JP2003518916A - Instruction method and instruction kit for bad breath odor - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for rapidly assessing the odor of bad breath, comprising the steps of: a) collecting a liquid and / or tissue sample from the oral cavity of a subject; b) β-galactosidase present in the sample. C) measuring the odor of halitosis of the subject by comparing the result obtained in step b) with the corresponding control value.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to methods for assessing the presence and odor level of halitosis. More particularly, β
-A method for measuring halitosis based on evaluation of galactosidase activity is disclosed along with a diagnostic kit used for this method.

BACKGROUND OF THE INVENTION The malodor of the mouth [halitosis, malodor in the mouth (fetor ex ore)] is a common human symptom dating back to the present day. Halitosis is usually generated in the oral cavity itself, generally by the production of offensive odorous gases due to microbial deposition under anaerobic conditions. Bad breath is thought to be primarily caused by the proteolytic activity of Gram-negative organisms. When gram-positive bacteria from the mouth are cultured in the presence of amino acids, a slight odor is assured or odorless is ensured. However, when Gram-negative bacteria are cultured in the presence of amino acids, they are full of malodor.

[0003] One of the practical problems associated with this condition is that self-measurement of malodor in the mouth is not reliable. This leads, on the one hand, to patients suffering from halitosis but not being aware of it, and on the other hand to those worried about this problem, although they do not actually suffer from halitosis. A simple test device is of particular importance because of the difficulties inherent in self-rating mouth malodor. Various test methods have been proposed for measuring associated parameters of halitosis and post-treatment improvement. One of the commonly used means is to measure volatile sulfide levels using electronic equipment [eg Halimeter, Interscan Corp., Chatsworth California] or visible means (eg by precipitation of lead acetate). It is a means.

However, the first technique is economically expensive and the second technique is time consuming. Another test method to measure proteolysis, the BANA test, involves a heating period at elevated temperatures in specialized equipment and requires excessive precision. A conventional patent, US 5,270,174, describes a technique for measuring microbial activity with halitosis. This technique involves swishing the oral cavity with sterile fluid, followed by introduction of sputum into a container containing an indicator of oxygen consumption. This test method is cumbersome and can take a long time to change color.

At the present time, there is no quick, easy way to allow an individual to objectively test the odor degree of their own halitosis. In a considerable social situation,
Such test methods may not be useful in order to avoid disorders or to warn individuals in advance that treatment of a condition requires some therapeutic treatment.
Such test methods may also be of value in the clinical setting, where a rapid objective test of halitosis aids in the assessment of periodontal disease and the measurement of the effects of various treatments.

An object of the present invention is to provide a simple and convenient evaluation method for detecting malodor in the mouth. Another object of the present invention is to provide a method of detecting malodor in the mouth which is useful both for home and health care professionals for assessing oral diseases and their response to treatment. Other objects and advantages of the invention will be apparent from the following description.

SUMMARY OF THE INVENTION It has now been unexpectedly found that the presence and degree of odor of halitosis can be obtained by measuring β-galactosidase enzyme levels in samples taken from the oral cavity. . It is not known that these enzymes are directly involved in halitosis odors, so it is surprising that their detection and measurement can be used as an indicator of the presence and severity of halitosis. Is. The invention firstly relates to a method for the rapid assessment of the odor level of halitosis, which comprises the steps of: a) collecting a sample of liquid and / or tissue from the subject's mouth, b) β present in the sample -Evaluating the amount of galactosidase, c) measuring the odor of the subject's halitosis by comparing the results obtained in step b) with the corresponding control values.

The term “appropriate reference values” refers to any quantitative or qualitative number that is indicative of the amount of β-galactosidase found in a sample, which is a series of discrete numbers. Alternatively, it is a graph in which a reference curve, for example, β-galactosidase level related to a sample collected from a target group, is drawn with respect to the result of another measurement of halitosis, and for example, an evaluation value of the odor of the whole mouth (odor determination based on the malodor intensity scale (Based on evaluation value). The term also refers to the use of mathematical equations to describe the above baseline curve. Furthermore, the term also provides a comparison of the β-galactosidase rating with a predetermined odor intensity scale based on such semi-quantitative β-galactosidase rating in the case of a semi-quantitative β-galactosidase level result. Include.

