Means and methods for preventing and/or treating caries

Abstract

The present invention relates to a microorganism belonging to the group of lactic acid bacteria characterized in that it is capable of specifically binding to Streptococcus mutans, wherein the specific binding is (i) resistant to heat treatment; and/or (ii) resistant to protease treatment; and/or (iii) calcium-dependent; and/or (iv) formed within a pH range between 4.5 and 8.5; and/or (v) formed in the presence of saliva. Preferably, the specific binding can be assayed as follows: (a) growing said microorganism to stationary phase; (b) mixing said microorganism with Streptococcus mutans which has been grown to stationary phase; (c) incubating the mixture obtained in step (b) under conditions allowing the formation of aggregates of said microorganism and Streptococcus mutans and (d) detecting aggregates by the occurrence of a pellet. Another aspect of the present invention is an analog or fragment of said microorganism which is thermally inactivated or lyophilized, wherein said analog or fragment retains the capability of specifically binding to Streptococcus mutans. In addition, the present invention encompasses compositions and additives for food, feed or drinks comprising the microorganism belonging to the group of lactic acid bacteria which specifically bind to Streptococcus mutans or an analog or fragment thereof. Moreover, uses of said microorganism or said analog or fragment thereof for the preparation of an anticariogenic or pharmaceutical composition or anticariogenic food or feedstuff as well as methods for producing said compositions or food or feedstuff are provided by the present invention.

Claims

1. A liquid or solid composition comprising cells of at least one microorganism belonging to Lactobacillus paracasei which is thermally inactivated by heating at more than 95° C. for at least 20 minutes, wherein all cells of at least one microorganism belonging to Lactobacillus paracasei in the liquid or solid composition are dead or inactivated cells, wherein said cells bind to Streptococcus mutans in the presence of saliva or at a pH range between 4.5 and 8.5, wherein the binding between said cells and Streptococcus mutans is calcium-dependent, resistant to heat treatment at more than 95° C. for at least 20 minutes, and resistant to protease treatment, and wherein the composition comprises the cells in an amount of at least 0.001% by weight based on the weight of the composition; wherein said composition optionally comprises less than 1% (w/w) lactose or more than 6% (w/w) lactose based on the composition; wherein the composition is an animal feed which further comprises at least one orally acceptable carrier or excipient; and wherein the microorganism is selected from the group consisting of L. paracasei DSM 16667, L. paracasei DSM 16668, L. paracasei DSM 16669, L. paracasei DSM 16670, and L. paracasei DSM 16671.

2. The composition of claim 1, wherein the feed composition is a pet feed for dogs, cats, and rats, a cattle feed for cows and pigs, chicken feed for chicken and turkeys, or a fish cultivation feed for porgy and yellowtail.

3. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises a raw feed material selected from the group consisting of cereals, brans, oil-seed meals, animal-derived raw feed materials, and combinations thereof.

4. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises a cereal selected from the group consisting of mile, wheat, barley, oats, rye, brown rice, buckwheat, fox-tail millet, Chinese millet, Deccan grass, corn, soybean, and combinations thereof.

5. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises a bran selected from the group consisting of rice bran, defatted rice bran, bran, lowest-grade flour, wheat germ, barley bran, screening pellet, corn bran, corn germ, and combinations thereof.

6. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises an oil-seed meal selected from the group consisting of soybean meal, soybean powder, linseed meal, cottonseed meal, peanut meal, safflower meal, coconut meal, palm meal, sesame meal, sunflower meal, rapeseed meal, kapok seed meal, mustard meal, and combinations thereof.

7. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises an animal-derived raw feed material selected from the group consisting of fish powders, meat powder, meat and bone powder, blood powder, bone powder, byproducts from butchery, feather meal, silkworm pupa, skim milk, casein, dry whey, hill, and combinations thereof.

8. The composition of claim 1, wherein the at least one orally acceptable carrier or excipient comprises a raw feed material selected from the group consisting of plant stems and leaves, byproducts from corn processing industries, starch, sugar, yeast, byproducts from fermentation, agricultural byproducts, and combinations thereof.

