WO2009010766A1

WO2009010766A1 - Method

Info

Publication number: WO2009010766A1
Application number: PCT/GB2008/002466
Authority: WO
Inventors: John Greenman; Saliha Saad
Original assignee: University Of The West Of England, Bristol
Priority date: 2007-07-19
Filing date: 2008-07-18
Publication date: 2009-01-22
Also published as: GB0714135D0

Abstract

This invention relates to a method of determining oral malodour in a subject, the method comprising determining the pH level of the tongue biofilm of the subject, and in which an elevated pH level is indicative of oral malodour. The invention also relates to apparatus for determining oral malodour in a subject, the apparatus comprising means for determining the pH of the tongue biofilm of the subject.

Description

METHOD

This invention relates to a method of determining oral malodour, to apparatus for determining oral malodour; to kits for determining oral malodour, to methods of treatment of oral malodour; to methods of identifying compounds useful in the treatment of oral malodour; and to the use of compounds in the treatment of oral malodour.

Background to the invention

Chronic oral malodour is often caused by oral microbes, in particular anaerobic types, that live on the upper (dorsum) surface of the tongue. In this environment the microbes exist together as mixed cultures (ecological consortia) where they grow as adherent (to each other and to other surfaces) layers generally described as biofϊlms. There is no single microbial species responsible for bad breath since many species have the ability to (1) attack proteins (2) enzymatically biotransform amino acids and peptides (cysteine, methionine, tryptophan, lysine, arginine, ornithine) into an output of odourous volatile compounds (putrefaction gases) hydrogen sulphide, methyl mercaptan, indole, skaltole, organic acids and amines. It is the concentration levels of these gases that dictate the degree of oral malodour that will be found in bad breath.

Oral malodour can also be caused by a metabolic disease or other host-related conditions.

Chronic oral malodour cannot be cured in the sense that it will go away permanently. However, it can be successfully treated, giving relief for considerable periods of time by taking chemical products known to be effective in controlling malodour or by mechanically removing the tongue biofilm by other means (e.g. tongue scraping or brushing). A significant problem with oral malodour is that it is almost impossible to self assess one's own level of breath odour.

Because having a high degree of oral malodour can have psycho-social effects on humans, there is a great need for understanding its aetiology and developing methods for its measurement. Existing methods of measuring bad breath include (1) human breath judges (using an organoleptic approach, see for example Greenman, J et al Journal Dental Research 83(1): 81-85, 2004), (2) sulphide monitors (e.g. a Halimeter (trade mark)) for example as disclosed in United States patent no. 6264615; European patent application 0819940; and United States patent application no. 20010056246A; and (3) gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS). These methods are usually expensive to perform and typically require specialist knowledge or facilities (laboratories). None can be easily conducted by individual subjects (self- assessment). What is needed is a very cheap and simple but reliable test (e.g. one that ends with a colour change) that can correlate (or co-vary) with the oral malodour status of the individual.

Previous workers have tried to search for relationships between salivary pH and malodour. However, the pH of saliva is notorious for changing with relationship to its flow rate which can vary enormously throughout the day (e.g. stimulated or unstimulated) and there is no obvious correlation with bad breath. Some workers have tried to show a relationship between the pH of incubated saliva and malodour but correlation is low and it is a smelly procedure which is unsuitable for consumer use. The accompanying Figure 1 illustrates the results of experiments on trying to determine a connection between oral malodour and the pH of saliva. In the underlying experiment, the pH of the saliva of 20 volunteers was determined and an organoleptic score determined for the breath of each volunteer by judges using the methodology of Greenman et al (2004) supra. Using the organoleptic approach the integer descriptives were: 0, no odour (below the smell threshold); 1 , barely noticeable odour; 2, slight odour; 3, moderate odour; 4, strong odour; and 5, extremely strong odour, close to saturation. Judges were allowed to give fractional scores if they thought the strength of the smell was between the integer categories (e.g., 2.5 if between 2 and 3). As shown in Figure 1 there is only a weak correlation (R²<0.5) between the pH of saliva and oral malodour. In the same experiment, the pH of the saliva was related to the degree of oral malodour by means of Halimeter measurements. As shown in Figure 2, there is also only a weak relationship (R² = 0.427) between the pH of saliva and the degree of oral malodour as reflected by the Halimeter reading. All previous workers in this field have chosen saliva as being the main sample to "take" in order to do quick measures or test for malodour generation. The reasoning to support the sampling of saliva is based on the view that bad breath processes occur in parts of the mouth generally described as the salivary domain. We believe that this view is incorrect.

