level of from about 0.1% to about 2%, preferably from about 0.25% to about 1.5%.

[0710] Also acceptable is that the amount of the solid form of the water-soluble fluoride compound present in the lozenges of the present invention is sufficient to give a fluoride concentration of from about 0.0025% to about 5.0% by weight, preferably from about 0.005% to about 2.0% by weight, to provide additional anticaries efficacy. Preferred fluorides are sodium fluoride, stannous fluoride, indium fluoride, and sodium monofluorophosphate. The lozenges can also contain various active ingredients such as antimicrobial agents, Zn salts, fluorides, and urea (see above).

[0711] Confectionery and candies

[0712] In a preferred embodiment, the present invention relates to oral care compositions in the form of confectionery or candies, which oral care compositions comprise invertase and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from colorants, sweetening agents, flavoring agents, and oil modifiers.

[0713] The preparation of confectionery formulations has been well known historically and has changed little over the years. Confectionery products have been classified as "hard" confectionery or "soft" confectionery. The volatile oil modifiers of the present invention can be incorporated by mixing the modifier into conventional hard and soft confectionery.

[0714] Hard candies can be processed and formulated by conventional methods. Generally, hard candies have a matrix composed of a mixture of sugar and other carbohydrate bulking agents, which are maintained in an amorphous or glassy condition. This form is considered a solid syrup of sugar, which typically has from about 0.5% to about 1.5% moisture. Such materials generally contain up to about 92% corn syrup, up to about 55% sugar, and from about 0.1% to about 5% water by weight of the final composition. The syrup component is typically prepared from high fructose corn syrup, but may include other materials. Additional ingredients such as flavorings, sweeteners, acidulants, colorants, etc. may also be added.

[0715] Such candies can be conventionally prepared by conventional methods, which are, for example, conventional methods involving a fire cooker, a vacuum cooker, and a scraping surface cooker (also known as a high speed atmospheric cooker).

[0716] The fire cooker involves a traditional method of making a candy matrix. In this method, the desired amount of carbohydrate bulking agent is dissolved in water by heating the reagents in a pan until the bulking agent dissolves. Additional bulking agent can then be added and cooking continued until the final temperature reaches 145°C to 156°C. The batch is then cooled and processed as a plastic-like mass to incorporate additives such as flavors, colorants, etc.

[0717] The high speed atmospheric cooker uses a heat exchanger surface, which involves spreading a layer of candy on the heat exchange surface and heating the candy to 165°C to 170°C within a few minutes. The candy is then rapidly cooled to 100°C to 120°C and acts as a plastic-like mass capable of incorporating additives such as flavors, colorants, etc.

[0718] In the vacuum cooker, the carbohydrate bulking agent is boiled to 125°C to 132°C, a vacuum is applied, and additional water is evaporated without additional heating. When the cooking is complete, the mass is semi-solid and has a plastic-like consistency. At this point, flavors, colorants, and other additives are mixed into the mass by conventional mechanical mixing operations.

[0719] During conventional hard candy manufacture, the optimal mixing required to uniformly mix flavors, colorants, and other additives is determined by the time required to obtain a uniform distribution of the materials. Generally, a mixing time of 4 to 10 minutes has been found to be acceptable.

[0720] Once the confectionery mass has been properly tempered, it can be cut into workable pieces or formed into the desired shape. Depending on the shape and size of the desired final product, a variety of shaping techniques can be used. A general discussion of the composition and preparation of hard candies can be found in H.A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, N.Y.

[0721] According to the present invention, useful equipment includes cooking and mixing devices well known in the confectionery manufacturing art, and the choice of specific devices will be apparent to one of ordinary skill in the art. In contrast, compressed tablet confections contain specific materials and are formed under pressure.

[0722] These confections typically contain up to about 95% by weight of the composition of sugar, as well as typical tablet excipients such as binders and lubricants and flavoring agents, coloring agents, etc. Similar to hard candies, soft candies can be used in the present invention. The preparation of soft candies (such as nougat) involves conventional methods, such as the combination of two main components, namely (1) high-boiling syrups such as corn syrup, hydrogenated starch hydrolysates, etc., and (2) relatively light-textured frappes, which are typically prepared from egg white, gelatin, vegetable proteins (such as soy-derived compounds), milk-derived compounds without sugar (such as milk proteins), and mixtures thereof. Frappes are typically relatively light and can be, for example, in the density range of about 0.5 to about 0.7 g / cc.

[0723] The flavoring components of the confections are flavors having associated bitter or other unpleasant aftertastes. These flavoring components can be selected from natural and synthetic flavoring liquids such as volatile oils, synthetic flavoring oils, flavoring aromatics and oils, liquids, oleoresins, or extracts derived from plants, leaves, flowers, fruits, stems, and combinations thereof. 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, sweet pepper oil, sage oil, mace extract, bitter almond oil, and cassia oil. In addition, the confections can also contain artificial, natural, or synthetic flavors, including fruit flavors alone and in combination (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, etc.).

[0724] Other useful flavoring agents include aldehydes and esters, such as benzaldehyde (cherry, almond), citral (i.e., α-citral (lemon, lime)), neral (i.e., β-citral (lemon, lime)), decanal (orange, lemon), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrus fruits), aldehyde C-12 (citrus fruits), tolyl aldehyde (cherry, almond), 2,6-dimethyl-octanal (green fruits), and 2-dodecanal (citrus, mandarin), mixtures thereof, etc.