9. The composition of claim 1, wherein the composition has anticariogenic activity.

10. The composition of claim 1, wherein the formation of aggregates of the microorganism and Streptococcus mutans occurs in the presence of saliva or at a pH range between 4.5 and 8.5.

11. The composition of claim 1, wherein the composition comprises at least 0.01% by weight of the cells.

12. The composition of claim 1, wherein the composition comprises at least 0.1% by weight of the cells.

13. The composition of claim 1, wherein the microorganism binds to Streptococcus mutans serotype c (DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060).

14. A method for the production of the composition according to claim 1 comprising obtaining the cells of a microorganism belonging to the group of lactic acid bacteria, and adding said cells to a raw or cooked feed material, molding, and granulating to form the composition.

15. A liquid or solid composition comprising cells of at least one microorganism belonging to Lactobacillus paracasei which is thermally inactivated by heating at more than 95° C. for at least 20 minutes, the cells consisting of dead or inactivated cells, wherein said cells bind to Streptococcus mutans in the presence of saliva or at a pH range between 4.5 and 8.5, wherein the binding between said cells and Streptococcus mutans is calcium-dependent, resistant to heat treatment at more than 95° C. for at least 20 minutes, and resistant to protease treatment, and wherein the composition comprises the cells in an amount of at least 0.001% by weight based on the weight of the composition; wherein said composition optionally comprises less than 1% (w/w) lactose or more than 6% (w/w) lactose based on the composition; wherein the composition is a food or drink which further comprises at least one orally acceptable carrier or excipient; and wherein the microorganism is selected from the group consisting of L. paracasei DSM 16667b1946, L. paracasei DSM 16668b1946, L. paracasei DSM 16669b1946, L. paracasei DSM 16670b1946, and L. paracasei DSM 16671b1946.

16. The composition of claim 15, wherein the food or drink is selected from the group consisting of juices, refreshing drinks, soups, teas, sour milk beverages, dairy products, ices, butter, cheese, processed milk, skim milk, meat products, fish meat cake products, egg products, confectioneries, breads, noodles, pickles, smoked products, dried fishes, and seasonings.

17. The composition of claim 15, wherein the composition is a powder food, sheet-like food, bottled food, canned food, retort food, capsule food, tablet food, or fluid food.

18. The composition of claim 15, wherein the composition comprises at least 0.01% by weight of the cells.

19. The composition of claim 15, wherein the composition comprises at least 0.1% by weight of the cells.

20. The composition of claim 15, wherein the microorganism binds to Streptococcus mutans serotype c (DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060).

21. A method for the production of the composition according to claim 15 comprising obtaining the cells of a microorganism belonging to the group of lactic acid bacteria, and adding said cells to a raw or cooked feed material, molding, and granulating to form the composition.

22. A liquid or solid composition comprising cells of at least one microorganism belonging to Lactobacillus paracasei which is thermally inactivated by heating at more than 95° C. for at least 20 minutes, wherein all cells of at least one microorganism belonging to Lactobacillus paracasei in the liquid or solid composition are dead or inactivated cells, wherein said cells bind to Streptococcus mutans in the presence of saliva or at a pH range between 4.5 and 8.5, wherein the binding between said cells and Streptococcus mutans is calcium-dependent, resistant to heat treatment at more than 95° C. for at least 20 minutes, and resistant to protease treatment, and wherein the composition comprises the cells in an amount of at least 0.001% by weight based on the weight of the composition; wherein said composition optionally comprises less than 1% (w/w) lactose or more than 6% (w/w) lactose based on the composition; wherein the composition is an animal feed which further comprises at least one orally acceptable carrier or excipient; and wherein the microorganism is selected from the group consisting of L. paracasei DSM 16667b1946, L. paracasei DSM 16668b1946, L. paracasei DSM 16669b1946, L. paracasei DSM 16670b1946, and L. paracasei DSM 16671b1946; and wherein the at least one orally acceptable carrier or excipient is selected from the group consisting of fluoride ion sources, anticalculus agents, buffers, abrasive materials, peroxide sources, alkali metal bicarbonate salts, thickening materials, humectants, surfactants, titanium dioxide, flavor agents, sweetening agents, xylitol, coloring agents, starch, glucose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ions, dried skim milk, glycerol, propylene glycol, water, ethanol, and mixtures thereof.