In a study relating to the effect of tongue brushing on oral malodour in adolescents, Cicek et al (Cicek, Y. et al, Paediatrics International, 45, page 719-723 December 2003) measured the pH of saliva in the mouths of adolescent subjects with litmus indicator paper taking saliva from the tongue as opposed to tongue biofilm. It was found that tongue brushing reduced the measured pH more than brushing teeth alone; however, this was not found to be statistically significant.

We have now shown that, unexpectedly, there is a strong relationship between pH measurements of the tongue biofilm in individuals and their oral malodour status. The tongue biofilm (and the fluid material that surrounds the microbial cells) is sufficiently different from saliva to be considered as a separate sample site, one that will best reflect malodour processes. It is relatively easy to sample tongue biofilm oneself and it is easy to determine the pH of such a sample for example by placing it into distilled water with a pH indicator.

An inexpensive method whereby one could reliably self-assess malodour could be of great commercial importance.

Summary of the Invention

According to one aspect of the invention there is provided a method of determining oral malodour in a subject, especially that malodour caused by oral microbes, the method comprising determining the pH of the tongue biofilm, in which an elevated tongue biofilm pH level (i.e. compared to normal) is indicative of oral malodour.

A tongue biofilm pH of about 6.5 is indicative of little if any oral malodour. A pH level of ≥7 is indicative of moderate malodour, and a level of ≥ 7.3 is indicative of strong malodour. The method of the invention is advantageous in that it can provide an accurate indication of oral malodour whereas methods based on pH measurements of saliva do not sufficiently correlate with oral malodour to be accurate. In particular, the relationship against saliva is not as good as tongue biofilm and the sample itself is much more likely to be prone to interference (e.g. depending on salivary flow rate). Also, the relationship goes the opposite way (the more acid saliva, the worse the malodour). Furthermore, the tongue biofilm is the site where malodour is generated not saliva (unless it is exceptionally stagnant). The method of the invention may be used to monitor whether an existing treatment for oral malodour is working.

Preferably, the pH of a tongue biofilm sample taken from the subject is determined. Alternatively, it is possible to determine tongue biofilm pH in situ. For example, it is now possible to get fluorescent dyes whose fluorescent behaviour depends on the pH of a solution. A suitable non-toxic dye could then be applied to the tongue biofilm and laser emission spectroscopy or a similar technique could be used to measure epifluorescence and therefore the pH of the tongue. Suitable fluorescent pH indicators include biscarboxyethyl carboxyfluorescein which has previously been used to monitor plasma pH in animals (Russell, D.A. et al Journal Photochemistry Photobiology B. 1995 July, 29(1): 17-22), and carboxyfluoroscein which has been used in the detection of pH gradients in biofilms (Vroom, J. M., et al Applied and Environmental Microbiology August 1999, page 3502- 351 1).

In general, the biofilm can be distinguished from the highly mobile outer saliva coating of the tongue biofilm. Saliva on the tongue is not the same as the biofilm fluid in the biofilm. Saliva on the tongue is rapidly replaced; it is part of the bulk saliva fluid compartment and is separated from the biofilm fluid compartment by diffusion layers. Thus the tongue biofilm and its associated fluid should be considered to be a different compartment and hence a different type of sample. Although molecules exchange between the saliva and tongue biofilm their turnover time is different. The ones in the salivary layer move through quickly (fast residence time), the molecules in the tongue biofilm have slow turnover and slow residence time.

The tongue biofilm sample for pH measurement is preferably taken from the tongue dorsum. A sample or measurement is preferably taken at least 4, more preferably at least 6, centimetres from the tip of the tongue. The sample may be taken from an area of 0.1 - 20cm², preferably 1-10cm² , more preferably 2-10cm² of tongue surface.

After collection, the sample may be mixed with water, preferably distilled sterile water, prior to, or simultaneously with, determination of the sample pH. Alternatively, the sample may be contacted directly with a pH-indicating agent which may itself be in aqueous solution. Mixing may be facilitated, for example by vortexing.

The determination of the pH of the sample is preferably conducted within a few minutes of collection. Most preferably, the pH of the sample is determined within one minute of collection.

