WO2006117029A1

WO2006117029A1 - Use of polylysine in combination with either green tea or olive extracts or both for use against halitosis

Info

Publication number: WO2006117029A1
Application number: PCT/EP2006/000973
Authority: WO
Inventors: Lucas Huybrechts
Original assignee: Lucas Huybrechts
Priority date: 2005-04-29
Filing date: 2006-02-03
Publication date: 2006-11-09
Also published as: BE1016514A6

Abstract

The invention discloses ingredients and formulations with enhanced efficacy and long duration effect for use against halitosis (unwanted bad breath), resulting from the synergistic effect of the use in the formulation of a mixture of (epsilon)-polylysine with polyhydroxyphenolic compounds, such as green tea extracts (that contain polyphenolic compounds and/or epigallocatechingallate) or olive extracts (that contain oleuropein and/or hydroxytyrosol) or a combination of them. It discloses also a new production procedure for preventing the occurence of precipitation when mixing (epsilon) polylysine and green tea extracts. The ingredients and formulations can be used in mouth rinses, mouth sprays, gels, tablets that dissolve in the oral cavity, chewing gums, candies, artificial saliva.

Description

SE OF POLYLYSINE IN COMBINATION WITH EITHER GREEN TEA OR OLIVE EXTRACTS OR BOTH FOR USE AGAINST HALITOSIS

Field of Invention.

This invention is about oral care formulations, such as mouth rinse-, mouth spray- and gel formulations, lozenges, tablets for dissolution in the oral cavity, candies and chewing gums, that contain (epsilon)-polylysine and other anti-bacterial ingredients, for use in the treatment against halitosis.

Background of the invention.

Approximately 25 % of the population is occasionally confronted with bad mouth breath, also known as "halitosis". It is generally accepted that, in most cases, unwanted bad breath is caused by sulfur containing gases (VSC), among which hydrogen sulfide, methylmercaptane and methylsulfide play a substantial role, that are produced by, predominantly, anaerobic bacteria that, often, reside on the inner-side of the tongue area. Some diseases such as, mouth cancer, diabetics, parodontitis, liver- and kidney diseases and sinusitis can agravate the problem, but the bacterial colonies in the oral cavity remain the dominant factor. Good oral hygienic practises do contribute to improving the condition of breath but are seldomly sufficient to solve halitosis.

The effect of ingredients & formulations against halitosis can be determined by measurment of the sulfur containing gases in the oral cavity from volunteers who are using either blanco's or formulations with such ingredients. This can be achieved with sophisticated gas chromatographic equipment as well as with the less sophisticated devices that can pick up the simple sulfur containing VSC gases. Such equipment is available and registered under the name Halimeter. Several products have been developed for treatment of halitosis: metal salts, oxidizing agents, bactericides, chitosan and natural extracts.

As for metal salts, mostly zinc-, stannic- and bismut salts are used, but their efficacy is limited (US 5833952, US6426085,US6702999,US50053556); in some cases they are used in the form of complexes in order to increase solubility (polyamines: US5587147, inuline: US6511679, glycine: US6607711); however due to their toxicological profile, long term use is not adviced.

Oxidizing agents too have antiseptic competence; several agents are used such as hydrogen peroxide (US40234463), but especially also chlorinedioxide (US6582682, US 6696047) and chlorite (US 6375933, US 02114851, US 030129144). Oxidizing agents act in a non-selective way, they may create resistant bacteria after long-term use and appear to have a relatively unattractive, low LD50 value. They are rather unstable in formulations, influence the function of other ingredients, and often they provide an unwanted taste that is typical for oxidizing and chlorinated agents. Occasionally bactericides are used, such as cetylpyridinium chloride (US5972312, US 6344184), salicylaldehyde (US40258733), or phenolic components such as triclosan (US5882631), but their efficacy is limited and short lived.

More often, chlorohexidine (US20030165411) is used; it has a broad spectrum activity, creates resistant bacteria after the long-term use, discolours teeth, has a rather unpopular taste, requires alcohol for dissolution and may burden the stomach. It's long term use is not adviced. vωω uαiLuαi c s ave een sugges e suc as ex rac s a contain piieno ic compounds, quinones, flavones, tannines, coumarines, terpenoides, alcaloides or essential oils. The latter is used more often against halitosis: US 5401496, US 5891422, 5 US 6348187, US 6350435, US 6379652, US 6555093, US 6689342, US 20054859, US 40253192; but the efficacy is limited.

