Compositions comprising lactobacillus strain

Abstract

The present invention relates to Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or combinations thereof, for use as a medicament, preferably for use as a medicament in one or more human and / or animal subject; as well as oral care compositions, methods and kits comprising said species.

Description

[0001] COMPOSITIONS COMPRISING LACTOBACILLUS STRAIN

[0002] REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

[0003] This application contains references to deposits of biological material, which deposits are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or combinations thereof, for use as a medicament, preferably for use as a medicament in one or more human and / or animal subject; as well as oral care compositions, methods and kits comprising said Lactobacillus species.

[0006] BACKGROUND OF THE INVENTION

[0007] Biofilms are communities of bacteria that are found on solid surfaces in many different environments, including surfaces of the oral cavity. Oral biofilm, or dental plaque, are caused by aggregation of pathogenic bacteria, and oral biofilm of the bacteria that are associated with oral health issues such as oral malodor, demineralization, dental caries, tooth decay, potential loss of teeth and gum disease (gingivitis and periodontitis).

[0008] Two types of bacterial interactions contribute to the generation of oral biofilm. The first type of interaction is the interaction between the oral pathogens and the substrates available in the oral cavity, such as soft tissues, dentin, or bacteria already attached to a surface leading to adhesion and a shift to a sessile life form. The second interaction is based on recognition between genetically distinct cells in suspension, leading to coaggregation. This interaction is highly specific interaction and only occurs between coaggregation partners.

[0009] The formation of oral biofilm occurs in three stages known as the lag phase, growth phase, and steady state, respectively. In the lag phase, glycoproteins from saliva bind to an oral surface such as teeth and create a structure termed the pellicle that functions as attachment site for bacteria. In the growth phase, coaggregation occurs, i.e., secondary bacterial colonizers attach to the primary bacterial colonizers, causing the diversity of the biofilm to increase and the biofilm to grow and mature. In the steady state, the biofilm growth slows down and eventually stops. This stage-based formation cycle causes biofilms to exist in several consecutive layers, which makes physical abrasion of biofilm more difficult. SUMMARY OF THE INVENTION

[0010] The inventors made the surprising observation that certain isolated Lactobacillus strains were able to bind to or aggregate with one or more oral pathogen bacterial strain, including isolated strains selected from the group of Porphyromonas, Prevotella, and Fusobacterium, more particularly selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum. Two particularly interesting Lactobacillus strains were identified that were able to coaggregate with several of the oral pathogen bacterial strains: Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859. This observation immediately led the inventors to propose several commercially interesting applications of said deposited Lactobacillus strains, in particular within the oral care and oral medical care spaces.

[0011] Binding or coaggregating oral pathogens can be leveraged for health-promoting purposes, e.g., by decreasing the abundance of these unwanted bacterial species in the oral cavity and / or reducing their ability to form biofilms. In the context of oral microbiota, coaggregation of selected species could alter the dynamics of biofilm and / or plaque build-up, resulting in altered microbial composition and exclusion of pathogens from oral biofilm.

[0012] Accordingly, in a first aspect, the present invention relates to Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or combinations thereof, for use as a medicament, preferably for use as a medicament in one or more human and / or animal subject.

[0013] In a second aspect, the present invention relates to Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, for use in the treatment of infection, preferably for use in the treatment of oral infection, more preferably for use in the treatment of oral infection in one or more human and / or animal subject, even more preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis in one or more human and / or animal subject.

[0014] In a third aspect, the present invention relates to isolated Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860, preferably in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth.

[0015] In a fourth aspect, the present invention relates to oral care compositions comprising at least one strain of Lactobacillus paracasei and / or at least one strain of Lactobacillus rhamnosus in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, and at least one oral care ingredient, characterized in that said strain(s) is capable of: aggregating with at least one oral pathogen bacterial strain, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more prefereably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; preferably said at least one strain(s) is capable of coaggregating two or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; more preferably said at least one strain(s) is capable of coaggregating three or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; and most preferably said at least one strain(s) is capable of coaggregating four or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum.

[0016] In a fifth aspect, the present invention relates to methods for preventing, inhibiting, reducing, altering, and / or eliminating growth of one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, comprising administering: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or a combination thereof; and / or b) an oral care composition according to the fourth aspect, to one or more human and / or animal subject in need thereof.

[0017] In a sixth aspect, the present invention relates to methods for preventing, inhibiting, reducing, altering, and / or eliminating oral biofilm comprising one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, the method comprising contacting the oral biofilm with: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or a combination thereof; and / or b) an effective amount of an oral care composition according to the fourth aspect.

[0018] In a final aspect, the invention relates to kits of parts comprising: a) an oral care composition according to the fourth aspect; and b) instructions for use. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 shows an example of FC-based analysis of coaggregation assay; an FC-based coaggregation analysis of P. gingivalis and 1 B06 strains incubated alone (autoaggregation) and together (coaggregation). Gate highlighted in bold contains double-positive events representing coaggregates formed by both strains. Size distribution of coaggregates can be estimated by gating strategy based on beads of known size (1, 4.5 and 10 pm).

[0020] Fig. 2 shows the FC-based analysis of coaggregation between selected test and target strains of Example 2 below. Aggregation index (upper panel), % target (middle panel) and % test (lower panel) calculated for strain pairs forming coaggregates in SAGF.

[0021] Fig. 3 shows a positive correlation between test strain concentration and the fraction of target cells bound in coaggregates as done in Example 3 below, resulting in up to 70 % of target cells being involved in aggregation.

[0022] Fig. 4A+B show bright field microscopy images and analysis of aggregate size and distribution of test strain and target bacteria pairs. Test strains were incubated in artificial saliva (SAGF) + 0.1 % mucin either alone (self-aggregation) or mixed with target strains in a ratio of 10:1 testtarget OD600 ratio before imaging by brightfield microscopy. Scale bar = 50 pm. Bar graphs show the distribution of aggregates in size groups based on their area in the microscopy images.

[0023] Fig. 5 shows confocal images showing the distribution of dental and test strain cells in the aggregates. Target strains and test strains were stained separately before they were incubated in artificial saliva + 0.1 % mucin wither alone (self-aggregation) or mixed in test / target strain pairs (10:1 OD600 ratio, 1 h incubation) before imaging by CLSM. Test strain cells are green and target strain cells are red.

[0024] Fig. 6A+B show interactions of test or target strains with salivary bacteria analysed in FCbased coaggregation assay. Fig. 6A shows FC-based quantification of the relative cell number of test or target strain interacting with salivary microbiota. Fig. 6B shows FC-based analysis of coaggregates size distribution formed by salivary bacteria with test and target strains.

[0025] Fig. 7A+B+C show FC-based analysis of the interaction between test strain, target strain and salivary microbiota. Fig. 7A shows percentage of test, target and salivary microbiota cells forming coaggregates. Fig. 7B shows percentage of coaggregates formed by 2 or 3 different binding partners. Fig. 7C shows analysis of coaggregates' size distribution (% of total coaggregate number) depending on coaggregates’ composition.

[0026] DEFINITIONS

[0027] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] Lactobacillus strains: Throughout this specification shorthand designations are employed for the two deposited Lactobacillus strains: 1B06 and 8A12. The shorthand designations were derived from their respective microtiter plate positions in a screening assay. The two shorthand designations refer to the following deposited strains:

[0029] 1 B06: Lactobacillus rhamnosus HH03 OB-Lb-0655 (deposited herewith as DSM 34859); this strain was also disclosed in WO 2020 / 084051 as Lactobacillus strain HH03 (DSM 32911) in the context of probiotics only.

[0030] 8A12: Lactobacillus paracasei HH20 MXS_B2_71_3 (deposited herewith as DSM 34860).

[0031] Denture: The term “denture” is meant to cover dentures as such as well as braces, aligners, retainers, and the like.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Lactobacillus strains of the invention for use as medicaments

[0034] The inventors envision the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or as fermentation broth, or combinations thereof, for use as a medicament, preferably for use as a medicament in one or more human and / or animal subject. In some embodiments, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or the Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 for use as a medicament may also be in the form of cell fragments. In a preferred embodiment, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or the Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 for use as a medicament are in the form of non-viable cells.

[0035] In particular, the inventors envision the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or as fermentation broth, for use in the treatment of infection, preferably for use in the treatment of oral infection, more preferably for use in the treatment of oral infection in one or more human and / or animal subject, even more preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis in one or more human and / or animal subject. In some embodiments, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or the Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 for use in the treatment of infection may also be in the form of cell fragments. In a preferred embodiment, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or the Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 for use in the treatment of infection are in the form of non-viable cells.

[0036] A preferred embodiment relates to the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, for use in the treatment of infection, preferably for use in the treatment of oral infection, more preferably for use in the treatment of oral infection in one or more human and / or animal subject with one or more oral pathogen bacterial strain, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more prefereably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum, characterized in that said Lactobacillus strain(s) is capable of: aggregating with at least one oral pathogen bacterial strain, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more prefereably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; preferably said at least one strain(s) is capable of coaggregating two or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; more preferably said at least one strain(s) is capable of coaggregating three or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; and most preferably said at least one strain(s) is capable of coaggregating four or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum.

[0037] In a most preferred embodiment, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or the Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 are in the form of non-viable cells.

[0038] In another preferred embodiment, said medicament further comprises one or more enzyme activity selected from the group of a hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase; preferably selected from an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and a beta-xylosidase; preferably the oral care composition comprises the one or more enzyme activity in an effective amount; even more preferably in an amount of from about 1 ppm to about 500 ppm.

[0039] In another preferred embodiment, said medicament further comprises an oxidoreductase and a mediator. Oxidoreductases such as laccases, oxidases, and peroxidases in combination with suitable mediators are able to prevent, reduce, or remove bad breath (also known as halitosis) by scavenging foul-smelling volatile sulfur compounds (VSCs) produced by some species of oral bacteria. Examples of VSCs include methanethiol (CH3SH), dimethyl sulfide (CH3SCH3), hydrogen sulfide (H2S), and diallyl sulfide. The VSCs are released by the bacteria into the oral environment where they are mixed with air expired from the lungs, resulting in an unpleasant odor emanating from the oral cavity. The oxidoreductase is included in an effective amount, preferably from about 1 ppm to about 500 ppm. Preferred oxidoreductases are described in, e.g., WO 99 / 09143 and include microbial oxidoreductases such as laccases from Polyporus sp. (e.g., P. pinsitus or P. versicolor), Myceliophthora sp. (e.g., M. thermophila), Rhizoctonia sp. (e.g., R. praticola or R. solani), Scytalidium sp. (e.g., S. thermophilium), Pyricularia sp. (e.g., P. oryzae), or Coprinus sp. (e.g., C. cinereus), peroxidases from Coprinus sp. (e.g., C. cinereus or C. macrorhizus) or Bacillus sp. (e.g., 8. pumilus), and glucose oxidases from Aspergillus sp. (e.g., A. niger) or Cladosporium sp. (e.g., C. oxysporum). Preferred mediators are described in, e.g., WO 99 / 09143 and include phenolic compounds such as 4-hydroxybenzoic acid, L-tyrosine, syringic acid, ferulic acid, sinapinic acid, chlorogenic acid, caffeic acid, and esters thereof. In a particularly preferred embodiment, the oxidoreductase is a laccase (EC 1.10.3.2) and the mediator is chlorogenic acid. In a most preferred embodiment, the oxidoreductase is a M. thermophila laccase and the mediator is chlorogenic acid. Laccases from M. thermophila are described in, e.g., WO 95 / 33836.

[0040] Oral care ingredients and formats

[0041] The oral care compositions of the invention comprise at least one strain of Lactobacillus paracasei and / or at least one strain of Lactobacillus rhamnosus in the form of viable cells and / or non-viable cells and / or as fermentation broth, and at least one oral care ingredient, wherein said strain(s) is capable of: aggregating with at least one oral pathogen bacterial strain, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more prefereably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum preferably said at least one strain(s) is capable of coaggregating two or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum more preferably said at least one strain(s) is capable of coaggregating three or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; and most preferably said at least one strain(s) is capable of coaggregating four or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum.