[0725] In the case of using sweeteners, it is contemplated that the present invention includes 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 (e.g., sodium or calcium salts); cyclamate and its various salts, such as sodium salt; dipeptide sweeteners, such as aspartame, dihydrochalcone compounds, glycyrrhizin; Stevia (Stevia Rebaudiana) (stevioside); chlorine derivatives of sucrose; flavanone alcohols; hydroxyguaiacol esters; L-amino dicarboxylic acid diamides; L-amino dicarboxylic acid amino enoate amides; and sugar alcohols, such as sorbitol, sorbitol syrup, mannitol, xylitol, etc. The synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly its potassium salt (acesulfame potassium), sodium salt, and calcium salt, is also contemplated.

[0726] The confectionery may also include colorants. These colorants may be selected from any of a variety of dyes suitable for food, pharmaceutical, and cosmetic applications and are referred to as FD&C dyes, etc. The acceptable materials for the foregoing use spectra are preferably water-soluble. Illustrative examples include the indigo dye known as FD&C Blue No. 2, which is the disodium salt of 5,5'-indigodisulfonic acid. Similarly, the dye known as FD&C Green No. 1 contains a triphenylmethane dye and is the monosodium salt of 4-[4-N-ethyl-p-sulfobenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-sulfobenzyl)-2,5-cyclohexadienimine]. A complete description of all FD&C and D&C dyes and their corresponding chemical structures can be found in Volume 5 of the Kirk-Othmer Encyclopedia of Chemical Technology.

[0727] The confectionery may also include volatile oil modifiers, such as oleoresin capsicum. The oil modifiers are present in an amount that is not detected as a separate component in the mouth but can still alter the sensory perception of the volatile oil.

[0728] The oil modifier is present in the confectionery in an amount of from about 1 to about 150 ppm. Chili peppers can be obtained from Capsicum minimum, Capsicum fruttescens, Capsicum annuum, and similar varieties. Commercially, the fruits of chili peppers are known as chilies or peppers. These fruits are known for their great biting force, pungency, and unique odor.

[0729] Regarding confectionery compression tablet formulations, these will contain a tablet granulation matrix and various additives such as sweeteners and flavorings. The tablet granulation matrix used will vary depending on a number of factors such as the type of matrix used, the desired brittleness, and the other components used in the manufacture of the final product. These confectioneries typically contain sugar in an amount up to 95% by weight of the composition.

[0730] Confectionery compression tablets can additionally include tablet excipients such as binders or lubricants, as well as flavoring agents, coloring agents, and volatile oils and volatile oil modifiers.

[0731] With respect to these confectioneries, the variations that can be practiced are very wide and within the capabilities of those skilled in the art, particularly with regard to the use of additional composition fillers, flavoring agents, coloring agents, etc.

[0732] Oral care compositions for external use

[0733] Oral care formulations for external use (such as denture cleaning solutions, denture cleaning tablets, denture cleaning powders, etc.) can include ingredients and / or substances selected from the following categories:

[0734]

[0735]

[0736] In a preferred embodiment, at least one oral care ingredient is selected from the group consisting of: carrier liquids, disinfectants and bleaching agents, cleaning agents, detergents and surfactants, foaming agents, preservatives, and flavoring agents.

[0737] In one aspect, the oral care composition of the present invention can also be incorporated into filaments suitable for tooth cleaning (such as filaments used as dental floss). Preferably, the oral care composition is coated on the outside of the filaments. Thus, in a preferred embodiment, the present invention relates to filaments comprising an oral care composition containing invertase, β-glucosidase, and glucoamylase, wherein the filaments are suitable for tooth cleaning.

[0738] Applications of the oral care composition

[0739] The oral care composition of the present invention is suitable for use in the treatment of oral diseases, wherein it is desired to prevent or remove oral biofilm. The composition of the present invention is particularly suitable for the treatment of periodontal diseases and dental caries.

[0740] Periodontal disease, also known as gum disease, is a group of inflammatory conditions caused by bacterial infection and subsequent establishment of biofilm on the teeth and tissues surrounding the teeth. Periodontal disease can be classified into the following categories according to severity: gingivitis (including plaque-induced gingivitis), chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic disease, necrotizing ulcerative gingivitis / periodontitis, periodontal abscess, and endodontic-periodontal lesions. Depending on the extent of the affected area, periodontal disease can be further considered local or systemic.

[0741] Dental caries, also known as tooth decay or cavities, is caused by organic acids (such as lactic acid) released by certain biofilm-forming bacteria (including Streptococcus mutans and some Lactobacillus species) resident in the oral cavity. Dental caries can be associated with other complications, such as inflammation of the tissues surrounding the teeth, tooth loss, and infection or abscess formation. Dental caries can be classified by location, etiology, rate of progression, and the hard tissues affected, such as according to the G.V. Black classification (classes I, II, III, IV, V, and VI).

[0742] In one aspect, the present invention relates to an oral care composition comprising invertase and at least one oral care ingredient for use as a medicament.

[0743] In one aspect, the present invention relates to an oral care composition comprising invertase and at least one oral care ingredient for use in the treatment of oral diseases.

[0744] In a preferred embodiment, the present invention relates to an oral care composition comprising invertase and at least one oral care ingredient for use in the treatment of periodontal disease and / or dental caries.

[0745] In one aspect, the present invention relates to the use of an oral care composition comprising invertase and at least one oral care ingredient for the treatment or prophylactic treatment of a human subject.

