U.S. patent application number 10/776185 was filed with the patent office on 2004-08-19 for methods of judging mouth odor.
This patent application is currently assigned to TAKASAGO INTERNATIONAL CORPORATION. Invention is credited to Hanada, Minoru, Sakurai, Kazutoshi, Sawano, Kiyohito, Yamazaki, Sadahiko.
Application Number | 20040161472 10/776185 |
Document ID | / |
Family ID | 18894832 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040161472 |
Kind Code |
A1 |
Sakurai, Kazutoshi ; et
al. |
August 19, 2004 |
Methods of judging mouth odor
Abstract
convenient methods for judging mouth odor with the use of an
artificial mouth odor prepared by a convenient method which load
any burdens neither on the operator nor on he subjects. Saliva
collected immediately after wake-up is cultured under anaerobic
conditions and head space components of the thus cultured saliva
are analyzed, or anaerobic bacteria contained in the collected
saliva are counted.
Inventors: |
Sakurai, Kazutoshi;
(Kanagawa, JP) ; Yamazaki, Sadahiko; (Kanagawa,
JP) ; Hanada, Minoru; (Kanagawa, JP) ; Sawano,
Kiyohito; (Kanagawa, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
TAKASAGO INTERNATIONAL
CORPORATION
|
Family ID: |
18894832 |
Appl. No.: |
10/776185 |
Filed: |
February 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10776185 |
Feb 12, 2004 |
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10041455 |
Jan 10, 2002 |
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6723501 |
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Current U.S.
Class: |
424/537 |
Current CPC
Class: |
G01N 33/487 20130101;
C12Q 1/04 20130101 |
Class at
Publication: |
424/537 |
International
Class: |
A61K 035/37 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2001 |
JP |
P. 2001-030642 |
Claims
What is claimed is:
1. A method of judging mouth odor characterized by culturing saliva
immediately after wake-up under anaerobic conditions and analyzing
the head space components of the cultured saliva thus obtained.
2. The method of judging mouth odor as claimed in claim 1, wherein
cresol, indole or skatole in the head space components is
detected.
3. A method of judging mouth odor as claimed in claim 1 or 2,
wherein the head space components are adsorbed by a solid phase
microextraction unit and then analyzed.
4. A method of judging mouth odor characterized by counting
anaerobic bacteria contained in saliva immediately after
wake-up.
5. An artificial mouth odor characterized by comprising head space
components of cultured saliva obtained by culturing saliva
immediately after wake-up under anaerobic conditions.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel methods of judging mouth
odor. More particularly, it relates to a method of judging mouth
odor wherein the odor of cultured saliva obtained by collecting
saliva immediately after wake-up and culturing it under anaerobic
conditions is employed as the mouth odor, and another method of
judging mouth odor wherein anaerobic bacteria contained in
collected saliva are counted.
BACKGROUND OF THE INVENTION
[0002] With the recent preference to cleanliness, people worrying
about their own body odor, mouth odor and the like have been
increasing. Mouth odor ranks second among these offensive odors
following body odor. According to questionnaire data, mouth odor is
not so strong in young people but becomes more and more noticeable
with aging.
[0003] In general, mouth odor has a broad meaning including all
offensive smells generating from the mouth, for example, smells
simply generating from smelling foods remaining in the mouth and
smells caused by systemic diseases.
[0004] As major components of mouth odor, hydrogen sulfide, methyl
mercaptane and dimethyl disulfide may be cited. These
sulfur-containing compounds are main factors causative of mouth
odor. It is considered that the strength of mouth odor relate to
these three sulfur-containing compounds. Accordingly, studies have
been made on mouth odor and prevention thereof by using these three
compounds.
[0005] However, hydrogen sulfide is in the form of a gas at
ordinary temperature while an aqueous solution or a solution in a
solvent of methyl mercaptane should be prepared in situ because of
its very low boiling point. Therefore, it takes a lot of trouble to
prepare an artificial mouth odor by using these compounds. Although
various odor-collection methods have been proposed so far to
analyze mouth odor, there are few methods which can be carried out
while loading little burden on subjects.