Description

(1) The Figures show:

(2) FIG. 1: Aggregation of Streptococcus mutans by Lactobacillus species

(3) The figure shows a mixture of an aggregating Lactobacillus with S. mutans (left tube) in comparison with a mixture of a non-aggregating Lactobacillus with S. mutans (right tube). The experiment has been performed as described in Example 3 and the tubes were left undisturbed for 20 minutes to allow the aggregates to settle.

(4) FIG. 2: Microscopic picture of an aggregating Lactobacillus species with Streptococcus mutans

(5) The figure shows a microscopic picture of the aggregate between Lactobacillus and S. mutans shown in FIG. 1 (left tube). The picture was taken at a 1000-fold magnification using a phase-contrast microscope.

(6) A better understanding of the present invention and of its many advantages will be had from the following examples, offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLE 1: STORAGE AND GROWTH

(7) Storage and growth of strains can occur according to ordinary procedures. For example, strains can be stored as frozen stocks at −80° C. 1 ml of a culture can be grown to stationary phase (OD600/mL 4-8) in MRS-Medium and mixed with 500 μl of a sterile 50% glycerine solution and frozen. Cultures of S. mutans can be grown in TSY-media to stationary phase (OD600/mL 1-2) and treated as mentioned above.

(8) Cultivation of S. mutans (DSMZ 20523, serotype c; NCTC 10923, serotype e; NCTC 11060, serotype f as well as non serotyped isolates) as well as cultivation of lactobacilli can be done in 5 ml in closed FALCON™ centrifuge tubes at 37° C. without shacking over night.

(9) In particular, the strains used in the present application were stored as frozen stocks at −80° C. 1 ml of a culture grown to stationary phase (OD600/mL 4-8) in MRS-broth was mixed with 500 μl of a sterile 50% glycerol solution and frozen.

(10) In particular, cultures of S. mutans were grown in TSY-broth to stationary phase (OD600/mL 1-2) and treated as mentioned above.

(11) Cultivation of S. mutans (DSMZ 20523, serotype c; NCTC 10923, serotype e; NCTC 11060, serotype f and other non serotyped—isolates isolated by OrganoBalance) and cultivation of lactobacilli was done in 5 ml in closed FALCON™ centrifuge tubes at 37° C. without shacking over night.

EXAMPLE 2: TAXONOMIC CLASSIFICATION OF STRAINS

(12) The taxonomic classification of the strains was done according to their carbohydrate fermentation pattern. This was determined using the API® 50 CH strips (bioMerieux, France) system and analyzed using APILAB PLUS software version 3.3.3 (bioMerieux, France).

EXAMPLE 3: TEST ON AGGREGATION OF STREPTOCOCCUS MUTANS

(13) Mixing of the lactobacilli with S. mutans was done in volumetric ratios of 3:1 to 60:1 (S. mutans:lactobacilli), this corresponds to a ratio of colony forming units from 1:50 to 1:2,5. An optical density measured at a wavelength of 600 nm in 1 ml means preferably for S. mutans 3×10.sup.8 colony forming units and for lactobacilli preferably 7×10.sup.9 colony forming units. Mixing was done in 2 mL volume in 15 mL FALCON™ centrifuge tubes. The culture suspensions were diluted with PBS-buffer to obtain the volumetric ratios mentioned above while keeping the final volume at 2 ml. The mixture was vortexed for 15 seconds. An aggregation is visible as an immediate turbidity of the suspension. The tubes were left undisturbed for 20 min, after that period of time the aggregates settle as a visible pellet whereas non-aggregating mixtures stay in suspension.

(14) As a control, self-aggregation of the respective Lactobacillus strain and the S. mutans strains was always investigated by performing the test with only the Lactobacillus or the S. mutans strain added to the tube. An aggregation of S. mutans by Lactobacillus is shown in FIGS. 1 (left tube) and 2.