Suitable methods for assessing the pH of the sample include chemical and non-chemical methods such as electronic methods. Suitable methods for assessing the pH of the sample chemically include methods in which the pH of the sample is determined by contact with a chemical reagent. The chemical reagent should be suitable for measuring pH values within the range pH 6-8. Suitable chemical reagents include Methyl red, Bromocresol purple, Chlorphenol red, Bromophenol blue, /j-Nitrophenol, Azolitmin, Phenol red, Neutral red, Rosolic acid, Cresol red, α-Naphtholphthalein, Tropeolin OOO, Thymol blue, Bromocresol Blue, Brilliant yellow, metacresol purple, paranitrophenol, cochineal, BDH Universal, and litmus. The chemical reagent may be impregnated in a suitable substrate such as paper e.g. as litmus paper, or universal pH paper. Preferably, the chemical reagent undergoes a visible colour change indicating pH values within the range pH 6-8 to a user.

Suitable methods for assessing the pH of tongue biofilm samples by electronic methods include the use of pH meters, probes, ion-sensitive field effect transistors and metal or glass-electrode-based apparatus.

According to another aspect of the invention there is provided apparatus for determining oral malodour in a subject, particularly caused by oral microbes, the apparatus comprising means for measuring the tongue biofilm pH of the subject.

Preferably, the apparatus includes means for taking a tongue biofilm sample. The means for taking a tongue biofilm sample may be in the form of a hygienic brush or a scraper. Where a brush is used it is preferably relatively soft. Preferably the apparatus includes means for measuring the pH of a tongue biofilm sample. The means for measuring the pH of the sample may embody the use of chemical or electronic methods of determining pH as described above.

Such apparatus is advantageous in that it can be provided in a format which is suitable for use by ordinary consumers. For example, the means for taking a sample of tongue biofilm may be a simple brush or scraper, and the means for measuring the pH of the sample may be a simple container including sterile distilled water into which the sample is placed, and a chemical reagent for determining the pH of the sample. Apparatus for determining oral malodour has not been available in such a format previously.

According to another aspect of the invention there is provided a kit for determining oral malodour, especially oral malodour due to oral microbes, the kit comprising means for taking a tongue biofilm sample from a subject, means for determining the pH of the sample, and instructions for use of the said means.

The instructions may include a score card correlating pH values determined by the means for determining the pH of the sample with degrees of oral malodour. The score card may include for example colour samples relating to standard tongue biofilm pH values as indicated by the colour of a pH-indicator chemical reagent and corresponding indicated oral malodour levels.

According to another aspect of the invention there is provided a method of treating oral malodour in a subject, especially oral malodour due to oral microbes, the method comprising determining that the subject has oral malodour and treating the subject with an oral malodour treatment agent or apparatus. The determination of oral malodour may be done by a method in accordance with the first aspect of the invention.

Preferably, the subject is a human subject.

A variety of oral malodour treatment agents may be used. Suitable chemical agents include chlorhexidine gluconate, proteases such as papain, zinc or copper-containing compounds, triclosan, CPC, active plant extracts, peroxides, ClO₂ and oxidising agents that produce or induce reactive oxygen species (ROS) including superoxide, singlet oxygen and hydroxyl radicals (hydroxyl anion). Further agents may include a microemulsion that can penetrate into biofilm and slowly release protons; esters of organic acids which can be hydrolysed in the tongue biofilm by microbial esterases; and carboxymethylcellulose or other ion-exchange resin (in its fully protonated form) which can be made into very small beads that could locate into the biofilm. The oral malodour treatment agent may work on the basis of affecting pH homeostasis or encouraging the biofilm to one that is more acid.

The oral malodour treatment agent may be provided in the form of a mouthwash, toothpaste, lozenge, oral spray or film.

According to a further aspect of the invention there is provided a method of identifying compounds which are useful in the treatment of oral malodour, the method comprising providing a subject with oral malodour, or a model of such a subject, treating the subject with a test compound, and determining the effect of the test compound on the oral malodour by determining the effect of treatment with the compound on the pH of the tongue biofilm of the subject as generally described above.

The subject may be a human or other animal. Where a model is used it may be a physical in vitro perfusion model of tongue biofilm using a real inoculum, or it may use a pure or mixed culture of volatile sulphur compound producing species, to establish a tongue biofilm-like ecological mix (a microcosm) that can simulate the pH behaviour of a real human tongue biofilm in situ.