[0006] To judge mouth odor, it has been a practice to analyze the
mouth odor by sensory tests or gas chromatography. Because of
depending on human olfactory perception, sensory tests suffer from
some problems such as olfactory fatigue, difficulties in numerical
indication and poor reproducibility. Although gas chromatography is
superior in objectivity, troublesome-operations are needed in
collecting and analyzing odor by this method. Moreover, gas
chromatography has an additional problem of being restricted in the
test site.
[0007] To collect mouth odor, use has been recently made of a
portable sulfide monitor and the like by which volatile
sulfur-containing compounds can be perceived in the oral cavity. In
this method, however, a number of burdens (resting quietly before
the measurement, being provided with a plastic tube for feeding
air, breathing through the nose while keeping his/her mouth open,
etc.) are loaded on a subject. Thus, this method is not applicable
to those who cannot breathe through the nose. In this case,
furthermore, trouble some operations should be performed by an
operator.
SUMMARY OF THE INVENTION
[0008] Accordingly, an object of the invention is to provide a
convenience method of judging mouth odor by using an artificial
mouth odor prepared by a convenient method.
[0009] In order to achieve the above object, the inventors have
conducted extensive studies. As a result, they have successfully
found out that mouth odor can be very excellently judged by
culturing saliva immediately after wake-up under anaerobic
conditions and using the head space components of the cultured
saliva thus obtained as an artificial mouth odor without resort to
any troublesome operations to be carried out by the operator for
preparing an artificial mouth odor and without loading any burdens
to the subject; and that the anaerobic bacterial count in the
saliva thus collected correlates to the components detected
therefrom and thus mouth odor can be very excellently judged by
counting the anaerobic bacteria without loading any burdens to the
operator or subject. The invention has been completed based on
these findings.
[0010] Accordingly, the invention relates to a method of judging
mouth odor characterized by culturing saliva immediately after
wake-up under anaerobic conditions and analyzing the head space
components of the cultured saliva thus obtained, and another method
of judging mouth odor characterized by counting anaerobic bacteria
contained in the collected saliva.
BRIEF DESCRIPTION OF THE DRAWING
[0011] FIG. 1 provides gas chromatograms obtained in Example 1-b
which show the relations between the anaerobic bacterial count in
saliva and odor components in the head space components after
culturing.
DETAILED DESCRIPTION OF THE INVENTION
[0012] To collect the saliva to be used in the invention, the
subject may brush his/her teeth before going to bed. However, it is
important to collect the saliva immediately before wake-up without
smoking or rinsing the mouth with water. The reason for collecting
the saliva immediately after wake-up resides in that since the
secretion of saliva has been controlled, there are many anaerobic
bacteria producing volatile compounds causing mouth odor in the
mouth in a stable state.
[0013] The saliva immediately after wake-up may be collected by an
arbitrary method without restriction. For example, the subject may
spit out saliva directly into a culture container. In this method,
it is not necessary to make the subject to rest quietly or to
provide a plastic tube for induction. Namely, the saliva can be
collected without loading any burdens to the subject.
[0014] The saliva thus collected may be cultured under arbitrary
conditions without any restriction. Namely, it may be introduced
into a thermostat maintained at 37 to 40.degree. C. still
preferably to about 37.degree. C. similar to the human body
temperature, and maintained therein for a definite period of time,
for example, from 24 to 72 hours. As the culture period is
prolonged, the compounds causing the mouth odor can be detected in
larger amounts and thus the judgment of the mouth odor can be more
advantageously carried out. The saliva as such may be cultured
under the anaerobic conditions.