Others suggest the use of extracts from Mouton Bark (US40224287) or the Citrus Vitus plant (US 6706256), or enzymes such as oxidoreductases (US 6074631), living organisms such as yeast (US 5866116), chitosan (US 4512968) or calcium hydroxyde i0 (US 6071500).

The use of cationic antimicrobial peptides against halitosis has been limited, although their use as anti-cavity agents is better known. The use against bacteria that cause caries does not automatically suggest it's efficacy against bacteria that cause halitosis. Caries 1 S and paradontisis are clinical states that are caused by other bacteria, such as streptococus mutans, than those that cause halitosis. It is known in the art, that bacteria that cause halitosis, predominantly reside on the inner side of the tongue rather than around the teeth (ref. S Roldan et al., Clin.Oral Investtig. 2003 Dec; 7 (4): 189-97; ref. M.Tanaka, Microbes Infect. 2004 Oct; 6 (12): 1078-83; ref. WJ.Loesche, Curr Infect. Dis. Rep. 2003 0 June; 5 (3): 220-226; ref. DV Messadi, Dermatol Clin 2003 Jan; 21 (1): 147-55; ref. U Cicek, Pediatr. Int. 2003 Dec; 45 (6): 719-23). Many people have halitosis without showing signs of paradontitis or vice versa. A number of cationic antimicrobial peptides are known to have a marginal effect only on halitosis, such as Iyso2yme and lactoferrin. This may be due to several reasons: either the intrinsic competence is limited, the 5 efficacy is neutralized from products naturally present in the oral cavity, bacteria are hidden deep in the caves of the surface of the tongue or the residence time in the oral cavity is too limited as a result of being removed with the saliva. Peptides against paradontitis and caries require competence on and around teeth, while products against halitosis require efficacy in the tongue area. Cationic peptides for use against halitosis 0 should, therefore, be evaluated specifically for such use.

Patent application (JP 2004107310) refers to the use of a "synergistic" mixture of epsilon-polylysine and inactive siloxane against bacterial plaque, caries, paradontitis and halitosis. The patent emphasizes the unsatisfactory efficacy of pure e-polylysine against a build-up of plaque and does not claim nor does it provide any experimental prove that 5 the peptide may work against halitosis; in fact siloxanes, which may be beneficial for use as abrasive in toothpastes that act in the vicinity and on teeth, cannot be used in a meaningful way in formulations ( mouth rinses, oral sprays, dissolving tablets) that act closer to the tongue area, which is an essential condition for the treatment of halitosis. Siloxanes do not degrade, as is the case with peptides, and are problematic for use in 0 dosing concepts (mouth washes, rinses, tablets....) that enter the body system more readily. It is preferable, as far as halitosis is concerned, to use only ingredients in such formulations that are well tolerated and degradable in the human body. The use of lysozyme is limited to gum formulations (CN1318314) and it's anti-halitosis competence is marginal; this is also the case for lactoferrin that has only been patented in 5 combination with other ingredients: lactoferrin and epigallocatechin (WO2004012522), lactoferrin or lysozyme and polyvinylalcohol (JP2002322088), lactoferrin or lysozyme and immunoglobulines (JP2003137809).

The patent application (EP2004013549) describes the discovery of the selective activity of epsilon polylysine against anaerobic bacteria, the main culprits in the occurence of halitosis. At a given dosage of the peptide, 70% and 11% of respectively anaerobes and aerobic bacteria are inhibited on the basis of which it's efficient and safe use against halitosis is claimed. The superior efficacy against halitosis has been confirmed by the determination of the content of sulfur gasses in the oral cavity of volunteers that have used bianco- and/or e-polylysine containing formulations. The duration of the effect was above 12 hours and even up to several days, in those cases where the product has been used for at least several days. The absence of siloxanes was a clear advantages in those formulations, such as mouth rinses, sprays, and dissolvable tablets, that are preferred for the treatment of halitosis since they can target the tongue area easily. However, new formulations are needed with enhanced advantages in use such as the degree of efficacity, duration of the effect, safety, lack of side effects such as tooth coloration and especially a combination of those.

Description of the invention

The experimental data that are included in this document (exp. 3) confirm the lack of efficacity of several antimicrobial peptides such as lysozyme, lactoferrin and histatine dh-5 (a synthetic clone of a part of histatine, an antimicrobial peptide that is naturally present in saliva), as well as the superior efficacity of epsilon-polylysine, as claimed in patent application EP2004013549.