(15) The lactobacilli strains of the present invention, in particular those deposited with the DSMZ exhibited aggregation of all S. mutans serotypes without showing a self-aggregation behaviour.

(16) Media:

(17) TABLE-US-00002 MRS-broth: MRS-mixture (Difco, USA) 55 g/L pH: 6.5 TSY-broth: TSY-mixture (Difco, USA) 30 g/L Yeast extract (Deutsche Hefewerke, Germany) 3 g/L

(18) Buffer:

(19) TABLE-US-00003 PBS-buffer: Na.sub.2HPO.sub.4*2H.sub.20 1.5 g/L KH.sub.2PO.sub.4 0.2 g/L NaCl 8.8 g/L pH adjusted with HCl

EXAMPLE 4: SPECIFICITY OF THE AGGREGATION TOWARDS TYPICAL MEMBERS OF THE ORAL FLORA

(20) The Lactobacillus cultures were grown as in Example 1,

(21) The oral bacteria—namely: Streptococcus salivarius subsp. thermophilus (isolated by OrganoBalance, identified by API® 50 CH strips (Biomerieux, France) according to manufacturers instructions); Streptococcus oralis (DSMZ 20066); Streptococcus oralis (DSMZ 20395); Streptococcus oralis (DSMZ 20627); Staphylococcus epidermidis (DSMZ 1798); Staphylococcus epidermidis (DSMZ 20044); Streptococcus mitis (DSMZ 12643); Streptococcus sanguinis (DSMZ 20567)—were grown in 5 mL BHI-medium in closed 15 mL FALCON™ centrifuge tubes at 37° C. over night. Each of the oral bacteria were preferably mixed in a volumetric ratio of 3:1 with Lactobacillus cultures and aggregation was assayed as in Example 3. For each testing of aggregation/non-aggregation only one of the aforementioned bacteria is preferably used to immediately determine the outcome of the testing.

(22) As a control, a self-aggregation of the respective oral bacteria as well as the tested Lactobacillus strains was always investigated by performing the test with only the lactobacilli or the oral flora strains added to the tube.

(23) The mentioned L. paracasei subsp. paracasei strains did not aggregate the oral bacteria mentioned above. The L. rhamnosus strains aggregated Streptococcus salivarius subsp thermophilus.

(24) TABLE-US-00004 BHI-broth: BHI-mixture (Difco, USA) 37 g/L pH: 7.2

EXAMPLE 5: TEMPERATURE RESISTANCE OF THE AGGREGATING CAPACITY OF THE LACTOBACILLI

(25) The bacteria were grown as in Example 1.

(26) The grown lactobacilli cultures were incubated at 121° C. at 2 bar in satured steam for 20 min (autoclaved). After cooling of the autoclaved cultures to room temperature, the lactobacilli were mixed in a volumetric ratio of 1:3 with grown S. mutans cultures and aggregation was assayed as in example 3 including the control experiments. Aggregation was also assayed using the oral bacteria as outlined in Example 4.

(27) It was found that the aggregation behaviour of the lactobacilli was not changed by the autoclaving procedure towards the tested S. mutans serotypes or towards the oral bacteria.

EXAMPLE 6: DEPENDENCY OF THE AGGREGATION ON PH-VALUE

(28) The bacteria were grown as in Example 1.

(29) 0.5 ml of the lactobacilli and 1.5 ml of S. mutans were harvested by centrifugation at 3200*g for 10 min and the supernatant was discarded. The cells were resuspended in their original volume (0.5 ml and 1.5 ml, respectively) in different PBS-buffers adjusted to different pH-values. The pH-values of the buffers were adjusted to values from 7.0 to 3.0 in steps of 0.5 pH-units. Cultures were resuspended in buffers of the respective pH-value that was to be used for the aggregation behaviour assay.

(30) Afterwards the lactobacilli were preferably mixed in a volumetric ratio of 1:3 with S. mutans cultures and aggregation was assayed as in example 3 including the control experiments. No visible aggregation of S. mutans by the lactobacilli occurred at pH values lower than 4.5.