[0746] In one aspect, the present invention relates to a method of treating a human subject, the method comprising administering to the human subject an oral care composition comprising invertase and at least one oral care ingredient. In a preferred embodiment, the oral care composition is administered to the oral cavity of the human subject.

[0747] In one aspect, the present invention relates to a method for preventing or removing oral biofilm, the method comprising contacting the biofilm with an oral care composition comprising invertase and at least one oral care ingredient. In one embodiment, the oral care composition is an external oral care composition and the biofilm is located on a target; preferably the target is a denture. In one embodiment, the target is located inside or outside the oral cavity.

[0748] Examples

[0749] Materials and Methods

[0750] Culture medium and solution

[0751] The YP + 2% maltose medium consists of 1% yeast extract, 2% bacto peptone, and 2% maltose.

[0752] The YP + 2% glucose medium consists of 1% yeast extract, 2% bacto peptone, and 2% dextrose.

[0753] The DAP4C-1 medium consists 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, 0.05% CaCO3.

[0754] The PDA plate consists of: 39 g potato dextrose agar, 50 mL glycerol, 20 g agar, and deionized water made up to 1 L. The medium is sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0755] The LB agar plate consists of: 37 g LB agar (Sigma Aldrich L3027), 5 g 0.5% soluble starch (Merck 101252), 10 mL of 1 M K2PO4, 20 mL of 20% glucose solution, and deionized water made up to 1 L.

[0756] The LB medium consists of 25 g LB broth (Fluka L3152) and deionized water made up to 1 L. The medium is sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0757] The COVE sucrose plates for Aspergillus transformant selection consist of: 342 g of sucrose, 20 g of agar powder, 20 mL of COVE salt solution, and deionized water to make up 1 L. The medium is sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The medium is cooled to 60 °C and 10 mM NaNO3 is added.

[0758] The COVE salt solution consists of: 26 g of MgSO4 / 7H2O, 26 g of KCl, 76 g of KH2PO4, 50 mL of COVE trace metal solution, and deionized water to make up 1 L.

[0759] The COVE trace metal solution consists of: 0.04 g of Na2B4O7 / 10H2O, 0.4 g of CuSO4 / 5H2O, 1.2 g of FeSO4 / 7H2O, 0.7 g of MnSO4 / H2O, 0.8 g of Na2MoO4 / 2H2O, 10 g of ZnSO4 / 7H2O, and deionized water to make up 1 L.

[0760] Example 1: Cloning and expression of invertase, β-glucosidase, and glucoamylase

[0761] SEQ ID NO:1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34 were identified in their respective donor organisms and cloning was completed using the DSMS system (described in US 2019 / 0225988) according to the strategy described in US2019 / 0225988, where three overlapping fragments were used for integration at the niiA / niiD locus of the ColS1300 strain.

[0762] ColS1300 protoplasts were prepared and transformed according to the method described in WO 2012 / 003379, where each overlapping DNA fragment was approximately 200 to 500 ng. The transformants were plated on COVE sucrose 10 mM NaNO3 plates and transformants capable of producing the corresponding mature polypeptides (SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 39 respectively) (as determined by SDS-PAGE electrophoresis) were selected.

[0763] Fermentation was carried out in a 250 mL baffled shake flask filled with 100 mL of fermentation medium (DAP4C-1) and inoculated with spores of a selected strain expressing the corresponding mature polypeptide. Fermentation was carried out at 30 °C with stirring at 150 rpm for four days. The culture broth was harvested by filtration using a 0.2 μm filtration device, and the mature polypeptide was purified by hydrophobic interaction chromatography. The integrity of the mature polypeptide was examined by SDS-PAGE electrophoresis, and the concentration of the purified enzyme was determined by absorbance at 280 nm.

[0764] Example 2: Activity assay

[0765] β-Fructofuranosidase (invertase) activity assay

[0766] β-Fructofuranosidase activity can be determined as follows: 50 ppm invertase (0.05 mg / mL, in 25 mM universal buffer (pH 6) containing acetic acid, MES, HEPES, and glycine) was incubated with a sucrose substrate (1% w / v) at 37 °C at 1400 rpm for 60 min, and a sample without added enzyme was used as a control. After incubation, the samples were centrifuged at 16100 x G for 5 min at room temperature, and the supernatant was analyzed by liquid chromatography using a Dionex TM -IC300 system (ThermoFisher Scientific) using the following gradient, and fructose and glucose (Sigma) were used as standard references:

[0767] Time (min) Flow rate (mL / min) % water % NaOH 0.5M % NaOAc 0.5M 0 0.8 97 3 0 4.5 0.8 96 4 0 7 0.8 93 7 0 10 0.8 80 15 5 25 0.8 50 15 35 28.1 0.8 85 15 0 29 0.8 90 10 0 30.1 0.8 97 3 0 33 0.8 97 3 0 33.1 0 - - -

[0768] For SEQ ID NO:40, the following table describes the fructose release after incubation measured as the area under the curve in nanocoulombs per minute (nC*min) by liquid chromatography, where the area under the curve is proportional to the fructose release. Liquid chromatography analysis showed that invertase could degrade sucrose into its constituent monosaccharides (fructose and sucrose), indicating β-fructofuranosidase activity.