Use of any suitable liquid or tissue in practicing the methods of the present invention (eg, surface mucosal cells harvested by scraping the mucous membrane of the tongue or oral cavity, periodontal pocket exudate, etc.) However, saliva is a preferred species sample. . In another aspect, the invention provides a kit for rapidly measuring halitosis, which kit comprises: a) means for collecting liquid and / or tissue samples from the oral cavity; b) on a solid support. Substrates of β-galactosidase that have been adsorbed and, when degraded by β-galactosidase, undergo a change in color or other distinguishable property; c) to determine the odor of halitosis from the change in color of the solid support. Color or intensity chart or instructions;

Any suitable β-galactosidase substrate having the above properties can be used in the preparation of the kit of the present invention. However, the preferred substrate is X-gal (5-
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside). In a preferred embodiment of the kit of the invention, the solid support also comprises a gratuitous inducer of β-galactosidase. Although any of these gratuitous inducers can be used, in a preferred embodiment of the kit of the present invention, the inducer is IPTG (
Isopropyl β-D-thiogalactoside). All of the above and other features and advantages of the present invention are considered to be further understood from the following description and non-limiting examples illustrating the preferred embodiments thereof.

Detailed Description of the Preferred Embodiments Example 1 Beta-Galactosidase as an Indicator of Halitosis Active 4 subjects were tested for their odor intensity (increasing intensity scale from 0 to 5). Odor judgment evaluation value and sulfide monitor (Interscan
Corp., Chatsworth, Ca model 1170) based on measurements of volatile sulfides). In addition, beta-galactosidase activity levels were measured colorimetrically by the following method: A sample on the underside of the tongue was taken with a plastic spoon. The sample was separated from the spoon by washing one sample with 0.2 ml water and washing twice. X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and IPTG (isopropylthiogalactopyranoside) were added (0.05 ml of 20 mg / ml solution and 50 ml, respectively).
mg / ml solution 0.05 ml), then these samples were incubated in an ELISA plate at 37 degrees Celsius for 1 hour. The relative amount of enzyme activity,
Recorded as OD at 650 nm.

[0012]   The results of these measurements are shown in Table I.

[Table 1]

It is clear that the increase in the ELISA reading of beta-galactosidase activity is accompanied by an increase in the odor determination rating and the volatile sulfide leveler. Simply moisten the adsorbent disc containing the β-galactosidase activity detector,
It was also found that β-galactosidase activity in saliva or other oral samples can then be measured by incubating them at room temperature for a short time. By comparing the intensity of the color developed to a color standard, a semi-quantitative assessment of β-galactosidase activity in this oral sample can be determined.

Example 2 Colorimetric test using a paper disc A 5 mm adsorptive paper disc was cut from a sheet of Whatman Chromatographic Paper (Whatman Ltd., Maidstone, England). X-g
100 mg of al was dissolved in 5 ml of dimethylformamide. IP in 2 ml of water
A second solution of 100 mg TG was prepared. The two solutions were combined. 20 microliters of this collected solution was applied to each of the above paper discs. The disks were then dried for 24 hours prior to use and then fixed to a plastic backing material. To use this test, a small amount of saliva was collected from a subject and used to thoroughly moisten the treated disc. These discs were left at room temperature for 10 minutes. The color developed was evaluated using the standard color scale provided for the subject. Breath odor rating from each subject was also measured using a sulfide monitor. A significant degree of correlation was found between the test evaluation values.

Example 3 Use of Paper Discs for Surveillance Treatments with Oral Detergents Adsorbent paper discs moistened with X-al 20 mg / ml and IPTG 50 mg / ml were used in a self-administration test and contained the active ingredient. Respiratory odor was evaluated before and after the use of the respiratory cleansing oral cleanser by comparison with Pselabo oral cleanser. Disks were saturated with their saliva samples immediately before and 1 hour, 2 hours, and 3 hours after use of the treatment detergent or Puselab detergent. After standing for 5 minutes at room temperature, the intensity of the color developed on these discs was measured. Respiratory odor rating was also measured with a sulfide monitor and and expert panel. The results obtained with these three different assays confirmed the breath-cleansing effect of the treated product compared to the control.