Although the efficacity of polyhydroxyphenolic compounds as such has proven to be limited, we have surprisingly discovered the existence of a synergistic effect between e- polylysine and such polyhydroxyphenolic compounds. For this reason it is appropriate and preferable to use such mixtures against halitosis.

Several different types of polylysine can be used such as polylysine (exp. 3: degree of polymerisation 5-7) as well as epsilon-polylysine (so-called e-polylysine), another peptide-polymer based on lysine (exp.3: degree of polymerisation 30). The degree of polymerisation can vary between 5 and 10000 lysine units. Optionally spray dried mixtures can be used for convenience, such as e-polylysine/dextrin mixtures. The reduction in sulfur gas content in the oral cavity after use of a formulation with e- polylysine in comparison to the bianco (oral cavity which is not treated with the product nor with hygienic products such as toothpaste) is between 60% and 80% when using a dosage between 0.5% and 1%. (Epsilon)-polylysine can be dosed at between 0.01% to 10% and more in formulations, but preferably 0.1 to 1% in liquid formulations and preferably between 0.5% and 3% in dry formulations (tablet), whereby the daily intake of epsilon polylysine should be limited to below the accepted safe daily intake dosage of 10 mg/kg bodyweigth. They can be used and will be effective under acid, neutral and basic pH conditions.

The availability of multiple hydroxygroups on polyhydroxyphenolic compounds is contributing to the low non-toxic nature of the products which render them as prime candidates for safe use in the oral cavity. Sources of such products can be found in extracts from green tea (Camellia Sinensis) or from olive leafs and fruit (Olea Europaea); green tea extracts contain varying (depending on the production procedures) levels of polyhydroxyphenolic compounds and polymers, among which BCCG (epigallocatechingallate) is one of the most active antimicrobial ingredients.

As alternative to green tea, extracts from white tea (sourced from the same plant as green tea extracts: camellia sinensis) and from red tea ( sourced from the Rooibos plant) can also be used. Oleuropein and hydroxytyrosol, which are structurally related, belong to the most important polyhydroxyphenolic compounds in olive leaf and fruit extracts. Their dosage can vary according to the extraction procedure. The use of Green tea extracts, including those with high BCCG content, appear to reduce modestly the sulfur gas content in the oral cavity in comparison to the bianco (oral cavity not treated with the product nor with hygienic products such a toothpaste), more specifically between +3% and -6% (experiment 1). Olive extracts exhibit an equal modest reduction of sulfur gas content ,of -3% to -6%. They can be used at a dosage of between 0.01% and with no upper limit set for safety reasons.

Experiment 1 demonstrates the synergistic effect that results from the combined use of polylysines or epsilon-polyly sines with polyhydroxyphenolic compounds such as olive extracts (with hydroxytyrosol) or green tea extracts (with BCCG). At a shared dosage of 0.5% the sulfur gas content reduction in comparison to the bianco is between 70% and 87%. The beneficial effect of the reduction of sulfur gasses persists after long-term use (experiment 2). Depending on the length of the period of use, the duration of the remaining effect after terminating the use of the product will be between more than 12hours to several days. The main advantages in comparison to existing antihalitosis products include: high efficacy and long duration time, extreem low toxicity (compared to synthetic cationic antiseptics such as chlorohexidine), no discoloration of teeth, no need for dissolution in alcohol, no bad taste, no known risk for creating resistant bacteria. In another aspect of the invention we noticed the difficulty in mixing green tea extracts and (epsilon) polylysine in the basic form. They immediately form a precipitate that may reduce it's effectiveness. We have surprisingly discovered that precipitation can be avoided if the products are mixed at a slight acid pH, preferably in the range of 5 to 6.9, more preferable around 5.5 to 5.8. This can be achieved for example by using (e) polylysine that has been adjusted to slightly acidic pH in solution before addition of the green tea extract. A product with a pH below 5 is less preferred as it may damage teeth.

Different forms of end formulations The new active ingredients can be used with other ingredients, known in the field, in different forms of end formulations for use in the oral cavity: mouth refreshing solutions, mouth rinses, mouth sprays, gels, chewing gum, candies, dissolving tablets and other food systems, artificial saliva and medical products for the treatment against halitosis. An overview of such end formulation is provided in US 6238648. Experiment 4 provides data on tablets, alike mint tablets, that dissolve in the oral cavity.