[0769]

[0770] Example 3: Thermal stability measurement

[0771] Preparation of oral care formulations for thermal stability measurement

[0772] In the presence of oral care components within the concentration ranges commonly used in oral care product formulations and selected commercial oral care products, the thermal stability or the midpoint of the thermal unfolding transition (Tm) of selected invertases (SEQ ID NOs: 12, 15, and 36) was measured. The Tm parameter is used to evaluate thermal stability because this is the temperature at which the populations of folded and unfolded protein molecules are equal and is a widely accepted parameter used in assessing thermal stability. High-purity and biotech-grade reagents were obtained from different suppliers and stock solutions were freshly prepared using MilliQ water. These formulated chemicals and their stock solutions, as well as the final concentrations used in the Tm measurements, are listed in Table 1.

[0773]

[0774]

[0775] The purified preparation of the enzyme sample was diluted to a stock concentration of 2 mg / ml and then further diluted 10-fold in an oral care formulation consisting of individual formulated chemicals, citrate phosphate buffer (McIlvaine buffer, see below), and MilliQ water, corresponding to a final protein concentration of 0.2 mg / ml. Using a robotic arm, all dilutions were performed in 384-well small-volume deep-well plates (Greiner Bio-One International, product number 784201) with a final volume of 70 μl and were used for thermal stability measurements. Tm measurements were performed on each enzyme at pH 5.0 and pH 6.0, in the physiological pH range close to the oral cavity. 100 ml of McIlvaine buffer (pH 5.0) was prepared by mixing 51.50 ml of 0.2 M Na2HPO4 + 48.50 ml of 0.1 M citric acid, while 100 ml of McIlvaine buffer (pH 6) was prepared by mixing 63.15 ml of 0.2 M Na2HPO4 + 36.85 ml of 0.1 M citric acid.

[0776] Determination of Tm

[0777] Thermal stability measurements were performed using a capillary-based nano differential scanning fluorometer (nanoDSF), Prometheus NT.Plex (NanoTemper Technologies GmbH, Munich, Germany). Standard nanoDSF-grade capillary action slides (from NanoTemper Technologies, catalog number: PR-AC002) were used. Enzyme samples were loaded into the capillaries by capillary action (in triplicate for each sample). The emission intensities at 330 nm and 350 nm were optimized by varying the LED power of the instrument to ensure sufficient signal. The fluorescence signals at 330 nm and 350 nm were continuously monitored as a function of temperature (heating rate for thermal unfolding was 3.3 °C per minute from 20 °C to 95 °C). The data were analyzed using the PR.StabilityAnalysis1.1.0.11077 software provided by the manufacturer. This analysis is model-independent and only uses the peak maximum of the first derivative, which corresponds to the approximate midpoint of the thermal unfolding transition, defined as Tm (see Figure 1 ).

[0778] Reproducibility of thermal stability data

[0779] Figure 1 An example of thermal stability data generated using the nanoDSF instrument is shown. Panel A is an example of the data of SEQ ID NO:40 (ratio of fluorescence emission at 350 nm to 330 nm) obtained in triplicate as a function of temperature. Panel B shows the first derivative of the raw data in Panel A. The peak maximum in the first derivative plot corresponds to the midpoint of the thermal unfolding transition, called Tm. In this example, the Tm at pH 6.0 corresponds to 61.9 °C and is highly reproducible within three replicates.

[0780] Figure 1 The data shown in

[0781] In the presence of oral care formulation components, the Thermal stability of invertase, β-glucosidase, glucoamylase and α-amylase

[0782] are examples of the types of data generated using nanoDSF for various enzymes in different formulations. In all cases, the data show a distinct unfolding transition and a clearly discernible peak in the first derivative, and are highly reproducible.

[0783] It is evident from these data that these components individually have no adverse effect on the thermal stability of the invertase, and under conditions similar to oral conditions, these enzymes have comparable or even improved stability in the presence of these components, making these enzymes suitable for oral care formulations and applications in the oral cavity.

[0784]

[0785]

[0786]

[0787]

[0788] Example 4: Human Salivary Biofilm Prevention Assay with Supernatants

[0789] The human salivary biofilm prevention assay was performed using the method described in WO 2020 / 099490 with some modifications. Briefly, biofilms were grown in 96-well plates in the presence of 50 mM HEPES buffer (pH 7) with 100 mM NaCl (as a control) or an enzyme solution containing invertases (SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, and 39). Samples of the supernatants from the recombinant Aspergillus oryzae strains expressing each enzyme were evaluated using 10 μl.

[0790] Under microaerophilic conditions, in a Thermo Scientific TM Rectangular AnaeroBox TM container (Thermo Scientific AnaeroGen 2.5L, #AN0025A), the plates were incubated at 37 °C without shaking for 24 hours. The enzyme and control samples were evaluated eight times in duplicate.

[0791] After incubation, planktonic bacteria were removed by two gentle washes with 100 μl of 0.9% NaCl, and the biofilms were stained with 0.095% crystal violet solution at room temperature for 15 min. The plates were rinsed twice with 100 μL of 0.9% NaCl, and the attached dye was solubilized with a solution of 96% ethanol and 0.1% acetic acid. The absorbance at 600 nm was measured using a microplate reader (SpectraMax M3, Molecular Devices).

[0792] For data processing, the absorbance is proportional to the extent of the remaining biofilm after enzyme or control treatment. The results are expressed as the percentage of biofilm prevention and are calculated as follows:

[0793] 100 - ((A600 nm of the enzyme - treated sample) / (A600 nm of the buffer - control - treated sample)×100)

[0794] Where A600 nm refers to the average of eight absorbance measurements at 600 nm of the enzyme - or control - treated sample. The results are listed in Table 4 and show that all evaluated invertases exhibit potent biofilm - preventing effects.