[0042] In some embodiments, the oral care compositions of the invention may comprise the at least one strain of Lactobacillus paracasei and / or the at least one strain of Lactobacillus rhamnosus in the form of cell fragments.

[0043] In a preferred embodiment, the oral care compositions of the invention comprise the at least one strain of Lactobacillus paracasei and / or the at least one strain of Lactobacillus rhamnosus in the form of non-viable cells.

[0044] The oral care ingredients may be varied according to the different types of oral care composition as well as the desired characteristics and / or activities of the oral care compositions. For the purpose of the present invention, the terms “ingredient” and “component” are used interchangeably in relation to oral care compositions.

[0045] A preferred embodiment relates to the oral care composition of the invention, wherein the at least one strain of Lactobacillus paracasei comprises a Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, and / or wherein the at least one strain of Lactobacillus rhamnosus comprises a Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth. In a most preferred embodiment, the at least one strain of Lactobacillus paracasei and / or the at least one strain of Lactobacillus rhamnosus are in the form of non-viable cells.

[0046] In another preferred embodiment, the oral care composition of the invention also comprises one or more enzyme activity selected from the group of a hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase; preferably selected from an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alphagalactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and a beta-xylosidase; preferably the oral care composition comprises the one or more enzyme activity in an effective amount; even more preferably in an amount of from about 1 ppm to about 500 ppm.

[0047] In another preferred embodiment, the oral care composition of the invention also comprises an oxidoreductase and a mediator. Oxidoreductases such as laccases, oxidases, and peroxidases in combination with suitable mediators are able to prevent, reduce, or remove bad breath (also known as halitosis) by scavenging foul-smelling volatile sulfur compounds (VSCs) produced by some species of oral bacteria. Examples of VSCs include methanethiol (CH3SH), dimethyl sulfide (CH3SCH3), hydrogen sulfide (H2S), and diallyl sulfide. The VSCs are released by the bacteria into the oral environment where they are mixed with air expired from the lungs, resulting in an unpleasant odor emanating from the oral cavity. The oxidoreductase is included in an effective amount, preferably from about 1 ppm to about 500 ppm. Preferred oxidoreductases are described in, e.g., WO 99 / 09143 and include microbial oxidoreductases such as laccases from Polyporus sp. (e.g., P. pinsitus or P. versicolor), Myceliophthora sp. (e g., M. thermophila), Rhizoctonia sp. (e g., R. praticola or R. solan!) , Scytalidium sp. (e.g., S. thermophilium), Pyricularia sp. (e.g., P. oryzae), or Coprinus sp. (e.g., C. cinereus), peroxidases from Coprinus sp. (e.g., C. cinereus or C. macrorhizus) or Bacillus sp. (e.g., B. pumilus), and glucose oxidases from Aspergillus sp. (e.g., A. niger) or Cladosporium sp. (e.g., C. oxysporum). Preferred mediators are described in, e.g., WO 99 / 09143 and include phenolic compounds such as 4-hydroxybenzoic acid, L-tyrosine, syringic acid, ferulic acid, sinapinicacid, chlorogenic acid, caffeic acid, and esters thereof. In a particularly preferred embodiment, the oxidoreductase is a laccase (EC 1.10.3.2) and the mediator is chlorogenic acid. In a most preferred embodiment, the oxidoreductase is a M. thermophila laccase and the mediator is chlorogenic acid. Laccases from M. thermophila are described in, e.g., WO 95 / 33836.

[0048] An oral care composition of the invention may be in the form of or as a part of a liquid mouthwash, mouth rinse, dental care formulation, chew tablet, melting sugar tablet, lozenge, hard candy, lollipop, chewing gum, toothpaste, dental paste toothpick, or dental floss.

[0049] An oral care composition of the invention may be an internal oral care composition such as toothpaste or toothpaste tablet, dental cream, mouthwash or mouthwash tablet, mouth rinse, lozenges, pastilles, chewing gum, confectionary, candy, and the like, which is designed to remove biofilm inside the oral cavity, e.g., biofilm residing on teeth, on soft tissues of the oral cavity, and on dentures residing in the oral cavity.

[0050] An oral care composition of the invention may also be an external oral care composition such as denture cleaning solution, denture cleaning tablet, denture cleaning powder, and the like, which is designed to remove biofilm from dentures that have been removed from the oral cavity for cleaning. In a preferred embodiment, the oral care composition is an internal oral care composition, and the at least one oral care component is selected from the group consisting of abrasives, humectants, solvents, thickening agents, binding agents, buffering agents, foaming agents, foaming modulators, sweetening agents, softening agents, plasticizing agents, flavoring agents, coloring agents, therapeutic agents, anti-microbial agents, tartar-controlling agents, fluoride sources, preservatives, detergents, surfactants, coloring agents, buffering agents, softeners, plasticizers, whitening agents, bleaching agents, gum-base ingredients, and bulking agents.

[0051] Although the oral care ingredients mentioned herein are categorized by a general header according to a functionality, this is not to be construed as a limitation, as an ingredient may comprise additional functionalities as will be appreciated by the skilled person.

[0052] In a preferred embodiment, an oral care composition of the invention may also be for use as a medicament, preferably for use as a medicament in the treatment of oral infection, preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis, more preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis in one or more human and / or animal subject.

[0053] Toothpaste, dental cream, mouthwash, and mouth rinse

[0054] Internal oral care compositions of the invention in the form of toothpaste, dental cream, mouthwash, and mouth rinse may include ingredients and / or substances selected from the following categories: Toothpaste

[0055] Toothpastes and dental creams / gels typically include as oral care ingredients abrasives, solvents, humectants, detergents / surfactants, thickening and binding agents, buffering agents, flavoring agents, sweetening agents, fluoride sources, therapeutic agents, coloring agents, and preservatives.

[0056] In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a toothpaste or dental cream, and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0057] In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a toothpaste or dental cream, and at least one oral care ingredient.

[0058] An oral care composition of the invention may be a toothpaste comprising the following ingredients (in weight % of the final toothpaste composition):

[0059] Abrasive: 10 to 70%

[0060] Humectant: 0 to 80%

[0061] Thickening agent: 0.1 to 20%

[0062] Binding agent: 0.01 to 10% Sweetening agent: 0.1 to 5%

[0063] Foaming agent: 0 to 15%

[0064] Lactobacillus of the invention: 0.01 to 20%

[0065] Mouthwash

[0066] Mouthwashes and mouth rinses of the invention, including plaque removing liquids, typically include as oral care ingredients a carrier liquid, detergents / surfactants, buffering agents, flavoring agents, humectants, sweetening agents, therapeutic agents, fluoride sources, coloring agents, and preservatives. In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a mouthwash or mouth rinse, and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients: In a preferred embodiment, the present invention relates to oral care compositions in the form of a mouthwash or mouth rinse, and at least one oral care ingredient.

[0067] An oral care composition of the invention may be a mouthwash comprising the following ingredients (in weight % of the final mouthwash composition):

[0068] Water: 0 to 70% Ethanol: 0 to 20%

[0069] Humectant: 0 to 20% Surfactant: 0 to 2%

[0070] Lactobacillus of the invention: 0.01 to 20%

[0071] Other ingredients: 0 to 2% (e.g., flavors, sweeteners, fluoride sources).

[0072] The mouthwash composition may be buffered with an appropriate buffer, e.g., sodium citrate or phosphate in the pH range 6-7.5.

[0073] Relevant oral care components suitable for toothpastes, dental creams, mouthwashes, and mouth rinses is further detailed below. The skilled person is capable of varying the oral care components according to the type of oral care composition as well as the desired characteristics and / or activities of the specific oral care composition. An oral care composition may not necessarily comprise all the mentioned ingredients.

[0074] Abrasives

[0075] Abrasive polishing material might be incorporated into the oral care composition of the invention. According to the invention said abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, bentonite, silicium dioxide, sodium bicarbonate, and also powdered plastics, such as polyvinyl chloride, polyamides, polymethyl methacrylate, polystyrene, phenolformaldehyde resins, melamine-formaldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels, and the like.

[0076] Also suitable as abrasive agents are calcium pyrophosphate, water-insoluble alkali metaphosphates, poly-metaphosphates, dicalcium phosphate and / or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxyapatite, and the like. It is also possible to employ mixtures of these substances.

[0077] Silica dental abrasives of various types are preferred because of their unique benefits of exceptional dental cleaning and polishing performance without unduly abrading tooth enamel or dentine, and which have a good compatibility with other possible ingredients, like metal ions and fluoride.

[0078] Dependent on the oral care composition, the abrasive product may be present in from 0 to 70% by weight, preferably from 1% to 70%.

[0079] For toothpastes the abrasive material content typically lies in the range of from 10% to 70% by weight of the final tooth-paste product. Humectants

[0080] Humectants are employed to prevent loss of water from, e.g., toothpastes and to avoid hardening of toothpastes upon exposure to air. Some humectants also give a desirable sweetness of flavor to toothpaste and mouthwash compositions. Suitable humectants for use in oral care compositions according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, xylitol, maltitol, lactitol, polyoxyethylene, polyethylene glycols (PEG), polypropylene glycols, propylene glycol, 1 ,3-propanediol, 1,4-butanediol, hydrogenated partially hydrolyzed polysaccharides and the like, coconut fatty acid, amide of N-methyl-taurine, and Pluronic®.

[0081] Humectants are in generally present in from 0% to 80%, preferably 5 to 70% by weight.

[0082] Thickening / bindinq agents

[0083] Suitable thickening and / or binding agents include silica, starch, tragacanth gum, xanthan gum, karaya gum, carrageenans (extracts of Irish moss), gum arabic, alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose and hydroxyethyl propyl cellulose, polyacrylic acid and its salts, polyvinylpyrrolidone and carboxyvinyl polymers, as well as inorganic thickeners such as amorphous silica compounds. These agents stabilize the oral care compositions of the invention.

[0084] Thickeners may be present in toothpaste, dental creams and gels as well as in mouthwashes in an amount of from 0.1 to 20% by weight, and binders to the extent of from 0.01 to 10% by weight of the final product.

[0085] Foaming agents and foaming modulators

[0086] As foaming agent soap, anionic, cationic, non-ionic, amphoteric and / or zwitterionic surfactants can be used, either alone or in combinations. These may be present at levels of from 0% to 15%, preferably from 0.1% to 13%, more preferably from 0.25% to 10% by weight of the final product. Surfactants are only suitable to the extent that they do not exert an inactivation effect on the enzymes and other components included in the oral care composition. Useful surfaceactive agents include anionic, nonionic, and ampholytic compounds, with anionic compounds being preferred.

[0087] Examples of suitable surfactants include salts of the 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 of 10 to 18 carbon atoms; salts of amides of higher fatty acid, e.g., 12 to 16 carbon atom acids, with lower aliphatic amino acids, such as sodium-N-methyl-N-palmitoyl tauride, sodium N-lauroyl-, N- myristoyl- and N-palmitoyl sarcosinates; salts of the esters of such fatty acids with isotopic acid or with glycerol monosulfate; such as the sodium salt of monosulfated monoglyceride of hydrogenated coconut oil fatty acids; salts of olefin sulfonates, e.g., alkene sulfonates or alkene 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 of 12 to 18 carbon atoms, e.g., coconut fatty acids.

[0088] The cation of the salt may be sodium, potassium or mono-, di or triethanol amine. The nonionic surfactants include sucrose / fatty acid esters, maltose / fatty acid esters, maltitol / fatty acid esters, maltotri itol / 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 polyglycerin / fatty acid esters.

[0089] Most preferred are sodium lauryl sulphate, sodium dodecylbenzene sulphonate and sodium lauryl sarcosinate.

[0090] Preferred foaming modulators include polyethylene glycols.

[0091] Foaming agents and foaming modulators may be present from in an amount of from 0% to 15% by weight, preferably from 0.01% to 10% by weight.

[0092] Sweetening agents

[0093] Suitable sweeteners include, but are not limited to, saccharin and water-soluble salts thereof, dextrose, sucrose, lactose, maltose, levulose, aspartame, cyclamate salts, D-tryptophan, dihydrochalchones, acesulphame, stevioside, levaudioside, glycyrrhizins, pellartine, thaumatin, p-methoxycinnamic aldehyde, hydrogenated starch hydrolysates, xylitol, sorbitol, erythritol, mannitol, and mixtures thereof.