Example 4 Enzymatic Evaluation Based on Paper Discs for Assessing Malodorous Odors The purpose of this test was to test the correlation with other types of oral malodorous parameters, such as odor assessment and sulfide monitor measurements. In addition, it was to test a simple enzymatic colorimetric method for detecting malodor in the mouth. In addition to colorimetric analysis, (functionality) measurements were made by two types of odor determination. Sulfide monitor measurements, microbial counts, BANA tests and indole tests were performed similarly. Subjects This study included 60 healthy young adult volunteers (mean age 23 ± 2 years, 35 women). Smokers or subjects who took antibiotics within a month prior to the study were not allowed to participate. The study was conducted according to an approved human subject plan, and participants signed a notice approval document.

Participants were asked to refrain from food or beverages for 2 hours before measurement. At first,
Subjects were tested for malodor related parameters: odor determination measurements, sulfide monitor levels, colorimetric and microbial counts. Subjects were randomly divided into 3 groups:
1) Treatment group: Active chewing gum [Breathanol (registered trade name)
, 2) placebo chewing gum [without Breathanol (registered trade name)], 3) control group (no treatment). Subjects were given chewing gum (or no treatment) and were asked to chew for 15 minutes. Subjects were reexamined at 1.5 and 3 hours after use. At the beginning of the study, subjects were asked for their own breathing opinions by measuring using the same scale as the odor rating (see below).

Measurement Parameters Color assay (OK2KA) paper discs (6 mm) were impregnated with an enzyme substrate as shown below: A 6 mm diameter disk was placed on a chromatographic paper [Whatman paper, Whatman paper, no. 3] to prepare a test paper. Two solutions were prepared. One of these solutions is N, N-dimethylformamide (Sigma) 2m
5-bromo-4-chloro-3-indolyl-D-galactopyranoside 10 in 1
Prepared by dissolving 0 mg (X-Gal, Sigma) and another one is 100 mg isopropyl β-D-thiogalactoside (I) in 2 ml double distilled water.
It was prepared by dissolving PTG, Sigma). 100 μl from each solution were combined together and swirled, then 2 μl of this mixture was impregnated on each paper disc. The discs were dried overnight at 37 ° C. Saliva (whole, unstimulated) was collected from each subject at the start of the study (before treatment) and at 1.5 and 3 hours. A 20 μl drop of each saliva sample was applied to a paper disc, then incubated for 10 minutes at room temperature and after 10 minutes the results were recorded as follows: 0-colorless, 1-light color, 2-dark color.

BANA and Indole Generation Analysis BANA Reagent Card [Perioscan ™, Oral-B Laboratories,
Redwood city, CA] and indole generated slides [DrySlide ™ INDOLE, Difco Laboratories, Detroit, MI] were used according to the manufacturer's instructions. Samples for these analyzes were obtained from the same posterior dorsal scraping used to measure tongue odor ratings by odor determination. Results were recorded by either strong response = 2, weak response-1 or no color change = 0.

Sulfide monitor The volatile sulfide compound (VSC) in the upper oral cavity was measured using a sulfide monitor (Model 1170, Interscan). Subjects were asked to refrain from speaking for 5 minutes before measurement. The monitor was zeroed in air and then inserted by inserting a disposable 1/4 inch plastic straw up to about 4 cm into the partially opened oral cavity. During the measurement period, subjects were asked to breathe through their nose. Results were recorded as peak ppb sulfide equivalents.

Organoleptic measurements Two odor ratings were measured for malodor over the mouth and malodor on the tongue. The subject evaluation of the entire mouth required short breathing through the mouth at a distance of about 10 cm from the nose of the judge. Tongue malodor was assessed by scraping the material from a remote backside area of the back of the tongue using a plastic spoon, scooping, and then assessing the malodor from this spoon by both judgments. The judgment evaluation value is 0 to
Recorded using a half-integer number of 5: 0, no perceptible odor; 1, barely perceptible odor; 2, slight but not clearly perceptible odor; 3, moderate odor; 4
, Strong odor; 5, exceptionally unclean odor.

Microbial Counting The number of organisms from saliva samples was determined by trypsin soy agar (TSA) and mild saliva agar (MSA) containing Diaslide (Diasl).
ide) (Savyon Diagnostics, Ashdod, Israel). Diasteride was incubated at 370C for 72 hours under anaerobic conditions. The number of organisms is T
All of SA and MSA, and the number of blue colonies formed on MSA were included.

