FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2006
PROVISIONAL SPECIFICATION
(See Section 10 and Rule 13)
A METHOD FOR MEASURING ORAL MALODOUR
HINDUSTAN UNILEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification describes the invention

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Technical Field
This invention relates to a method of measuring oral malodour. In particular, it relates to a method for measuring oral malodour that is suitable for field use.
The invention has been developed primarily for a method for measuring oral malodour suitable for field studies or demonstration and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.
Background and Prior Art
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Oral malodour or halitosis, commonly known as "bad breath" is a serious and widespread malady predominantly caused by volatile sulphur compounds produced by gram-negative bacteria residing in oral cavity. Maintaining oral hygiene is an effective remedy against bad breath. One of the simplest ways of maintaining oral hygiene is frequent brushing with toothpastes containing antibacterial ingredients.
Capability of measuring oral malodour is crucial in diagnosing oral malodour problem as well as in evaluating and demonstrating efficacy of treatment or oral hygiene products such as toothpastes and mouthwash in reducing oral malodour.
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Oral malodour is typically evaluated qualitatively by means of organoleptic scores assigned by examiners. JP60004859 (Lion Corp) discloses use of a test-piece made of water-absorbable material to which saliva is transferred for testing. However, this method requires trained and skilled personnel to evaluate organoleptic scores and the results are subjective.
Instrumental methods of analysis reduce the problems associated with subjective organoleptic testing. One of the instrumental methods for measuring oral malodour is based on quantitative evaluation of volatile sulphur compounds (hydrogen sulphide, methyl mercaptan and dimethyl sulphide) which are known to be related to oral malodour in breath, by using gas chromatography. For example, Halimeter®, InterScan Corp, USA,is one of the most common methods used in clinical study to measure VSC (Volatile sulphur compounds). It is a portable sulphide meter that uses an electrochemical, voltametric sensor that generates a signal when exposed to sulfide and mercaptans and measures the concentration of H2S gas in ppb. However, some of the components of malodour such as low molecular weight amines can not be measured by this method. Further, special tools are required for collecting saliva samples from exhaled air.
Another method for detecting oral malodour is by making use of ninhydrin colorimetric reaction which allows measuring amino acids and low molecular weight amines. Iwanicka-Grzegorek et al (Oral Diseases, 11 (1) p 37-39, 2005) have disclosed that salivary amine levels correlate well with volatile sulphur compounds and organoleptic scores. This method requires use of spectrophotometer for analysing colour changes.
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The methods described above are relatively expensive and require use of cumbersome laboratory instruments which limit their use in field studies or demonstrations. Therefore, researchers have tried to develop test methods suitable for field studies and demonstrations.
JP62115364 (Seisui Chemical Comapany, 1987)) describes use of test papers impregnated with a reagent which changes colour by reacting with mercaptan (a component of malodour) and a dye that does not change colour when contacted with saliva. Presence of non-reactive dye is said to help in easier identification of colour change. The test paper is contacted with varying levels of mercaptan concentration to prepare a colour calibration chart. JP11248701 (Kyoriso Yakuhin Kogyo, 1999) describes test papers or carrier sheets having enhanced storage stability. These taste papers are made with a material comprising siliceous material and are impregnated with a reagent which changes colour when reacted with mercaptan. Both the patents described above require transfer and contact of saliva from mouth of subject onto the test paper impregnated with reagent which is classified as irritant, necessitating use of special means to collect and transfer saliva, which causes difficulties particularly in field-use or in demonstrations. These patents do not disclose a convenient, safe and hygienic method for collecting and handling saliva samples.
Thus none of the prior art teaches a method for detection for oral malodour in an objective manner suitable for field studies or demonstrations whilst providing a convenient, safe and hygienic collection and handling of saliva samples.
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It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
Another object of the present invention is to provide a method for measuring oral malodour suitable for field use and demonstrations.
Yet another object of the present invention is to provide a method for measuring oral malodour that allows safe, convenient and hygienic collection and handling of saliva samples.