[0015] The culture can be carried out in a closed container without
particular restriction (for example, a glass bottle or a plastic
bottle) in which anaerobic conditions can be maintained. In this
case, the head space may be 5 to 10 times by volume as much as the
volume of the saliva, though the invention is not restricted
thereto. It is favorable to use a definite amount of saliva and a
definite volume of the head space.
[0016] The term "anaerobic conditions" as used herein means such
conditions that the culture can be carried out under purging with
an inert gas such as argon or nitrogen so that the saliva is not
brought into contact with oxygen. In case where there is no
analytical instrument nearby, the collected saliva is stored in a
dark and cold place and then the space in the container is purged
with an inert gas followed by the culture.
[0017] During the culture period, anaerobic bacteria grow in the
saliva under the culture and produce volatile compounds causing
mouth odor. Thus the head space is filled up with these compounds.
In the judgment method according to the invention, the volatile
compounds constituting the head space components are collected and
analyzed. Thus, mouth odor is judged with the use of these head
space components as an artificial mouth odor.
[0018] By using the head space components of the cultured saliva as
an artificial mouth odor, mouth odor can be conveniently judged
without any trouble of handling dangerous gaseous hydrogen sulfide
or preparing solutions of standard reagents (methyl mercaptane,
etc.) in situ for preparing an artificial mouth odor as in the
conventional cases. Thus, the burdens on the operator can be
relieved and no burden is loaded on the subject. Although a
conventional artificial mouth odor has a strong smell at the point
of the preparation, it is unstable and undergoes changes within a
short period of time due to the low boiling points of the
constituting compounds. Thus, it cannot sustain the smell at the
preparation. In contrast, the artificial mouth odor to be used in
the invention remains stable with showing little change for 1 to 2
hours when stored at a low temperature after taking out from the
culture container.
[0019] To collect the head space components to be analyzed, it is
adequate to employ a method of adsorbing by a solid phase
microextraction (SPME) unit as a static head space method. This
solid phase microextraction unit (SPME unit) consists of an SPME
holder like a microsyringe and a fiber assembly, which has a fused
silica fiber coated with a liquid phase (polydimethylsiloxane,
styrene divinylbenzene porous polymer, polyethylene glycol, carbon
molecular sieve adsorbent, etc.) at the tip, contained in a syringe
connected to a stainless plunger. As the plunger moves vertically,
the fiber is contained the syringe and exposed outside. To collect
the head space components, the plunger is pushed down and thus the
fiber is exposed to the head space. Then the components are
adsorbed by the fiber. The adsorption may be completed by exposing
the fiber to the head space for 2 to 30 minutes. This SPME unit is
available from SUPELCO Co.
[0020] The head space components thus adsorbed are desorbed from
the fiber liquid phase by heating and then the components contained
therein can be analyzed and detected. By using a gas chromatograph
provided with a mass spectrograph (GM-MS) in the analysis,
substances causing mouth odor can be identified and the strength of
the mouth odor can be judged merely by observing a chromatogram or
the monitor.
[0021] Since the head space components are collected by using the
solid phase microextraction unit, the operator can introduce the
head space components into the analyzer, make them to be adsorbed
therein and then desorbed and analyze them without touching them
with hands or smelling the offensive odor. Thus, the mouth odor can
be judged while loading no burden to the operator. Furthermore, the
solid phase microextraction unit is highly advantageous in that the
head space components can be very conveniently adsorbed and easily
handled.
[0022] It is considered that the above-described three components,
i.e., hydrogen sulfide, methyl mercaptane and dimethyl disulfide
cause mouth odor and the contents of these sulfur-containing
compounds in the saliva relate to the mouth odor strength. In the
present invention, various compounds such as phenol, cresol, indole
and skatole were detected in a large amount, in addition to the
three components as described above, from the head space of the
cultured saliva collected from a subject showing a strong mouth
odor, while only relatively small amounts of phenol and indole and
almost no cresol and skatole were detected from a subject showing a
weak mouth odor.