Other ingredients in end formulations

The end formulations can also contain components for the protection against caries: fluorides (US 2946725 and US 3678154; for example sodium fluoride, sodium monofluorophosphate and stannous fluoride or encapsulated fluoride ingredients (for protection against deactivating components such as calcium or orthophosphates). Fluorides are used at a concentration between 0.1% to 1% w/w, preferably between 0.25 and 0.5 % on weight basis. The end formulation can contain also other protecting anticavity compounds such as natural bactericides, synthetic bactericides, plant extracts, peptides with immunological activity, antibodies against S. Mutans, bacteriophages, sugars to reduce the production of acid (xylitol, erythritol), enzymes ( e.g. glucanases and dextranases) or a vaccin against glucosyltransferase or peptide analogues to the S. Mutans antigen I/H, ingredients for repair work (eg. calcium, phosphate, casein,, non- denaturated casein, casein hydrolysates (CPP), buffering components such as chitosan, polyethyleneimine fluorofosfaat, arginine and arginine containing peptides (with 2-4 amino acids).

Calcium salts can include calcium chloride, calcium acetate, calcium citrate, calcium butylate, calcium lactate, calcium salicylate or another non toxic anorganic or organic calcium salt at a concentration between 0.1 % to 5% w/w. ^, -, -, -, zwitterionic detergents as described in US 3988433, US 4051234, US 3959458. Non ionic detergents are condensates from hydrophilic alkylene oxide groups with hydrophobic organic components. For example: poloxamers (sold under the name Pluronic), polyoxyethylene sorbitan esters (Tween), polyethylene oxide condensates of alkyl phenols, condensates of ethylene oxide with reaction products from propylene oxide and ethylene diamine, ethylene oxide condensates from aliphatic alcohols, tertiary amine oxides with a long chain, tertiary phosphine oxide with a long chain, dialkylsulfoxides with a long chain and mixtures. Amphoteric detergents are aliphatic secondary and tertiary amines, with an aliphatic chain and with the presence of an anionic group (e.g. carboxylate, sulfonate, sulfate, phoshate, phosphonate).

Anionic detergents are salts of alkylsulfates with 8 to 20 carbon atoms ( for example sodium alkyl sulfate) and salts of sulfonated monoglycerides from fatty acids with 8 to 20 carbon atoms. Examples: sodium lauryl sulfate and sodium coconut monoglyceride sulfonate, sarcosinates such as sodium lauroyl sarcosinate, sodium lauryl sulfoacetate, sodium lauroyl isethionate, sodium laureth carboxylate, sodium dodecylbenzenesulfoiiate or mixtures. Often the dosage of an anionic detergent is between 0.025% to 9% and preferably between 0.1% and 5% w/w.

Thickeners can be used in the end formulation to provide the desired rheological profile: guar gum, carboxyvinyl polymers, carageenan, Koηjac, scleroglucan, carboxymethyl cellulose, hydroxyethyl cellulose, polyoxyethylene polyoxypropylene glycol copolymers, gum karaya, gum arabic, gum tragacanth and xanthan in a concentration of 0.1 % to 15%. Cross-linked polymers from acrylic acid, such as Carbopol from BF Goodrich are known in the sector.

The end formulation can contain a humidifier. Polyalcohols provide a wet feeling and prevent the product from becoming hard upon contact with air.

They include glycerin, sorbitol, butylene glycol, polyethylene glycol, sorbitol.

The end formulation can contain products against tooth-stone such as pyrophosphate salts such as Na.sub.4 P.sub.2 O.sub.7, K.sub.4 P.sub.2 O.sub.7, Na.sub.2 K.sub.2 P.sub.2 O.sub.7, Na.sub.2 H.sub.2 P.sub.2 O.sub.7 and K.sub.2 H.sub.2 P.sub.2 O.sub.7, sodium hexamethaphosphate, sodium tripolyphosphate and cyclic phoshphates such as sodium trimetaphosphate. The dosage is about 0.5% to 10% w/w. Anionic polycarboxylates or carboxylated chitosan could be used eventually in order to increase the anti-tooth stone effect. Copolymers of maleic anhydride with other ethylenic monomers such as methyl vinyl ether with a molecular weight between 30.000 and 1.000.000 and preferably between 30.000 and 500.000 are known under the name Gantrez (US4627977). The concentration in the end formulation is between 0.5% and 5%. Other possibilities include zinc citrate trihydrate, polyphosphates, diphosphonates (EHDP). The end formulation can contain aroma's, often at a concentration between 0.001% and 5% and preferably between 0.5% and 1.5% w/w. Examples are: spearmint, peppermint, menthol, anethole, methyl salicylate, cassia, 1-menthyl acetate, eugenol, parsley oil, oxanone, alpha-irisone, marjoram, propenyl guaethol, vanillin, thymol, linalool, cinnamaldehyde glycerol acetal, wintergreen, sassfras clove, sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit, orange. The end formulation can also contain sweeteners; besides the known anticariogenic sweeteners the following products are valuable: sucrose, glucose, saccharin, dextrose, levulose, lactose, mannitol, sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin, aspartame, D-tryptophan, dihydrochalcone, acesulfame and cyclamate salts.