[0795] Table 4. Prevention of biofilm formation (using supernatant samples).

[0796]

[0797] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

[0798] The invention is further defined by the following numbered paragraphs:

[0799] 1. An oral care composition, the oral care composition comprising an invertase selected from the group consisting of:

[0800] a) a polypeptide having 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 with SEQ ID NO:3;

[0801] b) a polypeptide having 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 with SEQ ID NO:6;

[0802] c) a polypeptide having 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 with SEQ ID NO:9;

[0803] d) a polypeptide having 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 with SEQ ID NO:12;

[0804] e) a polypeptide having 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 with SEQ ID NO:15;

[0805] f) a polypeptide having 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 with SEQ ID NO:18;

[0806] g) a polypeptide having 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 with SEQ ID NO:21;

[0807] h) a polypeptide having 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 with SEQ ID NO:24;

[0808] i) a polypeptide having 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 with SEQ ID NO:27;

[0809] j) a polypeptide having 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 with SEQ ID NO:30;

[0810] k) a polypeptide having 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;

[0811] l) a polypeptide having 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; and

[0812] m) a polypeptide having 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;

[0813] wherein the polypeptide has β - fructofuranosidase activity, and wherein the oral care composition further comprises at least one oral care ingredient.

[0814] 2. The oral care composition according to paragraph 1, wherein the invertase is selected from the group consisting of:

[0815] a) a polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3;

[0816] b) a polypeptide comprising SEQ ID NO:6, consisting essentially of SEQ ID NO:6, or consisting of SEQ ID NO:6;

[0817] c) a polypeptide comprising SEQ ID NO:9, consisting essentially of SEQ ID NO:9, or consisting of SEQ ID NO:9;

[0818] d) a polypeptide comprising SEQ ID NO:12, consisting essentially of SEQ ID NO:12, or consisting of SEQ ID NO:12;

[0819] e) a polypeptide comprising SEQ ID NO:15, consisting essentially of SEQ ID NO:15, or consisting of SEQ ID NO:15;

[0820] f) a polypeptide comprising SEQ ID NO:18, consisting essentially of SEQ ID NO:18, or consisting of SEQ ID NO:18;

[0821] g) a polypeptide comprising SEQ ID NO:21, consisting essentially of SEQ ID NO:21, or consisting of SEQ ID NO:21;

[0822] h) a polypeptide comprising SEQ ID NO:24, consisting essentially of SEQ ID NO:24, or consisting of SEQ ID NO:24;

[0823] i) a polypeptide comprising SEQ ID NO:27, consisting essentially of SEQ ID NO:27, or consisting of SEQ ID NO:27;

[0824] j) a polypeptide comprising SEQ ID NO:30, consisting essentially of SEQ ID NO:30, or consisting of SEQ ID NO:30;

[0825] k) a polypeptide comprising SEQ ID NO:33, consisting essentially of SEQ ID NO:33, or consisting of SEQ ID NO:33;

[0826] l) a polypeptide comprising SEQ ID NO:36, consisting essentially of SEQ ID NO:36, or consisting of SEQ ID NO:36; and

[0827] m) a polypeptide comprising SEQ ID NO:39, consisting essentially of SEQ ID NO:39, or consisting of SEQ ID NO:39.

[0828] 3. The oral care composition according to any one of paragraphs 1 - 2, wherein the invertase is present in an effective amount; preferably present in an amount of about 1 ppm to about 500 ppm.

[0829] 4. The oral care composition according to any one of paragraphs 1 - 3, wherein the invertase has comparable or improved thermal stability in the presence of at least one, such as 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.

[0830] 5. The oral care composition according to paragraph 4, wherein the thermal stability is determined at pH 5 and / or pH 6, preferably at pH 5 or pH 6, according to Example 3 herein.

[0831] 6. The oral care composition according to any one of paragraphs 1-5, which is in the form of an internal oral care composition; preferably in the form of toothpaste or toothpaste tablets, tooth cream, mouthwash or mouthwash tablets, oral cleanser, lozenges, soft lozenges, chewing gum, confectionery, or candies.

[0832] 7. The oral care composition according to any one of paragraphs 1-5, which is in the form of an external oral care composition; preferably in the form of a denture cleaning solution, denture cleaning tablets, or denture cleaning powder.

[0833] 8. The oral care composition according to any one of paragraphs 1-7, for use as a medicament.

[0834] 9. The oral care composition according to any one of paragraphs 1-7, for use in the treatment of oral diseases; preferably for use in the treatment of periodontal diseases (e.g., gingivitis) and / or dental caries.

[0835] 10. Use of the oral care composition according to any one of paragraphs 1-7 for the treatment or prophylactic treatment of a human subject.

[0836] 11. A method of treating a human subject, the method comprising administering the oral care composition according to any one of paragraphs 1-7; preferably administering the oral care composition to the oral cavity of the human subject.

[0837] 12. A method for preventing and / or removing oral biofilm, the method comprising contacting the oral biofilm with the oral care composition according to any one of paragraphs 1-7.

[0838] 13. The method according to paragraph 12, wherein the oral biofilm is located on a target, preferably a denture.

[0839] 14. The method according to paragraph 13, wherein the denture is located inside or outside the oral cavity.

[0840] 15. A kit, the kit comprising:

[0841] (1) The oral care composition according to any one of paragraphs 1-7; and

[0842] (2) Instructions for use.