[0023] Therefore, these components such as phenol, cresol, indole
and skatole are usable in judging mouth odor. As will be shown in
the following Examples, these compounds were detected in large
amounts from the head space components of the cultured saliva of a
subject showing a strong mouth odor but scarcely from the cultured
saliva of another subject showing a mouth odor. Accordingly, use of
these compounds provides an easy and convenient method for judging
mouth odor. These components have never been employed as
indications in judging mouth odor hitherto.
[0024] The present invention further provides a method of judging
mouth odor which comprises counting anaerobic bacteria contained in
cultured saliva collected immediately after wake-up. This method is
based on the fact that the contents of the above-described
components detected by analyzing the head space components of
cultured saliva samples obtained by culturing saliva collected a
number of subjects by the MS-GC method correlate to the anaerobic
bacterial counts in the saliva prior to culturing.
[0025] Mouth odor can be judged by determining the total anaerobic
bacterial count among bacteria in the oral cavity, in particular,
Porphyromonas gingivalis (Pg bacterium) which is seemingly
causative of morbid mouth odor and Fusobacterium nucleatum (Fn
bacterium) which is one of the bacteria commonly occurring in the
oral cavity and seemingly causative of physiological mouth odor.
When the all of the anaerobic bacteria in the saliva such as
Porphyromonas gingivalis (Pg bacterium) which is one of anaerobic
bacteria contained in the saliva and Fusobacterium nucleatum (Fn
bacterium) which is one of the bacteria commonly occurring in the
oral cavity are counted, a great number of bacteria are detected
from the saliva of subjects having strong mouth odor. Thus, it is
intended to apply this fact to the judgment of mouth odor.
[0026] The anaerobic bacteria may be counted by a publicly known
method. For example, the counting may be carried out as follows. A
definite amount of saliva diluted with physiological saline is
spread on an agar plate prepared by adding 2% of agar (DIFCO) and
0.5 .mu.g/ml portions of Hemin (Tokyo Kasei) and Menadione sodium
bisulfite (Sigma) to a GAM liquid medium (Nissui Seiyaku). After
culturing in an aerobic jar at 37 to 40.degree. C. for 24 to 48
hours, the colonies thus formed are counted with a colony counter
or the like. Thus, the bacterial count in the saliva can be
calculated.
[0027] Although the present invention has been described, an
embodiment thereof will be illustrated in greater detail. 2 to 5 ml
of saliva is collected from a subject immediately after wake-up and
introduced into a sterilized test tube of about 50 ml in volume
provided with a plastic screw cap. Then a 2 ml portion of the
saliva is collected into a sterilized test tube (bottle) of 10 to
20 ml in volume provided with a screw cap. The inner space of the
bottle is purged with an inert gas such as nitrogen gas and then
the saliva is cultured in an incubator at 37 to 40.degree. C. for
24 to 72 hours to give cultured saliva. After culturing the saliva,
head space components in the bottle are adsorbed by the fiber part
of a solid phase microextraction unit (SPME) and introduced into a
GC-MS. Then the head space components are analyzed and substances
causing mouth odor are identified from the gas chromatogram or the
monitor, thereby judging the strength of the mouth odor.
[0028] Alternatively, the mouth odor strength is judged by
collecting a portion of saliva immediately after wake-up and
determining the total anaerobic bacterial count. By using such a
method, the operator can easily judge mouth odor without resort to
any troublesome operations in preparing artificial mouth odor or
collecting mouth odor and without smelling any offensive odor.
Also, no burden is loaded on the subjects.
[0029] According to the invention, mouth odor can be conveniently
judged by collecting saliva immediately after wake-up and
determining the bacterial count therein (claim 4) or culturing the
saliva and analyzing the head space components of the cultured
saliva thus obtained (claim 1). Namely, mouth odor can be very
easily judged without resort to any troublesome operations for
preparing an artificial mouth odor as in the conventional cases.