The end formulation can also contain ingredients against over-sensitivity (for example potassium nitrate or potassium citrate), whitening agents (hydrogen peroxide, calcium peroxide, urea peroxide), preservatives, cooling agents (carboxamides, menthol, ketals), anti-inflammatory ingredients (aspirin, ibuprofen, naproxen....).

The compositions of end formulations are known and are added for reference: for example for mouthrinses US3988433, for candies US 4083955, for chewing gum US 4083955.

Mouth rinses and sprays often contain water (45% to 95%), ethanol (0% to 25%), a humidifier (0% to 50%), a tensio-active agent (0.01% to 7%), an aroma (0.04% to 2%), a sweetener (0.1% to 3%) a coloring agent (0.001% to 0.5%) and eventually an anticariogenic product (fluoride; 0.05% to 0.3%) or a product against tooth stone (0.1% to 3%).

Another formulation concerns non-abrasive gels (subgingival gels). They contain a thickener (0.1% to 20%), a humidifier (0.1% to 910%), an aroma

(0.04% to 2%), a sweetener (0.1% to 3%), a coloring agent (0.01% to 0.5%), water (2% to 45%) and an anticariogenic or anti-tooth stone product.

Chewing gum formulations often contain gum (50% to 99%), an aroma (0.4% to 2%), a sweetener (0.01% to 20%) and optionally an anticariogenic product. Candies, mints, capsules, tablets and other food systems have been described in US 4642903, US 4946684, US 4305502, US 4371516, US 5188825, US 5215756, US 5298261, US 3882228, US 4687662, US 4642903.

A. Method and materials Products

Epsilon polylysine (so-called e-polylysine; degree of polymerisation; dp 30) and epsilon polylysine/dextrin spray dried powder are available from Chisso Corporation (Japan). Polylysine with a dp of 2-7 can be obtained from Solabia (France). Green tea extract can be sourced from companies such as Shanghai Lithy Foods Material Corp. (China), Ningbo Hanpharm Biotech Co. (China) or Shaanxi Jiahe Phytochem Co (China) with varying levels of polyphenolic compounds and EGCG (epigallocatechmgallate); DSM corp. (The Netlierlands) offers a tea extract with 94% EGCG. Olive leaf extracts with more than 20% of oleuropein can be obtained from Guilin Layn Natural Ingredients corp. (China) and Genosa (Spain) offers olive fruit extracts with 45% hydroxytyrosol (Brandname: Hytolive). Egg based lysozyme and lactoferrin are sourced respectively from Belovo corp. (Belgium) and DMV corp. (The Netherlands). Xylitol and sorbitol are sourced from Roquette (France). Histatin dh-5 is the synthetic fungicidal histatin analogue from the C-terminal part, residues 11-24, and sourced from the Vrije Universiteit Amsterdam (The Netherlands). The menthol/thymol/eucalyptol alcohol mouth rinse can be obtained on the market under the trade name Listerine (Pfizer). Products such as glycine and lactic acid can be purchased from Aldrich (USA). Mint aroma's are from Firmenich (Germany).

Determination of content of sulfur gases in the oral cavity The vast majority of oral malodor originates from the anaerobic bacterial degradation of sulfur containing amino acids within the oral cavity, resulting in the emission of hydrogen sulfide (H₂S), methyl mercaptan (CH₃SH), and dimethyl sulfide (CH₃SCH₃), collectively referred to as volatile sulfur compounds (VSC). The Halimeter^® (InterScan Corp., USA) , the equipment that is used, responds to all three volatile sulfur compounds, and gives a reading of total VSCs.