[0843] 16. A polypeptide having β-fructofuranosidase activity, the polypeptide selected from the group consisting of:

[0844] (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;

[0845] (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;

[0846] (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: 9;

[0847] (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: 12;

[0848] (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: 15;

[0849] (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: 18;

[0850] (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: 21;

[0851] (h) a polypeptide having 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 with SEQ ID NO:24;

[0852] (i) a polypeptide having 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 with SEQ ID NO:27;

[0853] (j) a polypeptide having 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 with SEQ ID NO:30;

[0854] (k) a polypeptide having 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 with SEQ ID NO:33;

[0855] (l) a polypeptide having 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 with SEQ ID NO:36; and

[0856] (m) a polypeptide having 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 with SEQ ID NO:39.

[0857] 17. A polypeptide having β - fructofuranosidase activity, the polypeptide being selected from the group consisting of:

[0858] (a) a polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3;

[0859] (b) A polypeptide comprising SEQ ID NO:6, consisting essentially of SEQ ID NO:6, or consisting of SEQ ID NO:6;

[0860] (c) A polypeptide comprising SEQ ID NO:9, consisting essentially of SEQ ID NO:9, or consisting of SEQ ID NO:9;

[0861] (d) A polypeptide comprising SEQ ID NO:12, consisting essentially of SEQ ID NO:12, or consisting of SEQ ID NO:12;

[0862] (e) A polypeptide comprising SEQ ID NO:15, consisting essentially of SEQ ID NO:15, or consisting of SEQ ID NO:15;

[0863] (f) A polypeptide comprising SEQ ID NO:18, consisting essentially of SEQ ID NO:18, or consisting of SEQ ID NO:18;

[0864] (g) A polypeptide comprising SEQ ID NO:21, consisting essentially of SEQ ID NO:21, or consisting of SEQ ID NO:21;

[0865] (h) A polypeptide comprising SEQ ID NO:24, consisting essentially of SEQ ID NO:24, or consisting of SEQ ID NO:24;

[0866] (i) A polypeptide comprising SEQ ID NO:27, consisting essentially of SEQ ID NO:27, or consisting of SEQ ID NO:27;

[0867] (j) A polypeptide comprising SEQ ID NO:30, consisting essentially of SEQ ID NO:30, or consisting of SEQ ID NO:30;

[0868] (k) A polypeptide comprising SEQ ID NO:33, consisting essentially of SEQ ID NO:33, or consisting of SEQ ID NO:33;

[0869] (l) A polypeptide comprising SEQ ID NO:36, consisting essentially of SEQ ID NO:36, or consisting of SEQ ID NO:36; and

[0870] (m) A polypeptide comprising SEQ ID NO:39, consisting essentially of SEQ ID NO:39, or consisting of SEQ ID NO:39.

[0871] 18. A polypeptide according to any one of paragraphs 16 - 17, wherein the polypeptide is purified.

[0872] 19. A polypeptide according to any one of paragraphs 16 - 17, wherein the polypeptide is isolated.

[0873] 20. A particle comprising:

[0874] (a) a core containing a polypeptide according to any one of paragraphs 16 - 19, and optionally

[0875] (b) a coating consisting of one or more layers surrounding the core.

[0876] 21. A particle comprising:

[0877] (a) a core, and

[0878] (b) a coating consisting of one or more layers surrounding the core, wherein the coating contains a polypeptide according to any one of paragraphs 16 - 19.

[0879] 22. An oral composition comprising a polypeptide according to any one of paragraphs 16 - 19 or a particle according to paragraph 20 or 21, and at least one oral care ingredient.

[0880] 23. A liquid composition comprising a polypeptide according to any one of paragraphs 16 - 19, and an enzyme stabilizer such as a polyol like propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative such as an aromatic borate or a phenylboric acid derivative like 4 - formylphenylboric acid.

[0881] 24. The liquid composition according to paragraph 23, wherein the liquid composition further comprises a filler or a carrier material.

[0882] 25. The liquid composition according to paragraph 23 or 24, wherein the liquid composition further comprises a preservative.

[0883] 26. A composition comprising a polypeptide according to any one of paragraphs 16 - 19, a particle according to paragraph 20 or 21, or a liquid composition according to any one of paragraphs 23 - 25.

[0884] 27. The composition according to paragraph 26, wherein the composition is a liquid composition, a solid composition, a solution, a dispersion, a paste, a powder, a particle, a particulate matter, a coated particle, a tablet, a cake, a crystal, a crystal slurry, a gel or a pellet.

[0885] 28. A polynucleotide encoding a polypeptide according to any one of paragraphs 16 - 19.

[0886] 29. The polynucleotide according to paragraph 28, the polynucleotide comprising:

[0887] (a) SEQ ID NO:1 or its mature polypeptide coding sequence;

[0888] (b) SEQ ID NO:4 or its mature polypeptide coding sequence;

[0889] (c) SEQ ID NO:7 or its mature polypeptide coding sequence;

[0890] (d) SEQ ID NO:10 or its mature polypeptide coding sequence;

[0891] (e) SEQ ID NO:13 or its mature polypeptide coding sequence;

[0892] (f) SEQ ID NO:16 or its mature polypeptide coding sequence;

[0893] (g) SEQ ID NO:19 or its mature polypeptide coding sequence;

[0894] (h) SEQ ID NO:22 or its mature polypeptide coding sequence;

[0895] (i) SEQ ID NO:25 or its mature polypeptide coding sequence;

[0896] (j) SEQ ID NO:28 or its mature polypeptide coding sequence;

[0897] (k) SEQ ID NO:31 or its mature polypeptide coding sequence;

[0898] (l) SEQ ID NO:34 or its mature polypeptide coding sequence; and

[0899] (m) SEQ ID NO:37 or its mature polypeptide coding sequence.