According to a further aspect of the invention there is provided a method of treating oral malodour in a subject having oral malodour, the method comprising treating the subject with a compound which lowers the tongue biofilm pH of the subject.

According to another aspect of the invention there is provided the use of a compound which lowers the tongue biofilm pH of a subject in the preparation of a medicament for use in the treatment of oral malodour. A suitable compound may be in the form of a microemulsion that can penetrate into the tongue biofilm and slowly release protons, thus lowering the tongue biofilm pH. Alternatively, the compound may comprise esters of organic acids which can be hydrolysed in the tongue biofilm by existing microbial esterases again to lower the tongue biofilm pH. Brief description of the drawings

Methods and apparatus in accordance with the invention will now be described, by way of example only, with reference to the further accompanying drawings, Figures 3 to 6 in which:

Figure 3 is a graph which shows the relationship between organoleptic score and pH of the tongue biofϊlm as determined by methods in accordance with the invention;

Figure 4 is a graph which shows the relationship between log Halimeter readings and pH of tongue biofϊlm as determined by methods in accordance with the invention;

Figure 5 is a graph which illustrates the results of experiments in determining the correlation between pH and amount of sample as determined by optical density of tongue biofilm samples; and

Figure 6 is a graph which illustrates the effect of time from sampling on the pH of tongue biofilm samples.

Specific disclosure

Determination of oral malodour in human subjects

1. Human subjects After preliminary work in which it was indicated that there was a moderately strong relationship between the pH of tongue biofilm and oral malodour, further studies were conducted to look at this relationship.

A group of 20 volunteers (8 male and 12 female) from the University of the West of England, Bristol participated in this investigation. All were healthy adults, initial age between 18 and 60 years (mean age male= 39.08, mean age female=35.9), dentate and in good dental health. Subjects with gingivitis or periodontitis were excluded as were subjects who had taken antibiotics within the previous month of the study. Also subjects with diabetes mellitus, bronchitis, tonsillitis, sinusitis or other conditions that may contribute to oral malodour were excluded. The volunteers were advised not to wear any scented products on assessment days, not to consume highly seasoned food or food associated with oral malodour (e.g. garlic) on the day prior to the study and during the study day. The volunteers were instructed not to consume alcohol for 24 hours before the study. They were advised to keep their normal oral hygiene routine but not use oral mouthwashes or medicated lozenges within a week prior to the study. Moreover, the subjects were non- smokers and asked not to eat, drink, suck sweets or smoke at least one hour prior to the assessment. All subjects gave written consent to volunteer, and all studies concerning subject volunteers received ethical approval from the University's Ethical Approval Board. 2. Estimation of oral malodour

2.1 The organoleptic method

Each volunteer had the intensity of their mouth odour ranked by a trained odour judge using the standard organoleptic scale of 0 to 5 as outlined by Rosenberg and McCulloch (Rosenberg, M., McCulloch, C. A. (1992). Measurement of oral malodour: Current methods and future prospects. J Periodontal 63: 776-782. Greenman, J., Duffield, J., Spencer, P., Rosenberg, M., Corry, D., Saad, S., Lenton., P., Majerus, G., Nachnani, S., El-Maaytah, M. (2004). Study on the Organoleptic Intensity Scale for Measuring Oral Malodor. J Dent Res 83 (1): 81-85.) but modified in terms of odour descriptives according to Greenman et al, (Greenman, J. et al J. Dent Res. 83(1) 81-85, 2004; 2005), (0 = no odour, 1 = barely noticeable, 2 = slight odour, 3 = moderate odour, 4 = strong odour, 5 = extremely strong odour, close to saturation). The odour judge had previously demonstrated the ability to determine odours and their intensities (Hartley, G., El-Maaytah, M. A., Mckenzie, C, Greenman, J. (1996a). The tongue microbiota of low odour and malodorous individuals. Microbial Ecology in Health and Disease 9: 215-223.). The judge was also instructed to refrain from drinking coffee, smoking or wearing scented personal care products prior to the examination (Rosenberg, M., Kulkarni, G. V., Bosy, A., McCulloch, C.A.(1991). Reproductibility and sensitivity of oral malodour measurements with a portable sulphide monitor. J Dent Res 70: 1436-1440). The judge was not a smoker. Each volunteer was comfortably sat on a chair and the judge explained the procedure of "sniffing" from the oral cavity. Each subject was instructed to close his/her mouth for 2 minutes and to breathe through the nose. After that time, upon instruction, the volunteer opened his/her mouth and the gas was "sniffed" from a distance of 2 to 3 cm (nose to mouth). A score on the 0-5 scale was then recorded for each subject.