Yet another object of the present invention is to provide a method for measuring oral malodour suitable for evaluating and/or demonstrating efficacy of treatment or oral hygiene products such as toothpastes and mouthwash in reducing oral malodour.
Summary of the invention
According to the present invention, there is provided a method for measuring oral malodour comprising the steps of:
(a) collecting saliva from a subject using a saliva-collection unit, said unit including a licking section made of water-absorbable material to which the subject transfers the saliva;
(b) contacting said licking section with a reagent for
a predetermined contact time; said reagent capable
of changing colour by reacting with malodour-
indicating component in saliva;
(c) allowing a pre-determined post-contact time for
colour change;
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(d) comparing colour of the licking section with a precalibrated colour chart for measuring oral malodour.
According to a preferred aspect, said water-absorbable material is selected from paper, cotton or non-woven fabric.
According to a preferred aspect, the saliva-collection unit includes a holder attached to the licking section.
According to a further preferred aspect, surface of said holder is made of substantially water-impermeable material.
According to a particularly preferred aspect, said reagent is ninhydrin.
Detailed description of the invention
According to the present invention, there is provided a
method for measuring oral malodour comprising the steps of: (a) collecting saliva from a subject using a saliva-
collection unit, said unit including a licking section made of water-absorbable material to which the subject transfers the saliva;
(b) contacting said licking section with a reagent for
a pre-determined contact time; said reagent
capable of changing colour by reacting with
malodour-indicating component in saliva;
(c) allowing a pre-determined post-contact time for colour change;
(d) comparing colour of the licking section with a precalibrated colour chart for measuring oral malodour.
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It is preferred that predetermined contact time is from 2 to 300 seconds more preferably from 5 to 100 seconds, most preferably from 5-50 seconds.
It is preferred that predetermined post-contact time is from 2 to 600 seconds, more preferably from 5 to 300 seconds, most preferably from 10-200 seconds.
The water-absorbable material forming the licking section is preferably selected from paper, cotton or non-woven fabric. The licking section is preferably made from a food-grade material. It is preferable that the licking section is a flat sheet.
According to a preferred aspect, the saliva-collection unit includes a holder attached to the licking section. It is particularly preferred that the surface of said holder is made of substantially water-impermeable material. Preferably, the holder is a plastic laminate sheet.
According to a preferred aspect, the saliva-collection unit is made from an elongate strip of a water-absorbable material. The strip is partly laminated using a substantially water impermeable lamination material with the laminated portion defining the holder and the un-laminated portion defining the licking section. Preferably the area and the shape of the licking section is chosen so that a subject can conveniently transfer the saliva by licking. The shape of the licking section can be square, rectangular or circular. The area of the licking section is between 1-20, preferably 2-15, and more preferably between 4-10 cm2.
According to another preferred aspectm the saliva-collection unit is in form of ear-bud which are commercially available
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for cleaning ears, with the cotton bud forming the licking section and the plastic handle forming the holder.
Any reagent capable of changing colour by reacting with malodour-indicating component in saliva can be used. Preferably the reagent is selected from ninhydrin, bis (4-diethyl aminophenyl)methanol or bis(4-diethyl aminophenyl)methanol. Ninhydrin is capable of reacting with malodour-indicating component including amino acids and low molecular weight amines, whereas the reagents bis(4-diethyl aminophenyl)methanol and bis(4-diethyl aminophenyl)methanol are capable of reacting with a malodour-indicating component including mercaptan. It is particularly preferred that the reagent is ninhydrin.
In one of the preferred aspects, the licking section is subjected to drying by a heating means during the post-contact time. Any convenient heating means such as electrical strip heater and hair dryer can be used. Preferably the heating means is a hair dryer.
The invention and its method of use will now be described in further detail with the help of the following figures, which form non-limiting embodiments of the invention.
Brief Description of the drawings
Figure 1 is a schematic view of one of the preferred embodiments of a saliva collection-unit according to the present invention.
Figure 2 is a schematic view of another preferred embodiment of a saliva collection unit according to the present invention.
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Figure 3 is a precalibrated colour chart for visual identification and matching of colour for estimation of malodour, which is particularly suitable in field studies.