To be rigorous, the Halimeter does not respond equally to all three compounds, nor are all three compounds present in the same proportion in all patients — or even in the same patient — at all times. The relative amounts of these compounds are affected by diet, and the extent of anaerobic bacterial growth. Elucidating the precise breakdown of the three compounds in an oral malodor sample requires a gas chromatograph. Fortunately, the additive reading provided by the Halimeter correlates extremely well with organoleptic panels of trained human evaluators, and compares admirably with chromatographic testing. The Halimeter reads out in parts-per-billion (ppb) of volatile sulfur compounds. Normal readings, for subjects with no oral malodor, are generally in the range of 80-140 parts per billion (ppb). At levels of 200-300 ppb, oral malodor is noticeable by an observer standing close to the patient. At 350-400 ppb, the odor is noticeable by an observer standing several feet away from the patient. At 500-700 ppb the odor is more noticeable not because it is "stronger," but because it is more foul. At over 1000 ppb, the odor will linger for several minutes after the patient leaves the room. In many of these cases, odor will continue to emanate from the tongue during the entire sampling process, and the Halimeter reading will keep climbing, and may not truly peak, as the sample pump seems to draw more VSCs off the tongue surface. Before any antihalitosis product is used, the volunteer abstains from using hygienic products such as toothpaste over a period of several days, untill the average reading (of at least 3 measurements) is above 400 ppb. This is considered to be the "bianco" situation. The measurements are mostly carried out just after wakening up, before breakfast. This avoids contamination with the varying paramaters that could affect readings during or at the end of the day (foods, drinks, tobacco....). The volunteer is using the experimental product twice per day, in the morning after breakfast and in the evening just before going asleep. The measurement of sulfur gases is carried out about twelve hours after use of the product (the next morning). Each time 3 measurements are taken and the average is used.

B. Results Experiment 1: Determination of sulfur containing gases in the oral cavity over a period of 4 days; synergy between e-polylysine and polyhvdroxyphenolics in pump- spray formulations.

A volunteer was deprived for several days from using hygienic products (toothpaste's, moutwashes,...) in order to stimulate production of bad breath gases in the oral cavity and this untill the gas level was above 400 ppb (average of 3 measurements in the morning, just after wakening up on an empty stomach). This value was established three times, each time before the use of the formulation with polyhydroxyphenolic ingredient, or with e-polylysine or with a mixture of both; the average of the three values is set to be the "bianco". Subsequently, the volunteer used an oral pump spray formulation after breakfast ( 4 x 0.14 ml dosage) and repeated the treatment a second time just before going to bed in the evening.

The measurement of sulfur-gases was carried out after a residence time of above 12 hours (overnight; this is the time between the use of the product and the measurement); the measurement was done just after wakening up the following morning (on an empty stomach). The measurement was carried out three consecutive times (at the same moment) per day and was repeated daily over a period of 4 days (the given data are an average of 12 gas determinations over 4 days). Apart from the active ingredients, the aqueous solutions contained also 3% weight percent of erythritol and 3 drops of Mint-aroma per 40 gr of water. Formulations where made respectively with the following active ingredients: natural extracts that contain a polyhydroxyphenolic component, such as olive extract (with 40% hydroxytyrosol) or green tea extract, or epsilon-polylysine (dp 30) or a mixture of epsilon polylysine with such natural extracts. 0.2% Glycine was also added to all formulations which contained e-polylysine. The pH was adjusted with lactic acid to 6.8-7.0 in all formulations except those that contained a mixture of e-polylysine and a green tea extract ( pH:5.75) Active ingredient(s) in the formulation / dosage of active ingredient weight %/ measurement of Sulfur-gases in ppb (Avg of 3 determinations) / variation with the bianco/ pH: bianco / 0%/ 474 / -% / - olive extract (hytolive) / 0.5% / 440 / -7.2% / 6.8-7.0 e-polylysine dp30 / 0.5% / /172 / -63.7% / 6.8-7.0 e-polylysine dp30 + olive extract (hytolive) / 0.5% + 0.5% / -77.4% / 6.8-7.0 bianco /0%/ 402/-%/ - green tea extract A / 0.5% / 392 / -2.5% / 6.8-7.0 e-polylysine dp 30 / 0.5% / 90 / -77.6% / 6.8-7.0 5 e-polylysine dp30 + green tea extract A/ 0.5% + 0.5% / 52 / -87.1% / 5.75 bianco /0%/ 456/-%/ - green tea extract B/ 0.5% / 432 / -5.3% / 6.8-7.0 e-polylysine dp 30 / 0.5% / 172 / -62.3% / 6.8-7.0 ϊd e-polylysine dp30 + green tea extract B / 0.5% + 0.5% / 105 / -77.0% / 5.75 bianco /0%/ 415/-%/ -

0.5% green tea (94% EGCG) / 430 / 3.6% / 6.8-7.0 e-polylysine dp 30 / 0.5% / 172 / -58.6% / 6.8-7.0 IS e-polylysine dp30 + green tea (94% EGCG) / 0.5% + 0.5% / 126 / -69.6% / 5.75

One can notice the synergy between e-polylysine and polyhydroxyphenolic compounds.

