BACKGROUND

[0001] The oral cavity contains many different species of bacteria. Some species of oral pathogenic bacteria (e.g. Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans) have been implicated in the development of periodontal diseases, such as periodontitis, gingivitis, necrotizing periodontitis, necrotizing gingivitis and peri-implantitis. Certain species of oral pathogenic bacteria have been implicated in tooth decay (e.g. Streptococcus mutans).

[0002] It is believed that certain species of oral bacteria are beneficial for maintaining the health of the periodontium. Without being bound by any theory, it is believed that these beneficial oral bacteria can interfere with colonization by pathogenic oral bacteria of the oral epithelium. For example, studies have shown that Streptococcus sanguinis, Streptococcus mitis and Streptococcus salivarius have inhibitory effects on A. actinomycetemcomitans colonization of epithelial cells in vitro (W. Teughels et al, J Dent Res 86(7), 611-617, 2007). It has also been shown, using a canine model, that the application of beneficial bacteria to periodontal pockets following root planing delays and reduces recolonization of the periodontal pockets by pathogenic bacteria (W. Teughels, et al, J Dent Res, 86(11), 1078-1082, 2007). The beneficial bacteria Streptococcus sanguinis, Streptococcus mitis and Streptococcus salivarius have also been shown to inhibit A. actinomycetemcomitans-mduced production of the inflammatory cytokine interleukin-8 (IL-8) by the human oral keratinocyte cell line HOK-18A, which inflammatory response is implicated in periodontitis-related tissue destruction (I. Sliepen et al, J Dent Res 88(11), 1026-1030, 2009).

[0003] It is believed that selective stimulation of beneficial oral bacteria may provide a valid preventative approach for oral health, for example in the prevention of periodontitis.

BRIEF SUMMARY

[0004] In a first aspect, the present invention provides an oral care composition comprising a carboxylic acid of the formula:

wherein:

Ri is H, or is a substituted or unsubstituted alkyl group; and

R ₂ is H, OH, or NH ₂ ;

for use in:

(a) selectively promoting, in an oral cavity: growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria;

(b) selectively promoting, in an oral cavity, biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria; or

(c) maintaining and/or re-establishing a healthy oral microbiota.

[0005] Optionally, the carboxylic acid is present in the composition in an amount of from 0.01 weight % to 10 weight %, based on the weight of the oral care composition.

[0006] Optionally, the carboxylic acid is present in the composition in an amount of from 5 μιηοΐ/ml to 30 μιηοΐ/ml, based on the volume of the oral care composition.

[0007] Optionally, R ₂ is H.

[0008] Optionally, R ₂ is OH.

[0009] Optionally, R ₂ is NH ₂ .

[0010] Optionally, the carboxylic acid is an acidic amino acid. Further optionally, the acidic amino acid is an L-amino acid. Still further optionally, the acidic amino acid is aspartic acid or glutamic acid.

[0011] Optionally, Ri is H.

[0012] Optionally, Ri is an unsubstituted alkyl group. Further optionally, Ri is an unsubstituted C ₁ -C4 alkyl group. Still further optionally, the carboxylic acid is L-lactic acid.

[0013] Optionally, Ri comprises a saccharide residue. Further optionally, the saccharide residue is a galactopyranose residue. Still further optionally, the galactopyranose residue is an a-D- galactopyranose residue. Yet further optionally, the carboxylic acid is melibionic acid.

[0014] Optionally, Ri comprises at least one COOR ₃ group, wherein R ₃ is H or Ci_ ₄ alkyl. [0015] Optionally, Ri comprises at least one C(0)COOH group.

[0016] Optionally, Ri is (CH ₂ ) _n COOR ₃ , wherein n is from 1 to 5. Further optionally, n is 1. Still further optionally, the carboxylic acid is succinic acid or L-malic acid.

[0017] Optionally, the carboxylic acid is monomethyl succinate.

[0018] Optionally, Ri is (CH ₂ ) _m C(0)COOH, wherein m is from 1 to 4. Further optionally, the carboxylic acid is a-keto glutaric acid.

[0019] Optionally, Ri comprises two COOH groups. Further optionally, the carboxylic acid is citric acid.

[0020] Optionally, the composition is a dentifrice, a toothpaste, a gel, a tooth powder, a mouthwash, a mouthrinse, a lozenge, a tablet, a spray, a gum, or a film.

[0021] Optionally, the composition further comprises at least one species of bacteria that has beneficial effects on oral health. Further optionally, the species of bacteria that has beneficial effects on oral health is selected from Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula, Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundii and combinations thereof.

[0022] Optionally, the oral care composition is for use in preventing one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries.

[0023] In a second aspect, the present invention provides a method of selectively promoting, in an oral cavity of a subject:

(a) growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria; or

(b) biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria;

the method comprising contacting an oral cavity of the subject with an oral care composition according to the present invention.

[0024] In a third aspect, the present invention provides a method of maintaining and/or reestablishing a healthy oral microbiota in a subject, the method comprising contacting an oral cavity of the subject with an oral care composition according to the present invention. [0025] In a fourth aspect, the present invention provides a method of preventing one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries in a subject, the method comprising contacting an oral cavity of the subject with an oral care composition according to the present invention.

[0026] In a fifth aspect, the present invention provides the use, in an oral care composition, of a carboxylic acid of the formula:

wherein:

Ri is H, or is a substituted or unsubstituted alkyl group; and

R ₂ is H, OH, or NH ₂ ;

to:

(a) selectively promote growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria;

(b) selectively promote biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria;

(c) maintain and/or re-establish a healthy oral microbiota in a subject; or

(d) prevent one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries in a subject.

[0027] Optionally, the carboxylic acid selectively promotes growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria, after 24 hours incubation with the bacteria that have beneficial effects on oral health and the pathogenic oral bacteria.

[0028] Optionally, the carboxylic acid selectively promotes growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria, after 48 hours incubation with the bacteria that have beneficial effects on oral health and the pathogenic oral bacteria. [0029] Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

DETAILED DESCRIPTION

[0030] The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0031] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. In addition, all references cited herein are hereby incorporated by referenced in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.

[0032] Unless otherwise specified, all percentages and amounts expressed herein and elsewhere in the specification should be understood to refer to percentages by weight. The amounts given are based on the active weight of the material.