2.2 Halimeter measurements After the assessment of the oral malodour using the method as described in 2.1, the breath of each subject was assessed using a Halimeter (Rosenberg et al., 1991) which uses a sulphide sensitive voltametric sensor. This instrument is based on a liquid electro-chemical cell, through which the gas sample passes at a constant flow rate. The monitor was zeroed at ambient air and measurements performed by inserting a short length of Teflon (trade mark) tube connected to a drinking straw in the mouth. A plastic sleeve was included in order to ensure consistent straw placement in the oral cavity. The subject was comfortably sat on a chair and instructed to close his/her mouth for 2 minutes and breathe through the nose only. Following this period, the straw with the sleeve was inserted in the oral cavity and samples removed by gas suction through the Halimeter. The results were recorded as peak parts per billion of sulphide equivalents. Two readings were taken and an average value was calculated.

3. Sampling of saliva

Unstimulated saliva was collected from each individual (2ml) in a universal bottle and the pH was immediately measured within 1 minute of collection using a Pye Unicam model 290 pH meter and electrode. 4. Sampling of tongue biofilm

Samples were taken by the volunteers themselves from the dorsum of the tongue at least 7 cm from the tip, as far back as was possible without inducing a gagging response using a sterile toothbrush (Wisdom, Haventill, UK) medium soft nylon bristles, head area - lcm²). The toothbrush was used to sample the whole upper surface of the tongue - typically an area of about 15-20 cm² and the samples were processed immediately into 10 ml of sterile distilled water, vortexed for 30 seconds and the pH measured within 1 minute of collection.

5. Results The results of the experiments described above are illustrated in the accompanying Figures 3 and 4. It is apparent from those figures, from which it can be seen that R² = 0.7489;P< 0.0001 and R²= 0.8619; PO.0001 respectively, that there is a clear relationship between tongue biofilm pH and malodour parameters whether measured by organoleptic score or by Halimeter.

In contrast there was no such clear relationship between oral malodour and salivary pH when comparing the results of these experiments with those represented by Figures 1 and 2.

6. Further studies

Further studies in relation to oral malodor were conducted with four individuals. The methods used were as described above (2 Estimation of oral malodor; 2.1 The organoleptic method; Halimeter measurements; and 4 Sampling of tongue biofilm). The results are shown below.

It can be seen that there is a correlation between the pH of the tongue biofilm and oral malodour as determined by the organoleptic and halimeter scores.

7. Determination of the effect of dilution on the pH measurement of tongue biofilm samples Figure 5 shows how the pH of a tongue biofilm sample taken as described above changes as the sample is diluted. OD₅₄₀ was measured using a spectrophotometer (Cecil Instruments, UK). The more dilute the sample, the more the pH reflects that of the diluent (distilled water). The data suggest that a sample of OD₅₄₀ of >0.5 is desirable. Samples of less than this thickness or density may not give meaningful readings. For example, a tongue scraper can remove sufficient biofilm material into a volume of 2.0 ml distilled water to normally give an OD₅₄₀ above 1.0 OD units from about 10 cm² area of tongue surface. 8. Determination of the effect of delay in pH measurement of tongue biofilm samples

The data presented in Figure 6 shows the effects of leaving tongue biofilm or saliva samples taken as described above for various time periods at room temperature, as may be done by a consumer, on their pH measurement. It can be seen that the pH of saliva starts to rise quickly, but then flattens out after an hour. Conversely, the pH of the tongue biofilm starts to fall and flattens out after the first hour.

The data from Figure 6 suggests that pH readings of tongue biofilm samples should be taken as soon as possible after removing the sample, preferably within the first minute.

9. Treatment of oral malodour and monitoring of improvements in oral malodour

In another experiment, a single volunteer was assessed for malodour and a sample of tongue biofilm taken and added to 2.0 ml of distilled water. The biofilm sample was taken by a sterile brush removing biofilm from as much of the tongue surface as possible (probably around 15-20cm²). This gave a sample with an ODs_4O of 1.4.

The procedure for collecting tongue biofilm by scraping to get samples also served to clean the tongue, and to see what effect this would have on subsequent malodour parameters as measured by organoleptic score and Halimeter. A further sampling and cleaning procedure was repeated twice although the sample OD's achieved on the second and third occasions (0.9 and 0.4 respectively) were less than that obtained initially. The results are presented in Table 1.