[0094] Sweeteners may be present from in an amount of from 0.001% to 60% by weight, preferably from 0.01 % to 50% by weight.

[0095] Flavoring agents

[0096] Flavoring agents are usually present in low amounts, such as from 0.01 % to about 5% by weight, especially from 0.1 % to 5%. The flavors that may be used in the invention include, but are not limited to, Wintergreen oil, peppermint oil, spearmint oil, clove bud oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, 1-inenthvl acetate, sage, eugenol, parsley oil, oxanone, alpha-irisone, marjoram, lemon, orange, cranberry, propenyl guaethol, cinnamon, vanillin, ethyl vanillin, heliotropine, 4-cis-heptenal, diacetyl, methylpara-tert-butyl phenyl acetate, carvone, cineole, menthone, cinnamic aldehyde, limonene, ocimene, n-decyl alcohol, citronellol, alpha- terpineol, methyl acetate, citronellyl acetate, methyl eugenol, linalool, thymol, rosemary oil, pimento oil, diatomaceous oil, eucalyptus oil, and mixtures thereof.

[0097] Coolants may also be part of the flavor system or added separately to the composition. Preferred coolants in the present compositions are the paramenthan carboxyamide agents such as N-ethyl-p-menthan-3-carboxamide (known commercially as 'WS-3"), menthol, 3-1- menthoxypropanc-1,2-diol ("TK-10"), menthone glycerol acetal ("MGA"), menthyl lactate and mixtures thereof.

[0098] Whitening / bleachinq agents

[0099] Whitening / bleaching agents include H2O2 and may be added in amounts less than 5%, preferably from 0.05 to 4%, calculated on the basis of the weight of the final composition.

[0100] Other bleaching components which might be comprised by the present invention include, peroxydiphosphate, urea, peroxide, metal peroxides such as calcium peroxide, sodium peroxide, stronthium peroxide, magnesium peroxide, hypochlorite salts such as sodium hypochlorite, and the salts of perborate, persilicate, perphosphate and percarbonate such as sodium perborate, potassium persilicate and sodium percarbonate. The peroxide compounds can be stabilized by addition of a triphenylmethane dye, a chelating agent or antioxidants such as butylated hydroxy anisole (BHA) or butylated hydroxy toluene (BHT).

[0101] Solvent

[0102] A solvent may be added to a composition of the invention in an amount sufficient for giving the composition a flowable form in case the composition is, e.g., a toothpaste, dental cream, or gel, or to dissolve the other components of the composition, in case of, e.g., a mouthwash or mouth rinse.

[0103] Suitable solvents include water, ethanol, and water / ethanol mixtures, which may be present in an amount of from 0.1 % to 70%.

[0104] Anti-microbial agents

[0105] The present invention also includes water-soluble anti-microbial agents, such as chlorhexidine, triclosan, digluconate, hexetidine, alexidine, quaternary ammonium antibacterial compounds, and water-soluble sources of certain metal ions such as zinc, copper, silver and stannous (e.g., zinc, copper and stannous chloride, and silver nitrate) may also be included.

[0106] Sparingly soluble zinc salts such as zinc citrate, zinc C14-alkyl maleate, zinc benzoate, zinc caproate, zinc carbonate might also be included used in the compositions of the present invention to prolong the anti-microbial effectiveness of zinc ions due to the slow dissolution of these zinc salts in saliva. Anti-microbial agents may be present in an amount of from 0% to 50% by weight, preferably from 0.01 % to 40% by weight, most preferably from 0.1% to 30% by weight.

[0107] Tartar-controlling agent

[0108] Compositions of the invention may comprise a tartar-controlling agent such as inorganic phosphorous tartar-controlling agents including any of the pyrophosphates such as disodium pyrophosphate, dipotassium pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, and mixtures thereof.

[0109] Organic phosphorous compounds that may serve as tartar-controlling agents include polyphosphonates such as disodium ethane-1- hydroxy-1 , 1 -diphosphonate (EHDP), methanediphosphonic acid, and 2-phosphonobutane-1 2,4-tricarboxylic acid.

[0110] Tartar-controlling agents may be present in an amount of from 0% to 10% by weight, preferably from 0.1% to 5% by weight.

[0111] Preservatives

[0112] Suitable preservatives include sodium benzoate, potassium sorbate, p-hydroxybenzoate esters, methyl paraben, ethyl paraben, propyl paraben, citric acid, calcium citrate, and mixtures thereof.

[0113] Preservatives may be present in an amount of from 0% to 40% by weight, preferably from 0.01% to 30% by weight.

[0114] Fluoride sources

[0115] Compositions of the invention may also comprise ingredients that can be used as fluoride source. Preferred soluble fluoride sources include sodium fluoride, potassium fluoride, stannous fluoride, indium fluoride, sodium monofluorophosphate, sodium hexafluorosilicate, zinc fluoride, lithium fluoride, aluminum fluoride, acidulated phosphate fluoride, ammonium bifluoride, titanium tetrafluoride, and amine fluoride.

[0116] Especially preferred are sodium fluoride and sodium monofluorophosphate.

[0117] Fluoride sources may be present in an amount of from 0% to 20% by weight, preferably from 0.01 % to 15% by weight, most preferably from 0.1% to 10% by weight.

[0118] In a preferred embodiment, the at least one oral care ingredient is a fluoride source; preferably the fluoride source is selected from the group consisting of sodium fluoride, calcium fluoride, stannous fluoride, or sodium monofluorophosphate Coloring agents

[0119] Coloring agents or pigments suitable for oral care compositions of the invention include nontoxic, water-insoluble inorganic pigments such as titanium dioxide and chromium oxide greens, ultramarine blues and pinks and ferric 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.

[0120] A preferred opacifier is titanium dioxide.

[0121] Coloring agents may be present in an amount of from 0% to 20% by weight, preferably from 0.01% to 15% by weight, most preferably from 0.1% to 10% by weight.

[0122] Buffering agents

[0123] The oral care compositions of present invention may also include buffering agents, i.e., pH- adjusting agents, such as alkali metal hydroxides, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazole, and mixtures thereof.

[0124] Specific buffering agents include monosodium phosphate, trisodium phosphate, sodium hydroxide, potassium hydroxide, alkali metal carbonate salts, sodium carbonate, imidazole, pyrophosphate salts, sodium citrate, hydrochloric acid, sodium hydroxide, triethanolamine, triethylamine, lactic acid, malic acid, fumaric acid, tartaric acid, phosphoric acid, and mixtures of these.

[0125] Buffering agents may be present in an amount of from 0% to 10% by weight, preferably from 0.01 % to 5% by weight.

[0126] Chewing Gum

[0127] When the oral composition according to the invention is a chewing gum, it can be any known type of chewing gum, such as chewing gum pieces optionally coated, as well as sticks or chewing gum provided with an arbitrary desired shape in response to the intended use. The chewing gum preparation can be of any quality including the bubble gum quality.

[0128] In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a chewing gum comprising at least one oral care ingredient, wherein the at least one oral care ingredient is selected from elastomer, softening agent, plasticizing agent, emulsifier, wax, coloring agent, sweetening agent, flavoring agent, bulking agent, and thickening agent. In a preferred embodiment, the oral care composition further comprises an alpha-amylase. Gum base ingredients

[0129] Chewing gum is traditionally considered as being comprised of a water-insoluble or base portion and a water-soluble portion that contains flavoring agents, sweetening agents, and coloring agents. The gum base part of the gum is a masticatory substance which imparts the chew characteristics to the final product. It defines the release profile of flavors and the sweeteners and plays a significant role in the gum product. The flavors, sweeteners and colors can be thought of as providing the sensory appeal aspects of the chewing gum. No limitations as to the chewing gum bases used in a chewing gum preparation according to the invention exist. Conventional chewing gum bases available for instance from Dansk Tyggegummi Fabrik A / S, L.A. Dreyfus or Cafasa Gum SIA, are usually suitable, but specially made formulations can also be used. The formulation depends on the desired type of chewing gum or the desired type of structure. Suitable raw materials for gum bases include the substances according to the U.S. Chewing Gum Base Regulations - Code of Federal Regulations, Title 21 , Section 172,615 and in accordance with other national and international lists (or positive lists) and include elastomers, resins, waxes, polyvinyl acetates, oils, fats, emulsifiers, fillers, and antioxidants.

[0130] The gum base usually comprises from 15 to 90% by weight, preferably from 30 to 40% by weight, more preferably from 5 to 25% of the final product.

[0131] Elastomers provide the chew, springiness or bounce to the base and control bubble and flavor release in the final chewing gum. They may be any water-insoluble polymer known in the art. They include styrene butadiene copolymers (SBR) and non-SBR types, both natural and synthetic. Examples of natural elastomers include, without limitation, rubbers such as rubber latex (natural rubber) and guayule, and gums such as chicle, jelutong, balata, guttapercha, lechi capsi, sorva, crown gum, nispero, rosidinha, perillo, niger gutta, tunu, gutta kay, pendare, leche de vaca, chiquibul, crown gum, and the like, and mixtures thereof. Examples of synthetic elastomers include, without limitation, polyisobutylene, isobutylene-isoprene copolymers (butylrubber), polyethylene, polybutadiene, styrenebutadiene copolymers, polyisoprene, and the like, and mixtures thereof.

[0132] The amounts of elastomer (rubbers) employed in the gum base composition will vary greatly depending upon various factors such as the type of gum base used (adhesive, or conventional, bubble or standard) the consistency of the gum base composition desired, and the other components used in the composition to make the final chewing gum product. In general, the elastomer is present in the gum base composition in an amount of from about 15% to about 60%, preferably from about 25% to about 30%, by weight based on the total weight of the gum base composition.

[0133] Elastomer solvents aid in softening or plasticizing the elastomer component. In doing so they provide a bulkiness to the chew. Elastomer solvents include, but are not limited to, natural rosin esters and synthetic derivatives of, e.g., terpenes. Examples of elastomer solvents suitable for use herein include tall oil rosin ester; partially hydrogenated wood and gum rosin; the glycerol esters of wood and gum rosin, partially hydrogenated wood / gum rosin, partially dimerized wood and gum rosin, polymerized wood and gum rosin, and tall oil rosin; the deodorized glycerol ester of wood rosin; the pentaerythritol esters of wood and gum rosin; partially hydrogenated wood and gum rosin; the methyl ester of partially hydrogenated wood rosin; methyl, glycerol and pentaerythritol esters of rosins and modified rosins such as hydrogenated, dimerized and polymerized rosins; terpene resins such as polymers of alpha-pinene or beta-pinene, terpene hydrocarbon resins; polyterpene; and the like, and mixtures thereof. The elastomer solvent may be employed in the gum base composition in an amount of from about 2% to about 40%, and preferably from about 7% to about 15% by weight of the gum base composition.

[0134] Polyvinyl acetates provide stretch or elasticity to the gum base. They also affect chew bulkiness, softness and bubble, hydrophilic character, and flavor release.

[0135] The amounts of the different molecular weight polyvinyl acetates present in the gum base composition should be effective to provide the finished chewing gum with the desired chew properties, such as integrity, softness, chew bulkiness, film-forming characteristic, hydrophilic character, and flavor release. The total amount of polyvinyl acetate used in the gum base composition is usually from about 45% to about 92% by weight based on the total gum base composition. The vinyl polymers may possess a molecular weight ranging from about 2000 Da up to about 95,000 Da.

[0136] Typically, the low molecular weight polyvinyl acetate has a weight average molecular weight of from about 2,000 Da to about 14,000 Da. The medium molecular weight polyvinyl acetate typically has a weight average molecular weight of from about 15,000 Da to 55,000 Da. The high molecular weight polyvinyl acetate typically has a weight average molecular weight of from 55,000 Da to about 95,000 Da but may range as high as 500,000 Da.

[0137] Waxes, fats, and oils plasticize the elastomer mixture and improve the elasticity of the gum base. Waxes can provide a soft or firm chew, affect the flavor release, and provide bulkiness and smoothness to the gum base. Fats and oils provide a soft chew. The fats, oils and waxes may be use individually or in combination or the gum base may be a wax free gum base.