Thus, the burden on the operator can be lessened and no burden is
loaded on the subjects in the judgment of mouth odor. According to
claim 2, an additional novel method of judging mouth odor can be
provided. According to claim 3, furthermore, the head space
components can be collected and analyzed by convenient operations
and thus mouth odor can be judged while loading no burden on the
operator. Moreover, the artificial mouth odor as claimed in claim 5
is closely similar in the composition to natural mouth odor and can
be easily prepared. Therefore, it is easily usable in studies on
mouth odor such as masking of mouth odor.
[0030] Now, the present invention will be described in greater
detail by reference to the following Examples. However, it is to be
understood that the present invention is not construed as being
restricted thereto. Instruments employed:
[0031] GC-MS: manufactured by Agilent Technology (former
Hewlett-Packard) GC: HP-6890 plus MSD: HP-5973
1 Gas chromatography Column: DB-5MS (0.25 mm i.d. .times. 30 m,
0.25 um film, manufactured by J&W) Injection splitless: (1 min)
250.degree. C. Carrier gas: helium 1.5 ml/min Column temp.
35.degree. C. (5 min, hold), rate: 3.5.degree. C./min, 130.degree.
C. and up to 300.degree. C. (15.degree. C./min).
EXAMPLE 1
[0032] a. Preparation of Artificial Mouth Odor
[0033] 3 to 5 ml portions of saliva were collected from male
volunteers aged 20 to 60 immediately after wake-up without rinsing
the mouth with water or smoking and each introduced into a
sterilized 50 cc plastic test tube provided with a screw cap. Then
a 2 ml portion of the saliva was collected into a sterilized 12 ml
glass test tube (bottle) provided with a screw cap. The space in
the bottle was purged with nitrogen and then the bottle was closely
capped. Then the saliva was cultured in an incubator at 37.degree.
C. for 72 hours to give cultured saliva. After culturing the
saliva, head space components in the bottle and the cultured saliva
(referred to each as a panel) were employed as artificial mouth
odors in judging mouth odor.
[0034] b. Judgment of Mouth Odor (Analysis of Components)
[0035] Use was made of an SPME unit (manufactured by SUPELCO). The
fiber part of the SPME unit was exposed to the head space of each
bottle as described above and maintained as such for about 10
minutes to thereby adsorb the head space components. Then the head
space components were analyzed with a GC-MS. After uncapping the
bottles, the samples (panels 1 to 7) were each evaluated in 6
grades by 10 skilled panelists. The degrees of odor strength were
as follows: 1. no odor; 2. very weak odor; 3. somewhat weak odor;
4. weak odor; 5. strong odor; and 6. very strong odor. Table 1
shows the results.
2 TABLE 1 Sample No. Degree of odor strength Panel 1 3.8 Panel 2
4.2 Panel 3 2.8 Panel 4 3.9 Panel 5 4.3 Panel 6 6.0 Panel 7 5.2
[0036] FIG. 1 provides chromatograms of the panels 6 and 3,
evaluated respectively as 6.0 and 2.8 in mouth odor strength,
obtained by the GC-MS analysis.
[0037] As Table 1 and FIG. 1 show, not only hydrogen sulfide,
methyl mercaptane and dimethyl disulfide but also phenol, cresol,
indole and skatole were detected from the head space of the
cultured saliva (panel 6) showing a strong odor. High peaks
assignable to these components are observed. It is therefore
considered that the cultured saliva of a person having strong mouth
odor is rich in these components which cause together strong mouth
odor. In contrast thereto, less components were detected from the
head space components of the cultured saliva (panel 3) showing a
weak odor compared with the panel 6. In particular, peaks
assignable to cresol and skatole are low in this case. Based on
these results, the correlation between the strength of mouth odor
and the existence of phenol, cresol, indole and skatole can be
understood. Therefore, it can be also understood that these
compounds are reasonably usable in judging mouth odor.