Experiment 2: determination of sulfur containing gases in the oral cavity over a 0 period of one month; the longer term effect of an e-polylvsine containing pump- spray formulation.

The occurence of sulfur containing gases has been determined in the oral cavity of 2 volunteers over a period of sixteen days during which no hygienic product such as toothpaste has been used. The measurement was carried out immediately after wakening 5 up (on an empty stomach) and just before going to bed in the evening.

As from day 18, the following formulation (an oral pump spray recipe) has been used twice daily: after breakfast and after evening dinner (each time 4 x 0.14 ml dosage). Formulation (on % weight basis): 1% epsilon polylysine dp 30, 0.2% lactoferrin, 0.1% egg lysozym, 3% xylitol, 0.2% glycine, 2 drops of mint aroma, lactic acid up to a pH of 0 6.8 -7.0 and remainder is water. The head of the spray is targetted to the tongue surface. No alcohol is consumed during a 6 hour period proceeding the measurement; and no food is taken within 2 hours prior to the measurement. The time gap between the use of the product and the measurement is +12 hours. 5

Number of the day / volunteer 1: determination of sulfur gases in the morning ppb/ volunteer 2: determination of sulfur gases in the morning / volunteer 1: determination of gases in the evening / volunteer 2: determination of gases in the evening: 0 1 / - / - /296.3/-

2 /352.3/205.0/341.3/90.0

3 /245.3/256.3/206.0/35.7

4 / 213.7 / 202.7 / 294.3 / 109.7

5 /550.7/260.0/-/- 5 6/ / /351.3/120.3

7 /382.0/276.7/198.7/218.3

8 /484.0/327.7/144.7/182.3

9 /352.3/238.0/ / 10/427.3/261.0/ / 0 11/ / /387.7/91.7

12/631.7/439.3/298.0/128.3 14/515.3 / 259.3 / 606.0 / 72.7

15/488.7/306.7/374.7/83.0

16/368.3/302.0/ / 5

18/ / /73.3 /58.7

19/47.0 /33.0 /36.7 /-

20/79.0 /80.0 /71.7 /43.0

21/82.0 /96.7 /122.3/49.0 ib 22/72.0 /120.3/46.3 /65.0

23/85.3 /106.3/ /

24/ / /109.7/93.3

25/111.3/88.3 /81.3 /49.7

26/ /96.3 / /45.3

15 27/ /106.0/ /133.0

28/ /53.3 / /95.7

29/ /52.7 / /

Average of number of days / volunteer 1: average of determination of sulfur gases in the 0 morning (stand, dev.) / volunteer 2: average of determination of sulfur gases in the morning (stand, dev.) / volunteer 1: average of determination of gases in the evening (stamd. dev.)/ volunteer 2: average of determination of gases in the evening (stand, dev.):

Average Day 1 to 16 / 409.6 (79.4) / 264.7 (86.3) / 321.9 (78.3) / 109.9 (59.8) 5 Average Day 18 to 29 /121.2 (20.8)/ 37.6 (30.2) / 125.9 (31.2) /51.8 (18.2)

One can notice a substantial and continuous reduction in sulfur gases during the period that the product is consumed. 0 Experiment 3: The effect of a variety of active ingredients in a pump-sp ray- formulation over a period of 4 days.

A volunteer was deprived for several days from using hygienic products (toothpaste's, moutwashes,...) in order to stimulate production of bad breath gases in the oral cavity 5 and this untill the gas level was above 400 ppb (average of 3 measurements in the morning, just after wakening up on an empty stomach); the average of the three values is selected as the "bianco".

Subsequently, the volunteer used an oral pump spray formulation after breakfast ( 4 x 0.14 ml dosage) and repeated the treatment a second time just before going to bed in the 0 evening.