[0138] Waxes when used, may be of mineral, animal vegetable or synthetic origin. Non-limiting examples of mineral waxes include petroleum waxes such as paraffin and microcrystalline waxes, animal waxes include beeswax, vegetable waxes include carnauba, candellila, rice bran, esparto, flax and sugarcane, and synthetic waxes include those produced by the Fischer-Tropsch synthesis, and mixtures thereof. Suitable oils and fats usable in gum compositions include hydrogenated or partially hydrogenated vegetable or animal fats, such as cottonseed oil, soybean oil, coconut oil, palm kernel oil, beef tallow, hydrogenated tallow, lard, cocoa butter, lanolin, and the like; fatty acids such as palmitic, oleic, stearic, linoleic, lauric, myristic, caproic, caprylic, decanoic or esters and salts as sodium stearate and potassium stearate. These ingredients when used are generally present in amounts up to about 7% by weight of the gum composition, and preferably up to about 3.5% by weight of the gum composition.

[0139] Preferred as softeners are the hydrogenated vegetable oils and include soybean oil and cottonseed oil which may be employed alone or in combination. These softeners provide the gum base composition with good texture and soft chew characteristics. These softeners are generally employed in an amount from about 5% to about 14% by weight of the gum base composition.

[0140] Emulsifiers aid in dispersing the immiscible components of the gum base composition into a single stable system. They provide hydrophilic character to a gum base and aid in plasticizing the resins and polyvinyl acetates. They also affect the softness of the base and the bubble character of the base. Typical emulsifiers include acetylated monoglyceride, glyceryl monostearate, lecithin, fatty acid monoglycerides, diglycerides, propylene glycol monostearate, lecithin, triacetin, glyceryl triacetate and the like, and mixtures thereof.

[0141] Preferred emulsifiers are glyceryl monostearate and acetylated monoglycerides. These serve as plasticizing agents. The emulsifiers may be employed in an amount of from about 2% to about 15% by weight of the gum base composition, and preferably from about 7% to about 11 % by weight of the gum base composition.

[0142] The fats, oils, waxes, emulsifiers, and certain sugar bulking agents are often grouped together and referred to as softening agents. Because of the low molecular weight of these ingredients, the softeners can penetrate the fundamental structure of the gum base making it plastic and less viscous. Useful plasticizers and softeners of the above include lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate, propylene glycol nonastearate, acetylated monoglyceride, glycerin, fully unsaturated vegetable oils such as nonhydrogenated cottonseed oil, hydrogenated vegetable oils, petroleum waxes, sorbitan monostearate, tallow, and the like, and mixtures thereof and also include high fructose corn syrup, corn syrup, sorbitol solution, hydrogenated starch hydrolysate, and the like, and mixtures thereof.

[0143] The amount of softener present should he an effective amount to provide a finished chewing gum with the desired chew bulkiness and softness. When used as softeners these materials are generally employed in the gum base composition in an amount of up to about 25%, and preferably in an amount of from about 1% to about 17%, by weight of the gum base composition. The gum base may further contain a surfactant. Examples of suitable surfactants include polyoxyethylene (20) sorbitan monoleate, polyoxyethylene (20) sorbitan monolaurate, polyethylene (4) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene, (4) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, polyoxyethylene (5) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, sorbitan monolaurate, and the like. The amount of surfactant present should be effective to provide the finished chewing gum with the desired softness. Typically, the surfactant is employed in the base in an amount of from about 0.5% to about 3.0% by weight based on the total weight of the gum base.

[0144] The gum base composition of this invention may also include effective amounts of fillers sometimes referred to as bulking agents. These materials add firmness and bulk and affect the texture and the flavor release of the chewing gum. Useful fillers include organic and inorganic compounds (mineral adjuvants) such as calcium carbonate, magnesium carbonate, ground limestone, magnesium silicate, calcium phosphate, cellulose polymers, clay, alumina, aluminum hydroxide, aluminum silicate, tale, tricalcium phosphate, dicalcium phosphate, and the like, and mixtures thereof. These fillers or adjuvants may be used in the gum base compositions in various amounts. The amount of the filler present should be effective to provide a finished chewing gum with the desired flavor release and integrity. Typically, the filler is employed in the gum base composition in an amount from about 1 % to about 40%, and preferably from about 5% to about 20%, by weight of the gum base composition.

[0145] The gum base may also comprise an antioxidant to provide improved stability, lessen any oil-taste and provide longer shelf life. Typical non-limiting examples of antioxidants are butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), propyl gallate. Mixtures thereof may also be used.

[0146] Other gum ingredients

[0147] The remaining ingredients in chewing gum compositions are conventional and usually comprise from 10 to 85% by weight of the final product.

[0148] Examples thereof are sweetening agents, softeners, coloring agents, bulking agents, thickening agents, and flavoring agents of the type and in the amounts conventionally used for chewing gum.

[0149] Suitable flavoring agents those flavors known to the skilled artisan such as natural and artificial flavors. These flavorings may be chosen from synthetic flavor oils and flavoring aromatics and / or oils, oleoresins and extracts derived from plants, leaves, flowers, fruits, and so forth, 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, oil of nutmeg, allspice, oil of sage, mace, oil of bitter almonds, and cassia oil. Other useful flavorings 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 and so forth. These flavoring agents may be used in liquid or solid form and may be used individually or in admixtures. Commonly used flavors include mints such as peppermint, menthol, artificial vanilla, cinnamon derivatives, and various fruit flavors, whether employed individually or in admixture.

[0150] Other useful flavoring agents include aldehydes and esters such as cinnamyl acetate, cinnamaldehyde, citrate diethylacetal, dihydrocarvyl acetate, eugenyl formate, p-methyl anisole, and so forth may be used. Generally, any flavoring or food additive may be used.

[0151] Further examples of aldehyde flavorings include, but are not limited to, acetaldehyde (apple), benzaldehyde (cherry, almond), anisic aldehyde (licorice, anise), cinnamic aldehyde (cinnamon), citral, i.e., alpha-citral (lemon, lime), neral, i.e., beta-citral (lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, cream), heliotrope, i.e., piperonal (vanilla, cream), vanillin (vanilla, cream), alpha-amyl cinnamaldehyde (spicy fruity flavors), butyraldehyde (butter, cheese), valeraldehyde (butter, cheese), citronellal (many 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, i.e., trans-2-hexenal (berry fruits), tolyl aldehyde (cherry, almond), veratraldehyde (vanilla), 2,6-dimethyl-5-heptenal, i.e., melonal (melon), 2,6-dimethyloctanal (green fruit), and 2-dodecenal (citrus, mandarin), cherry, grape, strawberry shortcake, mixtures thereof and the like.

[0152] The amount of flavoring agent employed herein is normally a matter of preference subject to such factors as the type of final chewing gum composition, the individual flavor, the gum employed, and the strength of flavor desired. Thus, the amount of flavoring may be varied to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. In gum compositions, the flavoring agent is generally present in amounts from about 0.02% to about 5% by weight of the chewing gum composition.

[0153] The chewing gum compositions generally include bulking agents. These bulking agents (carders, extenders) may 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 (a racemic mixture of alpha-D-glucopyranosyl-1,6-mannitol and alpha-D-glucopyranosyl-1 ,6-sorbitol manufactured under the tradename Palatinit™ by Suddeutsche Zucker), glycerol, aspartame, Lycasin® glycerol, galactitol acesulphame K, saccharine and salts thereof, cyclamate and salts thereof, neohesperidine dihydrochalcone, glycyrrhizinic acid and salts thereof, thaumantine and sucralose as well as mixtures thereof or mixtures thereof with other suitable sweeteners, maltodextrins; hydrogenated starch hydrolysates; hydrogenated hexoses; hydrogenated disaccharides; minerals, such as calcium carbonate, talc, titanium dioxide, dicalcium phosphate, celluloses and the and the like, and mixtures thereof. Bulking agents may be used in amounts up to about 60%, and preferably in amounts from about 25% to about 60%, by weight of the chewing gum composition.

[0154] The chewing gum compositions may also include a high intensity sweetening agent (sweeteners). High intensity sweetening agents have a sweetness intensity substantially greater than that of sucrose. Examples of suitable intense sweeteners include: a) water-soluble naturally occurring intense sweeteners such as dihydrochalcones, monellin, steviosides, glycyrrhizin, dihydroflavenol, and L-aminodicarboxylic acid aminoalkonoic acid ester amides, such as those disclosed in in United States patent no. 4,619,834, and mixtures thereof; b) water-soluble artificial sweeteners including the soluble saccharin salts such as sodium or calcium saccharin salts, cyclamate salts, the sodium, ammonium or calcium salts of 3,4- dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassium salt of 3,4-dihydro-6- methyl-1 ,2,3-oxathiazine-4-one-2,2-dioxide (Acesulfam-K), the free acid form of saccharin, and the like, and mixtures thereof; c) dipeptide based sweeteners including L-aspartic acid derived sweeteners such as 1- aspartyl-L-phenylalanine methyl ester (Aspartame) and materials described in United States patent no. 3,492,131 , L-alpha-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate (Alitame), methyl esters of L-aspartyl-L-phenylglycerine and L-aspartyl-L-2,5-dihydrophenyl- glycine, L-aspartyl-2.5-dihydro-L-phenylalanine, L-aspartyl-L-(1-cyclohexen)-alanine, and the like, and mixtures thereof; d) water-soluble intense, sweeteners derived from naturally-occurring water-soluble sweeteners, such as chlorinated derivatives of ordinary sugar (sucrose), e.g., chlorodeoxysugar derivatives such as derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose, known, for example, under the product designation of Sucralose®; examples of chlorodeoxysucrose and chlorodeoxygalactosucrose derivatives include but are not limited to: to 1-chloro-1'-deoxysucrose; 4-chloro-4-deoxy-alpha-D-galactopyranosyl-alpha-D-fructofuranoside, or 4- chloro-4- deoxygalactosucrose; 4-chloro-4-deoxy-alpha-D-galactopyranosyl-1-chloro-ldeoxy- beta-D- fructo-furanoside, or 4,1'-dichloro-4,T-dideoxygalactosucrose; 1',6'-dichloro-1',6'- dideoxysucrose; 4-chloro-4-deoxy-alpha-D-galactopyranosy 1 -1 ,6-dichloro-1 ,6-dideoxy-beta-D- fructofuranoside, or 4,T,6'-trichloro-4,1',6'-trideoxygalactosucrose; 4,6-dichloro-4,6-dideoxy- alpha-D-galactopyranosyl-6-chloro-6-deoxy-beta-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-alpha- D-galactopyranosyl-1 ,6-dichloro-1,6-dideoxy-beta-D-fluctofuranoside, or 4,6,1',6'-tetrachloro- 4,6,1',6'-tetradeoxygalacto-sucrose; and 4,6,1',6'-tetradeoxy-sucrose, and mixtures thereof; and e) protein based intense sweeteners such as Thaumaoccous daniclii (Thaumatin I and II). The amount of sweetener employed in the chewing gum composition will vary with the sweetener selected for a particular chewing gum. Thus, for any given sweetener, a sufficient amount of sweetener is used to provide the level of sweetness desired. The saccharide sweeteners and sugar alcohols described above are usually used in an amount of from about 1 % to about 70% and preferably in an amount of from about 40% to about 50%, by weight based on the total weight of the chewing gum composition. The intense sweeteners described above are usually used in an amount of up to about 1 %, preferably from about 0.05% to about 0.4%, by weight based on the total weight of the chewing gum composition.

[0155] The coloring agents useful in the present invention are used in amounts effective to produce the desired color. These coloring agents include pigments, which may be incorporated in amounts up to about 6%, by weight of the gum composition. A preferred pigment, titanium dioxide, may be incorporated in amounts up to about 2%, and preferably less than about 1%, by weight of the gum composition. The colorants may also include natural food colors and dyes suitable for food, drug, and cosmetic applications. These colorants are known as F.D.& C. dyes and lakes. The materials acceptable for the foregoing uses are preferably water-soluble. Illustrative non-limiting examples include the indigoid dye known as F.D.& C. Blue No.2, which is the disodium salt of 5,5- indigotindisulfonic acid. Similarly, the dye known as F.D.& C. Green No.1 comprises a triphenylmethane dye and is the monosodium salt of 4-[4-(N-ethyl-N-p- sulfoniumbenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclo- hexadieneimine].