Statistical Analysis The Spearman correlation coefficient was used to determine the degree of association between various parameters. One way analysis of variance (ANOVA)
Was used to compare the colorimetric results (0, 1 and 2) in terms of other parameters. Perform stepwise multiple regression analysis,
The contribution of the expected colorimetric test results and sulfide monitor results to the odor assessment value was tested. Results The Spearman correlation coefficient comparing the colorimetric test evaluation value at three measurements (time point 0, 1.5 hour time point and 3 hour time point) with another parameter is shown in the table.
Shown in II. In this table, the apparent p-values are shown below each r-value.

[0024]

[Table 2]

Among various tests, the OK2KS evaluation value was most significantly associated with the odor of the whole mouth (p <0.007) and the odor of the tongue (p <0.005). A significant correlation
Between monitor measurements and OK2KS at the last two time points (p <0.00
5), and between the number of microorganisms and OK2KS at the first two time points (p <0.
． 013), a significant correlation was found. In contrast, OK2KS and BAN
No significant correlation was found with A or indole formation analysis (
p <0.055).

The correlation between OK2KS rating, sulfide monitor level, BANA test result and microbial count (MSA) was compared to the odor assessment rating in Table III below.
The correlation between the odor determination rating and the OK2KS rating is in all cases as great as or even greater than the corresponding correlation between the sulfide monitor measurements. The correlation between the sensory rating and the BANA test was almost insignificant, as was the correlation between the bacterial count on MSA and the odor determination rating. Indole measurements were not significantly associated with odor rating (not shown)
.

[0027]

[Table 3]

From the viewpoint of the colorimetric analysis evaluation value and the reading of the log monitor, the sequential multiple regression analysis of the odor determination evaluation value (at time 0) relating to the odor of the whole mouth and tongue is shown in Table IV.

[0029]

[Table 4]

Both the sulphide monitor readings and the OK2KS rating were significantly factored to a regression equation of the odor determination rating for both whole mouth odor and tongue odor, 0.47 (decision 2, tongue p. = 0.0007) to 0.60 (judgment 2, whole mouth p <
Multiple r values in the range up to 0.0001) were obtained.

The above results show that at all three time points during the test, OK2KS has a highly significant correlation with the odor assessment values related to the odor of the whole mouth and the k odor of the tongue. . Furthermore, the correlation between OK2KS and sensory rating was as significant or more significant as the corresponding correlation between sulfide monitor measurements and sensory rating. If you try multiple regression analysis, OK2
Considering the odor evaluation value by KS and sulfide monitor, both of these parameters were inserted into the regression equation to obtain a multiple r value up to 0.6. These results show that (1) OK2KS can be used as a test method associated with odor assessment values; and (2) OK2KS is used in conjunction with sulfide monitor assay methods to correlate with odor assessment values. Sex can be improved; O
The K2KS method is expected to be convenient and inexpensive, thus making it a useful method for both clinical and home settings.

While particular embodiments of the present invention have been described for purposes of illustration, the present invention may be practiced by those skilled in the art with many modifications, changes and adaptations without departing from the scope of the claims or departing from the spirit thereof. It should be understood that what can be done to.

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Claims (6)

[Claims] 1. A method for rapidly assessing the degree of bad breath odor, comprising: a) collecting a sample of liquid and / or tissue from the subject's oral cavity; b) the amount of β-galactosidase present in the sample. And c) measuring the degree of bad breath odor in the subject by comparing the result obtained in step b) with a corresponding control value. 2. The method of claim 1, wherein the liquid sample is saliva. 3. A kit for rapid assessment of halitosis, comprising: a) means for collecting a liquid and / or tissue sample from the oral cavity; b) adsorbed on a solid support, A substrate for β-galactosidase which, when degraded by galactosidase, undergoes a change in color or other discernible property; c) from the change in color and / or intensity of the solid support to determine the odor of halitosis. A color or intensity chart or instructions; 4. The β-galactosidase substrate is 5-bromo-4-chloro-3.
The kit according to claim 3, which is -indolyl-β-D-galactopyranoside (X-gal). 5. The kit of claim 3, wherein the solid support further comprises a gratuitous inducer of β-galactosidase. 6. The gratuitous inducer of β-galactosidase is isopropyl β
The kit according to claim 5, which is -D-thiogalactoside (IPTG).