EXAMPLE 2
[0038] (Judgment of Mouth Odor (Viable Bacterial Count in
Saliva))
[0039] Anaerobic bacteria contained in the saliva collected
immediately after wake-up as in Example 1-a were counted. By using
the saliva remaining after preparing the artificial mouth odor,
viable bacteria contained therein were counted. An agar medium was
prepared by adding 2% of agar (DIFCO) and 0.5 .mu.g/ml portions of
Hemin (Tokyo Kasei) and Menadione sodium bisulfite (Sigma) to a GAM
liquid medium (Nissui Seiyaku). After sterilizing, the agar medium
was pipetted in 10 ml portions into Petri dishes of 9 cm in
diameter and solidified at room temperature before using.
[0040] The saliva was diluted 5.times.10.sup.5-fold with
physiological saline and further diluted in 2 steps (.times.4). 50
.mu.l portions of the diluted samples of these 3 series were spread
on agar plates with a spreader and cultured in an anaerobic jar
(BBL) at 37.degree. C. for 2 days. The colonies thus formed were
counted with a colony counter and the bacterial count per 1 ml of
the saliva was calculated. Table 2 shows the results. Aerobic
bacteria were also counted by way of reference. Aerobic bacteria
were cultured by using a Mueller Hinton Medium (DIFCO) at
37.degree. C. for 3 days.
3TABLE 2 Bacterial count in saliva (CFU/ml) Panel Anaerobic count
Aerobic count 1 1.39 .times. 10.sup.8 2.98 .times. 10.sup.7 2 8.30
.times. 10.sup.7 4.60 .times. 10.sup.7 3 <1.60 .times. 10.sup.6
<6.00 .times. 10.sup.5 4 3.74 .times. 10.sup.8 8.15 .times.
10.sup.7 5 3.93 .times. 10.sup.8 1.43 .times. 10.sup.8 6 6.72
.times. 10.sup.8 1.67 .times. 10.sup.8 7 1.11 .times. 10.sup.8 5.32
.times. 10.sup.7
[0041] As Tables 1 and 2 show, the anaerobic bacterial count of the
saliva of the panel 3 showing a weak mouth odor immediately after
wake-up is much smaller than that of the panel 6 showing a strong
mouth odor. Therefore, it can be understood that the mouth odor
strength correlates to the anaerobic bacterial count and the
anaerobic bacterial count is thus usable as an indication in
judging mouth odor.
COMPARATIVE EXAMPLE 1
[0042] In accordance with a known method (Kouku Eiseigakkai Zasshi,
vol. 21, No. 1, March, 1979), an artificial mouth odor was prepared
by using hydrogen sulfide, methyl mercaptane and dimethyl
disulfide. An aqueous hydrogen sulfide solution (a) was prepared by
blowing hydrogen sulfide gas into 200 ml of purified water at
15.degree. C. for about 1 hour so as to dissolve 1 g of hydrogen
sulfide. Separately, methyl mercaptane (b) was prepared by weighing
10 g of a 15% aqueous solution of methyl mercaptane soda and
diluting with 100 cc of purified water. 14 ml of (b) was added to
100 ml of (a). Further, 200 mg of dimethyl disulfide was added to
the resultant mixture and thoroughly mixed. The obtained aqueous
solution was diluted 100-fold before using.
[0043] This artificial mouth odor had a very strong smell. When
refrigerated in a capped state, it suffered from no change over
several hours. In case of allowed to stand at room temperature
without capping, however, it failed to sustain the odor immediately
after preparation within several minutes due to the low boiling
points. In contrast thereto, the cultured saliva which had been
taken out from the incubator and stored at room temperature in
Example 1 showed no large change in the odor even after allowing to
stand at room temperature for about 1 to 2 hours.
[0044] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
[0045] This application is based on Japanese patent application No.
2001-30642 filed on Feb. 7, 2001, the entire contents thereof being
hereby incorporated by reference.
* * * * *