[0156] Examples of thickening agents include methyl cellulose, alginates, carrageenan, xanthan gum, gelatin, carob, tragacanth, and locust bean, emulsifiers, such as lecithin and glyceryl monostearate, acidulants such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid, and mixtures thereof.

[0157] The plasticizers, softening agents, emulsifiers, waxes, and antioxidants discussed above as being suitable for use in the gum base may also be used in the chewing gum composition.

[0158] Active gum ingredients

[0159] Oral care compositions of the invention in the form of a chewing gum may also contain various active ingredients such as antimicrobial agents, Zn salts, fluorides, and urea.

[0160] Moreover, the oral composition according to the invention may, if desired, include any other active ingredients, such as anti-caries agents, anti-calculus agents, anti-plaque agents, anti- periodontal agents, anti-fungal agents, anti-smoking agents, anti-cold agents, agents against gingivitis, etc.

[0161] The antimicrobials used in the compositions can be any of a wide of cationic antimicrobial agents such as quaternary ammonium compounds (e.g., cetyl pyridinium chloride) and substituted guanidines such as chlorhexidine and the corresponding compound alexidine. Mixtures of cationic anti-microbials may also be used in the present invention.

[0162] Antimicrobial quaternary ammonium compounds include those in which one or two of the substituents on the quaternary nitrogen has a carbon chain length (typically alkyl group) of some 8 to 20, typically 10 to 18 carbon atoms while the remaining substituents (typically alkyl or benzyl group) have a lower number of carbon atoms, such as 1 to 7 carbon atoms, typically methyl or ethyl groups. Dodecyl trimethyl ammonium bromide, tetradecyl pyridinium chloride, tetradecyl ethyl pyridinium chloride, dodecyl dimethyl (2-phenoxyethyl) ammonium bromide, benzyl dimethylstearyl ammonium chloride, cetyl pyridinium chloride, quaternized 5-amino-1 ,3-bis 2- ethyl-hexyl)-5-methyl hexa hydropyrimidine and benzethonium chloride are exemplary of typical quaternary ammonium antibacterial agents. Other compounds are the bis[4-(R-amino)-1- pyridinium] alkanes as disclosed in U.S. Patent 4,206,215, June 3, 1980, to Bailey incorporated herein by reference. The pyridinium compounds are the preferred quaternary ammonium compounds.

[0163] The cationic antimicrobial is generally used in the present compositions at a level of from about 0.02% to about 1%, preferably from about 0.3% to about 0.7% most preferably from about 0.3% to about 0.5%.

[0164] As easily soluble zinc salt it is in principle possible to use any physiologically acceptable, easily soluble zinc salt of an inorganic or organic acid, said salt being able to release zinc ions and being approved for the intended use, such as in foodstuffs, cosmetics, or pharmaceutical products. Non-limiting examples are for instance zinc citrate, zinc sulphate, zinc lactate, zinc chloride, zinc acetate as well as mixtures thereof. Among these salts zinc acetate is preferred.

[0165] The zinc salt used must be easily soluble such that a release is ensured in the oral cavity of an amount of zinc ions efficient for the purpose aimed at within a suitable period of time.

[0166] Advantageously, the zinc salt is present in the oral composition in an amount of from 0.001 to 1 .25% by weight. The amount used depends on the administration form and the intended use and is adapted such that an amount of zinc ions efficient for the intended use is released.

[0167] As taste-masking salt is used at least one salt selected among sodium chloride, ammonium chloride and physiologically acceptable alkali metal, alkaline earth metal and / or ammonium carbonates. The alkali metal is in particular sodium or potassium, whereas the alkaline earth metal advantageously is calcium or magnesium. Particularly preferred taste-masking salts are sodium, potassium and magnesium carbonates, sodium chloride, ammonium chloride as well as mixtures thereof.

[0168] The taste-masking salt is advantageously used in the oral composition in an amount of from 0.05 to 6.25% by weight, more preferred from 0.25 to 3.50% by weight, such as from 0.50 to 2.50% by weight.

[0169] The amount used of taste-masking salt for masking the taste of zinc can in each case be determined by a person skilled in the art and depends on the particular zinc salt in question and the selected administration form.

[0170] Urea is used as an anticariogenic product for neutralizing the acid produced in dental plaque after eating or drinking. Beyond urea the composition also can contain pharmacologically acceptable substances capable of releasing urea under the conditions prevailing in the mouth. Examples thereof are salts and addition compounds between urea and inorganic compounds such as magnesium sulphate, calcium phosphate, sodium chloride, etc.

[0171] The urea content of the composition according to the invention varies between 0.05% by weight and 80% by weight, preferably between 0.2% by weight and 25% by weight.

[0172] The chewing gum compositions may be prepared using standard techniques and equipment known to those skilled in the art. The apparatus useful in accordance with the present invention comprises mixing and beating apparatus as well.

[0173] Lozenges and pastilles

[0174] Lozenges are flavored medicated dosage forms intended to be sucked and held in the mouth or pharynx. They may contain vitamins, antibiotics, antiseptics, local anesthetics, antihistamines, decongestants, corticosteroids, astringents, analgesics, aromatics, demulcents, or combinations of these ingredients. Lozenges may take various shapes, the most common being the flat, circular, octagonal, and biconvex forms. Another type, called bacilli, are in the form of short rods or cylinders. A soft variety of lozenge, called a pastille, consists of medicament in a gelatin or glycerogelatin base or in base of acacia, sucrose, and water (H. A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, N.Y.).

[0175] In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a lozenge or pastille comprising at least one oral care ingredient, wherein the at least one oral care ingredient is selected from lubricant, bulking agent, sweetening agent, and flavoring agent. In a preferred embodiment, the oral care composition further comprises an alpha-amylase. Lubricants

[0176] The use of a lubricant in the manufacture of compressed lozenges is to facilitate the release of the lozenge from the die in which it is formed. The lubricant used in the present invention is a solid material which is not charged, and which will not interfere (e.g., complex) with the cationic antimicrobial. The material should preferably be water insoluble. One type of suitable material meeting these requirements is a non-toxic hydrocarbon fat or derivative. Examples include hydrogenated tallow and hydrogenated vegetable oil. Polyethylene glycols may also be used as a lubricant so long as they are solid materials which generally means having a molecular weight in the 4000 Da to 6000 Da range. These materials can also be used as a filler as noted below.

[0177] Mixtures of lubricants may also be used in the present invention. The lubricant is used at level of from about 0.1% to about 4.0% preferably from about 0.5% to about 2%.

[0178] Lozenge vehicle

[0179] The term “lozenge vehicle” is used herein to denote the material(s) which carries the active ingredients, i.e., the enzymes, as well as the lubricant. These materials are also known as bulking agents or fillers. Since the vehicle is non-cariogenic, the vehicle should be free of sucrose and similar materials.

[0180] Acceptable filler materials include mannitol, sorbitol, xylitol, polyethylene glycol and non- cariogenic dextrans. The fillers may be used alone or in combination.

[0181] Mannitol is a naturally occurring sugar alcohol and is available as a fine powder. It has a sweetness of only about 50% of that of sucrose. However, mannitol's negative heat of solution enables it to impart a pleasant, cooling sensation in the mouth as the lozenge dissolves.

[0182] Sorbitol is a chemical isomer of mannitol and possesses a similar degree of sweetness. Its heat of solution, being negative, also provides for a pleasant, cooling sensation in the mouth. Sorbitol is available either as free flowing granules or as a crystalline powder. Polyethylene glycols (PEG'S) can also be used in the present compositions. These materials are polymers of ethylene oxide with the generalized formula HOCH2(CH2OCH2)nCH2OH. The use of PEG'S alone is not favored but their use in combination with other fillers is acceptable. The molecular weights found most desirable are between 4000 Da and 6000 Da.

[0183] Fillers are generally used in the present invention at a level of from about 85% to about 99.8%, preferably from about 90% to about 98%, most preferably from about 94% to about 97%.

[0184] Other lozenge components

[0185] Acceptable lozenges may be manufactured using just an active ingredient, the lubricant and the filler material as outlined above. However, to make the lozenges more acceptable from an aesthetic viewpoint, generally included are materials such as spray-dried or encapsulated flavors or liquid flavors adsorbed onto a suitable diluent. Spray-dried or encapsulated flavors are preferred. Suitable flavors include oil of peppermint, oil of Wintergreen, oil of sassafras, oil of spearmint and oil of clove. Sweetening agents are also acceptable for use in the present compositions. Suitable agents include aspartame, acesulfame, saccharin, dextrose and levulose. Sweetening and flavoring agents are generally used in the compositions of this invention at levels of from about 0.1 % to about 2%, preferably from about 0.25% to about 1.5%.

[0186] It is also acceptable to have a solid form of a water-soluble fluoride compound present in the present lozenges in an amount 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 additionally anticaries effectiveness. Preferred fluorides are sodium fluoride, stannous fluoride, indium fluoride and sodium monofluorophosphate. The lozenges may also contain various active ingredients such as anti-microbial agents, Zn salts, fluorides, and urea (supra).

[0187] Confectionaries and candy

[0188] In a preferred embodiment, the present invention relates to oral care compositions of the invention in the form of a confectionary or candy comprising at least one oral care ingredient wherein the at least one oral care ingredient is selected from coloring agent, sweetening agent, flavoring agent, and oil-modifying agent. In a preferred embodiment, the oral care composition further comprises an alpha-amylase.

[0189] The preparation of confectionery formulations is historically well known and has changed little through the years. Confectionery items have been classified as either "hard" confectionery or "soft" confectionery. The volatile oil-modifying agent of the present invention can be incorporated by admixing the modifying agent into conventional hard and soft confections.

[0190] Hard confectionery may be processed and formulated by conventional means. In general, a hard confectionery has a base composed of a mixture of sugar and other carbohydrate bulking agents kept in an amorphous or glassy condition. This form is considered a solid syrup of sugars generally having from about 0.5% to about 1.5% moisture. Such materials normally 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 generally prepared from corn syrups high in fructose but may include other materials. Further ingredients such as flavorings, sweeteners, acidulants, colorants and so forth may also be added.

[0191] Such confectionery may be routinely prepared by conventional methods such as those involving fire cookers, vacuum cookers, and scraped-surface cookers also referred to as highspeed atmospheric cookers. Fire cookers involve the traditional method of making a candy base. In this method, the desired quantity of carbohydrate bulking agent is dissolved in water by heating the agent in a kettle until the bulking agent dissolves. Additional bulking agent may then be added, and cooking continued until a final temperature of 145 to 156 °C. is achieved. The batch is then cooled and worked as a plastic-like mass to incorporate additives such as flavor, colorants, and the like.

[0192] A high-speed atmospheric cooker uses a beat-exchanger surface, which involves spreading a film of candy on a heat exchange surface, the candy is heated to 165 to 170 °C. in a few minutes. The candy is then rapidly cooled to 100 to 120 °C. and worked as a plastic-like mass enabling incorporation of the additives, such as flavors, colorants, and the like.

[0193] In vacuum cookers, the carbohydrate bulking agent is boiled to 125 to 132 °C, vacuum is applied, and additional water is boiled off without extra heating. When cooking is complete, the mass is a semi-solid and has a plastic-like consistency. At this point, flavors, colorants, and other additives are admixed in the mass by routine mechanical mixing operations.

[0194] The optimum mixing required to uniformly mix the flavors, colorants, and other additives during conventional manufacturing of hard confectionery is determined by the time needed to obtain a uniform distribution of the materials. Normally, mixing times of from 4 to 10 minutes have been found to be acceptable.

[0195] Once the candy mass has been properly tempered, it may be cut into workable portions or formed into desired shapes. A variety of forming techniques may be utilized depending upon the shape and size of the final product desired. A general discussion of the composition and preparation of hard confections may be found in H. A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1980), Marcel Dekker, Inc., New York, N.Y.