[0033] As used herein, the expression "oral cavity" includes not only the cavity itself but also the saliva, teeth, gingiva, periodontal pockets, cheeks, tongue, mucosa, tonsils, any implants, and any device or structure which is placed into the oral cavity. The tonsils provide a reservoir (tonsil stones) for growth of anaerobic bacteria which may generate bad breath.

[0034] As used herein, the phrase "a healthy oral microbiota" refers to the microbial population of the oral cavity when the oral cavity is in a non-diseased state (for example, when there is no periodontal disease present, e.g. gingivitis, periodontitis, caries, peri-implantitis, peri-implant mucositis, necrotizing gingivitis and/or necrotizing periodontitis), i.e. a health-associated oral microbiota.

[0035] The term "beneficial bacteria" encompasses those bacteria which are present in the oral cavity in higher numbers or proportions in a healthy oral cavity, but which are present in lower numbers or proportions in conditions of oral disease (such as, for example, gingivitis, periodontitis, caries, peri-implantitis, peri-implant mucositis, necrotizing gingivitis and necrotizing periodontitis). This term also includes bacteria from oral or non-oral origins which have proven beneficial effects on oral health by preventing or treating oral diseases, which may be already present in the oral cavity or may be intentionally introduced into the oral cavity (for example as probiotics). Beneficial bacteria may, by their presence or metabolic activity, result in: lowering the number or proportion of pathogenic oral bacteria; lowering inflammation and inflammatory processes; lowering the metabolic activity of pathogenic species; lowering the production or inhibiting virulence factors produced by pathogenic bacteria; lowering or inhibiting biofilm formation; occupying a niche which may otherwise be colonized by pathogens; limiting a pathogen's ability to adhere to oral surfaces; affecting the viability, metabolic activity or growth of a pathogen; lowering the ability of a pathogen to produce virulence factors; degrading virulence factors produced by the pathogen or the oral microbiota; and/or attenuating the host response to pathogenic species. As discussed above, it is believed that these beneficial oral bacteria can interfere with colonization of the oral epithelium by pathogenic oral bacteria. It is believed that selective stimulation of beneficial oral bacteria may provide a valid preventative approach for oral health, for example in the prevention of periodontitis.

[0036] Bacteria require certain substrates in order to enable them to grow, multiply, be metabolically active and to colonize. Certain substrates can be used selectively by certain microorganisms (e.g. bacteria) to favor their growth, metabolic activity, multiplication and colonization, and thereby directly or indirectly suppress the growth of other microorganisms. The present inventors investigated the possibility of modifying the oral microbiota of a subject by selectively increasing the subject's indigenous beneficial oral bacterial population, by providing the beneficial oral bacteria with appropriate substrates for growth/multiplication.

[0037] Examples of beneficial bacteria include Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula, Streptococcus gordonii, Capnocytophaga sputigena and Actinomyces naeslundii. Examples of pathogenic bacteria include Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Streptococcus sobrinus.

[0038] The present inventors have found that certain carboxylic acids are able to cause an increase in growth, metabolic activity and biofilm formation of beneficial bacteria, while not causing an increase in growth, metabolic activity and biofilm formation of pathogenic oral bacteria.

[0039] The present invention therefore provides an oral care composition comprising a carboxylic acid of the formula:

wherein:

Ri is H, or is a substituted or unsubstituted alkyl group; and

R ₂ is H, OH, or NH ₂ ;

for use in:

(a) selectively promoting, in an oral cavity: growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria;

(b) selectively promoting, in an oral cavity, biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria; or

(c) maintaining and/or re-establishing a healthy oral microbiota.

[0040] In some embodiments, R ₂ is H. In other embodiments, R ₂ is OH. In other embodiments, R ₂ is NH ₂ .

[0041] In some embodiments wherein R ₂ is NH ₂ , the carboxylic acid is an acidic amino acid. The acidic amino acid may be an L-amino acid. In some embodiments, the acidic amino acid is aspartic acid or glutamic acid. In certain embodiments, the acidic amino acid is L-aspartic acid. In other embodiments, the acidic amino acid is L-glutamic acid.

[0042] In some embodiments, Ri is H. In certain embodiments, the carboxylic acid is acetic acid.

[0043] In some embodiments, Ri is a Ci-Cio substituted or unsubstituted alkyl group. In some embodiments, Ri is a linear or a branched alkyl group. In some embodiments, Ri is a cycloalkyl group.

[0044] In some embodiments, Ri is an unsubstituted alkyl group. In certain embodiments, Ri is an unsubstituted Ci-Cio; Ci-C ₆ ; or C ₁ -C4 alkyl group. In some embodiments, Ri is an unsubstituted methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), isobutyl (i-Bu) or tert-butyl (t-Bu) group. In some embodiments, the carboxylic acid is lactic acid. In certain embodiments, the carboxylic acid is L-lactic acid.

[0045] In some embodiments, Ri is a substituted Ci-Cio; Ci-C ₆ ; or C ₁ -C4 alkyl group. In certain embodiments, Ri is a substituted methyl (Me), ethyl (Et), n-propyl (n-Pr), or n-butyl (n-Bu) group. In certain embodiments, Ri is a substituted isopropyl (i-Pr), isobutyl (i-Bu) or tert-butyl (t-Bu) group. In some embodiments, Ri is substituted with at least one hydroxyl group. Further examples of groups with which Ri may be substituted include (but are not limited to) keto groups, carboxylic acid groups, saccharide residues, and ester groups.

[0046] In some embodiments, Ri comprises a saccharide residue. In certain embodiments, the saccharide residue is a galactopyranose residue. In some embodiments, the galactopyranose residue is an a-D-galactopyranose residue. In certain embodiments where Ri is a substituted alkyl group (including, but not limited to, a substituted methyl, ethyl, n-propyl or n-butyl group; or a substituted isopropyl, isobutyl or tert-butyl group), Ri is substituted with the saccharide residue of any of the above embodiments. In some such embodiments, Ri may be further substituted with at least one hydroxyl group. In certain embodiments, Ri is a saccharide residue linked by a glycosidic bond to a chain of at least three carbon atoms (i.e. the carboxylic acid has the structure (saccharide residue)-(chain of at least three carbon atoms)-CHR ₂ -COOH). The chain of at least three carbon atoms may be substituted with hydroxyl groups. In certain embodiments, the carboxylic acid is melibionic acid.