EXAMPLE 7: SENSITIVITY OF THE AGGREGATION BEHAVIOUR TO LYOPHILISATION

(31) The bacteria were grown as in Example 1.

(32) Aliquots of 1 ml of the lactobacilli cultures were harvested by centrifugation at 3200*g for 10 minutes. The supernatant was discarded and the pellets were lyophilised at room temperature under vacuum for two hours. Resulting dry pellets of each tested Lactobacillus strain were stored at room temperature and at 4° C., respectively, for 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks. After the storage time, lyophilised pellets were resuspended in 1 ml PBS-buffer, pH 7.0. The resuspended lactobacilli were mixed in a volumetric ratio of 1:3 with freshly grown S. mutans cultures and aggregation was assayed as in example 3 including the control experiments.

(33) The aggregation behaviour of the mentioned lactobacilli towards S. mutans was not changed by the lyophilization or the storage procedures.

EXAMPLE 8: TEST ON PROTEASE RESISTANCE

(34) The bacteria were grown as in Example 1.

(35) Proteases used were Pronase E, Proteinase K, Trypsin, Chymotrypsin (all obtained from Sigma, Germany). Aliquots of 1 ml of the lactobacilli were washed in PBS-buffer by harvesting the cells by centrifugation at 3200*g for 10 minutes and resuspending the pellet in 1 ml PBS-buffer (pH 7.0). Afterwards the cells were harvested again as described above and resuspended in PBS-buffer (pH 7.0) containing the respective protease at a final concentration of 2.5 mg/mL. The suspension was incubated for 1 hour at 37° C. Afterwards the cells were washed and resuspended in PBS-buffer (pH 7.0) as described above.

(36) The aggregation was assayed as in example 3 including the control experiments.

(37) The aggregation behaviour of the mentioned lactobacilli towards S. mutans was not changed by treatment with any of the mentioned proteases.

EXAMPLE 9: ION DEPENDENCY OF THE AGGREGATION BEHAVIOUR

(38) The bacteria were grown as in Example 1.

(39) Aliquots of 1 ml of the lactobacilli were washed in 1 ml 200 mM EDTA solution twice as described above. Afterwards the cells were harvested and resuspended in 1 ml PBS-buffer (pH 7.0).

(40) The aggregation was assayed as in Example 3 and a complete loss of the aggregation ability was observed. Resuspension of the lactobacilli in 1 mi of a 2 mM calcium solution after the two times washing in 200 mM EDTA-solution restored the ability to aggregate S. mutans. Resuspension of the EDTA washed cells in up to 100 mM magnesium solution did not restore the ability to aggregate S. mutans.

EXAMPLE 10: TEST OF AGGREGATION IN THE PRESENCE OF SALIVA

(41) The bacteria were grown as in Example 1.

(42) 2 ml aliquots of S. mutans cultures were harvested as described above and resuspended in 2 ml of saliva. The saliva was provided by two volunteers and used immediately after winning.

(43) The aggregation was assayed as in Example 3.

(44) The aggregation behaviour of the mentioned lactobacilli towards S. mutans did not change in the presence of saliva.

EXAMPLE 11: LOZENGE COMPOSITION (I)

(45) The lozenge composition is preferably prepared as is described in Example 4 on page 8 of DE-C236 45 147, wherein, in addition to the ingredients mentioned in said Example 4, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the lozenge.

EXAMPLE 12: LOZENGE COMPOSITION (II)

(46) The lozenge composition is preferably prepared as is described in Example 5 on page 8 of DE-C2 36 45 147, wherein, in addition to the ingredients mentioned in said Example 4, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the lozenge.

EXAMPLE 13: DENTIFRICE COMPOSITION

(47) The dentifrice composition is preferably prepared as is described in Example 3 on page 8 of DE-C2 36 45 147, wherein, in addition to the ingredients mentioned in said Example 4, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the dentifrice.