[0196] The apparatus useful in accordance with the present invention comprises cooking and mixing apparatus well known in the confectionery manufacturing arts, and election of the specific apparatus will be apparent to the artisan. In contrast, compressed tablet confections contain particular materials and are formed into structures under pressure.

[0197] These confections generally contain sugars in amounts up to about 95%, by weight of the composition, and typical tablet excipients such as binders and lubricants as well as flavoring agent, colorants and so forth. Like hard confectionery, soft confectionery may be utilized in this invention. The preparation of soft confections, such as nougat, involves conventional methods, such as the combination of two primary components, namely (1) a high boiling syrup such as corn syrup, hydrogenated starch hydrolysate or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gelatin, vegetable proteins, such as soy derived compounds, sugarless milk derived compounds such as milk proteins, and mixtures thereof. The frappe is generally relatively light, and may, for example, range in density from about 0.5 to about 0.7 grams / cc. The flavoring components of the confection are flavors having an associated bitter taste or other unpleasant after taste. These flavoring components may be chosen from natural and synthetic flavoring liquids such as volatile oils, synthetic flavor oils, flavoring aromatic and oils, liquids, oleoresins, or extracts derived from plants, leaves, flowers, fruits, stew, and combinations thereof. Non-limiting representative examples of volatile oils include spearmint oil, cinnamon oil, oil of Wintergreen (methyl salicylate), peppermint oil, menthol, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, oil of nutmeg, allspice oil, oil of sage, mace extract, oil of bitter almonds, and cassia oil. In addition, the confection may also contain artificial, natural, or synthetic flavors including fruit flavors such as vanilla, and citrus oils including lemon, orange, grape, lime and grapefruit and fruit essences including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot and so forth individual and mixed.

[0198] Other useful flavorings include aldehydes and esters such as benzaldehyde (cherry, almond), citral, / .e., alpha-citral (lemon, lime), neral, i.e., beta-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 fruit), and 2-dodecenal (citrus, mandarin), mixtures thereof and the like.

[0199] In the instance where sweeteners are utilized, the present invention contemplates the inclusion of those sweeteners well known in the art, including both natural and artificial sweeteners. The sweeteners may be chosen from the following non-limiting list: sugars such as sucrose, glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof, saccharin and its various salts such as the sodium or calcium salt; cyclamic acid and its various salts such as the sodium salt; the dipeptide sweeteners such as aspartame, dihydrachalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro-derivatives of sucrose; dihydroflavinol; hydroxyguaiacol esters; L-amino dicarboxylic acid gem-diamines; L-aminodicarboxylic acid aminoalkenoic acid ester amides; and sugar alcohols such as sorbitol, sorbitol syrup, mannitol, xylitol, and the like. Also contemplated is the synthetic sweetener 3,6-dihydro-6-methyl-1 ,2,3- oxathiazin-4-one-2,2-dioxide, particularly the potassium (acesulfame-K), sodium and calcium salts thereof.

[0200] The confection may also include a colorant. The colorants may be selected from any of the numerous dyes suitable for food, drug, and cosmetic applications, and known as FD&C dyes and the like. The materials acceptable for the foregoing spectrum of use are preferably water-soluble. Illustrative examples include indigoid dye, known as FD&C Blue No. 2, which is the disodium salt of 5,5'-indigotindisulfonic acid. Similarly, the dye known as FD&C Green No. 1 comprises a triphenylmethane dye and is the monosodium salts of 4-[4-N-ethyl-p-sulfobenzylami no)diphenylmethylane]-[1-(N-ethyl-N-p-sulfoniumbenzyl)-2-5-cyclohexadieneimine]. A full recitation of all FD&C and D&C dyes and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5. The confectionary may also include a volatile oil-modifying agent such as capsicum oleoresin. An oil-modifying agent is present in an amount, which is undetected as a separate ingredient in the oral cavity, but nevertheless can modify sensory perception of the volatile oil. The oil-modifying agent is present in an amount of from about 1 to about 150 ppm of the confection. The capsicum is available from Capsicum minimum, Capsicum frutescens, Capsicum annuum, and similar varieties. Commercially, the fruits of capsicum are referred to as chilies or as peppers. These fruits are known for their extreme potency of bite, pungency, and characteristic odor.

[0201] With respect to confectionery compressed tablet formulations, such will contain a tablet granulation base and various additives such as sweeteners and flavors. The tablet granulation base employed will vary depending upon factors such as the type of base used, friability desired and other components used to make the final product. These confections generally contain sugars in amounts up to 95% by weight of the composition.

[0202] The confectionery compressed tablet may additionally include tablet excipients such as binders or lubricants, as well as flavoring agents, coloring agents, and volatile oils and volatile oilmodifying agents.

[0203] The variations that one may practice with regard to these confections are wide ranging and within the ability of those skilled in the art particularly with regard to the use of additional composition fillers, flavoring agents, the use of coloring agents, etc.

[0204] External oral care compositions

[0205] An external oral care formulation, e.g., denture cleaning solution, denture cleaning tablet, denture cleaning powder, and the like, may include ingredients and / or substances selected from the following categories:

[0206] In a preferred embodiment the at least on oral care ingredient is selected from the group consisting of carrier liquids, disinfectant and bleaching agents, cleaning agents, detergents and surfactants, foaming agents, preservatives, and flavoring agents.

[0207] In one aspect, the oral care compositions of the invention may also be included in filaments suitable for use in dental cleaning, e.g., filaments useful as dental floss. Preferably, the oral care composition is coated onto the exterior of the filament. Thus, in a preferred embodiment, the present invention relates to a filament comprising an oral care composition comprising an invertase, a beta-glucosidase, and a glucoamylase, wherein the filament is suitable for dental cleaning.

[0208] Application of oral care compositions

[0209] The oral care compositions of the invention are suitable for use in the treatment of oral disease, wherein prevention, modification or removal of oral biofilm is desired. The compositions of the invention are particularly suitable for treating periodontal diseases and dental caries.

[0210] Periodontal disease, also known as gum disease, is a set of inflammatory conditions caused by bacterial infection and subsequent biofilm build-up on the test and the tissues surrounding the teeth. Periodontal disease may be divided in terms of severity into the following categories: gingivitis (including plaque-induced gingivitis), chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic disease, necrotizing ulcerative gingivitis / periodontitis, abscesses of the periodontium, and combined periodontic-endodontic lesions. Periodontal disease may further be considered either localized or generalized depending on the extent of the affected area.

[0211] Dental caries, also known as tooth decay or cavities, is caused by organic acids, such as lactic acid, being released by certain biofilm-forming bacteria residing in the oral cavity, including Streptococcus mutans and some Lactobacillus species. Dental caries may be associated with further complications such as inflammation of the tissue around the teeth, tooth loss, and infection or abscess formation. Dental caries may be classified by location, etiology, rate of progression, and affected hard tissues, for instance according to the G.V. Black classification (class I, II, III, IV, V, and VI). In one aspect, the present invention relates to a method of treatment of a human subject, the method comprising administering an oral care composition of the invention comprising at least one oral care ingredient to the human subject. In a preferred embodiment, the oral care composition is administered to the oral cavity of the human subject.

[0212] In another aspect, the invention relates to methods for preventing, inhibiting, reducing, altering, and / or eliminating growth of one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, comprising administering: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments, and / or as fermentation broth, or a combination thereof; and / or b) an oral care composition according to the invention, to one or more human and / or animal subject in need thereof.

[0213] In a preferred embodiment, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 are in the form of non-viable cells.

[0214] In yet another aspect, the invention relates to methods for preventing, inhibiting, reducing, altering, and / or eliminating oral biofilm comprising one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, the method comprising contacting the oral biofilm with: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or a combination thereof; and / or b) an effective amount of an oral care composition according to the invention.

[0215] In a preferred embodiment, the Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 are in the form of non-viable cells.

[0216] EXAMPLES

[0217] Materials and Methods

[0218] Cultivation and preparation of test and target strains

[0219] 719 test strains were selected for screening from the proprietary strain collection of Novozymes A / S. All strains belong to genera of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, and Weisella. All test strains were pre-cultured in YDA medium (25 g / l yeast extract, 20 g / l D(+)glucose, 1 g / l Tween 80, 2 g / l diammonium hydrogen citrate, 5 g / l sodium acetate anhydrous, 0.1 g / l magnesium sulfate heptahydrate, 0.05 g / l manganese(ll) sulfate monohydrate, and 2 g / l di-potassium hydrogen phosphate) for 24 hours at 37 °C, before 50 pL of the pre-culture was inoculated to fresh YDA medium incubated for 72 hours. Test strains were heat-inactivated for 10 min at 70°C and stored in 16% glycerol at - 80°C until needed.

[0220] Three target strains: Prevotella intermedia, Porphyromonas gingivalis and Fusobacterium nucleatum were grown in anaerobic jars supplemented with an AnaeroGen packet (Thermo Scientific™) to achieve an anaerobic environment. The target strains were grown for 48-72 hours at 37 °C in plaque medium (10 g / l tryptone, 10 g / l yeast extract, 2.5 g / l potassium phosphate, 2.5 g / l magnesium sulfate, 2 g / l starch from potato, 2 g / l D(+)glucose, 1.4 g / l L-cysteine HCI, 1 pg / l menadione, 1 pg / l hemin, and triethanolamine) to obtain “pre-cultures”. The pre-cultures were then adjusted to ODsoo =0.05 and incubated for 48 hours to obtain “main cultures”. The plaque medium was stored for maximum 4 days after preparation.

[0221] Measurement of aggregation index

[0222] The target strains were harvested by centrifugation (3200 x g, 15 min), resuspended, washed, and resuspended to ODSOo = 3 in SAGF + 0.1 % mucin (0.63 g / l sodium bicarbonate, 0.18 g / l ammonium chloride, 0.13 g / l sodium chloride, 0.96 g / l potassium chloride, 0.19 g / l potassium thiocyanate, 1.3 g / l potassium dihydrogen phosphate, 1.53 g / l sodium sulfate decahydrate, 0.4 g / l urea, 0.46 g / l calcium chloride dehydrate, and 0.1% mucin). The target strain (OD6oo = 3) and test strain (OD6oo = 3) were then mixed 1 :1 in a 96-well round-bottomed microtiter plate in a total volume of 200 pL. The microtiter plate was sealed with a plate sealing tape and vortexed for 1 minute on a Genie 2T Vortexer (Sigma-Aldrich) with a microplate attachment at max speed. The microtiter plate was subsequently incubated at room temperature for 60 minutes.

[0223] Aggregates were gently pelleted by centrifugation (300 x g, 10 s), and the supernatant (80 pL) was carefully removed, using a pipette positioning device to ensure that pipette tips were inserted to the same position in each well. The supernatant was transferred to another microwell plate, mixed with 120 pL PBS before reading the OD6oo. Self-aggregation of the test and target strains were included as controls and used for calculation of the aggregation index, which reflects the decrease in optical density when the strains are mixed: aggregation index [%] =

[0224] (OD600 (Target strain) + OD600 (Test strain)) — OD600 (Target strain + Test strain) - 7 - 7 - 7 \ - x 100

[0225] (OD600 (Target strain) + OD600 (Test strain)) Flow cytometry analysis of coaqqreqation

[0226] Target and test strains were harvested by centrifugation (4500 x g, 10 min), resuspended, washed, and ODsoo- adjusted as indicated in the Examples.

[0227] Target strains, test strains and salivary microbiota were stained with the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Thermo Fisher), 1 / 10 dilution of LIVE / DEAD™ Fixable Green Dead Cell Stain Kit (Thermo Fisher) and LIVE / DEAD™ Fixable Far Red Dead Cell Stain Kit (Thermo Fisher), respectively, for 30 minutes at room temperature in the dark. The samples were subsequently blocked with 10% yeast extract solution (25 g / L) and incubated 10 minutes, centrifuged at 4500 x g for 10 minutes and resuspended in SAGF + 0,1% mucin. In some experiments mucin was left out from the coaggregation buffer to avoid autoaggregation of P. gingivalis.