[0047] In certain embodiments, Ri comprises at least one COOR ₃ group, wherein R ₃ is H or Ci_ ₄ alkyl. In certain embodiments, R ₃ is selected from methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), isobutyl (i-Bu), or t-butyl (t-Bu).

[0048] In certain embodiments, Ri is (CH ₂ ) _n COOR ₃ , wherein n is from 1 to 5. In certain embodiments, n is from 1 to 4; from 1 to 3; or from 1 to 2. In certain embodiments, n is 1. In any of these embodiments, R3 may be methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), isobutyl (i-Bu), or t-butyl (t-Bu). In some embodiments, the carboxylic acid is succinic acid or L-malic acid. In some embodiments, the carboxylic acid is mono methyl succinate.

[0049] In some embodiments, Ri comprises at least one C(0)COOH group. In certain embodiments, Ri is (CH ₂ ) _m C(0)COOH, wherein m is from 1 to 4. In certain embodiments, m may be from 1 to 3; from 1 to 2; or m may be 1. In some embodiments, the carboxylic acid is a- keto glutaric acid.

[0050] In some embodiments, Ri comprises two COOH groups. In certain embodiments, the carboxylic acid is citric acid.

[0051] In some embodiments, Ri comprises a COOH group and a COOR ₃ group, wherein R ₃ is H or Ci_4 alkyl. In certain embodiments, R ₃ is selected from methyl (Me), ethyl (Et), n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), isobutyl (i-Bu), or t-butyl (t-Bu).

[0052] In some embodiments, the carboxylic acid is present in the composition in an amount of from 0.01 weight % to 10 weight %, from 0.05 weight % to 7.5 weight %, from 0.1 weight % to 5 weight %, or from 1 weight % to 2.5 weight %, based on the weight of the oral care composition.

[0053] In any of the above embodiments, the carboxylic acid may be present in the composition in an amount of from 1 μιηοΐ/ιηΐ to 50 μιηοΐ/ιηΐ, from 2 μιηοΐ/ιηΐ to 40 μιηοΐ/ιηΐ, from 5 μιηοΐ/ιηΐ to 30 μιηοΐ/ιηΐ, from 10 μιηοΐ/ιηΐ to 25 μιηοΐ/ιηΐ, from 20 μιηοΐ/ιηΐ to 25 μιηοΐ/ιηΐ; or about 25 μιηοΐ/ιηΐ, about 20 μιηοΐ/ιηΐ, about 10 μιηοΐ/ιηΐ, or about 5 μιηοΐ/ιηΐ. In some embodiments the carboxylic acid is present in the composition in an amount of from 22 μιηοΐ/ιηΐ to 30 μιηοΐ/ιηΐ.

[0054] The oral care composition as described in any of the above embodiments may be a dentifrice, a toothpaste, a gel, a tooth powder, a mouthwash, a mouthrinse, a lozenge (which may be dissolvable or chewable), a tablet, a spray, a gum, or a film (which may be wholly or partially dissolvable, or indissolvable).

[0055] The oral care compositions as described in any of the above embodiments may further comprise at least one species of bacteria that has beneficial effects on oral health. In these embodiments, the composition may be referred to as a synbiotic composition, as it contains both a probiotic (the bacteria which has beneficial effects on oral health) and a prebiotic (the carboxylic acid which stimulates the growth, metabolic activity and/or colonization of the probiotic bacteria). In certain embodiments, the species of bacteria that has beneficial effects on oral health is selected from Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula, Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundii and combinations thereof. [0056] The oral care composition as described in any of the above embodiments may also be for use in preventing one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries.

[0057] The present invention also provides a method of selectively promoting, in an oral cavity of a subject:

(a) growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria; or

(b) biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria;

the method comprising contacting an oral cavity of the subject with an oral care composition according to any of the above-described embodiments.

[0058] The present invention also provides a method of maintaining and/or re-establishing a healthy oral microbiota in a subject, the method comprising contacting an oral cavity of the subject with an oral care composition according to any of the above-described embodiments.

[0059] The present invention also provides a method of preventing one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries in a subject, the method comprising contacting an oral cavity of the subject with an oral care composition according to any of the above-described embodiments.

[0060] In the above methods, the subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, for example a companion animal (e.g. a cat or dog).

[0061] In certain embodiments, the contacting of the oral cavity with the oral care composition comprises applying the oral care composition to the oral cavity using a brush, rinsing the oral cavity with the oral care composition in the form of a mouthwash, or spraying the oral care composition into the oral cavity using, for example, an atomizer.

[0062] The present invention also provides the use, in an oral care composition, of a carboxylic acid as described in any of the above embodiments, to: (a) selectively promote growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria;

(b) selectively promote biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria;

(c) maintain and/or re-establish a healthy oral microbiota in a subject; or

(d) prevent one or more of gingivitis, periodontitis, peri-implantitis, peri-implant mucositis, necrotizing gingivitis, necrotizing periodontitis and caries in a subject.

[0063] In any of the above embodiments of the compositions, methods and uses, the carboxylic acid may selectively promote growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria, after 24 hours incubation with the bacteria that have beneficial effects on oral health and the pathogenic oral bacteria.

[0064] In any of the above embodiments of the compositions, methods and uses, the carboxylic acid may selectively promote growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria, after 48 hours incubation with the bacteria that have beneficial effects on oral health and the pathogenic oral bacteria.

[0065] In any of the above embodiments of the compositions, methods and uses, the bacteria that have beneficial effects of oral health may be selected from Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula, Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundii and combinations thereof. Additionally or alternatively, the pathogenic bacteria may be selected from Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Streptococcus sobrinus and combinations thereof.

[0066] In any of the above embodiments wherein the compositions, methods and uses concern the selective promotion (in an oral cavity of a subject) of growth, metabolic activity or colonization of bacteria that have beneficial effects on oral health, relative to growth, metabolic activity or colonization of pathogenic oral bacteria, the bacteria that have beneficial effects of oral health may be selected from Streptococcus mitis, Streptococcus sanguinis, Actinomyces viscosus, Veillonella parvula, Actinomyces naeslundii and combinations thereof, and in certain embodiments may be selected from Streptococcus mitis and Veillonella parvula. Additionally or alternatively, the pathogenic bacteria may be selected from Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Streptococcus sobrinus and combinations thereof.