Detailed Description of the drawings
Preferred embodiments of saliva-collection unit according to the present invention will now be described with reference to Figures 1 and 2.
Referring to Figure 1, a preferred embodiment of a saliva collection unit is made of a strip of filter paper (whatman no 41) of length of about 10 cm and width of about 2 cm. About 8 cm length of the filter paper is laminated or plastic-coated to form the holder (H) , surface of which is substantially water impenetrable. The un-laminated portion of the strip forms the licking section (L) . It is possible to bend the saliva collection unit such that the holder is at an angle of from about 10 to about 90° with the licking section to facilitate the saliva transfer by the subject. In use, the subject holds the holder (H) and transfers the saliva by to the licking section(L). Saliva is conveniently transferred by licking the licking section (L).
Now referring to Figure 2, another preferred embodiment of a saliva collection unit comprises a holder (H) attached to a licking section (L). The holder is made of a plastic rod of diameter of about 0.2 cm and length of about 6 cm. The licking section L comprises a strip of filter paper (Whatman no 41), glued to the holder forming a lollipop like shape. In use, the subject holds the holder (H) and transfers the saliva by to the licking section (L). Saliva is conveniently transferred by licking the licking section (L).
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Now referring to Figure 3, which is a precalibrated colour chart according to the present invention which can be used for visual identification and matching of colour for estimation of malodour. The chart is based on colour developed by the reaction of different concentrations of methionine with ninhydrin. Lighter shades of colour purple indicate low concentration of methionine, which corresponds to less oral malodour. On the other hand the darker shades indicate higher concentration of methionine, which corresponds to less oral malodour. The precalibrated colour chart can be conveniently carried in the field studies for measurement of oral malodour.
Examples
The invention will now be demonstrated with examples. The examples are by way of illustration only and do not limit the scope of the invention in any manner.
Materials and Methods
1.5% w/v Ninhydrin (sourced from Hi Media, India) in acetone was used. The ninhydrin was made freshly for every assay.
Methionine (SRL Labs, India) was used at concentration between 10—1000 ppm
Whatman's filter paper No 41 was partly laminated and cut into strips 10-cm x2 cm forming a saliva collection unit with laminated portion of about 8 cm x 2 cm area forming the holder (H) attached to a licking section (L) of dimension 2 cm x 2 cm, as shown in Figure 1. Strip was laminated using a food grade polypropylene material. The lamination material
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was subjected to the Global Migration Test using two solvents namely Alcohol and Distilled Water. The extraction value was within the overall limit of 10 mg/dm2 as specified for plastics in contact with foodstuffs, pharmaceuticals and drinking water, indicating that the lamination material is food-grade and accidental contact with mouth of the subject is not likely to be harmful. The saliva collection unit was autoclaved before use.
Method for Colour measurement
The basis of investigation of optical properties of an object is recording the intensity of light reflected from it at many narrow-band wavelength(s) throughout the spectra of visible light (400-700nm), namely, reflectance spectrophotometry. The term reflectance indicates the ratio of the intensity of reflected light to that of incident light. The L*a*b* colour system is a three-dimensional coordinate system with an L*-axis (brightness) and two orthogonal axes representing chromaticity, namely, an a*-axis (red-green) and a b*-axis (yellow-blue). L*a*b* values is most commonly used for quantification of skin colour. The Minolta chromameter was used for quantification of colour formed on the licking section and the L, a and b measurements were taken. The instrument was calibrated with the reference plate before taking the readings.
Halimeter measurements
Halimeter® (Model RH-17K, InterScan) was used for measuring malodour.
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Collection of saliva samples from subjects
The panellists deposited saliva by licking the licking section of the saliva collection unit whilst holding the holder in hand. The panellists held his/her head down for 30 seconds followed by licking for 10 sees.
The weight of saliva deposited on the saliva collection unit was between 45 - 60 mg.
The saliva collection unit allows for safe, convenient and reproducible transfer of saliva by the subjects to the saliva collection unit.