[0900] 30. The polynucleotide according to paragraph 28 or 29, the polynucleotide being purified.

[0901] 31. The polynucleotide according to paragraph 28 or 29, the polynucleotide being isolated.

[0902] 32. A nucleic acid construct or expression vector, the nucleic acid construct or expression vector comprising the polynucleotide according to paragraph 28 or 29, the polynucleotide being operably linked to one or more control sequences directing the production of the polypeptide in an expression host.

[0903] 33. A recombinant host cell comprising the nucleic acid construct or expression vector according to paragraph 32.

[0904] 34. The recombinant host cell according to paragraph 33, wherein the polypeptide is heterologous to the recombinant host cell.

[0905] 35. The recombinant host cell according to paragraph 33 or 34, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.

[0906] 36. The recombinant host cell according to any one of paragraphs 33 - 35, the recombinant host cell comprising at least two copies of the polynucleotide according to any one of paragraphs 28 - 29, for example, three, four, five, or more copies.

[0907] 37. The recombinant host cell according to any one of paragraphs 33 - 36, the recombinant host cell being a yeast recombinant host cell, for example, a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica cell.

[0908] 38. A recombinant host cell according to any one of paragraphs 33 - 36, which is a filamentous fungal recombinant host cell, such as a cell of the genus Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Monographella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma, in particular, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis xantha, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus versicolor, Fusarium bacilliforme, Fusarium cerealis, Fusarium culmorum, Fusarium crookwellense, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium lignicolum, Fusarium oxysporum, Fusarium proliferatum, Fusarium roseum, Fusarium sambucinum, Fusarium semitectum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thermoascus aurantiacus, Trichoderma longibrachiatum, Trichoderma versicolor, Trichoderma viride, or Trichoderma harzianum cells.

[0909] 39. A recombinant host cell according to any one of paragraphs 33 - 36, which is a prokaryotic recombinant host cell, such as a Gram - positive cell selected from the group consisting of the genus Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces, or a Gram - negative bacterium selected from the group consisting of the genus Campylobacter, Escherichia, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniforme, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0910] 40. A recombinant host cell according to any one of paragraphs 33 - 39, wherein the recombinant host cell is isolated.

[0911] 41. A recombinant host cell according to any one of paragraphs 33 - 40, wherein the recombinant host cell is purified.

[0912] 42. A method for producing a polypeptide having β - fructofuranosidase activity, the method comprising culturing a recombinant host cell according to any one of paragraphs 33 - 41 under conditions conducive to the production of the polypeptide.

[0913] 43. The method according to paragraph 42, further comprising recovering the polypeptide.

[0914] 44. A method for producing a polypeptide according to any one of paragraphs 16 - 19, the method comprising culturing a cell that produces the polypeptide in its wild - type form under conditions conducive to the production of the polypeptide.

[0915] 45. The method according to paragraph 44, further comprising recovering the polypeptide.

[0916] 46. A whole - culture medium formulation or cell - culture composition comprising a polypeptide according to any one of paragraphs 16 - 19.

Claims

1. An oral care composition, the oral care composition comprising an invertase selected from the group consisting of: a) a polypeptide having at least 70% sequence identity with SEQ ID NO: 3; b) a polypeptide having at least 70% sequence identity with SEQ ID NO: 6; c) a polypeptide having at least 70% sequence identity with SEQ ID NO: 9; d) a polypeptide having at least 70% sequence identity with SEQ ID NO: 12; e) a polypeptide having at least 70% sequence identity with SEQ ID NO: 15; f) a polypeptide having at least 70% sequence identity with SEQ ID NO: 18; g) a polypeptide having at least 70% sequence identity with SEQ ID NO: 21; h) a polypeptide having at least 70% sequence identity with SEQ ID NO: 24; i) a polypeptide having at least 70% sequence identity with SEQ ID NO: 27; j) a polypeptide having at least 70% sequence identity with SEQ ID NO: 30; k) a polypeptide having at least 70% sequence identity with SEQ ID NO: 33; l) a polypeptide having at least 70% sequence identity with SEQ ID NO: 36; and m) a polypeptide having at least 70% sequence identity with SEQ ID NO: 39; wherein the polypeptide has β-fructofuranosidase activity, and wherein the oral care composition further comprises at least one oral care ingredient.