[0067] In any of the above embodiments wherein the compositions, methods and uses concern the selective promotion of biofilm formation by bacteria that have beneficial effects on oral health, relative to biofilm formation by pathogenic oral bacteria, the pathogenic bacteria may be selected from Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and combinations thereof; and in certain embodiments may be selected from Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans. Additionally or alternatively, the bacteria that have beneficial effects of oral health may be selected from Streptococcus salivarius, Streptococcus sanguinis, Streptococcus gordonii, Capnocytophaga sputigena, and combinations thereof.

[0068] The oral care compositions of the present invention may further comprise additional ingredients. These additional ingredients may include, but are not limited to, abrasives, diluents, bicarbonate salts, pH modifying agents, surfactants, foam modulators, thickening agents, humectants, sweeteners, flavorants, pigments, antibacterial agents, anticaries agents, anticalculus or tartar control agents, and mixtures thereof.

[0069] In some embodiments, particularly (but not limited to) those embodiments, wherein the oral care composition is a toothpaste, the compositions of the present invention may further comprise an abrasive. Abrasives that may be used include silica abrasives such as precipitated or hydrated silicas having a mean particle size of up to about 20 microns, such as Zeodent 105 and Zeodent 114 marketed by J.M. Huber Chemicals Division, Havre de Grace, Md. 21078, or Sylodent 783 marketed by Davison Chemical Division of W.R. Grace & Company. Abrasives such as Sorbosil AC 43 from PQ Corporation may also be included. Other useful dentifrice abrasives include aluminium oxide, aluminum silicate, calcined alumina, bentonite or other siliceous materials, insoluble phosphates, and mixtures thereof. The abrasive may be present in an amount of from 5 to 30 wt% based on the weight of the composition, optionally from 10 to 20 wt% based on the weight of the composition. In certain embodiments, particularly (but not limited to) those embodiments wherein the oral care composition is a mouthwash or mouthrinse, the compositions may be free of abrasives.

[0070] In some embodiments, the oral care compositions of the present invention comprise at least one bicarbonate salt useful for example to impart a "clean feel" to teeth and gums due to effervescence and release of carbon dioxide. Any orally acceptable bicarbonate can be used, including without limitation, alkali metal bicarbonates such as sodium and potassium bicarbonates, ammonium bicarbonate and the like. The one or more additional bicarbonate salts are optionally present in a total amount of about 0.1 wt. % to about 50 wt. %, for example about 1 wt. % to 20 wt. %, by total weight of the composition.

[0071] In some embodiments, the oral care compositions of the present invention comprise at least one pH modifying agent. Such agents include acidifying agents to lower pH, basifying agents to raise pH, and buffering agents to control pH within a desired range. For example, one or more compounds selected from acidifying, basifying and buffering agents can be included to provide a pH of 2 to 10, or in various illustrative embodiments, 2 to 8, 3 to 9, 4 to 8, 5 to 7, 6 to 10, 7 to 9, etc. Any orally acceptable pH modifying agent can be used, including without limitation, phosphoric and sulfonic acids and salts thereof, alkali metal hydroxides such as sodium hydroxide, carbonates such as sodium carbonate, bicarbonates, sesquicarbonates, borates, silicates, phosphates (e.g., monosodium phosphate, trisodium phosphate), imidazole and the like. One or more pH modifying agents are optionally present in a total amount effective to maintain the composition in an orally acceptable pH range.

[0072] The oral care compositions of the invention may also comprise at least one surfactant. Any orally acceptable surfactant, most of which are anionic, nonionic or amphoteric, can be used. Suitable anionic surfactants include without limitation, water-soluble salts of C ₈ _ ₂ o alkyl sulfates, sulfonated monoglycerides of C ₈ _ ₂ o fatty acids, sarcosinates, taurates and the like. Illustrative examples of these and other classes include sodium lauryl sulfate, sodium coconut monoglyceride sulfonate, sodium lauryl sarcosinate, sodium lauryl isoethionate, sodium laureth carboxylate and sodium dodecyl benzenesulfonate. Suitable nonionic surfactants include without limitation, poloxamers, polyoxyethylene sorbitan esters, fatty alcohol ethoxylates, alkylphenol ethoxylates, tertiary amine oxides, tertiary phosphine oxides, dialkyl sulfoxides and the like. Suitable amphoteric surfactants include without limitation, derivatives of C ₈ _ ₂ o aliphatic secondary and tertiary amines having an anionic group such as carboxylate, sulfate, sulfonate, phosphate or phosphonate. Betaines may also be used, a suitable example of which is cocoamidopropyl betaine. One or more surfactants are optionally present in a total amount of about 0.01 wt.% to about 10 wt. %, for example, from about 0.05 wt. % to about 5 wt. %, or from about 0.1 wt. % to about 2 wt. % by total weight of the composition.

[0073] The oral care compositions of the invention may comprise at least one foam modulator, useful for example to increase amount, thickness or stability of foam generated by the composition upon agitation. Any orally acceptable foam modulator can be used, including without limitation, polyethylene glycols (PEGs), also known as polyoxyethylenes. High molecular weight PEGs are suitable, including those having an average molecular weight of 200,000 to 7,000,000, for example 500,000 to 5,000,000, or 1,000,000 to 2,500,000. One or more PEGs are optionally present in a total amount of about 0.1 wt. % to about 10 wt. %, for example from about 0.2 wt. % to about 5 wt. %, or from about 0.25 wt. % to about 2 wt.%, by total weight of the composition.

[0074] The oral care compositions of the present invention may comprise at least one thickening agent, useful for example to impart a desired consistency and/or mouth feel to the composition. Any orally acceptable thickening agent can be used, including without limitation, carbomers, also known as carboxyvinyl polymers, carrageenans, also known as Irish moss and more particularly i-carrageenan (iota-carrageenan), cellulosic polymers such as hydroxyethylcellulose, carboxymethylcellulose (CMC) and salts thereof, e.g., CMC sodium, natural gums such as karaya, xanthan, gum arabic and tragacanth, colloidal magnesium aluminum silicate, colloidal silica and the like. A preferred class of thickening or gelling agents includes a class of homopolymers of acrylic acid crosslinked with an alkyl ether of pentaerythritol or an alkyl ether of sucrose, or carbomers. Carbomers are commercially available from B. F. Goodrich as the Carbopol® series. Particularly preferred Carbopols include Carbopol 934, 940, 941, 956, 974P, and mixtures thereof. Silica thickeners such as DT 267 (from PPG Industries) may also be used. One or more thickening agents are optionally present in a total amount of from about 0.01 wt. % to 15 wt.%, for example from about 0.1 wt.% to about 10 wt.%, or from about 0.2 wt. % to about 5 wt.%), by total weight of the composition. [0075] The compositions of the invention may comprise at least one viscosity modifier, useful for example to help inhibit settling or separation of ingredients or to promote re-dispersibility upon agitation of a liquid composition. Any orally acceptable viscosity modifier can be used, including without limitation, mineral oil, petrolatum, clays and organomodified clays, silica and the like. One or more viscosity modifiers are optionally present in a total amount of from about 0.01 wt. % to about 10 wt. %, for example, from about 0.1 wt.% to about 5 wt.%, by total weight of the composition.