[0228] Coaggregation partners were mixed in a 96-well round-bottomed microtiter plate in a total volume of 200 pL. The microtiter plate was sealed with a lid, mixed for 30 seconds on microplate shaker with microplate attachment at 1300 rpm and incubated at room temperature for 60 minutes. Aggregates were gently resuspended by pipette and transferred (50 pL) to a new 96- well round-bottomed microtiter plate using a pipette positioning device. 950 pl PBS was added, and 40 pl of the cell suspension was diluted with 360 pl PBS in a new 96-well round-bottomed microtiter plate for flow cytometry analysis (Attune NxT Acoustic Focusing Cytometer, Attune NxT Autosampler, Thermo Fisher). Sample volume of 50 pl was analyzed using VL1 channel (440 / 50 nm), BL1 channel (530 / 30 nm) and RL1 channel (670 / 14 nm) with a flow rate of 100 pl / min.

[0229] Event counts corresponding to target strain, test strain and salivary microbiota were extracted with Boolean combination gates function (FlowJo 10) and applied to calculate aggregation index [%], [%] target, and [%] test: x. . , .. . . (Count (A) + Count (B)) - Count ((C) + (D)) aggregation index [%] = x lOO

[0230] (Count (A) + Count (B))

[0231] Count (D)\

[0232] [%] test = 100 - 777 x 100

[0233] Count (B)7 r / Count (C)\

[0234] [%] target = 100 - - 77 x 100

[0235] ACount (A) /

[0236] For coaggregate analysis, double and triple stained events were extracted with Boolean gates combination function (FlowJo 10) and summed up according to the presence of test or target strain, or salivary microbiota. Polystyrene beads (Polysciences Europe, 07310-15, 17135-5 and 17136-5) were used for gate setting to estimate size distribution of coaggregates, (see Figure 1).

[0237] Preparation of coagqregates for imaging

[0238] Aggregates were prepared as described above, but in a 10:1 testtarget OD-ratio (test strain at OD6oo=5, target strain at OD6oo=0.5). After the aggregates were gently pelleted, they were mixed with pipette tips that were shortened at the tip to avoid shearing the aggregates. 60 pL of each sample was sandwiched between a microscope slide and a coverslip using a GeneFrame (Thermo Fisher) and imaged using a bright field microscope.

[0239] 20 microscope images of two biological replicates for each sample type were analyzed for aggregate size distribution using image analysis software, in this case a standard Python script in Jupyterlab (ver. 3.0.14, Jupyter) run through Anaconda Navigator (ver. 2.0.3, Anaconda Inc.).

[0240] The script identified aggregates in an image and grouped them according to area size, before it calculated the contribution of each size group to the total aggregate number and the total biomass. For each analysis, the script ran each image through a number of parameters: a size filter followed by dilation, erosion and setting of threshold parameters. The size filter parameter excluded single cells from the analysis, the script parameter ‘DilateErode’ determined how substantial the dilation and erosion procedures were, while the parameter ‘Erosionincrease’ controlled how many times larger erosion was compared to the dilation. These three parameters were unique for each target strain. Lastly, the threshold parameter set the divide between the aggregate and background pixel populations. For each target-test strain combination a threshold was manually set.

[0241] In order to visualize the coaggregates in the confocal microscope, the target and test cells were washed as describe previously, before they were stained separately. The target strains were stained with the LIVE / DEAD™ Fixable Far Red Dead Cell Stain Kit (Thermo Fisher) and test strains were stained with 1 / 10 dilution of LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Thermo Fisher) for 30 minutes at room temperature. The samples were subsequently blocked with 10% yeast extract solution (25 g / L) and incubated for further 10 minutes before samples were centrifuged at 13,000 x g for 10 minutes and the supernatant exchanged with SAGF + 0.1% mucin. Coaggregates were prepared as described above in a 1 :10 targettest OD6oo ratio and imaged in the LSM700 confocal microscope (Zeiss) with an excitation / emission at 552 / 552 for stained test cells and 639 / 660 for stained target cells. Images were captured using the Axiocam HR camera (Zeiss) and the Zen Black software (Zeiss). The color for the test strains was manually changed to green to better distinguish the cells. Example 1 : Screening of lactic acid bacteria reveals few strains coaggregating multiple oral pathogens

[0242] Coaggregation between bacteria often relies on very specific interactions. Our goal was to identify bacteria that will coaggregate with several oral pathogens, and we therefore set out to screen a large number of lactic acid bacteria to identify those that coaggregated with one or more selected oral pathogens. In total, we selected 719 strains of lactic acid bacteria representing 69 species (the “test strains”) from the proprietary strain collection of Novozymes A / S and tested their ability to coaggregate with three well-known oral pathogens (the “target strains”): Fusobacterium nucleatum DSM 15642 (F. nucleatum), Porphorymonas gingivalis DSM 20907 (P. gingivalis) and Prevotella intermedia DSM 20706 (P. intermedia). In some tests, a strain of the oral pathogen Prevotella nigrecens (P. nigrecens), for example, P. nigrecens DSM 13386, was also included (data not shown).

[0243] Lactic acid bacteria were cultured, harvested, and heat-treated to generate non-viable cell mass. Coaggregation capacity was tested by mixing and incubating test and target strains (ODsoo ratio 3:1) in SAGF with 1% mucin to simulate conditions in the oral cavity. The interaction between test and target strain was quantified by the aggregation index, which reflects the decrease in optical density that occurs in the supernatant of gently pelleted samples as result of aggregation. It is calculated by comparing the optical density of samples in which target and test strains are mixed, relative to the optical density of the pure cultures. We used an aggregation index of 30% as the lower threshold to identify strains capable of coaggregation. We identified 103 such strains (Figure 1), representing 35 bacterial species. Most of the strains coaggregated with F. nucleatum (74 test strains) and fewest with P. intermedia (27 test strains). Only 21 test strains aggregated with more than one of the target strains, and only 5 strains coaggregated with all three target strains. 27 test strains were selected for further characterisation based on their aggregation index and ability to coaggregate with more than one target strain.

[0244] Example 2: Flow cytometry analysis provides detailed insight into coaggregation efficiency

[0245] The OD-based coaggregation assay used for screening is cheap, fast and of high throughput. However, it may deliver false positive results when one of the coaggregation partners auto-aggregates, or false negative results when binding partners are not applied in an optimal ratio. Additionally, the assay provides no information about the aggregate size, and how effectively one strain removes the other strain from suspension by capturing it in aggregates.

[0246] To overcome these drawbacks, we established a new method to quantify coaggregation based on flow cytometry, the so-called “FC-based coaggregation assay” or “the assay”. In addition to providing a coaggregation index, the assay also provides information about the aggregate size, and the proportion of cells captured in aggregates versus those remaining as single cells. In this assay, the test and target strains were stained with different fluorescent dyes prior to coaggregation. After coaggregation, the sample was analyzed by flow cytometry. Single-stained particles represent free, planktonic cells or autoaggregates, while double stained particles represent coaggregates. The difference between counts of cell populations incubated with and without a potential binding partner is used to calculate the % of bound cells.

[0247] In contrast to the OD-based assay, the flow cytometry-based assay analyzes the entire sample, including planktonic cells and coaggregates. This enables us to analyze the size distribution of aggregates, and to quantify the percentage of cells from test and target strains that participate in coaggregation ([%] test and [%] target, respectively). Using this method, the FCbased analysis of coaggregation between L. rhamnosus 1 B06 and P. gingivalis revealed that 97% of test strain cells and 56% of target strain cells formed coaggregates.

[0248] To estimate size distribution of coaggregates, double-stained events were divided into 4 groups based on gates set with size reference beads (Figure 1). The smallest possible coaggregates contain only two cells, while the largest aggregates may contain more than 200 cells if we assume an average bacterial cell volume of 2 pm3(Table 1; Figure 1). Although coaggregates are exposed to shear forces during flow cytometry, we observed coaggregates as large as 10 pm, indicating a very strong binding between the cells.

[0249] Table 1. Reference size beads applied in the flow cytometrical analysis of coaggregation. Based on the bead volume and average volume of a bacterial cell, the number of bacterial cells building a coaggregate was calculated. Per definition, the smallest coaggregate contains at least two cells.

[0250] Based on the OD6oo-based aggregation index, we selected 27 strains for further analysis by flow cytometry. Using the FC-based coaggreation assay, we searched for strains that bound the highest fraction of target strain within aggregates, leaving only few planktonic cells behind. The FC-based assay confirmed that the 27 test strains formed coaggregates, and two strains, L. paracasei 8A12 and L. rhamnosus 1 B06, stood out from the rest by capturing a very high percentage (up to 70%) of target strain bacteria in aggregates (Figure 2) and by the ability to bind all target strains tested. Figure 2 shows the results of the FC-based analysis of coaggregation between two selected test (1 B06 & 8A12) and three target strains (F. nucleatum, P. gingivalis, and P. intermedia) Aggregation index (upper panel), % target (middle panel) and % test (lower panel) calculated for strain pairs forming coaggregates in SAGF.

[0251] The majority of coaggregates containing F. nucleatum and P. intermedia were between 1 and 4.5 pm. In case of coaggregates formed between P. gingivalis and L rhamnosus 1 B06, the number of coaggregates was very high, but the coaggregate size was relatively small (<1 m) (data not shown).

[0252] The testtarget concentration ratio was kept constant at 3:1 in all coaggregation assays. However, as the OD6oo / cell conversion factor differs among bacterial species due to variation in cell size and shape, the concentration ratio between test and target strains measured as cell counts rather than optical density was not 3. In fact, our flow cytometry analysis revealed that the cell count ratios for each coaggregation pair varied between 3-9 (Table 2).

[0253] Table 2. Concentration ratios of test vs. target in Example 2.

[0254] Example 3: Coaggregation efficiency depends on the applied amount of test strain

[0255] The ability of two strains to coaggregate may not only depend on the specific mechanism of interaction between two bacterial strains, but also on the concentration ratio between the strains. We therefore investigated if coaggregation efficiency correlated with the cell concentration of the test strain (ODeoo = 0.25-2.5) while keeping the target strain constant (ODeoo = 0.5). We observed a positive correlation between test strain concentration and the fraction of target cells bound in coaggregates, resulting in up to 70 % of target cells being involved in aggregation (Figure 3). The ODeoo ratio between the test and target strains was 5:1 in the samples with the highest level of target coaggregation. Skewing this ratio even further could potentially capture even more of the target pathogens in coaggregates.

[0256] Inversely, the fraction of test cells participating in the coaggregation decreased, indicating saturation of the system where a further increase in the test strain concentration only captures a small amount of target cells in coaggregates (data not shown). It is thus difficult to envision that 100% capture of target cells can be achieved. The highest fraction of cells captured in coaggregates in any sample was achieved in coaggregation of 1B06 with P. gingivalis (test: target OD600 ratio = 1 :2, cell count ratio = 3.3:1) where 93% of 1 B06 cells were participating in coaggregation.

[0257] These results provide a basis for considering product dosage and they demonstrate that different targets pathogens are coaggregated with different efficiency. A 5:1 OD6oo ratio of test: target strain was needed to coaggregate > 70% of P. intermedia and F. nucleatum (cell count ratio = 3:1 and 1:1, respectively), while coaggregation of P. gingivalis happens already at ratio 1 :1 (cell count ratio 1 :1 for both test strains).

[0258] Example 4: Structure analysis reveals that L. rhamnosus 1B06 forms larger coaggregates with target cells embedded within

[0259] The desired outcome of coaggregation of oral pathogens with lactic acid bacteria is to neutralize their ability to colonize the oral cavity and form biofilms that lead to oral disease. We hypothesize that this outcome is more likely if the coaggregates are large, or if the lactic acid bacteria shield the target pathogen from interacting with other oral microorganisms and mucosal surfaces. We therefore proceeded to visualize the aggregates by microscopy. We used brightfield microscopy and image analysis to identify and count aggregates in seven size groups based on their area (data not shown). The contribution of each size group to the total biomass was then calculated based on area.

[0260] Autoaggregation of target strain cells was negligible, as only the three smallest size groups were represented (not shown) and coaggregation did not occur with the negative control strain Lactobacillus fermentum (not shown). When test and target strains were mixed, large coaggregates formed (Figure 4A+B). The aggregate structure was visibly different in the different samples, and test strain 1 B06 consistently formed larger aggregates than strain 8A12. It should be noted that in this assay, aggregates were not exposed to the sheer force of a flow cytometer, and the aggregate size was therefore larger than the size indicated by the FC-based aggregation assay.