The measurement of sulfur containing gases was carried out after a residence time of above 12 hours (overnight; this is the time between the use of the product and the measurement); the measurement was done just after wakening up the following morning (on an empty stomach). The measurement was carried out three consecutive times (at the 5 same moment) per day and was repeated daily over a period of 4 days (the given data are an average of 12 gas determinations over 4 days).

Ten different formulations where used respectively with one of the following active ingredients: olive extract (with either 20% oleuropein or 40% hydroxytyrosol depending on the extraction procedure), epsilon-polylysine (with a degree of polymerisation of dp 0 30 or 5 to 7), or a mixture of e-polylysine and dextrin, or other antimicrobial peptides such as lysozyme, lactoferrin, histatin dh-5 or a mix of etheric oils (menthol/thymol/eucalyptol) in 25% ethanol. 0.2% Glycine was also added to all formulations which contained e-polylysine (dp30). The pH was adjusted with lactic acid to 6.8-7.0.

Column: no. of formulation/ dosage of 16 ingredients(% on weight basis)/ sulfur gases in bianco experiment ..ppb / average s-gas content in ppb after four days of use of the formulation / % difference with the bianco

It is demonstrated that polylysine is active in a variety of forms, either as e-polylysine with a degree of polymerisation of dp 30 or polylysine with a dp of 5-7 or as an spray dried e-polylysine / dextrin 50/50 mixture; the activity is dose dependant. A variety of olive extracts can be used, such as one containing 20% oleuropein or +40% hydroxytyrosol. It is evident that not all antibacterial peptides demonstrate antihalitosis competence; as opposted to e-polylysine, the activity of lysozym, lactoferrin and histatin hv-5 is modest. Equally, the effect of a mixtures of etheric oils such as thymol in the presence of ethanol is limited. Formulation 5 has been stored for + 3 months before use, demonstrating it's resistance against degradation.

Experiment 4: Determination of sulfur containing gases in the oral cavit^y after the use of e-polylvsine and Dolvhvdroxvphenolic compounds in Mint-tablets After establishing the sulfur gas content of above 400 ppb for the determination of the bianco, as in experiment 3, the volunteer used one tablet in the morning, after breakfast and one tablet after evening dinner. The gas measurement was conducted before breakfast.

Column: no. of formulation / dosage of 8 ingredients in two formulations/ sulfur gases in the oral cavity in the bianco experiment ..ppb / average sulfur-gas content in ppb after four days of use of the formulation / % difference with the bianco

The next recipes has been enclosed as example only. As for other recipes, references is made to known formulations that have been described in the literature and that have been mentioned in this document. The dosage of ingredients is as weigth (%) of the complete formulation

chewing gum

Peptide mix : the use of epsilon-polylysine, or a mixture of e-polylysine and another antimicrobial peptide, such a lysozyme. Lozenge & mint 0.28 gram

Mouth rinse & Mouth spray

* Peptide mix : epsilon-polylysine, or a mixture of e-polylysine and another antimicrobial peptide, such a lysozyme.

Pasta & dentrifice

Claims

We claim,

1. The use of polylysine in combination with one or more ingredients that are selected from the group of polyhydroxyphenolic compounds, consisting of green tea extracts and Olea Europaea (olive) extracts, in formulations for use against halitosis.

2. The use according to claim 1. whereas polylysine means epsilon-polylysine.

3. The use according to claim 1. whereas the green tea extract contains epigallocatechingallate in a dosage between 0.01% and above 90%

4. The use according to claim 1. whereas the Olea Europaea extract contains a concentrated dosage of oleuropein and/or hydroxytyrosol between 0.01% and above 90%.

5. The use of ingredients according to claim 1. in the presence of one or more ingredients from the list consisting of lysozyme, lactoferrin, glycine, lactic acid, citric acid, malic acid, sorbitol,erythritol, mannitol,xylitol.

6. The use according to claim 1. with a formulation that contains epsilon-polylysine and green tea extract, whereas the pH has been adjusted to below 6, preferably between 5.0 and 5.8.

7. The use according to claim 1. of the ingredients in mouth rinses & washes, lozenges, mints, tablets, oral spray pump solutions, gums and candies.

8. The use of ingredients according to claim 1. to 5. in combination with traditionally known ingredients in the formulations of claim 7. such as those that are mentioned in this document, for example, fluorides, synthetic bactericides, saccharides, phosphates, enzymes, calcium complexing ingredients and peptides and proteins, calcium salts, detergents, thickening agents, humidifiers, anti calculus products, aroma's, sweeteners, anti cacking agents, whitening agents, anti sensitive ingredients and preservatives.