[0076] The compositions of the invention may also comprise at least one humectant. Any orally acceptable humectant can be used, including without limitation, polyhydric alcohols such as glycerin, sorbitol (optionally as a 70 wt.%> solution in water), xylitol or low molecular weight polyethylene glycols (PEGs). Most humectants also function as sweeteners. One or more humectants are optionally present in a total amount of from about 1 wt.% to about 70 wt.%, for example, from about 1 wt.% to about 50 wt.%, from about 2 wt.% to about 25 wt.%, or from about 5 wt.%) to about 15 wt.%>, by total weight of the composition.

[0077] The oral care compositions of the present invention may comprise at least one sweetener, useful for example to enhance taste of the composition. One or more sweeteners are optionally present in a total amount depending strongly on the particular sweetener(s) selected, but typically 0.005 wt.%) to 5 wt.%), by total weight of the composition, optionally 0.005 wt.%> to 0.2 wt.%>, further optionally 0.05 wt.% to 0.1 wt.% by total weight of the composition.

[0078] The compositions of the present invention may also comprise at least one flavorant, useful for example to enhance taste of the composition. Any orally acceptable natural or synthetic flavorant can be used, including without limitation tea flavours, vanillin, sage, marjoram, parsley oil, spearmint oil, cinnamon oil, oil of wintergreen (methylsalicylate), peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, citrus oils, fruit oils and essences including those derived from lemon, orange, lime, grapefruit, apricot, banana, grape, apple, strawberry, cherry, pineapple, etc., bean- and nut-derived flavors such as coffee, cocoa, cola, peanut, almond, etc., adsorbed and encapsulated flavorants and the like. Also encompassed within flavorants herein are ingredients that provide fragrance and/or other sensory effect in the mouth, including cooling or warming effects. Such ingredients illustratively include menthol, menthyl acetate, menthyl lactate, camphor, eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone, a-irisone, propenyl guaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-/?-menthan-3-carboxamine, N,2,3-trimethyl-2-isopropylbutanamide, 3-(l-menthoxy)- propane- 1,2-diol, cinnamaldehyde glycerol acetal (CGA), menthone glycerol acetal (MGA) and the like. One or more flavorants are optionally present in a total amount of from about 0.01 wt. % to about 5 wt. %, for example, from about 0.03 wt. % to about 2.5 wt.%, optionally about 0.05 wt.% to about 1.5 wt.%>, further optionally about 0.1 wt.%> to about 0.3 wt.%> by total weight of the composition.

[0079] The compositions of the invention may comprise at least one colorant. Colorants herein include pigments, dyes, lakes and agents imparting a particular luster or reflectivity such as pearling agents. Any orally acceptable colorant can be used, including without limitation talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silica, titanium dioxide, zinc oxide, red, yellow, brown and black iron oxides, ferric ammonium ferrocyanide, manganese violet, ultramarine, titaniated mica, bismuth oxychloride, and the like. One or more colorants are optionally present in a total amount of from about 0.001 wt.%) to about 20 wt.%>, for example, from about 0.01 wt.%> to about 10 wt. %>, or from about 0.1 wt. %> to about 5 wt.%), by total weight of the composition.

[0080] The compositions of the present invention may also comprise an antibacterial or preservative agent, such as chlorhexidine, triclosan, quaternary ammonium compounds (for example benzalkonium chloride) or parabens such as methylparaben or propylparaben. One or more antibacterial or preservative agent is optionally present in the composition in a total amount of from about 0.01 wt.%> to about 0.5 wt.%>, optionally about 0.05 wt.%> to about 0.1 wt.%> by total weight of the composition.

[0081] The oral care compositions may also comprise a fluoride ion source. Fluoride ion sources include, but are not limited to: stannous fluoride, sodium fluoride, potassium fluoride, potassium monof uorophosphate, sodium monofluorophosphate, ammonium monof uorophosphate, sodium f uorosilicate, ammonium f uorosilicate, amine fluoride such as olaf ur (Ν'- octadecyltrimethylendiamine-N,N,N'-tris(2-ethanol)-dihydrofl uoride), ammonium fluoride, and combinations thereof. In certain embodiments the fluoride ion source includes stannous fluoride, sodium fluoride, amine fluorides, sodium monofluorophosphate, as well as mixtures thereof. In certain embodiments, the oral care composition of the invention may also contain a source of fluoride ions or fluorine -providing ingredient in amounts sufficient to supply about 50 to about 5000 ppm fluoride ion, e.g., from about 100 to about 1000, from about 200 to about 500, or about 250 ppm fluoride ion. Fluoride ion sources may be added to the compositions of the invention at a level of about 0.001 wt. % to about 10 wt. %, e.g., from about 0.003 wt. % to about 5 wt. %, 0.01 wt. % to about 1 wt., or about 0.05 wt. %. However, it is to be understood that the weights of fluoride salts to provide the appropriate level of fluoride ion will obviously vary based on the weight of the counter ion in the salt, and one of skill in the art may readily determine such amounts. A preferred fluoride salt may be sodium fluoride.

[0082] The compositions of the present invention may comprise a saliva stimulating agent useful, for example, in amelioration of dry mouth. Any orally acceptable saliva stimulating agent can be used. One or more saliva stimulating agents are optionally present in saliva stimulating effective total amount.

[0083] The compositions of the present invention may include antisensitivity agents, e.g., potassium salts such as potassium nitrate, potassium bicarbonate, potassium chloride, potassium citrate, and potassium oxalate; capsaicin; eugenol; strontium salts; chloride salts and combinations thereof. Such agents may be added in effective amounts, e.g., from about 1 wt. % to about 20 wt. % by weight based on the total weight of the composition, depending on the agent chosen.