After transfer of saliva, 50 μL of ninhydrin reagent was added on the licking section and blow dried with hot air using a hair dryer (Philips Flex Cool No. 1200 at HOT setting.) for 1 min for the colour to develop. Heating was stopped if the paper turned purple in less than a minute. Care was taken to avoid over heating as it caused the paper to turn brown, and thus ineffective for oral malodour detection.
Standardization of the ninhydrin assay for saliva samples
Ninhydrin reagent reacts with amino acids to give a purple coloured compound. Volatile sulphur compounds are formed from sulphur containing amino acids. Methionine, a sulphur containing amino acid, was used as the amino acid for standardization. The predetermined amount of methionine at various concentrations was pipetted on to a filter paper. The filter paper was dipped in ninhydrin reagent and dried by means of a drier. The change in colour was measured using chromameter. The measured values of L were found to vary
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linearly with methionine concentration over a range of 10-100 ppm as well as for 100-1000 ppm. Observed correlation between the measured value of L using chromameter and the methionine concentration (M) in ppm is given in Table 1.
Table 1: Correlation between the measured value of L using chromameter and the methionine concentration (M)

Range of methionine concentration (ppm) Relation between measured value of L and methionine concentration (M)in ppm Correlation coefficient
10-100 L = -1.8 + 85.9 M 0.96
100-1000 L = -2.9 + 69.4 M 0.91
A precalibrated colour chart comprising the colour developed by the reaction of different concentrations of methionine with ninhydrin was prepared. The colour developed by saliva samples before brushing were in the range of 500-1000 ppm of methionine. The precalibrated colour chart is depicted in Figure 3. Lighter shades of purple, corresponding to high value of L, indicate low concentration of methionine, which corresponds to less oral malodour. On the other hand the darker shades, corresponding to low value of L, indicate higher concentration of methionine, which corresponds to less oral malodour. It was found that the extent of methionine concentration could be easily and correctly estimated by comparing the colour developed on the filter paper with the precalibrated colour chart and visually choosing the matching colour.
The precalibrated colour chart can be conveniently carried in the field studies for measurement of oral malodour.
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Correlation between halimeter readings and method of the invention
Zinc Sulfate was used as the active for malodour reduction. A panel of 18 volunteers who used the toothpaste containing 0.2 g Zinc Sulfate per 100 g of toothpaste was tested for malodour by halimeter as well as by the method according to the present invention in which the volunteers transferred saliva on the saliva collection unit according to the method described previously. After transferring saliva, the saliva collection units were reacted with ninhydrin and the colour change was measured.
The changes in malodour after 7 days usage of the toothpaste containing zinc sulfate as detected by the halimeter and the method according to the present invention are given in Table 2.
Table 2: Comparison between measurement of L value and halimeter readings (for 18 measurements)

Before brushing (Day 0) Before brushing (Day 7)
Halimeter Reading L value (Chromameter) Halimeter reading L value (Chromameter)
Mean 128.0 61.4 88.5 71.9
Standard deviation 41.9 7.2 34.5 6.1
Standard deviation as % of mean value 32.7 11.7 50.7 8.5
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It is clear that the method according to the present invention (measurements of L value) have less variability as compared to Halimeter measurements. It was found that the extent of oral malodour could be easily and correctly estimated by comparing the colour developed on the licking section of the saliva-collection unit with the precalibrated colour chart and visually choosing the matching colour.
Further, The changes in malodour after 7 days usage of the toothpaste containing zinc sulphate as detected by the halimeter and the method according to the present invention were found to correlate well with the correlation coefficient of ~ 0.7, which is higher than that reported in the prior art of about 0.54 (Iwanicka-Grzegorek et al (Oral Diseases, 11 (1) p 37-39, 2005)).
From the above, it is clear that the method for measuring oral malodour according to the present invention allows quantitative detection of oral malodour in an objective manner suitable for field studies or demonstrations whilst providing a convenient, safe and hygienic collection and handling of saliva samples.
Dated this 8th day of August 2007
HINDUSTAN UNILEVER LIMITED

(S. Venkatramani)
Senior Patents Manager
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