EXAMPLE 14: CHALK-BASED DENTIFRICE COMPOSITION

(48) The chalk-based dentifrice composition is preferably prepared as is described in chapter 7.1.4.4 “Rezepturbeispiel” on page 205 of the textbook “Kosmetik”, W. Umbach (editor), 2.sup.nd edition, Thieme Verlag, 1995, wherein, in addition to the ingredients mentioned in said chapter on page 205, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the chalk-based dentifrice.

EXAMPLE 15: GEL-DENTIFRICE ON BASIS OF SILICIC ACID/SODIUM FLUORIDE

(49) The gel-dentifrice on basis of silicic acid/sodium fluoride dentifrice composition is preferably prepared as is described in chapter 7.1.4.4 “Rezepturbeispiel” on page 205 of the textbook “Kosmetik”, W. Umbach (editor), 2.sup.nd edition, Thieme Verlag, 1995, wherein, in addition to the ingredients mentioned in said chapter on page 205, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the gel-dentifrice on basis of silicic acid/sodium fluoride.

EXAMPLE 16: DENTIFRICE COMPOSITION AGAINST TARTAR

(50) The dentifrice composition against tartar is preferably prepared as is described in chapter 7.1.4.4 “Rezepturbeispiel” on page 206 of the textbook “Kosmetik”, W. Umbach (editor), 2.sup.nd edition, Thieme Verlag, 1995, wherein, in addition to the ingredients mentioned in said chapter on page 206, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the dentifrice against tartar.

EXAMPLE 17: CHEWING GUM COMPOSITION

(51) The chewing gum composition is preferably prepared as is described in Example 6 on page 9 of DE-02 36 45 147, wherein, in addition to the ingredients mentioned in said Example 4, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg of the chewing gum.

EXAMPLE 18: CONCENTRATED MOUTHWASH COMPOSITION

(52) The concentrated mouth wash composition is preferably prepared as is described in chapter 7.1.4.4 “Rezepturbeispiel” on page 206 of the textbook “Kosmetik”, W. Umbach (editor), 2.sup.nd edition, Thieme Verlag, 1995, wherein, in addition to the ingredients mentioned in said chapter on page 206, the microorganism of the present invention is added in an amount of 10.sup.2 to 10.sup.13, cells per ml of the concentrated mouthwash composition.

EXAMPLE 19: FILM PREPARATION

(53) Preparation of Films:

(54) 1. water phase heat water to 60° C. aspartame (sweetener) is added under stirring aspartame is dissolved completely a polymeric water-soluble film former, like, for example, KOLLICOAT® IR copolymer (polyethylenglycol on polyvinylalcohol) or PVP (polyvinylpyrrolidon) or natural polymers such as alginates are added under stirring until they are dissolved after 10 min. the rest of the foam is removed the microorganism of the present invention in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per final aroma film is added after cooling down of the mixture; alternatively, the mutant or derivative of the microorganism of the present invention or an analog or fragment of the microorganism of the present invention can be added.

(55) 2. oily phase menthol is dissolved in peppermint-oil polysorbat 80 is added to the peppermint-oil menthol mix under stirring this mixture is then added to propylene-glykole under stirring optional colorants (such as pigments, lakes) can be added

(56) 3. under stirring the oily phase is slowly mixed with the water phase

(57) 4. the thin films are mechanically generated using a cutting device

(58) Sample Formulations:

(59) TABLE-US-00005 formu- formu- lation I lation II weight composition weight composition [g] in film [%] [g] in film [%] Phase I aspartame 0.7 1.4 0.7 1.8 KOLLICOAT® IR 35.0 68.5 25.0 65.8 ascorbic acid — — 1.0 2.6 cherry flavour 6.0 15.8 water demin. 85.0 — 80.0 Phase II menthol 1.4 2.7 — peppermint oil 5.6 11.0 — polysorbat 80 0.7 1.4 — propylene glykol 7.0 13.7 5.0 13.2 green lake 0.7 1.4 — azorubin lake — — 0.3 0.8 sum 136.1 100.0 118.0 100.0 solid content 51.1 38.0

(60) Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, for any reason, including all publications, all U.S: and foreign patents and all U.S. and foreign patent applications, are specifically and entirely incorporated by reference for all purposes. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.