[0084] The composition of the invention may further comprise an antioxidant. Any orally acceptable antioxidant can be used, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin A, carotenoids, vitamin E, flavonoids, polyphenols, herbal antioxidants, chlorophyll, melatonin, and mixtures thereof.

[0085] The compositions of the present invention may additionally optionally comprise a tartar control (anticalculus) agent as provided below. Tartar control agents among those useful herein include salts of the specified agents, including alkali metal and ammonium salts. The agents include: phosphates and polyphosphates, polyaminopropanesulfonic acid (AMPS), polyolefm sulfonates, polyolefm phosphates, diphosphonates such as azacycloalkane-2,2-diphosphonates (e.g., azacycloheptane-2,2-diphosphonic acid), N-methyl azacyclopentane-2,3-diphosphonic acid, ethane- 1 -hydroxy- 1,1-diphosphonic acid (EHDP) and ethane- 1 -amino- 1,1-diphosphonate. Useful inorganic phosphate and polyphosphate salts include monobasic, dibasic and tribasic sodium phosphates, sodium tripolyphosphate, tetrapolyphosphate, sodium trimetaphosphate, sodium hexametaphosphate and mixtures thereof. Other useful tartar control agents include polycarboxylate polymers and polyvinyl methyl ether/maleic anhydride (PVM/MA) copolymers, such as GANTREZ®.

EXAMPLES

Example 1

[0086] The metabolic activity of various beneficial oral bacteria and pathogenic oral bacteria in the presence of the carboxylic acids succinic acid, L-lactic acid, mono methyl succinate, L-malic acid, citric acid, melibionic acid, alpha-keto-glutaric acid, acetic acid and L-aspartic acid was investigated.

[0087] The beneficial oral bacteria tested were Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundii and Veillonella parvula. The pathogenic oral bacteria tested were Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Streptococcus sobrinus.

[0088] Substrates that were able to increase the metabolic activity of at least one of the above beneficial bacteria while not or only minimally increasing the growth of the pathogenic bacteria are considered to be prebiotic substrates.

[0089] The extent of metabolic activity of the tested bacteria in the presence of the above- mentioned acids after 24 hours and 48 hours was investigated through a high throughput phenotype microarray system (PM) for microbial cells (Biolog Inc.). Each PM can contain up to 95 different biochemical compounds (e.g. organic acids) that may act as substrates for bacteria as well as one negative control, not containing the substrates. Microarray plates were used in which the different wells of each plate were pre-loaded (by the manufacturer, Biolog Inc.) with different organic acids to be tested. Each bacterial species was tested using a separate microarray plate. Thus, a single bacterial species was tested with a variety of organic acids on each particular plate. The microarrays are based on redox technology, using cell respiration as a universal reporter. Active cell respiration results in the reduction of a tetrazolium dye and in the formation of a strong color - for example, when using the tetrazolium dye "Biolog Redox Dye Mix D", the color changes from transparent/colorless to purple when the dye is reduced. The observation of this color change indicates which of the substrates improve the metabolic activity and health of the cells.

[0090] Each bacterial species was collected from blood agar plates (incubated for 48 hours anaerobically at 37°C) and transferred to an IF-0 Base inoculation fluid (Biolog Inc.), which is a nutritionally poor medium. The resulting cell suspension was adjusted to a transmittance of 42% (relative to the transmittance of the base inoculation fluid with no bacterial species present) at 492nm using a BioRad SmartSpec 3000 Photometer (the adjustment being effected by adding either further inoculation fluid or further bacteria until the transmittance of 42% was achieved). A 1 :5 dilution of this suspension was prepared by mixing 3 mL of the 42% transmittance cell suspension with 15 mL of an inoculation fluid (which inoculation fluid was formed by mixing 11.6 mL sterile water, 62.5 mL IF-0 base inoculation fluid and 0.9 mL of the tetrazolium dye "Biolog Redox Dye mix D"), resulting in a final cell density which was equal to 85% transmittance using a BioRad SmartSpec 3000 Photometer. For each of the acids tested, the microarray plate (obtained from the supplier (Biolog Inc.) with acid already present in the relevant wells in powder form) was directly inoculated with 100 μίΛνεΙΙ of this 85% transmittance cell suspension.

[0091] All plates were incubated in an anaerobic atmosphere at 37°C. Color changes were measured spectrophotometrically at 492 nm (Thermo Scientific Multiskan Ascent) at 24 hrs and 48 hrs using the same plate at both time points. For each bacterium tested, a respective control was also used, the control being a well of the plate which contained the particular bacterium (cell suspension) but did not contain any carboxylic acid. After 24hrs and 48hrs under continuous shaking in a Multiscan microplate reader, the optical density (OD) of the synthetic medium at 24 hours and 48 hours was measured (using the microplate reader) at a wavelength of 492nm for each combination of bacterium/organic acid, and for each of their respective controls (i.e. which contained the particular bacterium but no carboxylic acid). For each bacterium, the OD value obtained at 24hrs and at 48hrs was divided by the OD value obtained for the respective control at 24hrs and 48hrs (respectively), so that the control had a value of 1. A value greater than 1 for a particular combination of bacterium with carboxylic acid therefore indicates that the increase in metabolic activity of the bacteria after 24hrs or 48hrs was greater than the increase observed for the control. [0092] The experiment was carried out three times for each combination of bacterium with carboxylic acid, with each repeat being carried out on a different day (thus providing three biological replicas). The controls were also carried out three times, as above. The values shown are the average (mean) of the three single values obtained (as detailed above) for each combination of bacterium with carboxylic acid, at 24hrs and at 48hrs. The results are shown in Tables 1 to 4, below:

Table 1: Pathogenic bacteria at 24 hrs

**A actinomycetemcomitans

Succinic acid

0.9767 1.0400 0.7960 0.9752 0.9019 0.9046 2.8247 0.9268

L-lactic acid

A 1.6747 1.4435 0.7156 1.5404 0.9183 0.9076 3.4930 1.0230

Mono methyl succinate

0.88 ^ldi naesun80 0.8328 0.8102 0.9911 0.9553 0.8806 2.0008 1.0231

L-malic acid

0.9796 0.8764 0.9161 0.9803 0.9740 0.8788 2.0256 0.9743

Citric acid C

0.8551 0.5992 0.9010 0.9333 0.9935 0.8938 1.2832 1.0814

Melibionic acid ^it spugena

1.2539 0.6213 1.0326 1.6342 0.9984 1.0430 0.8685 1.0443

Alpha-keto-glutaric S

acid

0.9093 0.6312 0.912 ^dii goron7 0.9915 1.1057 0.9629 1.4942 1.0211

Acetic acid

0.9735 1.0659 0.8805 1.1538 0.9882 0.9557 1.2465 1.0033

A

L-aspartic acid

0.8814 0.7060 0.8729 0.943 ⁱ vscosus4 0.9496 0.9451 1.8448 1.0323

Table 2: Beneficial bacteria at 24hrs

S

l ⁱⁱ savarus

S

i ⁱ sanguns l Vparvua

S ^iit ms

Table 3: Pathogenic bacteria at 48hrs

**A actinomycetemcomitans

L-aspartic acid

Table 4: Beneficial bacteria at 48hrs

[0093] In the above data, a value of 2 was taken as the threshold above which the organic acid caused markedly increased metabolic activity of the bacterium. This value was selected in order to exclude low-stimulating metabolites and avoid false positive results.

[0094] As can be seen from the above data, succinic acid, L-lactic acid, mono methyl succinate and L-malic acid exhibited prebiotic effects at 24hrs, and succinic acid, mono methyl succinate, citric acid, melibionic acid, alpha-keto-glutaric acid, acetic acid and L-aspartic acid exhibited prebiotic effects at 48hrs. Without being bound by any theory, it is believed that those substrates which exhibit prebiotic effects at 48hrs may provide beneficial effects to the oral cavity upon prolonged use.

[0095] As L-lactic acid is metabolized faster by the beneficial bacteria than by the pathogenic bacteria (shown by a value of 2 in the above data sets being reached at 24hrs for the beneficial bacteria but not for the pathogenic bacteria), the presence of L-lactic acid would be expected to cause the beneficial bacteria to suppress the growth of the pathogenic bacteria within a short timescale, thus maintaining a healthy oral microbiota. As L-lactic acid is metabolized faster by the beneficial bacteria than by the pathogenic bacteria, this organic acid is consumed/metabolized (and thus taken out of the environment) by the beneficial bacteria before the pathogenic bacteria can start using it. Without being bound by any theory, it is believed that the stimulatory effect of L-lactic acid on the pathogenic bacteria as seen at 48hrs might be abolished when a mixture of beneficial and pathogenic bacteria is present (such as in the oral cavity), as this organic acid substrate has already been metabolized by the beneficial bacteria and is therefore unavailable for use by the pathogenic bacteria. Thus, if the growth/metabolic activity/co Ionization of the beneficial bacteria is stimulated before that of the pathogenic bacteria, then the beneficial bacteria can multiply and exert an inhibitory effect on the pathogenic bacteria before the latter have the chance to grow/multiply.

Example 2

[0096] The extent of growth of various beneficial oral and pathogenic oral bacteria in the presence of the carboxylic acids L-aspartic acid and succinic acid, as examples, was investigated.

[0097] The beneficial oral bacteria tested were Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, Actinomyces viscosus, Streptococcus gordonii, Capnocytophaga sputigena, Actinomyces naeslundii and Veillonella parvula. The pathogenic oral bacteria tested were Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Streptococcus sobrinus.

[0098] Substrates that were able to increase the growth (or maximal growth density or extent of growth) of at least one of the above beneficial bacteria while not or only minimally increasing the growth (or maximal growth density) of the pathogenic bacteria are considered to be prebiotic compounds.

[0099] The extent of growth of the tested bacteria in response to selected carboxylic acids (L- aspartic acid, succinic acid) was investigated by setting up growth curves in a nutritionally rich medium (brain heart infusion broth (BHI), Oxoid), over 48 hours. Late exponential growth phase liquid cultures were prepared by transferring the respective bacterium from blood agar plates to BHI and overnight incubation at 37°C in an anaerobic atmosphere for A. viscosus, V. parvula, F. nucleatum, P. gingivalis, P. intermedia, T. forsythia, A. naeslundii, and C. sputigena, and in a 5% CC"2 environment for S. salivarius, S. sanguinis, S. mitis, A. actinomycetemcomitans, S. mutans, S. sobrinus and S. gordonii. Overnight cultures were transferred to BHI and adjusted to a concentration of 1 ^χ 10 ⁷ CFU/ml (colony forming units per ml) by measuring the optical density at 600nm (OD ₆ oo) (BioRad SmartSpec 3000). For each strain, 200 μΐ of the bacterial suspension was added to a 96-well plate containing 20 μΐ of the respective carboxylic acids. Final concentrations of the carboxylic acids were set to 5, 10, 20 and 25 μηιοΐ/ιηΐ. For each bacterium tested, a respective control was also used, which did not contain the carboxylic acid. Plates were incubated as previously described.

[0100] For each combination of bacterium/carboxylic acid, and for each of their respective controls (i.e. the particular bacterium in the nutritionally rich medium with no carboxylic acid), the optical density was measured at 630nm (OD ₆ 3o) at 0 h, 24 h and 48 h (Thermo Scientific Multiskan Ascent). Additionally, the OD ₆ 3o for combinations of bacterium/carboxylic acid grown in a 5% C0 ₂ environment were also measured every hour between 0 h and 9 h. For each combination of bacterium/carboxylic acid, the maximal OD value obtained over the 48hr time period was divided by the maximal OD value obtained for the respective control over the 48hr period, so that the control had a value of 1. A value greater than 1 for a particular combination of bacterium with carboxylic acid therefore indicates that the extent of bacterial growth over 48hrs was greater than extent of bacterial growth over 48hrs for the control.