Table 1 Breath monitoring before and after cleaning procedure to collect tongue biofilm sample. Salivary pH of this volunteer was 6.58 throughout.

The results of this experiment, which indicate that oral malodour was reduced as indicated by the three parameters measured, show that the relationship between pH measurements of the tongue biofilm and malodour parameters will hold in line with the improvement of malodour by treatment, (i.e. it is an immediate or instantaneous measurement of the malodour level at the time of sampling and through transition).

10. Method for determining oral malodour In one embodiment, a subject can determine oral malodour by taking a sample of tongue biofilm using a sterile brush, intimately mixing the sample with 1-10 mL sterile distilled water in a sterile container, and determining the pH of the sample using 1 drop of 0.1 % aqueous solution per 10ml water Bromophenol blue. The thus determined sample pH is then compared against a set of standard pH values indicating a range of degrees of oral malodour represented on a score card. For example a pH value of ≥7 is indicative of moderate malodour.

11. Apparatus for determining oral malodour In one embodiment, apparatus for determining oral malodour comprises a hygienic brush for taking a tongue biofϊlm sample, a container containing 10 mL sterile distilled water, a pH indicator such as Bromophenol Blue, and a colour card indicating a range of degrees of oral malodour corresponding to the determined pH for the tongue biofϊlm sample.

A suitable kit for personal use may also include instructions for use of the apparatus and reagents.

12. Method for treatment of oral malodour

In one embodiment, a method for treatment of oral malodour comprises first determining that a subject has oral malodour by a method in accordance with the present invention, and then treating with a suitable oral malodour treatment agent, such as chlorhexidine gluconate in a 0.12% solution. Such a treatment agent may be in a kit in accordance with the invention as described above.

Claims

1. A method of determining oral malodour in a subject, the method comprising determining the pH level of the tongue biofilm of the subject, and in which an elevated pH level is indicative of oral malodour.

2. A method according to claim 1 in which a tongue biofilm pH level of about 6.5 is indicative of little or any oral malodour.

3. A method according to claim 1 or 2 in which a tongue biofilm pH level of above about 7 is indicative of moderate oral malodour.

4. A method according to claim 1, 2 or 3 in which a tongue biofilm pH level of > 7.3 is indicative of strong malodour.

5. A method of determining oral malodour according to any preceding claim in which the pH level of the tongue biofilm is assessed in situ.

6. A method according to any preceding claim in which the pH level of the tongue biofilm of the upper surface of the tongue is determined.

7. A method according to claim 6 in which the pH level of the tongue biofilm of the rear of the tongue is determined.

8. A method according to any one of claims 1 to 7 in which the pH of the tongue biofϊlm is determined by taking a sample of the tongue biofilm and determining the pH of that sample.

9. A method according to claim 8 in which the sample is taken from the upper surface of the tongue.

10. A method according to claim 9 in which the sample is taken from the rear upper surface of the tongue.

11. A method according to claim 8 or 9 in which a sample or measurement is taken at least 4 centimetres from the tip of the tongue.

12. A method according to claim 11 in which the sample or measurement is taken at least 6 centimetres from the tip of the tongue.

13. A method according to any one of claims 5 to 12 in which the sample or measurement is taken from an area of 0.1 -20cm² of tongue surface.

14. A method according to claim 13 in which the sample or measurement is taken from an area of 1-15cm² of tongue surface.

15. A method according to claim 14 in which the sample or measurement is taken from an area of 2- 10cm² of tongue surface.

16. A method according to any one of claims 8 to 15 in which the sample is taken using a hygienic device.

17. A method according to any one of claims 8 to 16 in which the sample is placed in a solvent.

18. A method according to any one of claims 8 to 17 in which the OD₅₄₀ of the sample is >0.5.

19. A method according to claim 17 in which the solvent is water.

20. A method according to claim 19 in which the water is distilled.

21. A method according to claim 17, 18 or 19 in which the pH of the sample is determined after the sample is placed in the solvent.

22. A method according to any one of claims 5 to 21 in which the pH is determined by contacting the sample with a chemical reagent.

23. A method according to claim 22 in which the chemical reagent undergoes a visible colour change indicating a tongue biofilm pH value to a user.