[0261] We subsequently visualized the test and target cells within the largest aggregates using CLSM by fluorescently labeling strains before coaggregation. Coaggregates formed by 1 B06 were large with a relatively loose structure, containing voids between the filamentous lactic acid bacteria (Figure 5). The structure of coaggregates with F. nucleatum indicates that F. nucleatum is bridging the lactic acid bacteria. In contrast, the other target pathogens were situated as small aggregates within the larger clump of lactic acid bacteria, indicating that they cause the lactic acid bacteria to autoaggregate as well as coaggregating with the target pathogen. The structure of coaggregates with 8A12 appeared denser, particularly for F. nucleatum-QM2 coaggregates. Example 5: Coaggregates capture target cells in the presence of oral microbiota

[0262] The coaggregation experiments above provided a proof-of-concept that selected test strains are capable of forming coaggregates with selected oral pathogens. However, these oral pathogens exist in a complex and highly diverse microbial community in the oral cavity, and the use of coaggregation as a tool to capture and neutralize pathogens will require that coaggregates also form when the target pathogen is surrounded by other microorganisms. We therefore investigated the interaction of test and target strains in the presence of salivary microbiota. We investigated whether target strain cells would preferably bind to test strain cells or with the abundant salivary bacteria. Additionally, we investigated if coaggregates of test and target strain increase in size by incorporating salivary bacteria.

[0263] Salivary bacteria were isolated from saliva donated by healthy individuals and pooled before processing. Based on 16 S rRNA gene sequencing, P. intermedia constituted 1% of the salivary microbiome, while P. gingivalis and F. nucleatum could not be detected. Therefore, target strain cells were spiked to the salivary bacteria to determine the coaggregation effect of test strains in the presence of the oral microbiota.

[0264] For the coaggregation assay, the test strain, target strain, and salivary bacteria were fluorescently labeled with different fluorophores, mixed in SAGF at a cell count ratio of 1:1:1, and analyzed by flow cytometry. We first mixed individual strains with the saliva microbiota to quantify the coaggregation properties of the microbiota. Both target and test strains strongly coaggregated with salivary microbiota, resulting in >90 % of test strain cells and >80 % of target strain cells were located in coaggregates (Figure 6A), although the majority of coaggregates were small (<4,5 pm) (Figure 6B).

[0265] Next, we mixed both test and target strains with the salivary microbiota, and the binding dynamics changed significantly. The involvement of salivary microbiota in coaggregates appeared to be dictated by which target strain (rather than the test strain) was added to the mix. Coaggregates involving P. gingivalis captured the largest fraction (~ 50%) of the salivary microbiota, while coaggregates involving F. nucleatum captured less than 15%. The involvement of salivary microbiota in coaggregation appeared to positively impact the ability of test strains to coaggregate with the target strains, as the fraction of target strain cells that associated with aggregates (40-60%) was substantially higher (Figure 7A) than what we obtained in previous coaggregation experiments without salivary microbiota at 1 :1 ODsoo ratios (Figure 3, middle panel).

[0266] Applying Boolean gating strategy, we could determine if each coaggregate contained cells from all three bacterial populations, and if they only contained two, we could identify which they were. Aggregates were thus classified as containing 1) salivary microbiota and test strain cells, 2) salivary microbiota and target strain cells, 3) test and target strain cells, or 4) all three populations (Figure 7B). It should be noted that autoaggregation did occur among salivary bacteria, but these events were excluded from the analysis.

[0267] The first result that stands out from this analysis is that target strain cells are present in the vast majority (70-98%) of aggregates. The second result that stands out is that salivary microbiota is also present in the vast majority of aggregates (76-97%). Hence, the target pathogens have a high propensity to form aggregates, and most aggregates involve the salivary microbiota. Among the different target species, P. gingivalis and P. intermedia both aggregated strongly with the salivary microbiota, and a large fraction of aggregates did not even contain the test strain. In contrast, most of the aggregates that contained F. nucleatum also contained the test strain.

[0268] The fate of bacterial aggregates in the oral cavity is likely dependent on the size of the aggregates, and a fraction of aggregates that are few in numbers can be responsible for capturing a large part of the bacterial biomass if they are large. We therefore analyzed the size distribution of coaggregates containing target strains (Figure 7C). Overall, the coaggregates containing target strain cells and salivary microbiota without the involvement of the test strain were small (<4.5 p.m), which is consistent with our analysis of target strain and salivary microbiota in absence of test strain (Figure 6B). A similar result was obtained for aggregates that only contained test- and target strain cells. The largest coaggregates were those that contained all three bacterial populations, indicating that the test strain augmented coaggregate formation by target strain cells and the salivary microbiota.

[0269] Example 6: Test strains coaggregate with several clinical isolates of the target pathogens

[0270] As the ability to coaggregate has been described to be strain specific, we suspected different strains of the target pathogens would not coaggregate as well as the type of strain used in our screening assay. We therefore quantified coaggregation of test strains 8A12 and 1 B06 with clinical isolates in addition to the type of strain of the target pathogen (Table 3). As negative controls, we included two lactic acid bacteria strains belonging to the same species as 1 B06 and BA12 (L rhamnosus 3R73-NC and L paracasei 3P47-NC, respectively). As shown in Table 3, the coaggregation activity of 1 B06 and 8A12 is not limited to type strains used in the initial screening process but encompasses several strains of the same species.

[0271] Table 3. Heat map of coaggregation results displayed as % target. FC-based coaggregation assay of selected test strains and species-matching controls with type strains as well as clinical isolates of target pathogens was performed in SAGF + 1% mucin (P. gingivalis strains was tested in SAGF).

[0272] Example 7: Coaggregation ability is unique for the selected test strain

[0273] In the next step, we investigated whether coaggregation ability is a strain specific feature, or if other strains of the same species display similar properties. We selected 18 L. rhamnosus strains from the Novozymes A / S strain collection with the highest average nucleotide identity (ANI) when compared to 1B06 and performed FC-based coaggregation assays with P. gingivalis.

[0274] Only one out of the 18 additionally tested L. rhamnosus strains coaggregated with P. gingivalis to a similar extent as 1 B06 (Table 4). The two strains that were most similar to 1B06 were selected for further coaggregation tests with F. nucleatum and P. intermedia. Despite the genetic similarities to 1 B06, coaggregation by these strains was very poor (data not shown). The properties of 1 B06 are thus highly unique. Table 4. AN! and % target in coaggregates formed by P. gingivalis, B106 and 18 proprietary L. rhamnosus strains selected from the strain collection of Novozymes. Strains highlighted in bold were selected for the analysis of interaction with F. nucleatum and P. intermedia. We performed a pangenome analysis, where we compared the genome sequences of

[0275] 1 B06 to L. rhamnosus_ and rhamnosus_2. Genes unique to 1B06 could be identified (see Table 5 below) to support the discovery of the molecular mode of action behind the interaction of 1 B06 with target strains.

[0276] Table 5. List of genes present only in L. rhamnosus 1B06 and absent in L. rhamnosus_1 and L. rhamnosus_2. Hypothetical proteins were excluded.

[0277] We followed a similar analysis strategy for L paracasei 8A12. Phylogenetic analysis of L paracasei strains available in our proprietary strain collection revealed presence of another strain with ANI score of 99.978%. This strain was tested in coaggregation assays with all target strains, but it did not show any activity. We compared genomes of those 2 strains and generated a list of genes specific only to the selected test strain, which could be an interesting starting point for the analysis of molecular mode of action (Table 6 below).

[0278] Table 6. List of genes present only in L. paracasei 8A 12, but not in L. paracasei with ANI 99.978%.

[0279] DEPOSIT OF BIOLOGICAL MATERIAL

[0280] The following biological material has been deposited under the terms of the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 B, D-38124 Braunschweig, Germany, and given the following accession numbers: Deposit Accession Number Date of Depos

[0281] Lactobacillus rhamnosus HH03 OB-Lb-0655 DSM 34859 29-NOV-2023

[0282] Lactobacillus paracasei HH20 MXS_B2_71_3 DSM 34860 29-NOV-2023 The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0283] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this 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 the case of conflict, the present disclosure including definitions will control.

[0284] (Original in Electronic Form)

[0285] (This sheet is not part of and does not count as a sheet of the international application)

[0286] (Original in Electronic Form)

[0287] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0288] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0289] (This sheet is not part of and does not count as a sheet of the international application)

[0290] FOR RECEIVING OFFICE USE ONLY

[0291] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or combinations thereof, for use as a medicament, preferably for use as a medicament in one or more human and / or animal subject.

2. Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, for use in the treatment of infection, preferably for use in the treatment of oral infection, more preferably for use in the treatment of oral infection in one or more human and / or animal subject, even more preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis in one or more human and / or animal subject.

3. An isolated Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860.

4. The isolated Lactobacillus strain according to claim 3 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth.

5. An oral care composition comprising at least one strain of Lactobacillus paracasei and / or at least one strain of Lactobacillus rhamnosus in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, and at least one oral care ingredient, characterized in that said strain(s) is capable of: aggregating with at least one oral pathogen bacterial strain, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum preferably said at least one strain(s) is capable of coaggregating two or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatunr, more preferably said at least one strain(s) is capable of coaggregating three or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, and Fusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum; and most preferably said at least one strain(s) is capable of coaggregating four or more oral pathogen bacterial strains, preferably selected from the group of Porphyromonas, Prevotella, andFusobacterium, even more preferably selected from the species Porphyromonas intermedia, Prevotella gingivalis, Prevotella nigrecens, and Fusobacterium nucleatum.

6. The oral care composition according to claim 5, wherein the at least one strain of Lactobacillus paracasei comprises a Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, and / or wherein the at least one strain of Lactobacillus rhamnosus comprises a Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859 in the form of viable cells and / or non-viable cells, cell fragments, and / or as fermentation broth.

7. The oral care composition according to any of claims 5-6, which further comprises one or more enzyme activity selected from the group of a hydrolase, isomerase, ligase, lyase, oxi do reductase, and transferase; preferably selected from an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alphagalactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and a beta-xylosidase; preferably the oral care composition comprises the one or more enzyme activity in an effective amount; even more preferably in an amount of from about 1 ppm to about 500 ppm.

8. The oral care composition according to any of claims 5-6, which further comprises an oxi do reductase and a mediator.

9. The oral care composition according to claim 8, wherein the oxidoreductase is a laccase, an oxidase, or a peroxidase; preferably wherein the oxidoreductase is a laccase.

10. The oral care composition according to any of claims 8-9, wherein the mediator is a phenolic compound; preferably wherein the mediator is chlorogenic acid.

11. The oral care composition according to any of claims 5-10 in the form of or as a part of a liquid mouthwash, mouth rinse, dental care formulation, chew tablet, melting sugar tablet, lozenge, hard candy, lollipop, chewing gum, toothpaste, dental paste toothpick, or dental floss.

12. The oral care composition according to any of claims 5-11 for use as a medicament.

13. The oral care composition according to claim 12 for use as a medicament in the treatment of oral infection, preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis, more preferably for use in the treatment of gingivitis, periodontal disease, dental caries, and / or halitosis in one or more human and / or animal subject.

14. A method for preventing, inhibiting, reducing, altering, and / or eliminating growth of one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, comprising administering: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or a combination thereof; and / or b) an oral care composition according to any of claims 5-11 , to one or more human and / or animal subject in need thereof.

15. A method for preventing, inhibiting, reducing, altering, and / or eliminating oral biofilm comprising one or more oral pathogen bacterial species in the mouth of one or more human or animal subject, the method comprising contacting the oral biofilm with: a) an effective amount of Lactobacillus paracasei HH20 MXS_B2_71_3 strain DSM 34860 and / or Lactobacillus rhamnosus HH03 OB-Lb-0655 strain DSM 34859, in the form of viable cells and / or non-viable cells and / or cell fragments and / or as fermentation broth, or a combination thereof; and / or b) an effective amount of an oral care composition according to any of claims 5-11.

16. A kit of parts comprising: a) an oral care composition according to any of claims 5-11 ; and b) instructions for use.

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