2. The oral care composition according to claim 1, wherein the invertase is selected from the group consisting of: a) a polypeptide comprising SEQ ID NO: 3, consisting essentially of SEQ ID NO: 3, or consisting of SEQ ID NO: 3; b) a polypeptide comprising SEQ ID NO: 6, consisting essentially of SEQ ID NO: 6, or consisting of SEQ ID NO: 6; c) a polypeptide comprising SEQ ID NO: 9, consisting essentially of SEQ ID NO: 9, or consisting of SEQ ID NO: 9; d) a polypeptide comprising SEQ ID NO: 12, consisting essentially of SEQ ID NO: 12, or consisting of SEQ ID NO: 12; e) a polypeptide comprising SEQ ID NO: 15, consisting essentially of SEQ ID NO: 15, or consisting of SEQ ID NO: 15; f) a polypeptide comprising SEQ ID NO: 18, consisting essentially of SEQ ID NO: 18, or consisting of SEQ ID NO: 18; g) a polypeptide comprising SEQ ID NO: 21, consisting essentially of SEQ ID NO: 21, or consisting of SEQ ID NO: 21; h) a polypeptide comprising SEQ ID NO: 24, consisting essentially of SEQ ID NO: 24, or consisting of SEQ ID NO: 24; i) A polypeptide comprising SEQ ID NO:27, consisting essentially of SEQ ID NO:27, or consisting of SEQ ID NO:27; j) A polypeptide comprising SEQ ID NO:30, consisting essentially of SEQ ID NO:30, or consisting of SEQ ID NO:30; k) A polypeptide comprising SEQ ID NO:33, consisting essentially of SEQ ID NO:33, or consisting of SEQ ID NO:33; l) A polypeptide comprising SEQ ID NO:36, consisting essentially of SEQ ID NO:36, or consisting of SEQ ID NO:36; and m) A polypeptide comprising SEQ ID NO:39, consisting essentially of SEQ ID NO:39, or consisting of SEQ ID NO:

39.

3. The oral care composition according to any one of claims 1-2, wherein the invertase is present in an effective amount; preferably present in an amount of about 1 ppm to about 500 ppm.

4. The oral care composition according to any one of claims 1-3, wherein the invertase has comparable or improved thermal stability in the presence of at least one, such as 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.

5. The oral care composition according to any one of claims 1-4, wherein the invertase prevents the formation of oral biofilm by at least 5%, such as 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%.

6. The oral care composition according to any one of claims 1-5, for use as a medicament.

7. The oral care composition according to any one of claims 1-5, for use in the treatment of oral diseases; preferably for use in the treatment of periodontal disease and / or dental caries.

8. A method for preventing and / or removing oral biofilm, the method comprising contacting the oral biofilm with the oral care composition according to any one of claims 1-5.

9. A kit, the kit comprising: a) The oral care composition according to any one of claims 1-5; and b) Instructions for use.

10. A polypeptide having β-fructofuranosidase activity, the polypeptide selected from the group consisting of: a) A polypeptide having at least 70% sequence identity with SEQ ID NO:3; b) A polypeptide having at least 70% sequence identity with SEQ ID NO:6; c) A polypeptide having at least 70% sequence identity with SEQ ID NO:9; d) a polypeptide having at least 70% sequence identity with SEQ ID NO:12; e) a polypeptide having at least 70% sequence identity with SEQ ID NO:15; f) a polypeptide having at least 70% sequence identity with SEQ ID NO:18; g) a polypeptide having at least 70% sequence identity with SEQ ID NO:21; h) a polypeptide having at least 70% sequence identity with SEQ ID NO:24; i) a polypeptide having at least 70% sequence identity with SEQ ID NO:27; j) a polypeptide having at least 70% sequence identity with SEQ ID NO:30; k) a polypeptide having at least 70% sequence identity with SEQ ID NO:33; l) a polypeptide having at least 70% sequence identity with SEQ ID NO:36; and m) a polypeptide having at least 70% sequence identity with SEQ ID NO:

39.

11. A polypeptide having β-fructofuranosidase activity, said polypeptide being selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3; b) a polypeptide comprising SEQ ID NO:6, consisting essentially of SEQ ID NO:6, or consisting of SEQ ID NO:6; c) a polypeptide comprising SEQ ID NO:9, consisting essentially of SEQ ID NO:9, or consisting of SEQ ID NO:9; d) a polypeptide comprising SEQ ID NO:12, consisting essentially of SEQ ID NO:12, or consisting of SEQ ID NO:12; e) a polypeptide comprising SEQ ID NO:15, consisting essentially of SEQ ID NO:15, or consisting of SEQ ID NO:15; f) a polypeptide comprising SEQ ID NO:18, consisting essentially of SEQ ID NO:18, or consisting of SEQ ID NO:18; g) a polypeptide comprising SEQ ID NO:21, consisting essentially of SEQ ID NO:21, or consisting of SEQ ID NO:21; h) a polypeptide comprising SEQ ID NO:24, consisting essentially of SEQ ID NO:24, or consisting of SEQ ID NO:24; i) a polypeptide comprising SEQ ID NO:27, consisting essentially of SEQ ID NO:27, or consisting of SEQ ID NO:27; j) a polypeptide comprising SEQ ID NO:30, consisting essentially of SEQ ID NO:30, or consisting of SEQ ID NO:30; k) a polypeptide comprising SEQ ID NO:33, consisting essentially of SEQ ID NO:33, or consisting of SEQ ID NO:33; l) a polypeptide comprising SEQ ID NO:36, consisting essentially of SEQ ID NO:36, or consisting of SEQ ID NO:36; and m) a polypeptide comprising SEQ ID NO:39, consisting essentially of SEQ ID NO:39, or consisting of SEQ ID NO:

39.

12. A polynucleotide encoding a polypeptide according to any one of claims 10-11.

13. A nucleic acid construct or expression vector comprising the polynucleotide according to claim 12, the polynucleotide being operably linked to one or more control sequences directing the production of the polypeptide in an expression host.

14. A recombinant host cell comprising the nucleic acid construct or expression vector according to claim 13.

15. A method for producing a polypeptide having β-fructofuranosidase activity, the method comprising culturing the recombinant host cell according to claim 14 under conditions conducive to the production of the polypeptide and optionally recovering the polypeptide.

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