24. A method according to claim 23 in which the chemical reagent is selected from Methyl red, Bromocresol purple, Chlorphenol red, Bromophenol blue, p- Nitrophenol, Azolitmin, Phenol red, Neutral red, Rosolic acid, Cresol red, α- Naphtholphthalein, Tropeolin OOO, Thymol blue, Bromocresol Blue, Brilliant yellow, metacresol purple, paranitrophenol, cochineal, BDH Universal and litmus.

25. A method according to any one of claims 1 to 21 in which the pH of the tongue biofilm or samples is determined by non-chemical means.

26. A method according to claim 25 in which the pH is determined by electronic means.

27. A method according to claim 26 in which the electronic means includes a metal or glass pH electrode or an ion-sensitive field effect transistor.

28. A method according to any one of claims 8 to 27 in which the pH of the sample is determined within one minute of collection.

29. Apparatus for determining oral malodour in a subject, the apparatus comprising means for determining the pH of the tongue biofilm of the subject.

30. Apparatus according to claim 29 comprising means for taking a tongue biofilm sample.

31. Apparatus according to claim 30, comprising means for assessing the pH of the sample.

32. Apparatus according to any one of claims 29 to 31 in which the means for taking a tongue biofilm sample comprises a scraper or brush.

33. Apparatus according to any one of claims 29 to 32 in which the means for assessing the pH of the sample comprises a pH meter or probe, or chemical reagent.

34. Apparatus according to any one of claims 29 to 33 including means for displaying tongue biofilm pH level information and/or oral malodour information to a user.

35. A kit for assessing oral malodour for use by a subject, the kit comprising means for taking a tongue biofilm sample from the subject, means for assessing the pH level of the tongue biofilm sample, and means for indicating the presence or absence or degree of oral malodour on the basis of the thus determined pH level.

36. A kit according to claim 35, including oral malodour treatment agent and/or apparatus.

37. A kit according to claim 36 in which the oral malodour treatment agent is selected from chlorhexidine gluconate, proteases, zinc or copper-containing compounds and triclosan, CPC, active plant extracts, peroxides, ClO₂ and oxidising agents that produce or induce reactive oxygen species (ROS) including superoxide, singlet oxygen and hydroxyl radicals (hydroxyl anion).

38. A kit according to claim 36 or 37 in which the oral malodour treatment apparatus is a tongue scraper and/or brush.

39. A method for treating oral malodour in a subject, the method comprising determining that the subject has oral malodour, treating the oral malodour, and determining the effectiveness of the treatment by determining whether or not the subject still has oral malodour by a method according to any one of claims 1-28 and continuing treatment until effective.

40. A method according to claim 39 in which the effectiveness of the treatment is determined on a regular basis.

41. A method of treating oral malodour according to claim 39 or 40 comprising determining that the subject has oral malodour by a method according to any one of claims 1 to 28 or by use of apparatus according to any one of claims 29 to 34, in which the subject having oral malodour is then treated with an oral malodour treatment agent and/or apparatus.

42. A method according to any one of claims 39-41 in which the oral malodour treatment agent is selected from chlorhexidine gluconate, proteases, and zinc or copper-containing compounds, triclosan, CPC, active plant extracts, peroxides, ClO₂ and oxidising agents that produce or induce reactive oxygen species (ROS) including superoxide, singlet oxygen and hydroxyl radicals (hydroxyl anion).

43. A method according to claim 42 in which the oral malodour treatment agent is in the form of a mouthwash, toothpaste, lozenge, oral spray or film.

44. A method according to claim 42 in which the oral treatment apparatus is a tongue brush and/or scraper.

45. A method of identifying compounds which are useful in the treatment of oral malodour, the method comprising providing a subject or a model thereof with oral malodour, treating the subject with a test compound, and determining the effect of the test compound on the oral malodour by determining the effect of treatment with the compound on the pH of the tongue biofilm of the subject by a method according to any one of claims 1 to 28.

46. A method according to claim 45 in which the subject is a human or other animal.

47. A method according to claim 45 in which the model is an in vitro model of the tongue biofilm.

48. A method of treating oral malodour in a subject having oral malodour, the method comprising treating the subject with a compound which lowers the tongue biofilm pH of the subject.

49. Use of a compound which lowers the tongue biofilm pH of a subject in the preparation of a medicament for use in the treatment of oral malodour.

50. Use a compound which lowers the tongue biofilm pH of a subject in the treatment of oral malodour.