[0101] The experiment was carried out on three different days (thus providing 3 biological replicas) and on each day the experiment was carried out in quadruple (thus providing 4 technical replicas) for each combination of bacterium with carboxylic acid and for each control. For each day and for each combination, the average (mean) of the values obtained (as detailed above) for the four technical replicas was calculated to provide a single value for each combination on each day. The values shown in Tables 5 to 6, below, are the average (mean) of the three single values obtained for each combination of bacterium with carboxylic acid. The results are shown in Tables 5 to 6, below:

Table 5: Pathogenic bacteria over 48 hours

*A actinomycetemcomitans L- L- L- L-

Succinic Succinic Succinic Succinic aspartic aspartic aspartic aspartic

acid acid acid acid acid acid acid acid

25 20 10 5

25 20 10 5

μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml μηιοΐ/ml

A naeslundii 0.8435 0.9021 0.8774 0.8893 0.1840 0.2228 0.5885 0.9975

C sputigena 0.9489 0.9776 0.9529 0.9521 0.4571 0.6198 0.7668 0.9743

S gordonii 0.9466 0.9583 0.9400 0.9448 0.1860 0.2928 0.7021 0.8555

A viscosus 0.5023 0.5787 0.6163 0.6862 0.1586 0.1827 0.3693 0.5671

S salivarius 0.9943 1.0719 0.9975 0.9808 0.2197 0.2795 0.8904 1.0823

S sanguinis 0.8613 0.8622 0.8606 0.8662 0.2132 0.2724 0.6726 0.7980

V parvula 1.1498 1.1378 1.1168 1.0851 1.3566 1.4603 1.1922 1.1624

S mitis 1.1257 1.2536 1.2959 1.2333 0.1591 0.4339 0.7615 0.7273

Table 6: Beneficial bacteria over 48 hours

[0102] In the above data, a value of 1.25 was taken as the threshold above which the carboxylic acid caused markedly increased extent of growth of the bacterium relative to the control. This value was selected in order to exclude low-stimulating metabolites and avoid false positive results.

[0103] As can be seen from the above data, L-aspartic acid exhibited prebiotic effects at concentrations of 10 and 20 μιηοΐ/ιηΐ, and succinic acid exhibited prebiotic effects at concentrations of 20 and 25 μιηοΐ/ιηΐ.

Example 3

[0104] The effects of the carboxylic acids L-aspartic acid and succinic acid, as examples, upon biofilm growth of various beneficial oral bacteria and pathogenic oral bacteria, was also investigated.

[0105] Substrates that were able to increase the biofilm mass of at least one of the above beneficial bacteria while not or only minimally increasing the biofilm mass of the pathogenic bacteria are considered to be prebiotic compounds.

[0106] The increase in biofilm formation of the tested bacteria in response to selected carboxylic acids (L-aspartic acid and succinic acid) was investigated by setting up biofilm growth assays in a nutritionally rich medium (brain heart infusion broth (BHI), Oxoid), over 48 hours. Late exponential growth phase liquid cultures were prepared by transferring the respective bacterium from blood agar plates to BHI and overnight incubation at 37°C in an anaerobic atmosphere for A. viscosus, V. parvula, F. nucleatum, P. gingivalis, P. intermedia, T. forsythia, A. naeslundii, and C. sputigena, and in a 5% C0 ₂ environment for S. salivarius, S. sanguinis, S. mitis, A. actinomycetemcomitans, S. mutans, and S. gordonii. Overnight cultures were transferred to BHI and adjusted to a concentration of l x lO ⁷ CFU/ml by measuring the optical density at 600nm (OD ₆ oo) (BioRad SmartSpec 3000). For each strain, 200 μΐ of the bacterial suspension was added to a 96-well plate containing 20 μΐ of the respective carboxylic acids. Final concentrations of the carboxylic acids were set to 5, 10, 20 and 25 μιηοΐ/ιηΐ. For each bacterium tested, a respective control was also used, which did not contain the carboxylic acid. Additionally, for each bacterium tested, a background control, to correct for background staining, was added. This background control contained the bacterium tested and 0.03 weight % chlorhexidine, as an antiseptic. Plates were incubated as previously described. After 48hrs the supernatant was removed from the wells of the plates. The wells were washed twice with 1 x 100 μΙ_, PBS (phosphate buffered saline), fixed for 20 minutes with 96 %vol. ethanol (96 %vol. solution in water) and the biofilm retained at the bottom of the wells was stained with 1 weight% crystal violet (1 weight % solution in water). The bound dye was dissolved with 5 %vol. acetic acid (5 % vol. solution in water). Quantification of the stained biofilm was performed by measuring the absorbance at 630 nm using a Multiskan Ascent microplate reader (Thermo Scientific).

[0107] For each combination of bacterium/ carboxylic acid, the OD value obtained at 48hrs was divided by the OD value obtained for the respective control after subtracting the OD value of the background control, so that the control had a value of 1. A value greater than 1 for a particular combination of bacterium with carboxylic acid therefore indicates that the biofilm growth over 48hrs was greater than the biofilm growth over 48hrs for the control.

[0108] The experiment was carried out on three different days (thus providing 3 biological replicas) and each day the experiment was carried out in quadruple (thus providing 4 technical replicas) for each combination of bacterium with carboxylic acid and for each control. For each day and for each combination, the average (mean) of the values obtained (as detailed above) for the four technical replicas was calculated to provide a single value for each combination on each day. The values shown in Tables 7 to 8, below, are the average (mean) of the three single values obtained for each combination of bacterium with carboxylic acid. The results are shown in Tables 7 to 8, below: L- L- L- L-

Succinic Succinic Succinic Succinic aspartic aspartic aspartic aspartic

acid acid acid acid acid acid acid acid

25 20 10 5

25 20 10 5

μιηοΐ/ml μιηοΐ/ml μιηοΐ/ml μιηοΐ/ml μmol/ml μηιοΐ/ηιΐ μπιοΐ/ml μιηοΙΛηΙ

A actino* 0.5553 0.4857 0.5044 0.6182 0.0581 0.0593 0.5093 0.6941

F nucleatum 0.8549 0.9621 0.9710 1.2145 0.1087 0.5404 5.3887 2.7146

P gingivalis 0.9307 1.0779 0.9936 0.9713 0.1691 0.1065 1.0031 1.0520

P intermedia 1.5442 1.6295 1.6018 1.4322 0.1442 0.0589 0.7023 0.8344

S mutans 0.0000 0.1923 0.0000 0.0000 0.9686 1.9127 0.2498 0.0053

T forsythia 0.9659 1.0055 0.9131 1.0369 0.0000 0.0000 0.0000 0.3223

Table 7: Pathogenic bacteria at 48 hours

*A actinomycetemcomitans

Table 8: Beneficial bacteria over 48 hours

[0109] In the above data, a value of 1.6 was taken as the threshold above which the organic acid caused markedly increased biofilm growth of the bacterium relative to the control. This value was selected in order to exclude low- stimulating metabolites and avoid false positive results.

[0110] As can be seen from the above data, both L-aspartic acid and succinic acid exhibited prebiotic effects at 25 μιηοΐ/ηιΐ.